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Bonnie Bartel

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DOI: 10.1146/annurev.arplant.57.032905.105218
2006
Cited 2,439 times
MicroRNAs AND THEIR REGULATORY ROLES IN PLANTS
MicroRNAs (miRNAs) are small, endogenous RNAs that regulate gene expression in plants and animals. In plants, these approximately 21-nucleotide RNAs are processed from stem-loop regions of long primary transcripts by a Dicer-like enzyme and are loaded into silencing complexes, where they generally direct cleavage of complementary mRNAs. Although plant miRNAs have some conserved functions extending beyond development, the importance of miRNA-directed gene regulation during plant development is now particularly clear. Identified in plants less than four years ago, miRNAs are already known to play numerous crucial roles at each major stage of development-typically at the cores of gene regulatory networks, targeting genes that are themselves regulators, such as those encoding transcription factors and F-box proteins.
DOI: 10.1016/s0092-8674(02)00863-2
2002
Cited 2,110 times
Prediction of Plant MicroRNA Targets
We predict regulatory targets for 14 Arabidopsis microRNAs (miRNAs) by identifying mRNAs with near complementarity. Complementary sites within predicted targets are conserved in rice. Of the 49 predicted targets, 34 are members of transcription factor gene families involved in developmental patterning or cell differentiation. The near-perfect complementarity between plant miRNAs and their targets suggests that many plant miRNAs act similarly to small interfering RNAs and direct mRNA cleavage. The targeting of developmental transcription factors suggests that many plant miRNAs function during cellular differentiation to clear key regulatory transcripts from daughter cell lineages.
DOI: 10.1101/gad.1004402
2002
Cited 1,746 times
MicroRNAs in plants
MicroRNAs (miRNAs) are an extensive class of ~22-nucleotide noncoding RNAs thought to regulate gene expression in metazoans. We find that miRNAs are also present in plants, indicating that this class of noncoding RNA arose early in eukaryotic evolution. In this paper 16 Arabidopsis miRNAs are described, many of which have differential expression patterns in development. Eight are absolutely conserved in the rice genome. The plant miRNA loci potentially encode stem-loop precursors similar to those processed by Dicer (a ribonuclease III) in animals. Mutation of an Arabidopsis Dicer homolog, CARPEL FACTORY, prevents the accumulation of miRNAs, showing that similar mechanisms direct miRNA processing in plants and animals. The previously described roles of CARPEL FACTORY in the development of Arabidopsis embryos, leaves, and floral meristems suggest that the miRNAs could play regulatory roles in the development of plants as well as animals.
DOI: 10.1261/rna.2183803
2003
Cited 1,724 times
A uniform system for microRNA annotation
MicroRNAs (miRNAs) are small noncoding RNA gene products about 22 nt long that are processed by Dicer from precursors with a characteristic hairpin secondary structure. Guidelines are presented for the identification and annotation of new miRNAs from diverse organisms, particularly so that miRNAs can be reliably distinguished from other RNAs such as small interfering RNAs. We describe specific criteria for the experimental verification of miRNAs, and conventions for naming miRNAs and miRNA genes. Finally, an online clearinghouse for miRNA gene name assignments is provided by the Rfam database of RNA families.
DOI: 10.1105/tpc.108.064311
2008
Cited 1,118 times
Criteria for Annotation of Plant MicroRNAs
MicroRNAs (miRNAs) are approximately 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs play critical roles in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interactions with specific target mRNAs. miRNAs are not the only Dicer-derived small RNAs produced by plants: A substantial amount of the total small RNA abundance and an overwhelming amount of small RNA sequence diversity is contributed by distinct classes of 21- to 24-nucleotide short interfering RNAs. This fact, coupled with the rapidly increasing rate of plant small RNA discovery, demands an increased rigor in miRNA annotations. Herein, we update the specific criteria required for the annotation of plant miRNAs, including experimental and computational data, as well as refinements to standard nomenclature.
DOI: 10.1105/tpc.105.031716
2005
Cited 823 times
MicroRNA-Directed Regulation of Arabidopsis<i>AUXIN RESPONSE FACTOR17</i>Is Essential for Proper Development and Modulates Expression of Early Auxin Response Genes
Abstract The phytohormone auxin plays critical roles during plant growth, many of which are mediated by the auxin response transcription factor (ARF) family. MicroRNAs (miRNAs), endogenous 21-nucleotide riboregulators, target several mRNAs implicated in auxin responses. miR160 targets ARF10, ARF16, and ARF17, three of the 23 Arabidopsis thaliana ARF genes. Here, we describe roles of miR160-directed ARF17 posttranscriptional regulation. Plants expressing a miRNA-resistant version of ARF17 have increased ARF17 mRNA levels and altered accumulation of auxin-inducible GH3-like mRNAs, YDK1/GH3.2, GH3.3, GH3.5, and DFL1/GH3.6, which encode auxin-conjugating proteins. These expression changes correlate with dramatic developmental defects, including embryo and emerging leaf symmetry anomalies, leaf shape defects, premature inflorescence development, altered phyllotaxy along the stem, reduced petal size, abnormal stamens, sterility, and root growth defects. These defects demonstrate the importance of miR160-directed ARF17 regulation and implicate ARF17 as a regulator of GH3-like early auxin response genes. Many of these defects resemble phenotypes previously observed in plants expressing viral suppressors of RNA silencing and plants with mutations in genes important for miRNA biogenesis or function, providing a molecular rationale for phenotypes previously associated with more general disruptions of miRNA function.
DOI: 10.1038/338394a0
1989
Cited 671 times
The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis
DOI: 10.1016/j.cub.2004.06.022
2004
Cited 640 times
MicroRNA Regulation of NAC-Domain Targets Is Required for Proper Formation and Separation of Adjacent Embryonic, Vegetative, and Floral Organs
MicroRNAs (miRNAs) are approximately 21 nucleotide (nt) RNAs that regulate gene expression in plants and animals. Most known plant miRNAs target transcription factors that influence cell fate determination, and biological functions of miRNA-directed regulation have been reported for four of 15 known microRNA gene families: miR172, miR159, miR165, and miR168. Here, we identify a developmental role for miR164-directed regulation of NAC-domain genes, which encode a family of transcription factors that includes CUP-SHAPED COTYLEDON1 (CUC1) and CUC2.Expression of a miR164-resistant version of CUC1 mRNA from the CUC1 promoter causes alterations in Arabidopsis embryonic, vegetative, and floral development, including cotyledon orientation defects, reduction of rosette leaf petioles, dramatically misshapen rosette leaves, one to four extra petals, and one or two missing sepals. Reciprocally, constitutive overexpression of miR164 recapitulates cuc1 cuc2 double mutant phenotypes, including cotyledon and floral organ fusions. miR164 overexpression also leads to phenotypes not previously observed in cuc1 cuc2 mutants, including leaf and stem fusions. These likely reflect the misregulation of other NAC-domain mRNAs, including NAC1, At5g07680, and At5g61430, for which miR164-directed cleavage products were detected.These results demonstrate that miR164-directed regulation of CUC1 is necessary for normal embryonic, vegetative, and floral development. They also show that proper miR164 dosage or localization is required for separation of adjacent embryonic, vegetative, and floral organs, thus implicating miR164 as a common regulatory component of the molecular circuitry that controls the separation of different developing organs and thereby exposes a posttranscriptional layer of NAC-domain gene regulation during plant development.
DOI: 10.1016/s0092-8674(00)80842-9
2000
Cited 449 times
FKF1, a Clock-Controlled Gene that Regulates the Transition to Flowering in Arabidopsis
Plant reproduction requires precise control of flowering in response to environmental cues. We isolated a late-flowering Arabidopsis mutant, fkf1, that is rescued by vemalization or gibberellin treatment. We positionally cloned FKF1, which encodes a novel protein with a PAS domain similar to the flavin-binding region of certain photoreceptors, an F box characteristic of proteins that direct ubiquitin-mediated degradation, and six kelch repeats predicted to fold into a beta propeller. FKF1 mRNA levels oscillate with a circadian rhythm, and deletion of FKF1 alters the waveform of rhythmic expression of two clock-controlled genes, implicating FKF1 in modulating the Arabidopsis circadian clock.
DOI: 10.1104/pp.103.023630
2003
Cited 396 times
MicroRNAs: At the Root of Plant Development?
Although most genes use RNA in the form of mRNA as a coding intermediate for protein production, there are many genes whose final products are RNA. These noncoding RNAs range from the familiar transfer and ribosomal RNAs to the more recently discovered regulatory RNAs. One type of regulatory RNA was
DOI: 10.1105/tpc.112.096586
2012
Cited 376 times
Plant Peroxisomes: Biogenesis and Function
Peroxisomes are eukaryotic organelles that are highly dynamic both in morphology and metabolism. Plant peroxisomes are involved in numerous processes, including primary and secondary metabolism, development, and responses to abiotic and biotic stresses. Considerable progress has been made in the identification of factors involved in peroxisomal biogenesis, revealing mechanisms that are both shared with and diverged from non-plant systems. Furthermore, recent advances have begun to reveal an unexpectedly large plant peroxisomal proteome and have increased our understanding of metabolic pathways in peroxisomes. Coordination of the biosynthesis, import, biochemical activity, and degradation of peroxisomal proteins allows for highly dynamic responses of peroxisomal metabolism to meet the needs of a plant. Knowledge gained from plant peroxisomal research will be instrumental to fully understanding the organelle's dynamic behavior and defining peroxisomal metabolic networks, thus allowing the development of molecular strategies for rational engineering of plant metabolism, biomass production, stress tolerance, and pathogen defense.
DOI: 10.1105/tpc.13.3.465
2001
Cited 376 times
A Gain-of-Function Mutation in <i>IAA28</i> Suppresses Lateral Root Development
The phytohormone auxin is important in many aspects of plant development. We have isolated an auxin-resistant Arabidopsis mutant, iaa28-1, that is severely defective in lateral root formation and that has diminished adult size and decreased apical dominance. The iaa28-1 mutant is resistant to inhibition of root elongation by auxin, cytokinin, and ethylene, but it responds normally to other phytohormones. We identified the gene defective in the iaa28-1 mutant by using a map-based positional approach and found it to encode a previously uncharacterized member of the Aux/IAA gene family. IAA28 is preferentially expressed in roots and inflorescence stems, and in contrast to other Aux/IAA genes, IAA28 transcription is not induced by exogenous auxin. Studies of the gain-of-function iaa28-1 mutant suggest that IAA28 normally represses transcription, perhaps of genes that promote lateral root initiation in response to auxin signals.
DOI: 10.1002/j.1460-2075.1990.tb07516.x
1990
Cited 368 times
The recognition component of the N-end rule pathway.
Research Article1 October 1990free access The recognition component of the N-end rule pathway. B. Bartel B. Bartel Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. Search for more papers by this author I. Wünning I. Wünning Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. Search for more papers by this author A. Varshavsky A. Varshavsky Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. Search for more papers by this author B. Bartel B. Bartel Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. Search for more papers by this author I. Wünning I. Wünning Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. Search for more papers by this author A. Varshavsky A. Varshavsky Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. Search for more papers by this author Author Information B. Bartel1, I. Wünning1 and A. Varshavsky1 1Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. The EMBO Journal (1990)9:3179-3189https://doi.org/10.1002/j.1460-2075.1990.tb07516.x PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The N-end rule-based degradation signal, which targets a protein for ubiquitin-dependent proteolysis, comprises a destabilizing amino-terminal residue and a specific internal lysine residue. We report the isolation and functional analysis of a gene (UBR1) for the N-end recognizing protein of the yeast Saccharomyces cerevisiae. UBR1 encodes a approximately 225 kd protein with no significant sequence similarities to other known proteins. Null ubr1 mutants are viable but are unable to degrade the substrates of the N-end rule pathway. These mutants are partially defective in sporulation and grow slightly more slowly than their wild-type counterparts. The UBR1 protein specifically binds in vitro to proteins bearing amino-terminal residues that are destabilizing according to the N-end rule, but does not bind to otherwise identical proteins bearing stabilizing amino-terminal residues. Previous ArticleNext Article Volume 9Issue 101 October 1990In this issue RelatedDetailsLoading ...
DOI: 10.1016/j.pbi.2006.03.004
2006
Cited 332 times
Biosynthetic diversity in plant triterpene cyclization
Plants produce a wealth of terpenoids, many of which have been the tools of healers and chiefs for millennia. Recent research has led to the identification and characterization of many genes that are responsible for the biosynthesis of triterpenoids. Cyclases that generate sterol precursors can be recognized with some confidence on the basis of sequence; several catalytically important residues are now known, and the product profiles of sterol-generating cyclases typically reflect their phylogenetic position. By contrast, the phylogenetic relationships of cyclases that generate nonsteroidal triterpene alcohols do not consistently reflect their catalytic properties and might indicate recent and rapid catalytic evolution.
DOI: 10.1146/annurev.arplant.48.1.51
1997
Cited 277 times
AUXIN BIOSYNTHESIS
Indole-3-acetic acid (IAA) is the most abundant naturally occurring auxin. Plants produce active IAA both by de novo synthesis and by releasing IAA from conjugates. This review emphasizes recent genetic experiments and complementary biochemical analyses that are beginning to unravel the complexities of IAA biosynthesis in plants. Multiple pathways exist for de novo IAA synthesis in plants, and a number of plant enzymes can liberate IAA from conjugates. This multiplicity has contributed to the current situation in which no pathway of IAA biosynthesis in plants has been unequivocally established. Genetic and biochemical experiments have demonstrated both tryptophan-dependent and tryptophan-independent routes of IAA biosynthesis. The recent application of precise and sensitive methods for quantitation of IAA and its metabolites to plant mutants disrupted in various aspects of IAA regulation is beginning to elucidate the multiple pathways that control IAA levels in the plant.
DOI: 10.1074/jbc.m111955200
2002
Cited 274 times
Characterization of a Family of IAA-Amino Acid Conjugate Hydrolases from Arabidopsis
The mechanisms by which plants regulate levels of the phytohormone indole-3-acetic acid (IAA) are complex and not fully understood. One level of regulation appears to be the synthesis and hydrolysis of IAA conjugates, which function in both the permanent inactivation and temporary storage of auxin. Similar to free IAA, certain IAA-amino acid conjugates inhibit root elongation. We have tested the ability of 19 IAA-l-amino acid conjugates to inhibit <i>Arabidopsis</i> seedling root growth. We have also determined the ability of purified glutathione <i>S</i>-transferase (GST) fusions of four <i>Arabidopsis</i>IAA-amino acid hydrolases (ILR1, IAR3, ILL1, and ILL2) to release free IAA by cleaving these conjugates. Each hydrolase cleaves a subset of IAA-amino acid conjugates <i>in vitro</i>, and GST-ILR1, GST-IAR3, and GST-ILL2 have <i>K</i><sub>m</sub> values that suggest physiological relevance. <i>In vivo</i> inhibition of root elongation correlates with <i>in vitro</i> hydrolysis rates for each conjugate, suggesting that the identified hydrolases generate the bioactivity of the conjugates.
DOI: 10.1093/genetics/156.3.1323
2000
Cited 271 times
Genetic Analysis of Indole-3-butyric Acid Responses in<i>Arabidopsis thaliana</i>Reveals Four Mutant Classes
Abstract Indole-3-butyric acid (IBA) is widely used in agriculture because it induces rooting. To better understand the in vivo role of this endogenous auxin, we have identified 14 Arabidopsis mutants that are resistant to the inhibitory effects of IBA on root elongation, but that remain sensitive to the more abundant auxin indole-3-acetic acid (IAA). These mutants have defects in various IBA-mediated responses, which allowed us to group them into four phenotypic classes. Developmental defects in the absence of exogenous sucrose suggest that some of these mutants are impaired in peroxisomal fatty acid chain shortening, implying that the conversion of IBA to IAA is also disrupted. Other mutants appear to have normal peroxisomal function; some of these may be defective in IBA transport, signaling, or response. Recombination mapping indicates that these mutants represent at least nine novel loci in Arabidopsis. The gene defective in one of the mutants was identified using a positional approach and encodes PEX5, which acts in the import of most peroxisomal matrix proteins. These results indicate that in Arabidopsis thaliana, IBA acts, at least in part, via its conversion to IAA.
DOI: 10.1126/science.7792599
1995
Cited 262 times
ILR1, an Amidohydrolase That Releases Active Indole-3-Acetic Acid from Conjugates
In plants, the growth regulator indole-3-acetic acid (IAA) is found both free and conjugated to a variety of amino acids, peptides, and carbohydrates. IAA conjugated to leucine has effects in Arabidopsis thaliana similar to those of free IAA. The ilr1 mutant is insensitive to exogenous IAA-Leu and was used to positionally clone the Arabidopsis ILR1 gene. ILR1 encodes a 48-kilodalton protein that cleaves IAA-amino acid conjugates in vitro and is homologous to bacterial amidohydrolase enzymes. DNA sequences similar to that of ILR1 are found in other plant species.
DOI: 10.1104/pp.010550
2001
Cited 262 times
The Arabidopsis <i>pxa1</i> Mutant Is Defective in an ATP-Binding Cassette Transporter-Like Protein Required for Peroxisomal Fatty Acid β-Oxidation
Abstract Peroxisomes are important organelles in plant metabolism, containing all the enzymes required for fatty acid β-oxidation. More than 20 proteins are required for peroxisomal biogenesis and maintenance. The Arabidopsis pxa1 mutant, originally isolated because it is resistant to the auxin indole-3-butyric acid (IBA), developmentally arrests when germinated without supplemental sucrose, suggesting defects in fatty acid β-oxidation. Because IBA is converted to the more abundant auxin, indole-3-acetic acid (IAA), in a mechanism that parallels β-oxidation, the mutant is likely to be IBA resistant because it cannot convert IBA to IAA. Adultpxa1 plants grow slowly compared with wild type, with smaller rosettes, fewer leaves, and shorter inflorescence stems, indicating that PXA1 is important throughout development. We identified the molecular defect in pxa1 using a map-based positional approach. PXA1 encodes a predicted peroxisomal ATP-binding cassette transporter that is 42% identical to the human adrenoleukodystrophy (ALD) protein, which is defective in patients with the demyelinating disorder X-linked ALD. Homology to ALD protein and other human and yeast peroxisomal transporters suggests that PXA1 imports coenzyme A esters of fatty acids and IBA into the peroxisome for β-oxidation. The pxa1 mutant makes fewer lateral roots than wild type, both in response to IBA and without exogenous hormones, suggesting that the IAA derived from IBA during seedling development promotes lateral root formation.
DOI: 10.1073/pnas.88.16.7351
1991
Cited 256 times
The N-end rule is mediated by the UBC2(RAD6) ubiquitin-conjugating enzyme.
The N-end rule relates the in vivo half-life of a protein to the identity of its amino-terminal residue. Distinct versions of the N-end rule operate in all organisms examined, from mammals to bacteria. We show that UBC2(RAD6), one of at least seven ubiquitin-conjugating enzymes in the yeast Saccharomyces cerevisiae, is essential for multiubiquitination and degradation of the N-end rule substrates. We also show that UBC2 is physically associated with UBR1, the recognition component of the N-end rule pathway. These results indicate that some of the UBC2 functions, which include DNA repair, induced mutagenesis, sporulation, and regulation of retrotransposition, are mediated by protein degradation via the N-end rule pathway.
DOI: 10.1002/j.1460-2075.1992.tb05080.x
1992
Cited 252 times
Ubiquitin as a degradation signal.
Research Article1 February 1992free access Ubiquitin as a degradation signal. E.S. Johnson E.S. Johnson Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. Search for more papers by this author B. Bartel B. Bartel Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. Search for more papers by this author W. Seufert W. Seufert Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. Search for more papers by this author A. Varshavsky A. Varshavsky Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. Search for more papers by this author E.S. Johnson E.S. Johnson Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. Search for more papers by this author B. Bartel B. Bartel Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. Search for more papers by this author W. Seufert W. Seufert Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. Search for more papers by this author A. Varshavsky A. Varshavsky Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. Search for more papers by this author Author Information E.S. Johnson1, B. Bartel1, W. Seufert1 and A. Varshavsky1 1Department of Biology, Massachusetts Institute of Technology, Cambridge 02139. The EMBO Journal (1992)11:497-505https://doi.org/10.1002/j.1460-2075.1992.tb05080.x PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info For many short-lived eukaryotic proteins, conjugation to ubiquitin, yielding a multiubiquitin chain, is an obligatory pre-degradation step. The conjugated ubiquitin moieties function as a ‘secondary’ signal for degradation, in that their posttranslational coupling to a substrate protein is mediated by amino acid sequences of the substrate that act as a primary degradation signal. We report that the fusion protein ubiquitin--proline--beta-galactosidase (Ub-P-beta gal) is short-lived in the yeast Saccharomyces cerevisiae because its N-terminal ubiquitin moiety functions as an autonomous, primary degradation signal. This signal mediates the formation of a multiubiquitin chain linked to Lys48 of the N-terminal ubiquitin in Ub-P-beta gal. The degradation of Ub-P-beta gal is shown to require Ubc4, one of at least seven ubiquitin-conjugating enzymes in S.cerevisiae. Our findings provide the first direct evidence that a monoubiquitin moiety can function as an autonomous degradation signal. This generally applicable, cis-acting signal can be used to manipulate the in vivo half-lives of specific intracellular proteins. Previous ArticleNext Article Volume 11Issue 21 February 1992In this issue RelatedDetailsLoading ...
DOI: 10.1016/j.pbi.2004.07.011
2004
Cited 240 times
MicroRNA regulation of gene expression in plants
It has only been a few years since we began to appreciate that microRNAs provide an unanticipated level of gene regulation in both plants and metazoans. The high level of complementarity between plant microRNAs and their target mRNAs has allowed rapid progress towards the elucidation of their varied biological functions. MicroRNAs have been shown to regulate diverse developmental processes, including organ separation, polarity, and identity, and to modulate their own biogenesis and function. Recently, they have also been implicated in some processes outside of plant development.
DOI: 10.1007/s11103-008-9329-1
2008
Cited 226 times
Sucrose induction of Arabidopsis miR398 represses two Cu/Zn superoxide dismutases
DOI: 10.1105/tpc.11.3.365
1999
Cited 224 times
<i>IAR3</i> Encodes an Auxin Conjugate Hydrolase from Arabidopsis
Amide-linked conjugates of indole-3-acetic acid (IAA) are putative storage or inactivation forms of the growth hormone auxin. Here, we describe the Arabidopsis iar3 mutant that displays reduced sensitivity to IAA-Ala. IAR3 is a member of a family of Arabidopsis genes related to the previously isolated ILR1 gene, which encodes an IAA-amino acid hydrolase selective for IAA-Leu and IAA-Phe. IAR3 and the very similar ILL5 gene are closely linked on chromosome 1 and comprise a subfamily of the six Arabidopsis IAA-conjugate hydrolases. The purified IAR3 enzyme hydrolyzes IAA-Ala in vitro. iar 3 ilr1 double mutants are more resistant than either single mutant to IAA-amino acid conjugates, and plants overexpressing IAR3 or ILR1 are more sensitive than is the wild type to certain IAA-amino acid conjugates, reflecting the overlapping substrate specificities of the corresponding enzymes. The IAR3 gene is expressed most strongly in roots, stems, and flowers, suggesting roles for IAA-conjugate hydrolysis in those tissues.
DOI: 10.1104/pp.104.039677
2004
Cited 220 times
A Family of Auxin-Conjugate Hydrolases That Contributes to Free Indole-3-Acetic Acid Levels during Arabidopsis Germination
Auxins are hormones important for numerous processes throughout plant growth and development. Plants use several mechanisms to regulate levels of the auxin indole-3-acetic acid (IAA), including the formation and hydrolysis of amide-linked conjugates that act as storage or inactivation forms of the hormone. Certain members of an Arabidopsis amidohydrolase family hydrolyze these conjugates to free IAA in vitro. We examined amidohydrolase gene expression using northern and promoter-beta-glucuronidase analyses and found overlapping but distinct patterns of expression. To examine the in vivo importance of auxin-conjugate hydrolysis, we generated a triple hydrolase mutant, ilr1 iar3 ill2, which is deficient in three of these hydrolases. We compared root and hypocotyl growth of the single, double, and triple hydrolase mutants on IAA-Ala, IAA-Leu, and IAA-Phe. The hydrolase mutant phenotypic profiles on different conjugates reveal the in vivo activities and relative importance of ILR1, IAR3, and ILL2 in IAA-conjugate hydrolysis. In addition to defective responses to exogenous conjugates, ilr1 iar3 ill2 roots are slightly less responsive to exogenous IAA. The triple mutant also has a shorter hypocotyl and fewer lateral roots than wild type on unsupplemented medium. As suggested by the mutant phenotypes, ilr1 iar3 ill2 imbibed seeds and seedlings have lower IAA levels than wild type and accumulate IAA-Ala and IAA-Leu, conjugates that are substrates of the absent hydrolases. These results indicate that amidohydrolases contribute free IAA to the auxin pool during germination in Arabidopsis.
DOI: 10.1073/pnas.90.24.11628
1993
Cited 214 times
Isolation of an Arabidopsis thaliana gene encoding cycloartenol synthase by functional expression in a yeast mutant lacking lanosterol synthase by the use of a chromatographic screen.
Whereas vertebrates and fungi synthesize sterols from epoxysqualene through the intermediate lanosterol, plants cyclize epoxysqualene to cycloartenol as the initial sterol. We report the cloning and characterization of CAS1, an Arabidopsis thaliana gene encoding cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A yeast mutant lacking lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] was transformed with an A. thaliana cDNA yeast expression library, and colonies were assayed for epoxysqualene mutase activity by thin-layer chromatography. One out of approximately 10,000 transformants produced a homogenate that cyclized 2,3-epoxysqualene to the plant sterol cycloartenol. This activity was shown to be plasmid dependent. The plasmid insert contains a 2277-bp open reading frame capable of encoding an 86-kDa protein with significant homology to lanosterol synthase from Candida albicans and squalene-hopene cyclase (EC 5.4.99.-) from Bacillus acidocalcarius. The method used to clone this gene should be generally applicable to genes responsible for secondary metabolite biosynthesis.
DOI: 10.1073/pnas.91.14.6649
1994
Cited 195 times
Differential regulation of an auxin-producing nitrilase gene family in Arabidopsis thaliana.
Nitrilases (nitrile aminohydrolase, EC 3.5.5.1) convert nitriles to carboxylic acids. We report the cloning, characterization, and expression patterns of four Arabidopsis thaliana nitrilase genes (NIT1-4), one of which was previously described [Bartling, D., Seedorf, M., Mithöfer, A. & Weiler, E. W. (1992) Eur. J. Biochem. 205, 417-424]. The nitrilase genes encode very similar proteins that hydrolyze indole-3-acetonitrile to the phytohormone indole-3-acetic acid in vitro, and three of the four genes are tandemly arranged on chromosome III. Northern analysis using gene-specific probes and analysis of transgenic plants containing promoter-reporter gene fusions indicate that the four genes are differentially regulated. NIT2 expression is specifically induced around lesions caused by bacterial pathogen infiltration. The sites of nitrilase expression may represent sites of auxin biosynthesis in A. thaliana.
DOI: 10.1104/pp.104.049577
2005
Cited 186 times
An Auxin Transport Independent Pathway Is Involved in Phosphate Stress-Induced Root Architectural Alterations in Arabidopsis. Identification of <i>BIG</i> as a Mediator of Auxin in Pericycle Cell Activation
Arabidopsis (Arabidopsis thaliana) plants display a number of root developmental responses to low phosphate availability, including primary root growth inhibition, greater formation of lateral roots, and increased root hair elongation. To gain insight into the regulatory mechanisms by which phosphorus (P) availability alters postembryonic root development, we performed a mutant screen to identify genetic determinants involved in the response to P deprivation. Three low phosphate-resistant root lines (lpr1-1 to lpr1-3) were isolated because of their reduced lateral root formation in low P conditions. Genetic and molecular analyses revealed that all lpr1 mutants were allelic to BIG, which is required for normal auxin transport in Arabidopsis. Detailed characterization of lateral root primordia (LRP) development in wild-type and lpr1 mutants revealed that BIG is required for pericycle cell activation to form LRP in both high (1 mm) and low (1 microm) P conditions, but not for the low P-induced alterations in primary root growth, lateral root emergence, and root hair elongation. Exogenously supplied auxin restored normal lateral root formation in lpr1 mutants in the two P treatments. Treatment of wild-type Arabidopsis seedlings with brefeldin A, a fungal metabolite that blocks auxin transport, phenocopies the root developmental alterations observed in lpr1 mutants in both high and low P conditions, suggesting that BIG participates in vesicular targeting of auxin transporters. Taken together, our results show that auxin transport and BIG function have fundamental roles in pericycle cell activation to form LRP and promote root hair elongation. The mechanism that activates root system architectural alterations in response to P deprivation, however, seems to be independent of auxin transport and BIG.
DOI: 10.1007/s003440010025
2001
Cited 184 times
Inputs to the Active Indole-3-Acetic Acid Pool: De Novo Synthesis, Conjugate Hydrolysis, and Indole-3-Butyric Acid b-Oxidation
DOI: 10.1093/mp/ssr006
2011
Cited 184 times
Transport and Metabolism of the Endogenous Auxin Precursor Indole-3-Butyric Acid
Plant growth and morphogenesis depend on the levels and distribution of the plant hormone auxin. Plants tightly regulate cellular levels of the active auxin indole-3-acetic acid (IAA) through synthesis, inactivation, and transport. Although the transporters that move IAA into and out of cells are well characterized and play important roles in development, little is known about the transport of IAA precursors. In this review, we discuss the accumulating evidence suggesting that the IAA precursor indole-3-butyric acid (IBA) is transported independently of the characterized IAA transport machinery along with the recent identification of specific IBA efflux carriers and enzymes suggested to metabolize IBA. These studies have revealed important roles for IBA in maintaining IAA levels and distribution within the plant to support normal development.
DOI: 10.1105/tpc.109.065821
2009
Cited 184 times
The<i>Arabidopsis</i>PLEIOTROPIC DRUG RESISTANCE8/ABCG36 ATP Binding Cassette Transporter Modulates Sensitivity to the Auxin Precursor Indole-3-Butyric Acid
Abstract Plants have developed numerous mechanisms to store hormones in inactive but readily available states, enabling rapid responses to environmental changes. The phytohormone auxin has a number of storage precursors, including indole-3-butyric acid (IBA), which is apparently shortened to active indole-3-acetic acid (IAA) in peroxisomes by a process similar to fatty acid β-oxidation. Whereas metabolism of auxin precursors is beginning to be understood, the biological significance of the various precursors is virtually unknown. We identified an Arabidopsis thaliana mutant that specifically restores IBA, but not IAA, responsiveness to auxin signaling mutants. This mutant is defective in PLEIOTROPIC DRUG RESISTANCE8 (PDR8)/PENETRATION3/ABCG36, a plasma membrane–localized ATP binding cassette transporter that has established roles in pathogen responses and cadmium transport. We found that pdr8 mutants display defects in efflux of the auxin precursor IBA and developmental defects in root hair and cotyledon expansion that reveal previously unknown roles for IBA-derived IAA in plant growth and development. Our results are consistent with the possibility that limiting accumulation of the IAA precursor IBA via PDR8-promoted efflux contributes to auxin homeostasis.
DOI: 10.1073/pnas.1005878107
2010
Cited 176 times
<i>Arabidopsis PIS1</i> encodes the ABCG37 transporter of auxinic compounds including the auxin precursor indole-3-butyric acid
Differential distribution of the plant hormone auxin within tissues mediates a variety of developmental processes. Cellular auxin levels are determined by metabolic processes including synthesis, degradation, and (de)conjugation, as well as by auxin transport across the plasma membrane. Whereas transport of free auxins such as naturally occurring indole-3-acetic acid (IAA) is well characterized, little is known about the transport of auxin precursors and metabolites. Here, we identify a mutation in the ABCG37 gene of Arabidopsis that causes the polar auxin transport inhibitor sensitive1 ( pis1 ) phenotype manifested by hypersensitivity to auxinic compounds. ABCG37 encodes the pleiotropic drug resistance transporter that transports a range of synthetic auxinic compounds as well as the endogenous auxin precursor indole-3-butyric acid (IBA), but not free IAA. ABCG37 and its homolog ABCG36 act redundantly at outermost root plasma membranes and, unlike established IAA transporters from the PIN and ABCB families, transport IBA out of the cells. Our findings explore possible novel modes of regulating auxin homeostasis and plant development by means of directional transport of the auxin precursor IBA and presumably also other auxin metabolites.
DOI: 10.1111/j.1365-313x.2010.04373.x
2010
Cited 155 times
Ethylene directs auxin to control root cell expansion
Root morphogenesis is controlled by the regulation of cell division and expansion. We isolated an allele of the eto1 ethylene overproducer as a suppressor of the auxin-resistant mutant ibr5, prompting an examination of crosstalk between the phytohormones auxin and ethylene in control of root epidermal cell elongation and root hair elongation. We examined the interaction of eto1 with mutants that have reduced auxin response or transport and found that ethylene overproduction partially restored auxin responsiveness to these mutants. In addition, we found that the effects of endogenous ethylene on root cell expansion in eto1 seedlings were partially impeded by dampening auxin signaling, and were fully suppressed by blocking auxin influx. These data provide insight into the interaction between these two key plant hormones, and suggest that endogenous ethylene directs auxin to control root cell expansion.
DOI: 10.1104/pp.110.157461
2010
Cited 148 times
Conversion of Endogenous Indole-3-Butyric Acid to Indole-3-Acetic Acid Drives Cell Expansion in Arabidopsis Seedlings
Genetic evidence in Arabidopsis (Arabidopsis thaliana) suggests that the auxin precursor indole-3-butyric acid (IBA) is converted into active indole-3-acetic acid (IAA) by peroxisomal beta-oxidation; however, direct evidence that Arabidopsis converts IBA to IAA is lacking, and the role of IBA-derived IAA is not well understood. In this work, we directly demonstrated that Arabidopsis seedlings convert IBA to IAA. Moreover, we found that several IBA-resistant, IAA-sensitive mutants were deficient in IBA-to-IAA conversion, including the indole-3-butyric acid response1 (ibr1) ibr3 ibr10 triple mutant, which is defective in three enzymes likely to be directly involved in peroxisomal IBA beta-oxidation. In addition to IBA-to-IAA conversion defects, the ibr1 ibr3 ibr10 triple mutant displayed shorter root hairs and smaller cotyledons than wild type; these cell expansion defects are suggestive of low IAA levels in certain tissues. Consistent with this possibility, we could rescue the ibr1 ibr3 ibr10 short-root-hair phenotype with exogenous auxin. A triple mutant defective in hydrolysis of IAA-amino acid conjugates, a second class of IAA precursor, displayed reduced hypocotyl elongation but normal cotyledon size and only slightly reduced root hair lengths. Our data suggest that IBA beta-oxidation and IAA-amino acid conjugate hydrolysis provide auxin for partially distinct developmental processes and that IBA-derived IAA plays a major role in driving root hair and cotyledon cell expansion during seedling development.
DOI: 10.1105/tpc.111.083071
2011
Cited 138 times
Multiple Facets of<i>Arabidopsis</i>Seedling Development Require &amp;#x2028;Indole-3-Butyric Acid–Derived Auxin
Levels of auxin, which regulates both cell division and cell elongation in plant development, are controlled by synthesis, inactivation, transport, and the use of storage forms. However, the specific contributions of various inputs to the active auxin pool are not well understood. One auxin precursor is indole-3-butyric acid (IBA), which undergoes peroxisomal β-oxidation to release free indole-3-acetic acid (IAA). We identified ENOYL-COA HYDRATASE2 (ECH2) as an enzyme required for IBA response. Combining the ech2 mutant with previously identified iba response mutants resulted in enhanced IBA resistance, diverse auxin-related developmental defects, decreased auxin-responsive reporter activity in both untreated and auxin-treated seedlings, and decreased free IAA levels. The decreased auxin levels and responsiveness, along with the associated developmental defects, uncover previously unappreciated roles for IBA-derived IAA during seedling development, establish IBA as an important auxin precursor, and suggest that IBA-to-IAA conversion contributes to the positive feedback that maintains root auxin levels.
DOI: 10.1104/pp.17.01050
2017
Cited 135 times
Peroxisome Function, Biogenesis, and Dynamics in Plants
Recent advances highlight understanding of the diversity of peroxisome contributions to plant biology and the mechanisms through which these essential organelles are generated.
DOI: 10.1534/genetics.108.090399
2008
Cited 134 times
Identification and Characterization of Arabidopsis Indole-3-Butyric Acid Response Mutants Defective in Novel Peroxisomal Enzymes
Abstract Genetic evidence suggests that indole-3-butyric acid (IBA) is converted to the active auxin indole-3-acetic acid (IAA) by removal of two side-chain methylene units in a process similar to fatty acid β-oxidation. Previous studies implicate peroxisomes as the site of IBA metabolism, although the enzymes that act in this process are still being identified. Here, we describe two IBA-response mutants, ibr1 and ibr10. Like the previously described ibr3 mutant, which disrupts a putative peroxisomal acyl-CoA oxidase/dehydrogenase, ibr1 and ibr10 display normal IAA responses and defective IBA responses. These defects include reduced root elongation inhibition, decreased lateral root initiation, and reduced IBA-responsive gene expression. However, peroxisomal energy-generating pathways necessary during early seedling development are unaffected in the mutants. Positional cloning of the genes responsible for the mutant defects reveals that IBR1 encodes a member of the short-chain dehydrogenase/reductase family and that IBR10 resembles enoyl-CoA hydratases/isomerases. Both enzymes contain C-terminal peroxisomal-targeting signals, consistent with IBA metabolism occurring in peroxisomes. We present a model in which IBR3, IBR10, and IBR1 may act sequentially in peroxisomal IBA β-oxidation to IAA.
DOI: 10.1038/nchembio.1044
2012
Cited 120 times
A role for the root cap in root branching revealed by the non-auxin probe naxillin
The acquisition of water and nutrients by plant roots is a fundamental aspect of agriculture and strongly depends on root architecture. Root branching and expansion of the root system is achieved through the development of lateral roots and is to a large extent controlled by the plant hormone auxin. However, the pleiotropic effects of auxin or auxin-like molecules on root systems complicate the study of lateral root development. Here we describe a small-molecule screen in Arabidopsis thaliana that identified naxillin as what is to our knowledge the first non-auxin-like molecule that promotes root branching. By using naxillin as a chemical tool, we identified a new function for root cap-specific conversion of the auxin precursor indole-3-butyric acid into the active auxin indole-3-acetic acid and uncovered the involvement of the root cap in root branching. Delivery of an auxin precursor in peripheral tissues such as the root cap might represent an important mechanism shaping root architecture.
DOI: 10.1105/tpc.113.113407
2013
Cited 115 times
Disrupting Autophagy Restores Peroxisome Function to an<i>Arabidopsis lon2</i>Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation
Peroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic requirements change, and peroxisomal contents are remodeled. The resident peroxisomal protease LON2 is positioned to degrade obsolete or damaged peroxisomal proteins, but data supporting such a role in plants have remained elusive. Arabidopsis thaliana lon2 mutants display defects in peroxisomal metabolism and matrix protein import but appear to degrade matrix proteins normally. To elucidate LON2 functions, we executed a forward-genetic screen for lon2 suppressors, which revealed multiple mutations in key autophagy genes. Disabling core autophagy-related gene (ATG) products prevents autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation. We found that atg2, atg3, and atg7 mutations suppressed lon2 defects in auxin metabolism and matrix protein processing and rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during peroxisome content remodeling, provide evidence for the existence of pexophagy in plants, and indicate that peroxisome destruction via autophagy is enhanced when LON2 is absent.
DOI: 10.1007/s11103-012-9917-y
2012
Cited 108 times
A gain-of-function mutation in IAA16 confers reduced responses to auxin and abscisic acid and impedes plant growth and fertility
Auxin regulates many aspects of plant development, in part, through degradation of the Aux/IAA family of transcriptional repressors. Consequently, stabilizing mutations in several Aux/IAA proteins confer reduced auxin responsiveness. However, of the 29 apparent Aux/IAA proteins in Arabidopsis thaliana, fewer than half have roles established through mutant analysis. We identified iaa16-1, a dominant gain-of-function mutation in IAA16 (At3g04730), in a novel screen for reduced root responsiveness to abscisic acid. The iaa16-1 mutation also confers dramatically reduced auxin responses in a variety of assays, markedly restricts growth of adult plants, and abolishes fertility when homozygous. We compared iaa16-1 phenotypes with those of dominant mutants defective in the closely related IAA7/AXR2, IAA14/SLR, and IAA17/AXR3, along with the more distantly related IAA28, and found overlapping but distinct patterns of developmental defects. The identification and characterization of iaa16-1 provides a fuller understanding of the IAA7/IAA14/IAA16/IAA17 clade of Aux/IAA proteins and the diverse roles of these repressors in hormone response and plant development.
DOI: 10.1016/j.pbi.2016.07.008
2016
Cited 82 times
Plant peroxisomes: recent discoveries in functional complexity, organelle homeostasis, and morphological dynamics
Peroxisomes are essential for life in plants. These organelles house a variety of metabolic processes that generate and inactivate reactive oxygen species. Our knowledge of pathways and mechanisms that depend on peroxisomes and their constituent enzymes continues to grow, and in this review we highlight recent advances in understanding the identity and biological functions of peroxisomal enzymes and metabolic processes. We also review how peroxisomal matrix and membrane proteins enter the organelle from their sites of synthesis. Peroxisome homeostasis is regulated by specific degradation mechanisms, and we discuss the contributions of specialized autophagy and a peroxisomal protease to the degradation of entire peroxisomes and peroxisomal enzymes that are damaged or superfluous. Finally, we review how peroxisomes can flexibly change their morphology to facilitate inter-organellar contacts.
DOI: 10.1105/tpc.9.10.1781
1997
Cited 153 times
Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the nitrilase encoded by the NIT1 gene.
Indole-3-acetonitrile (IAN) is a candidate precursor of the plant growth hormone indole-3-acetic acid (IAA). We demonstrated that IAN has auxinlike effects on Arabidopsis seedlings and that exogenous IAN is converted to IAA in vivo. We isolated mutants with reduced sensitivity to IAN that remained sensitive to IAA. These mutants were recessive and fell into a single complementation group that mapped to chromosome 3, within 0.5 centimorgans of a cluster of three nitrilase-encoding genes, NIT1, NIT2, and NIT3. Each of the three mutants contained a single base change in the coding region of the NIT1 gene, and the expression pattern of NIT1 is consistent with the IAN insensitivity observed in the nit1 mutant alleles. The half-life of IAN and levels of IAA and IAN were unchanged in the nit1 mutant, confirming that Arabidopsis has other functional nitrilases. Overexpressing NIT2 in transgenic Arabidopsis caused increased sensitivity to IAN and faster turnover of exogenous IAN in vivo.
DOI: 10.1105/tpc.017046
2003
Cited 148 times
IBR5, a Dual-Specificity Phosphatase-Like Protein Modulating Auxin and Abscisic Acid Responsiveness in Arabidopsis
Auxin is an important plant hormone that plays significant roles in plant growth and development. Although numerous auxin-response mutants have been identified, auxin signal transduction pathways remain to be fully elucidated. We isolated ibr5 as an Arabidopsis indole-3-butyric acid-response mutant, but it also is less responsive to indole-3-acetic acid, synthetic auxins, auxin transport inhibitors, and the phytohormone abscisic acid. Like certain other auxin-response mutants, ibr5 has a long root and a short hypocotyl when grown in the light. In addition, ibr5 displays aberrant vascular patterning, increased leaf serration, and reduced accumulation of an auxin-inducible reporter. We used positional information to determine that the gene defective in ibr5 encodes an apparent dual-specificity phosphatase. Using immunoblot and promoter-reporter gene analyses, we found that IBR5 is expressed throughout the plant. The identification of IBR5 relatives in other flowering plants suggests that IBR5 function is conserved throughout angiosperms. Our results suggest that IBR5 is a phosphatase that modulates phytohormone signal transduction and support a link between auxin and abscisic acid signaling pathways.
DOI: 10.1091/mbc.e04-05-0422
2005
Cited 146 times
The<i>Arabidopsis</i>Peroxisomal Targeting Signal Type 2 Receptor PEX7 Is Necessary for Peroxisome Function and Dependent on PEX5
Plant peroxisomal proteins catalyze key metabolic reactions. Several peroxisome biogenesis PEROXIN (PEX) genes encode proteins acting in the import of targeted proteins necessary for these processes into the peroxisomal matrix. Most peroxisomal matrix proteins bear characterized Peroxisomal Targeting Signals (PTS1 or PTS2), which are bound by the receptors PEX5 or PEX7, respectively, for import into peroxisomes. Here we describe the isolation and characterization of an Arabidopsis peroxin mutant, pex7-1, which displays peroxisome-defective phenotypes including reduced PTS2 protein import. We also demonstrate that the pex5-1 PTS1 receptor mutant, which contains a lesion in a domain conserved among PEX7-binding proteins from various organisms, is defective not in PTS1 protein import, but rather in PTS2 protein import. Combining these mutations in a pex7-1 pex5-1 double mutant abolishes detectable PTS2 protein import and yields seedlings that are entirely sucrose-dependent for establishment, suggesting a severe block in peroxisomal fatty acid β-oxidation. Adult pex7-1 pex5-1 plants have reduced stature and bear abnormally shaped seeds, few of which are viable. The pex7-1 pex5-1 seedlings that germinate have dramatically fewer lateral roots and often display fused cotyledons, phenotypes associated with reduced auxin response. Thus PTS2-directed peroxisomal import is necessary for normal embryonic development, seedling establishment, and vegetative growth.
DOI: 10.1073/pnas.0304368101
2004
Cited 130 times
An <i>Arabidopsis</i> indole-3-butyric acid-response mutant defective in PEROXIN6, an apparent ATPase implicated in peroxisomal function
Genetic evidence suggests that plant peroxisomes are the site of fatty acid beta-oxidation and conversion of the endogenous auxin indole-3-butyric acid (IBA) to the active hormone indole-3-acetic acid. Arabidopsis mutants that are IBA resistant and sucrose dependent during early development are likely to have defects in beta-oxidation of both IBA and fatty acids. Several of these mutants have lesions in peroxisomal protein genes. Here, we describe the Arabidopsis pex6 mutant, which is resistant to the inhibitory effects of IBA on root elongation and the stimulatory effects of IBA on lateral root formation. pex6 also is sucrose dependent during early seedling development and smaller and more pale green than WT throughout development. PEX6 encodes an apparent ATPase similar to yeast and human proteins required for peroxisomal biogenesis, and a human PEX6 cDNA can rescue the Arabidopsis pex6 mutant. The pex6 mutant has reduced levels of the peroxisomal matrix protein receptor PEX5, and pex6 defects can be partially rescued by PEX5 overexpression. These results suggest that PEX6 may facilitate PEX5 recycling and thereby promote peroxisomal matrix protein import.
2001
Cited 127 times
The Arabidopsis pxa1 mutant is defective in an ATP-binding cassette transporter-like protein required for peroxisomal fatty acid beta-oxidation.
Peroxisomes are important organelles in plant metabolism, containing all the enzymes required for fatty acid beta-oxidation. More than 20 proteins are required for peroxisomal biogenesis and maintenance. The Arabidopsis pxa1 mutant, originally isolated because it is resistant to the auxin indole-3-butyric acid (IBA), developmentally arrests when germinated without supplemental sucrose, suggesting defects in fatty acid beta-oxidation. Because IBA is converted to the more abundant auxin, indole-3-acetic acid (IAA), in a mechanism that parallels beta-oxidation, the mutant is likely to be IBA resistant because it cannot convert IBA to IAA. Adult pxa1 plants grow slowly compared with wild type, with smaller rosettes, fewer leaves, and shorter inflorescence stems, indicating that PXA1 is important throughout development. We identified the molecular defect in pxa1 using a map-based positional approach. PXA1 encodes a predicted peroxisomal ATP-binding cassette transporter that is 42% identical to the human adrenoleukodystrophy (ALD) protein, which is defective in patients with the demyelinating disorder X-linked ALD. Homology to ALD protein and other human and yeast peroxisomal transporters suggests that PXA1 imports coenzyme A esters of fatty acids and IBA into the peroxisome for beta-oxidation. The pxa1 mutant makes fewer lateral roots than wild type, both in response to IBA and without exogenous hormones, suggesting that the IAA derived from IBA during seedling development promotes lateral root formation.
DOI: 10.1105/tpc.105.035691
2005
Cited 121 times
Identification and Functional Characterization of<i>Arabidopsis</i>PEROXIN4 and the Interacting Protein PEROXIN22[W]
Abstract Peroxins are genetically defined as proteins necessary for peroxisome biogenesis. By screening for reduced response to indole-3-butyric acid, which is metabolized to active auxin in peroxisomes, we isolated an Arabidopsis thaliana peroxin4 (pex4) mutant. This mutant displays sucrose-dependent seedling development and reduced lateral root production, characteristics of plant peroxisome malfunction. We used yeast two-hybrid analysis to determine that PEX4, an apparent ubiquitin-conjugating enzyme, interacts with a previously unidentified Arabidopsis protein, PEX22. A pex4 pex22 double mutant enhanced pex4 defects, confirming that PEX22 is a peroxin. Expression of both Arabidopsis genes together complemented yeast pex4 or pex22 mutant defects, whereas expression of either gene individually failed to rescue the corresponding yeast mutant. Therefore, it is likely that the Arabidopsis proteins can function similarly to the yeast PEX4–PEX22 complex, with PEX4 ubiquitinating substrates and PEX22 tethering PEX4 to the peroxisome. However, the severe sucrose dependence of the pex4 pex22 mutant is not accompanied by correspondingly strong defects in peroxisomal matrix protein import, suggesting that this peroxin pair may have novel plant targets in addition to those important in fungi. Isocitrate lyase is stabilized in pex4 pex22, indicating that PEX4 and PEX22 may be important during the remodeling of peroxisome matrix contents as glyoxysomes transition to leaf peroxisomes.
DOI: 10.1111/j.1365-313x.2005.02343.x
2005
Cited 107 times
Mutations in Arabidopsis acyl‐CoA oxidase genes reveal distinct and overlapping roles in β‐oxidation
Indole-3-butyric acid (IBA) is an endogenous auxin used to enhance rooting during propagation. To better understand the role of IBA, we isolated Arabidopsis IBA-response (ibr) mutants that display enhanced root elongation on inhibitory IBA concentrations but maintain wild-type responses to indole-3-acetic acid, the principle active auxin. A subset of ibr mutants remains sensitive to the stimulatory effects of IBA on lateral root initiation. These mutants are not sucrose dependent during early seedling development, indicating that peroxisomal beta-oxidation of seed storage fatty acids is occurring. We used positional cloning to determine that one mutant is defective in ACX1 and two are defective in ACX3, two of the six Arabidopsis fatty acyl-CoA oxidase (ACX) genes. Characterization of T-DNA insertion mutants defective in the other ACX genes revealed reduced IBA responses in a third gene, ACX4. Activity assays demonstrated that mutants defective in ACX1, ACX3, or ACX4 have reduced fatty acyl-CoA oxidase activity on specific substrates. Moreover, acx1 acx2 double mutants display enhanced IBA resistance and are sucrose dependent during seedling development, whereas acx1 acx3 and acx1 acx5 double mutants display enhanced IBA resistance but remain sucrose independent. The inability of ACX1, ACX3, and ACX4 to fully compensate for one another in IBA-mediated root elongation inhibition and the ability of ACX2 and ACX5 to contribute to IBA response suggests that IBA-response defects in acx mutants may reflect indirect blocks in peroxisomal metabolism and IBA beta-oxidation, rather than direct enzymatic activity of ACX isozymes on IBA-CoA.
DOI: 10.1534/genetics.106.061044
2006
Cited 101 times
An Arabidopsis Basic Helix-Loop-Helix Leucine Zipper Protein Modulates Metal Homeostasis and Auxin Conjugate Responsiveness
Abstract The plant hormone auxin can be regulated by formation and hydrolysis of amide-linked indole-3-acetic acid (IAA) conjugates. Here, we report the characterization of the dominant Arabidopsis iaa–leucine resistant3 (ilr3-1) mutant, which has reduced sensitivity to IAA–Leu and IAA–Phe, while retaining wild-type responses to free IAA. The gene defective in ilr3-1 encodes a basic helix-loop-helix leucine zipper protein, bHLH105, and the ilr3-1 lesion results in a truncated product. Overexpressing ilr3-1 in wild-type plants recapitulates certain ilr3-1 mutant phenotypes. In contrast, the loss-of-function ilr3-2 allele has increased IAA–Leu sensitivity compared to wild type, indicating that the ilr3-1 allele confers a gain of function. Microarray and quantitative real-time PCR analyses revealed five downregulated genes in ilr3-1, including three encoding putative membrane proteins similar to the yeast iron and manganese transporter Ccc1p. Transcript changes are accompanied by reciprocally misregulated metal accumulation in ilr3-1 and ilr3-2 mutants. Further, ilr3-1 seedlings are less sensitive than wild type to manganese, and auxin conjugate response phenotypes are dependent on exogenous metal concentration in ilr3 mutants. These data suggest a model in which the ILR3/bHLH105 transcription factor regulates expression of metal transporter genes, perhaps indirectly modulating IAA-conjugate hydrolysis by controlling the availability of metals previously shown to influence IAA–amino acid hydrolase protein activity.
DOI: 10.1074/jbc.m611831200
2007
Cited 95 times
Arabidopsis thaliana Squalene Epoxidase 1 Is Essential for Root and Seed Development
Squalene epoxidase converts squalene into oxidosqualene, the precursor of all known angiosperm cyclic triterpenoids, which include membrane sterols, brassinosteroid phytohormones, and non-steroidal triterpenoids. In this work, we have identified six putative <i>Arabidopsis</i> squalene epoxidase (SQE) enzymes and used heterologous expression in yeast to demonstrate that three of these enzymes, SQE1, SQE2, and SQE3, can epoxidize squalene. We isolated and characterized <i>Arabidopsis sqe1</i> mutants and discovered severe developmental defects, including reduced root and hypocotyl elongation. Adult <i>sqe1–3</i> and <i>sqe1–4</i> plants have diminished stature and produce inviable seeds. The <i>sqe1–3</i> mutant accumulates squalene, consistent with a block in the triterpenoid biosynthetic pathway. Therefore, SQE1 function is necessary for normal plant development, and the five <i>SQE</i>-like genes remaining in this mutant are not fully redundant with <i>SQE1</i>.
DOI: 10.1073/pnas.0811329106
2009
Cited 93 times
Peroxisome-associated matrix protein degradation in <i>Arabidopsis</i>
Peroxisomes are ubiquitous eukaryotic organelles housing diverse enzymatic reactions, including several that produce toxic reactive oxygen species. Although understanding of the mechanisms whereby enzymes enter peroxisomes with the help of peroxin (PEX) proteins is increasing, mechanisms by which damaged or obsolete peroxisomal proteins are degraded are not understood. We have exploited unique aspects of plant development to characterize peroxisome-associated protein degradation (PexAD) in Arabidopsis . Oilseed seedlings undergo a developmentally regulated remodeling of peroxisomal matrix protein composition in which the glyoxylate cycle enzymes isocitrate lyase (ICL) and malate synthase (MLS) are replaced by photorespiration enzymes. We found that mutations expected to increase or decrease peroxisomal H 2 O 2 levels accelerated or delayed ICL and MLS disappearance, respectively, suggesting that oxidative damage promotes peroxisomal protein degradation. ICL, MLS, and the β-oxidation enzyme thiolase were stabilized in the pex4–1 pex22–1 double mutant, which is defective in a peroxisome-associated ubiquitin-conjugating enzyme and its membrane tether. Moreover, the stabilized ICL, thiolase, and an ICL-GFP reporter remained peroxisome associated in pex4–1 pex22–1 . ICL also was stabilized and peroxisome associated in pex6–1 , a mutant defective in a peroxisome-tethered ATPase. ICL and thiolase were mislocalized to the cytosol but only ICL was stabilized in pex5–10 , a mutant defective in a matrix protein import receptor, suggesting that peroxisome entry is necessary for degradation of certain matrix proteins. Together, our data reveal new roles for PEX4, PEX5, PEX6, and PEX22 in PexAD of damaged or obsolete matrix proteins in addition to their canonical roles in peroxisome biogenesis.
DOI: 10.1007/s11103-007-9134-2
2007
Cited 92 times
IBR3, a novel peroxisomal acyl-CoA dehydrogenase-like protein required for indole-3-butyric acid response
DOI: 10.1105/tpc.108.065185
2009
Cited 86 times
Silver Ions Increase Auxin Efflux Independently of Effects on Ethylene Response
Silver nitrate and aminoethoxyvinylglycine (AVG) are often used to inhibit perception and biosynthesis, respectively, of the phytohormone ethylene. In the course of exploring the genetic basis of the extensive interactions between ethylene and auxin, we compared the effects of silver nitrate (AgNO(3)) and AVG on auxin responsiveness. We found that although AgNO(3) dramatically decreased root indole-3-acetic acid (IAA) responsiveness in inhibition of root elongation, promotion of DR5-beta-glucuronidase activity, and reduction of Aux/IAA protein levels, AVG had more mild effects. Moreover, we found that that silver ions, but not AVG, enhanced IAA efflux similarly in root tips of both the wild type and mutants with blocked ethylene responses, indicating that this enhancement was independent of ethylene signaling. Our results suggest that the promotion of IAA efflux by silver ions is independent of the effects of silver ions on ethylene perception. Although the molecular details of this enhancement remain unknown, our finding that silver ions can promote IAA efflux in addition to blocking ethylene signaling suggest that caution is warranted in interpreting studies using AgNO(3) to block ethylene signaling in roots.
DOI: 10.1093/mp/ssn063
2009
Cited 80 times
Disruption of Arabidopsis CHY1 Reveals an Important Role of Metabolic Status in Plant Cold Stress Signaling
To study cold signaling, we screened for Arabidopsis mutants with altered cold-induced transcription of a firefly luciferase reporter gene driven by the CBF3 promoter (CBF3-LUC). One mutant, chy1-10, displayed reduced cold-induction of CBF3-LUC luminescence. RNA gel blot analysis revealed that expression of endogenous CBFs also was reduced in the chy1 mutant. chy1-10 mutant plants are more sensitive to freezing treatment than wild-type after cold acclimation. Both the wild-type and chy1 mutant plants are sensitive to darkness-induced starvation at warm temperatures, although chy1 plants are slightly more sensitive. This dark-sensitivity is suppressed by cold temperature in the wild-type but not in chy1. Constitutive CBF3 expression partially rescues the sensitivity of chy1-10 plants to dark treatment in the cold. The chy1 mutant accumulates higher levels of reactive oxygen species, and application of hydrogen peroxide can reduce cold-induction of CBF3-LUC in wild-type. Map-based cloning of the gene defective in the mutant revealed a nonsense mutation in CHY1, which encodes a peroxisomal beta-hydroxyisobutyryl (HIBYL)-CoA hydrolase needed for valine catabolism and fatty acid beta-oxidation. Our results suggest a role for peroxisomal metabolism in cold stress signaling, and plant tolerance to cold stress and darkness-induced starvation.
DOI: 10.1016/j.bbamcr.2015.09.005
2016
Cited 55 times
Pexophagy and peroxisomal protein turnover in plants
Peroxisomes are dynamic, vital organelles that sequester a variety of oxidative reactions and their toxic byproducts from the remainder of the cell. The oxidative nature of peroxisomal metabolism predisposes the organelle to self-inflicted damage, highlighting the need for a mechanism to dispose of damaged peroxisomes. In addition, the metabolic requirements of plant peroxisomes change during development, and obsolete peroxisomal proteins are degraded. Although pexophagy, the selective autophagy of peroxisomes, is an obvious mechanism for executing such degradation, pexophagy has only recently been described in plants. Several recent studies in the reference plant Arabidopsis thaliana implicate pexophagy in the turnover of peroxisomal proteins, both for quality control and during functional transitions of peroxisomal content. In this review, we describe our current understanding of the occurrence, roles, and mechanisms of pexophagy in plants. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann.
DOI: 10.1016/s0031-9422(98)00366-5
1998
Cited 114 times
Cloning and characterization of the Arabidopsis thaliana lupeol synthase gene
A 2274 bp Arabidopsis thaliana cDNA was isolated that encodes a protein 57% identical to cycloartenol synthase from the same organism. The expressed recombinant protein encodes lupeol synthase, which converts oxidosqualene to the triterpene lupeol as the major product. Lupeol synthase is a multifunctional enzyme that forms other triterpene alcohols, including β-amyrin, as minor products. Sequence analysis suggests that lupeol synthase diverged from cycloartenol synthase after plants diverged from fungi and animals. This evolutionary order is the reason that fungi and animals do not make lupeol.
DOI: 10.1073/pnas.91.6.2211
1994
Cited 105 times
Molecular cloning, characterization, and overexpression of ERG7, the Saccharomyces cerevisiae gene encoding lanosterol synthase.
We report the cloning, characterization, and overexpression of Saccharomyces cerevisiae ERG7, which encodes lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme responsible for the complex cyclization/rearrangement step in sterol biosynthesis. Oligonucleotide primers were designed corresponding to protein sequences conserved between Candida albicans ERG7 and the related Arabidopsis thaliana cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A PCR product was amplified from yeast genomic DNA using these primers and was used to probe yeast libraries by hybridization. Partial-length clones homologous to the two known epoxysqualene mutases were isolated, but a full-length sequence was found neither in cDNA nor genomic libraries, whether in phage or plasmids. Two overlapping clones were assembled to make a functional reconstruction of the gene, which contains a 2196-bp open reading frame capable of encoding an 83-kDa protein. The reconstruction complemented the erg7 mutation when driven from either its native promoter or the strong ADH1 promoter.
DOI: 10.1105/tpc.12.12.2395
2000
Cited 100 times
Cloning and Characterization of <i>IAR1</i>, a Gene Required for Auxin Conjugate Sensitivity in Arabidopsis
Most indole-3-acetic acid (IAA) in higher plants is conjugated to amino acids, sugars, or peptides, and these conjugates are implicated in regulating the concentration of the free hormone. We identified iar1 as an Arabidopsis mutant that is resistant to the inhibitory effects of several IAA–amino acid conjugates but remains sensitive to free IAA. iar1 partially suppresses phenotypes of a mutant that overproduces IAA, suggesting that IAR1 participates in auxin metabolism or response. We used positional information to clone IAR1, which encodes a novel protein with seven predicted transmembrane domains and several His-rich regions. IAR1 has homologs in other multicellular organisms, including Drosophila, nematodes, and mammals; in addition, the mouse homolog KE4 can functionally substitute for IAR1 in vivo. IAR1 also structurally resembles and has detectable sequence similarity to a family of metal transporters. We discuss several possible roles for IAR1 in auxin homeostasis.
DOI: 10.1074/jbc.m104679200
2001
Cited 99 times
chy1, an Arabidopsis Mutant with Impaired β-Oxidation, Is Defective in a Peroxisomal β-Hydroxyisobutyryl-CoA Hydrolase
The Arabidopsis chy1 mutant is resistant to indole-3-butyric acid, a naturally occurring form of the plant hormone auxin. Because the mutant also has defects in peroxisomal β-oxidation, this resistance presumably results from a reduced conversion of indole-3-butyric acid to indole-3-acetic acid. We have cloned CHY1, which appears to encode a peroxisomal protein 43% identical to a mammalian valine catabolic enzyme that hydrolyzes β-hydroxyisobutyryl-CoA. We demonstrated that a human β-hydroxyisobutyryl-CoA hydrolase functionally complementschy1 when redirected from the mitochondria to the peroxisomes. We expressed CHY1 as a glutathioneS-transferase (GST) fusion protein and demonstrated that purified GST-CHY1 hydrolyzes β-hydroxyisobutyryl-CoA. Mutagenesis studies showed that a glutamate that is catalytically essential in homologous enoyl-CoA hydratases was also essential in CHY1. Mutating a residue that is differentially conserved between hydrolases and hydratases established that this position is relevant to the catalytic distinction between the enzyme classes. It is likely that CHY1 acts in peroxisomal valine catabolism and that accumulation of a toxic intermediate, methacrylyl-CoA, causes the altered β-oxidation phenotypes of the chy1 mutant. Our results support the hypothesis that the energy-intensive sequence unique to valine catabolism, where an intermediate CoA ester is hydrolyzed and a new CoA ester is formed two steps later, avoids methacrylyl-CoA accumulation. The Arabidopsis chy1 mutant is resistant to indole-3-butyric acid, a naturally occurring form of the plant hormone auxin. Because the mutant also has defects in peroxisomal β-oxidation, this resistance presumably results from a reduced conversion of indole-3-butyric acid to indole-3-acetic acid. We have cloned CHY1, which appears to encode a peroxisomal protein 43% identical to a mammalian valine catabolic enzyme that hydrolyzes β-hydroxyisobutyryl-CoA. We demonstrated that a human β-hydroxyisobutyryl-CoA hydrolase functionally complementschy1 when redirected from the mitochondria to the peroxisomes. We expressed CHY1 as a glutathioneS-transferase (GST) fusion protein and demonstrated that purified GST-CHY1 hydrolyzes β-hydroxyisobutyryl-CoA. Mutagenesis studies showed that a glutamate that is catalytically essential in homologous enoyl-CoA hydratases was also essential in CHY1. Mutating a residue that is differentially conserved between hydrolases and hydratases established that this position is relevant to the catalytic distinction between the enzyme classes. It is likely that CHY1 acts in peroxisomal valine catabolism and that accumulation of a toxic intermediate, methacrylyl-CoA, causes the altered β-oxidation phenotypes of the chy1 mutant. Our results support the hypothesis that the energy-intensive sequence unique to valine catabolism, where an intermediate CoA ester is hydrolyzed and a new CoA ester is formed two steps later, avoids methacrylyl-CoA accumulation. branched-chain amino acid β-hydroxyisobutyryl indole-3-butyric acid indole-3-acetic acid plant nutrient medium PN medium plus sucrose kanamycin HIBYL-CoA hydrolase polymerase chain reaction kilobase(s) base pair(s) glutathione S-transferase Germinating seedlings of oilseed plants β-oxidize long-chain fatty acids as an energy source until photosynthesis begins. Enzymes that catalyze each step of fatty acid β-oxidation have been identified, and different isozymes may participate during distinct developmental stages or in different tissues. Although mammals β-oxidize fatty acids in both the mitochondria and peroxisomes (reviewed in Refs. 1Tabak H.F. Braakman I. Distel B. Trends Cell Biol. 1999; 9: 447-453Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 2Lazarow P.B. Trends Cell Biol. 1993; 3: 89-93Abstract Full Text PDF PubMed Scopus (26) Google Scholar), plant fatty acid β-oxidation is exclusively peroxisomal (reviewed in Refs. 2Lazarow P.B. Trends Cell Biol. 1993; 3: 89-93Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 3Olsen L.J. Plant Mol. Biol. 1998; 38: 163-189Crossref PubMed Google Scholar, 4Kindl H. Biochime. 1993; 75: 225-230Crossref PubMed Scopus (66) Google Scholar, 5Gerhardt B. Prog. Lipid Res. 1992; 31: 417-446Crossref PubMed Scopus (97) Google Scholar). Peroxisomes are small organelles bounded by a single lipid bilayer that contains β-oxidation enzymes and catalases to inactivate H2O2 (reviewed in Refs. 3Olsen L.J. Plant Mol. Biol. 1998; 38: 163-189Crossref PubMed Google Scholar, 5Gerhardt B. Prog. Lipid Res. 1992; 31: 417-446Crossref PubMed Scopus (97) Google Scholar). Leaf peroxisomes also house the enzymes acting in photorespiration, and specialized peroxisomes in seedlings and senescing tissues called glyoxysomes contain the enzymes required for the glyoxylate cycle, which converts acetyl-CoA to succinate (reviewed in Refs. 3Olsen L.J. Plant Mol. Biol. 1998; 38: 163-189Crossref PubMed Google Scholar, 5Gerhardt B. Prog. Lipid Res. 1992; 31: 417-446Crossref PubMed Scopus (97) Google Scholar). The branched chain amino acids (BCAAs)1 valine, leucine, and isoleucine can be broken down to generate energy during germination, senescence, or carbon starvation; the end products of these reactions enter the citric acid cycle and fuel respiration (6Anderson M.D. Che P. Song J. Nikolau B.J. Wurtele E.S. Plant Physiol. 1998; 118: 1127-1138Crossref PubMed Scopus (77) Google Scholar, 7Dunford R. Kirk D. Rees T.A.P. Phytochemistry. 1990; 29: 41-43Crossref Scopus (11) Google Scholar, 8Mazelis M. Miflin B.J. The Biochemistry of Plants. Academic Press, New York1980: 542-567Google Scholar). The subcellular localization of BCAA catabolism in plants remains controversial. Although BCAA catabolism is mitochondrial in mammals, peroxisomes isolated from mung bean and sunflower cotyledons can convert labeled BCAAs to propionyl-CoA and acetyl-CoA (9Gerbling H. Gerhardt B. Plant Physiol. 1988; 88: 13-15Crossref PubMed Google Scholar, 10Gerbling H. Gerhardt B. Plant Physiol. 1989; 91: 1387-1392Crossref PubMed Google Scholar). The initial catabolic enzymes are shared by the three BCAAs. The amino acids are first oxidatively transaminated to form the α-keto acids (see Fig. 1; step 1), which undergo oxidative decarboxylation and esterification to form the acyl-CoA esters (step 2). Then, the catabolic mechanisms of the three BCAAs diverge. The isobutyryl-CoA made from Val is desaturated to form the α,β-unsaturated thioester methacrylyl-CoA (step 3), which can react with nucleophiles such as free thiols and cause damage within the organelle (11Brown G.K. Hunt S.M. Scholem R. Fowler K. Grimes A. Mercer J.F.B. Truscott R.M. Cotton R.G.H. Rogers J.G. Danks D.M. Pediatrics. 1982; 70: 532-538PubMed Google Scholar). Hydration to β-hydroxyisobutyryl-CoA (HIBYL-CoA; step 4) and thioester hydrolysis (step 5) forms diffusible, transportable β-hydroxyisobutyrate (12Shimomura Y. Murakami T. Fujitsuka N. Nakai N. Sato Y. Sugiyama S. Shimomura N. Irwin J. Hawes J.W. Harris R.A. J. Biol. Chem. 1994; 269: 14248-14253Abstract Full Text PDF PubMed Google Scholar, 13Shimomura Y. Murakami T. Nakai N. Huang B. Hawes J.W. Harris R.A. Methods Enzymol. 2000; 324: 229-240Crossref PubMed Google Scholar, 14Hawes J.W. Jaskiewicz J. Shimomura Y. Huang B. Bunting J. Harper E.T. Harris R.A. J. Biol. Chem. 1996; 271: 26430-26434Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This β-hydroxy acid is oxidized to methylmalonyl semialdehyde (step 6) and thioesterified to form propionyl-CoA (step 7). Leu and Ile catabolic pathways exclude the CoA hydrolysis step, perhaps because the corresponding intermediates are less reactive than methacrylyl-CoA. We are examining the metabolism and function of the phytohormone auxin, which affects virtually all plant developmental processes, including root elongation and lateral root initiation (15Davies P.J. Plant Hormones. 2nd Ed. Kluwer Academic Publishers, Dordrecht1995: 1-38Crossref Google Scholar). Indole-3-butyric acid (IBA) is a naturally occurring auxin that is converted to the more abundant auxin indole-3-acetic acid (IAA) in several plant species (reviewed in Ref. 16Ludwig-Müller J. Plant Growth Regul. 2000; 32: 219-230Crossref Scopus (263) Google Scholar). Because even- but not odd-chain length IAA derivatives have auxin activity (17Fawcett C.H. Wain R.L. Wightman F. Proc. R. Soc. Lond. Ser. B. 1960; 152: 231-254Crossref PubMed Scopus (48) Google Scholar), the mechanism of this conversion probably parallels fatty acid β-oxidation. Previously, we described a group of Arabidopsis thaliana mutants that are resistant to the inhibitory effects of IBA on root elongation but respond normally to IAA (18Zolman B.K. Yoder A. Bartel B. Genetics. 2000; 156: 1323-1337Crossref PubMed Google Scholar). Mutants in several β-oxidation enzymes, including an acyl-CoA oxidase (ACX3 (19Eastmond P.J. Hooks M.A. Williams D. Lange P. Bechtold N. Sarrobert C. Nussaume L. Graham I.A. J. Biol. Chem. 2000; 275: 34375-34381Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar)), a multifunctional protein (AIM1 (20Richmond T.A. Bleecker A.B. Plant Cell. 1999; 11: 1911-1923PubMed Google Scholar)), and a thiolase (PED1 (21Hayashi M. Toriyama K. Kondo M. Nishimura M. Plant Cell. 1998; 10: 183-195PubMed Google Scholar)) are IBA-resistant and IAA-sensitive (18Zolman B.K. Yoder A. Bartel B. Genetics. 2000; 156: 1323-1337Crossref PubMed Google Scholar). 2B. K. Zolman and B. Bartel, unpublished results. 2B. K. Zolman and B. Bartel, unpublished results. In addition, a mutant defective in PEX5, a receptor required to import peroxisomal matrix proteins, is IBA-resistant (18Zolman B.K. Yoder A. Bartel B. Genetics. 2000; 156: 1323-1337Crossref PubMed Google Scholar). The isolation and characterization of these mutants provide compelling evidence that Arabidopsisperoxisomes convert IBA to IAA in a mechanism similar to fatty acid chain shortening and demonstrate that screening for IBA resistance is a powerful method to identify mutants defective in β-oxidation and peroxisomal function. Here we describe the cloning and characterization of the gene defective in the Arabidopsis chy1 mutant, which is IBA-resistant both in root elongation and lateral root initiation and has defects in the peroxisomal β-oxidation of fatty acids. CHY1 encodes a β-hydroxyisobutyryl-CoA hydrolase, which may act in peroxisomal Val catabolism. We hypothesize that the loss of CHY1 indirectly disrupts both fatty acid β-oxidation and the conversion of IBA to IAA because the toxic intermediate, methacrylyl-CoA, accumulates in the peroxisome. A. thaliana accessions Columbia (Col-0), Landsberg erecta tt4 (Ler), and Wassilewskija (Ws) were used. Plants were grown in soil (Metromix 200, Scotts, Marysville OH) under continuous illumination by Sylvania Cool White fluorescent bulbs at 22–25 °C. Plants were grown aseptically on PNS (plant nutrient medium with 0.5% sucrose (22Haughn G.W. Somerville C. Mol. Gen. Genet. 1986; 204: 430-434Crossref Scopus (481) Google Scholar)) solidified with 0.6% agar, either alone or supplemented with hormones (from 1 or 100 mmstocks in ethanol), kanamycin (Kan; from a 25 mg/ml aqueous stock), or gluphosinate ammonium (Basta; from a 50 mg/ml stock in 25% ethanol; Crescent Chemical Co., Hauppauge, NY). Plates were wrapped with gas-permeable surgical tape (LecTec Corp., Minnetonka, MN) and grown at 22 °C under continuous yellow-filtered light to prevent breakdown of indolic compounds (23Stasinopoulos T.C. Hangarter R.P. Plant Physiol. 1990; 93: 1365-1369Crossref PubMed Scopus (116) Google Scholar). Isolation of the IBA-resistant mutants was described previously (18Zolman B.K. Yoder A. Bartel B. Genetics. 2000; 156: 1323-1337Crossref PubMed Google Scholar). chy1-1 (B17) andchy1-3 (B7) were generated by ethyl methanesulfonate in the Col-0 accession, and chy1-2 (B52) is an untagged transferred DNA-induced allele in the Ws accession (24Feldmann K.A. Plant J. 1991; 1: 71-82Crossref Scopus (448) Google Scholar). Mutants were backcrossed to the parental accession at least once prior to phenotypic analysis. Seeds were surface-sterilized and plated on PNS with the indicated hormone concentration. Seedlings were grown for the indicated time and removed from the agar, and the primary root was measured. Data are expressed as percent elongation on supplementedversus unsupplemented media. To assay lateral root initiation, seeds were germinated and grown on PNS for 4 days, then transferred to media containing IBA, IAA, or no hormone and grown for 4 additional days, after which roots were counted using a dissecting microscope. For hypocotyl elongation assays, seeds were plated on PN (without sucrose) or PNS and incubated for 24 h under white light before being transferred to the dark. Hypocotyl elongation was measured after 5 days in the dark and is expressed as percent elongation on medium without sucrose versus medium with sucrose. The mutants were mapped after outcrossing. The resulting F2 seeds were plated on 15 µmIBA, and DNA was isolated (25Celenza J.L. Grisafi P.L. Fink G.R. Genes Dev. 1995; 9: 2131-2142Crossref PubMed Scopus (375) Google Scholar) from resistant individuals. Mutants were mapped using published Simple SequenceLength Polymorphism (SSLP (26Bell C.J. Ecker J.R. Genomics. 1994; 19: 137-144Crossref PubMed Scopus (917) Google Scholar)) andCleaved Amplified PolymorphicSequence (CAPS (27Konieczny A. Ausubel F.M. Plant J. 1993; 4: 403-410Crossref PubMed Scopus (1367) Google Scholar)) markers. New markers were identified by PCR-amplifying and sequencing ∼1.3-kb genomic DNA fragments from different accessions and by identifying polymorphisms that altered fragment sizes or restriction enzyme recognition sites (TableI).Table INew CAPS markers used in CHY1 cloningMarkerEnzymeSize of productsOligonucleotidesCol-0WsbpMNA5MspAI135, 1102455′-GTTAGAGGCAACGAGATCAGATAG-3′5′-CCGAACCGAGATCGAACCAAGG-3′K9I9DraI285, 145, 40430, 405′-GTGTCTCCATTGTACTGCTCTGCTTG-3′5′-CGTGTGGTTGACCCCTTCGTTCTAC-3′MNA5 and K9I9 are CAPS markers (27Konieczny A. Ausubel F.M. Plant J. 1993; 4: 403-410Crossref PubMed Scopus (1367) Google Scholar), which reveal polymorphisms when cut with the indicated restriction enzymes following amplification. PCR conditions were 40 cycles of 15 s at 94 °C, 15 s at 55 °C, and 30 s at 72 °C. Products were visualized following electrophoresis on 4% agarose gels. In MNA5, Ler products are the same as Ws; in K9I9, Ler products are like Col-0. Open table in a new tab MNA5 and K9I9 are CAPS markers (27Konieczny A. Ausubel F.M. Plant J. 1993; 4: 403-410Crossref PubMed Scopus (1367) Google Scholar), which reveal polymorphisms when cut with the indicated restriction enzymes following amplification. PCR conditions were 40 cycles of 15 s at 94 °C, 15 s at 55 °C, and 30 s at 72 °C. Products were visualized following electrophoresis on 4% agarose gels. In MNA5, Ler products are the same as Ws; in K9I9, Ler products are like Col-0. Genomic DNA extracted from chy1 mutant plants was amplified using two pairs of oligonucleotides: K14B20-1 (5′-CCCACAGACGTAAACAATAGTGCTCC-3′) plus K14B20-2 (5′-GCGTGCGTCTATTTCGTAAGAC-3′) and K14B20-3 (5′-CAACTTCCTAAACTAAGCGGGTGAAG-3′) plus K14B20-4 (5′-GTCTTACGAAATAGACGCACGC-3′). Alternatively, DNA was amplified with K14B20-1 plus K14B20-2, K14B20-5 (5′-GCAGCTGAATGCTCTGTCCTTCCAC-3′) plus K14B20-6 (5′-GGGTGTATGCATCGAGAATTGTTGAGGC-3′), and K14B20-3 plus K14B20-7 (5′-CGAAATCGACCATGGACGGATACACC-3′). Primers were designed to amplify overlapping fragments, which covered the gene from 110 bp upstream of the putative translation start site to 40 bp downstream of the stop codon. Amplification products were purified by sequential ethanol, polyethylene glycol, and ethanol precipitations (28Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York1999Google Scholar) and sequenced directly using an automated DNA sequencer (Lone Star Laboratories, Houston, TX or Rice Sequencing Facility, Rice University, Houston, TX) with the primers used for amplification. A full-length CHY1 cDNA was isolated from a plasmid-based cDNA library (29Minet M. Dufour M.-E. Lacroute F. Plant J. 1992; 2: 417-422PubMed Google Scholar) by colony hybridization with a 600-bp probe amplified from genomic DNA with K14B20-5 plus K14B20-7. The resulting cDNA was subcloned into the NotI site of pBluescript II KS(+) (Stratagene) to form pKS-CHY1 and was sequenced with both internal and vector-derived primers (GenBank™ accession number AF276301). A CHY1 genomic clone was constructed by isolating a 5.9-kbBamHI fragment (2.4-kb 5′-untranslated region, 2.5-kb coding sequence, and 1-kb 3′-untranslated region) from the K14B20 TAC clone (30Sato S. Nakamura Y. Kaneko T. Katoh T. Asamizu E. Kotani H. Tabata S. DNA Res. 2000; 7: 31-63Crossref PubMed Scopus (30) Google Scholar) and subcloning it into the BamHI site of the plant transformation vector pBIN19 (31Bevan M. Nucleic Acids Res. 1984; 12: 8711-8721Crossref PubMed Scopus (1811) Google Scholar) to give pBIN-CHY1. This plasmid was electroporated (28Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York1999Google Scholar) into Agrobacterium tumefaciens strain GV3101 (32Koncz C. Schell J. Mol. Gen. Genet. 1986; 204: 383-396Crossref Scopus (1567) Google Scholar), which was used to infiltratechy1 mutant plants (33Clough S.J. Bent A.F. Plant J. 1998; 16: 735-743Crossref PubMed Google Scholar). Transformants were selected for the ability to develop on PNS containing 12 µg/ml Kan (chy1-1;chy1-3) or on PN in the dark (chy1-2, which contains an unlinked T-DNA conferring Kan resistance). A human HIBYL-CoA hydrolase (HIBCH) cDNA (14Hawes J.W. Jaskiewicz J. Shimomura Y. Huang B. Bunting J. Harper E.T. Harris R.A. J. Biol. Chem. 1996; 271: 26430-26434Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) in the Lafmid BA vector was obtained from Image Consortium, Lawrence Livermore National Laboratory (IMAGE Consortium clone ID 32783 (34Lennon G.G. Auffray C. Polymeropoulos M. Soares M.B. Genomics. 1996; 33: 151-152Crossref PubMed Scopus (1089) Google Scholar)). The mitochondrial targeting signal was removed by mutagenizing theHIBCH cDNA with the oligonucleotide 5′-GTGTGCTTGGACATCTCGCGGATCCCCGTC-3′ (altered residues underlined), which created a BamHI site 5′ of a natural ATG at the end of the mitochondrial targeting signal. A peroxisomal targeting signal was added at the C terminus of the human protein by first mutagenizing with the oligonucleotide 5′-CAAAGACTTAACCCGGGTATTCAAATCTTC-3′ (altered residues are underlined) to create an SmaI site 13 codons upstream of the stop codon. The 3′-end of the CHY1 cDNA was then excised with ScaI and NotI and inserted into the mutagenized HIBCH cDNA cut with SmaI and NotI, replacing the last 13 amino acids of the human protein with the C-terminal 28 amino acids from CHY1. The modified cDNA was excised with BamHI and NotI and ligated into pBluescript II KS(+) cut with the same enzymes, then digested with XhoI and NotI and ligated into the plant transformation vector 35SpBARN (35LeClere, S., and Bartel, B. (2001) Plant Mol. Biol., in press.Google Scholar) cut with the same enzymes to give the construct 35S-HIBCH-AKL. Three control plasmids also were constructed in 35SpBARN: the full-length human cDNA with the mitochondrial targeting signal (35S-mHIBCH), the human cDNA lacking the N-terminal mitochondrial targeting sequence (35S-HIBCH), and the full-length human cDNA with the CHY1 C-terminal region (35S-mHIBCH-AKL). We also subcloned the full-length Arabidopsis CHY1 cDNA as an NotI fragment into 35SpBARN to make 35S-CHY1. Inserts of all constructs were sequenced to verify that no unintended changes were introduced during the mutagenesis or subcloning. Each of the constructs was electroporated into Agrobacterium and transformed intochy1-1 mutant plants (see above). Transformants were selected after growth on PN in the dark for 5 days or on PNS plates supplemented with 7.5 µg/ml gluphosinate ammonium under white light for 10 days. Lines homozygous for the transgenes were selected by examining the pattern of gluphosinate ammonium resistance in the T3 generation. Mutagenesis was performed on pKS-CHY1 to add an NdeI site immediately 5′ of the initiator ATG using the oligonucleotide 5′-GGCCATCTCGACTGCCATATGTCTTACTGGTCAGATTCG-3′ (altered residues are underlined). In addition, two CHY1 amino acids were separately altered using oligonucleotide-directed mutagenesis: Glu-141 was changed to Ala using the oligonucleotide 5′-CCCAGAGCTGTCGCAGGCATGGCAAAAACC-3′, and Asp-149 was changed to Gly using the oligonucleotide 5′-GGAGGCGCCTACCCCGGGAAAGAGCCCCAG-3′ (altered residues are underlined). The NdeI-NotI fragments of the wild-type and mutagenized cDNAs were ligated into the protein expression vector pGEX-KTO (36Davies R.T. Goetz D.H. Lasswell J. Anderson M.N. Bartel B. Plant Cell. 1999; 11: 365-376Crossref PubMed Scopus (167) Google Scholar) cut with the same enzymes. Protein expression was performed as described previously (36Davies R.T. Goetz D.H. Lasswell J. Anderson M.N. Bartel B. Plant Cell. 1999; 11: 365-376Crossref PubMed Scopus (167) Google Scholar), except that cells were allowed to grow 4 h after induction with isopropyl-1-thio-β-d-galactopyranoside. Following purification, the free glutathione was removed by dialyzing proteins against phosphate buffer (25 mm KPO4, pH 6.0, 0.1 mm EDTA, 10% glycerol) overnight. Protein expression, purification, and quantification were confirmed by SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining (28Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York1999Google Scholar). CoA-ester hydrolysis was assayed essentially as described previously (12Shimomura Y. Murakami T. Fujitsuka N. Nakai N. Sato Y. Sugiyama S. Shimomura N. Irwin J. Hawes J.W. Harris R.A. J. Biol. Chem. 1994; 269: 14248-14253Abstract Full Text PDF PubMed Google Scholar, 13Shimomura Y. Murakami T. Nakai N. Huang B. Hawes J.W. Harris R.A. Methods Enzymol. 2000; 324: 229-240Crossref PubMed Google Scholar, 14Hawes J.W. Jaskiewicz J. Shimomura Y. Huang B. Bunting J. Harper E.T. Harris R.A. J. Biol. Chem. 1996; 271: 26430-26434Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). GST-CHY1 or GST was incubated at 25 °C with each substrate in 1.5 ml of 25 mm phosphate buffer, 50 mmNaCl, 1 mm EDTA, 0.1% Triton X-100. To assay HIBYL-CoA hydrolysis, 10 units of crotonase (Sigma Chemical Co.) was incubated with methacrylyl-CoA prior to addition of 60 ng of GST-CHY1 or 600 ng of GST. Other substrates were tested with 200 ng of GST-CHY1 or GST. At each time point, 450 µl was removed for spectrophotometric determination of liberated CoA. Determination of the pH optimum was done using 25 mm potassium phosphate buffers from pH 5 to 9. Substrate specificity assays were done either at pH 7.5 or 8.0. Methacrylyl-CoA was synthesized as described (13Shimomura Y. Murakami T. Nakai N. Huang B. Hawes J.W. Harris R.A. Methods Enzymol. 2000; 324: 229-240Crossref PubMed Google Scholar). IAA-N-acetylcysteamine, IBA-N-acetylcysteamine, and IAA-CoA were synthesized by benzotriazolyloxytris(pyrrolidine)phosphonium hexafluorophosphate-mediated coupling (37Coste J. Le-Nguyen D. Castro B. Tetrahedron Lett. 1990; 31: 205-208Crossref Scopus (642) Google Scholar) of IAA or IBA toN-acetylcysteamine or CoA using established protocols (38Belshaw P.J. Walsh C.T. Stachelhaus T. Science. 1999; 284: 486-488Crossref PubMed Scopus (263) Google Scholar). Acetyl-, benzoyl-, β-hydroxybutyryl-, butyryl-, crotonoyl-, isobutyryl-, isovaleryl-, malonyl-, methylmalonyl-, oleoyl-, phenylacetyl-, and propionyl-CoA esters were from Sigma. Kinetic values were determined using the Igor-Pro program (WaveMetrics). For reverse-transcription PCR analysis, wild-type, chy1-1 and chy1-2 mutant plants were grown on PNS plates covered with filter paper for 4 days under white light. Tissue was harvested by immersion in liquid nitrogen. RNA was isolated as described previously (39Nagy F. Kay S.A. Chua N.H. Gelvin S.B. Schilperourt R.A. Molecular Biology Manual. Kluwer Academic Publishers, Dordrect1988: B4/1-B4/29Google Scholar). Reverse transcription of the RNA was done using the Retroscript reverse transcription-PCR kit (Ambion) according to the instructions of the manufacturer. Amplification of wild-type and mutant cDNAs was performed using primers K14B20-5 plus K14B20-7 or K14B20-5 plus K14B20-6, which span the mutations inchy1-1 and chy1-2, respectively. FourteenArabidopsis IBA-response mutants were previously identified (18Zolman B.K. Yoder A. Bartel B. Genetics. 2000; 156: 1323-1337Crossref PubMed Google Scholar). We show here that three of these mutants (B7, B17, and B52) are alleles of the same gene, which we named CHY1 (for HIBYL-CoA hydrolase; see below). Whereas exogenous IBA and IAA both inhibit wild-type root elongation, the chy1 alleles are resistant to IBA (Fig.2 A) but remain sensitive to IAA (Fig. 2 B) over a range of concentrations. Similarly,chy1 mutants form wild-type numbers of lateral roots when induced on IAA but form few or no lateral roots when grown on IBA (Fig.2 C). In contrast, chy1 mutants respond like wild-type to other phytohormones tested, including the synthetic auxins 2,4-dichlorophenoxyacetic acid (18Zolman B.K. Yoder A. Bartel B. Genetics. 2000; 156: 1323-1337Crossref PubMed Google Scholar) and 1-naphthaleneacetic acid, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, and the cytokinin benzyladenine (data not shown). Thechy1 mutation is recessive; F1 progeny from a backcross of chy1-1 to Col-0 or chy1-2 to Ws respond normally to IBA (data not shown). We previously showed thatchy1 belongs to the subset of IBA-response mutants that develop poorly in the dark in the absence of supplemental sucrose and that this defect correlates with slower fatty acid β-oxidation during germination (18Zolman B.K. Yoder A. Bartel B. Genetics. 2000; 156: 1323-1337Crossref PubMed Google Scholar). This phenotype suggested that the IBA-response defects in chy1 resulted from a reduced ability to convert IBA to IAA in a peroxisomal chain-shortening process. To determine the molecular nature of this defect, we used positional information to clone the gene defective in the chy1 mutants. We mapped the three chy1 alleles to an ∼800-kb interval on the bottom of chromosome 5, between MNA5 (40Kaneko T. Kotani H. Nakamura Y. Sato S. Asamizu E. Miyajima N. Tabata S. DNA Res. 1998; 5: 131-145Crossref PubMed Scopus (29) Google Scholar) and K9I9 (41Kotani H. Nakamura Y. Sato S. Asamizu E. Kaneko T. Miyajima N. Tabata S. DNA Res. 1998; 5: 213-216Crossref Scopus (12) Google Scholar), south of the previously described peroxisome-defective pex5 (18Zolman B.K. Yoder A. Bartel B. Genetics. 2000; 156: 1323-1337Crossref PubMed Google Scholar) and ped2(21Hayashi M. Toriyama K. Kondo M. Nishimura M. Plant Cell. 1998; 10: 183-195PubMed Google Scholar, 42Hayashi M. Nito K. Toriyama-Kato K. Kondo M. Yamaya T. Nishimura M. EMBO J. 2000; 19: 5701-5710Crossref PubMed Scopus (91) Google Scholar) mutants (Fig. 3 A). Because the chy1mutant phenotype suggested peroxisomal defects (18Zolman B.K. Yoder A. Bartel B. Genetics. 2000; 156: 1323-1337Crossref PubMed Google Scholar), we scanned the sequence in this interval for genes that might be involved in peroxisomal biogenesis or function. The clone K14B20 (30Sato S. Nakamura Y. Kaneko T. Katoh T. Asamizu E. Kotani H. Tabata S. DNA Res. 2000; 7: 31-63Crossref PubMed Scopus (30) Google Scholar) contains a gene encoding a protein 43% identical to a mammalian enzyme that hydrolyzes HIBYL-CoA to β-hydroxyisobutyrate and CoA during Val catabolism (Fig. 1, step 5) (12Shimomura Y. Murakami T. Fujitsuka N. Nakai N. Sato Y. Sugiyama S. Shimomura N. Irwin J. Hawes J.W. Harris R.A. J. Biol. Chem. 1994; 269: 14248-14253Abstract Full Text PDF PubMed Google Scholar, 13Shimomura Y. Murakami T. Nakai N. Huang B. Hawes J.W. Harris R.A. Methods Enzymol. 2000; 324: 229-240Crossref PubMed Google Scholar, 14Hawes J.W. Jaskiewicz J. Shimomura Y. Huang B. Bunting J. Harper E.T. Harris R.A. J. Biol. Chem. 1996; 271: 26430-26434Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Unlike the mammalian enzyme, which is mitochondrial (12Shimomura Y. Murakami T. Fujitsuka N. Nakai N. Sato Y. Sugiyama S. Shimomura N. Irwin J. Hawes J.W. Harris R.A. J. Biol. Chem. 1994; 269: 14248-14253Abstract Full Text PDF PubMed Google Scholar, 13Shimomura Y. Murakami T. Nakai N. Huang B. Hawes J.W. Harris R.A. Methods Enzymol. 2000; 324: 229-240Crossref PubMed Google Scholar, 14Hawes J.W. Jaskiewicz J. Shimomura Y. Huang B. Bunting J. Harper E.T. Harris R.A. J. Biol. Chem. 1996; 271: 26430-26434Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), the Arabidopsis protein was predicted to be peroxisomal by the PSORT program (43Nakai K. Kanehisa M. Genomics. 1992; 14: 897-911Crossref PubMed Scopus (1366) Google Scholar) because of the presence of a type 1 (C-terminal) peroxisomal targeting signal (44Gould S.J. Keller G.-A. Hosken N. Wilkinson J. Subramani S. J. Cell Biol. 1989; 108: 1657-1664Crossref PubMed Scopus (886) Google Scholar). We hypothesized that disruption of Val catabolism might indirectly disrupt peroxisomal β-oxidation (see below) and cause an IBA-resistant, IAA-sensitive mutant phenotype. To determine if chy1 is defective in this HIBYL-CoA hydrolase homolog, we PCR-amplified and seq
DOI: 10.1038/nsmb0705-569
2005
Cited 86 times
MicroRNAs directing siRNA biogenesis
DOI: 10.1186/1471-2229-8-41
2008
Cited 84 times
The IBR5 phosphatase promotes Arabidopsis auxin responses through a novel mechanism distinct from TIR1-mediated repressor degradation
Abstract Background In Arabidopsis, I NDOLE-3- B UTYRIC ACID R ESPONSE5 (IBR5), a putative dual-specificity protein phosphatase, is a positive regulator of auxin response. Mutations in IBR5 result in decreased plant height, defective vascular development, increased leaf serration, fewer lateral roots, and resistance to the phytohormones auxin and abscisic acid. However, the pathways through which IBR5 influences auxin responses are not fully understood. Results We analyzed double mutants of ibr5 with other mutants that dampen auxin responses and found that combining ibr5 with an auxin receptor mutant, tir1 , enhanced auxin resistance relative to either parent. Like other auxin-response mutants, auxin-responsive reporter accumulation was reduced in ibr5 . Unlike other auxin-resistant mutants, the Aux/IAA repressor reporter protein AXR3NT-GUS was not stabilized in ibr5 . Similarly, the Aux/IAA repressor IAA28 was less abundant in ibr5 than in wild type. ibr5 defects were not fully rescued by overexpression of a mutant form of IBR5 lacking the catalytic cysteine residue. Conclusion Our genetic and molecular evidence suggests that IBR5 is a phosphatase that promotes auxin responses, including auxin-inducible transcription, differently than the TIR1 auxin receptor and without destabilizing Aux/IAA repressor proteins. Our data are consistent with the possibility that auxin-responsive transcription can be modulated downstream of TIR1-mediated repressor degradation.
DOI: 10.1104/pp.109.142505
2009
Cited 78 times
Arabidopsis LON2 Is Necessary for Peroxisomal Function and Sustained Matrix Protein Import
Relatively little is known about the small subset of peroxisomal proteins with predicted protease activity. Here, we report that the peroxisomal LON2 (At5g47040) protease facilitates matrix protein import into Arabidopsis (Arabidopsis thaliana) peroxisomes. We identified T-DNA insertion alleles disrupted in five of the nine confirmed or predicted peroxisomal proteases and found only two-lon2 and deg15, a mutant defective in the previously described PTS2-processing protease (DEG15/At1g28320)-with phenotypes suggestive of peroxisome metabolism defects. Both lon2 and deg15 mutants were mildly resistant to the inhibitory effects of indole-3-butyric acid (IBA) on root elongation, but only lon2 mutants were resistant to the stimulatory effects of IBA on lateral root production or displayed Suc dependence during seedling growth. lon2 mutants displayed defects in removing the type 2 peroxisome targeting signal (PTS2) from peroxisomal malate dehydrogenase and reduced accumulation of 3-ketoacyl-CoA thiolase, another PTS2-containing protein; both defects were not apparent upon germination but appeared in 5- to 8-d-old seedlings. In lon2 cotyledon cells, matrix proteins were localized to peroxisomes in 4-d-old seedlings but mislocalized to the cytosol in 8-d-old seedlings. Moreover, a PTS2-GFP reporter sorted to peroxisomes in lon2 root tip cells but was largely cytosolic in more mature root cells. Our results indicate that LON2 is needed for sustained matrix protein import into peroxisomes. The delayed onset of matrix protein sorting defects may account for the relatively weak Suc dependence following germination, moderate IBA-resistant primary root elongation, and severe defects in IBA-induced lateral root formation observed in lon2 mutants.
DOI: 10.1534/genetics.112.146100
2013
Cited 53 times
Genetic Dissection of Peroxisome-Associated Matrix Protein Degradation in <i>Arabidopsis thaliana</i>
Abstract Peroxisomes are organelles that sequester certain metabolic pathways; many of these pathways generate H2O2, which can damage proteins. However, little is known about how damaged or obsolete peroxisomal proteins are degraded. We exploit developmentally timed peroxisomal content remodeling in Arabidopsis thaliana to elucidate peroxisome-associated protein degradation. Isocitrate lyase (ICL) is a peroxisomal glyoxylate cycle enzyme necessary for early seedling development. A few days after germination, photosynthesis begins and ICL is degraded. We previously found that ICL is stabilized when a peroxisome-associated ubiquitin-conjugating enzyme and its membrane anchor are both mutated, suggesting that matrix proteins might exit the peroxisome for ubiquitin-dependent cytosolic degradation. To identify additional components needed for peroxisome-associated matrix protein degradation, we mutagenized a line expressing GFP–ICL, which is degraded similarly to endogenous ICL, and identified persistent GFP-ICLfluorescence (pfl) mutants. We found three pfl mutants that were defective in PEROXIN14 (PEX14/At5g62810), which encodes a peroxisomal membrane protein that assists in importing proteins into the peroxisome matrix, indicating that proteins must enter the peroxisome for efficient degradation. One pfl mutant was missing the peroxisomal 3-ketoacyl-CoA thiolase encoded by the PEROXISOME DEFECTIVE1 (PED1/At2g33150) gene, suggesting that peroxisomal metabolism influences the rate of matrix protein degradation. Finally, one pfl mutant that displayed normal matrix protein import carried a novel lesion in PEROXIN6 (PEX6/At1g03000), which encodes a peroxisome-tethered ATPase that is involved in recycling matrix protein receptors back to the cytosol. The isolation of pex6-2 as a pfl mutant supports the hypothesis that matrix proteins can exit the peroxisome for cytosolic degradation.
DOI: 10.1534/genetics.118.300755
2018
Cited 40 times
Biology in Bloom: A Primer on the <i>Arabidopsis thaliana</i> Model System
Abstract Arabidopsis thaliana could have easily escaped human scrutiny. Instead, Arabidopsis has become the most widely studied plant in modern biology despite its absence from the dinner table. Pairing diminutive stature and genome with prodigious resources and tools, Arabidopsis offers a window into the molecular, cellular, and developmental mechanisms underlying life as a multicellular photoautotroph. Many basic discoveries made using this plant have spawned new research areas, even beyond the verdant fields of plant biology. With a suite of resources and tools unmatched among plants and rivaling other model systems, Arabidopsis research continues to offer novel insights and deepen our understanding of fundamental biological processes.
DOI: 10.1080/15548627.2019.1569915
2019
Cited 34 times
A facile forward-genetic screen for<i>Arabidopsis</i>autophagy mutants reveals twenty-one loss-of-function mutations disrupting six<i>ATG</i>genes
Macroautophagy is a process through which eukaryotic cells degrade large substrates including organelles, protein aggregates, and invading pathogens. Over 40 autophagy-related (ATG) genes have been identified through forward-genetic screens in yeast. Although homology-based analyses have identified conserved ATG genes in plants, only a few atg mutants have emerged from forward-genetic screens in Arabidopsis thaliana. We developed a screen that consistently recovers Arabidopsis atg mutations by exploiting mutants with defective LON2/At5g47040, a protease implicated in peroxisomal quality control. Arabidopsis lon2 mutants exhibit reduced responsiveness to the peroxisomally-metabolized auxin precursor indole-3-butyric acid (IBA), heightened degradation of several peroxisomal matrix proteins, and impaired processing of proteins harboring N-terminal peroxisomal targeting signals; these defects are ameliorated by preventing autophagy. We optimized a lon2 suppressor screen to expedite recovery of additional atg mutants. After screening mutagenized lon2-2 seedlings for restored IBA responsiveness, we evaluated stabilization and processing of peroxisomal proteins, levels of several ATG proteins, and levels of the selective autophagy receptor NBR1/At4g24690, which accumulates when autophagy is impaired. We recovered 21 alleles disrupting 6 ATG genes: ATG2/At3g19190, ATG3/At5g61500, ATG5/At5g17290, ATG7/At5g45900, ATG16/At5g50230, and ATG18a/At3g62770. Twenty alleles were novel, and 3 of the mutated genes lack T-DNA insertional alleles in publicly available repositories. We also demonstrate that an insertional atg11/At4g30790 allele incompletely suppresses lon2 defects. Finally, we show that NBR1 is not necessary for autophagy of lon2 peroxisomes and that NBR1 overexpression is not sufficient to trigger autophagy of seedling peroxisomes, indicating that Arabidopsis can use an NBR1-independent mechanism to target peroxisomes for autophagic degradation.Abbreviations: ATG: autophagy-related; ATI: ATG8-interacting protein; Col-0: Columbia-0; DSK2: dominant suppressor of KAR2; EMS: ethyl methanesulfonate; GFP: green fluorescent protein; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; ICL: isocitrate lyase; MLS: malate synthase; NBR1: Next to BRCA1 gene 1; PEX: peroxin; PMDH: peroxisomal malate dehydrogenase; PTS: peroxisomal targeting signal; thiolase: 3-ketoacyl-CoA thiolase; UBA: ubiquitin-associated; WT: wild type
DOI: 10.1023/a:1011699722052
2001
Cited 84 times
A library of Arabidopsis 35S-cDNA lines for identifying novel mutants.
DOI: 10.1105/tpc.105.036236
2005
Cited 74 times
A Receptor for Auxin
A long-sought hormone receptor has been found. Two recent Nature articles reveal that the F-box protein TRANSPORT INHIBITOR RESPONSE1 (TIR1) binds auxin and responds to the phytohormone even when heterologously expressed in animal systems ([Dharmasiri et al., 2005a][1]; [Kepinski and Leyser, 2005][2
DOI: 10.1534/genetics.108.091512
2008
Cited 54 times
Arabidopsis <i>iba response5</i> Suppressors Separate Responses to Various Hormones
Auxin controls numerous plant growth processes by directing cell division and expansion. Auxin-response mutants, including iba response5 (ibr5), exhibit a long root and decreased lateral root production in response to exogenous auxins. ibr5 also displays resistance to the phytohormone abscisic acid (ABA). We found that the sar3 suppressor of auxin resistant1 (axr1) mutant does not suppress ibr5 auxin-response defects, suggesting that screening for ibr5 suppressors might reveal new components important for phytohormone responsiveness. We identified two classes of Arabidopsis thaliana mutants that suppressed ibr5 resistance to indole-3-butyric acid (IBA): those with restored responses to both the auxin precursor IBA and the active auxin indole-3-acetic acid (IAA) and those with restored response to IBA but not IAA. Restored IAA sensitivity was accompanied by restored ABA responsiveness, whereas suppressors that remained IAA resistant also remained ABA resistant. Some suppressors restored sensitivity to both natural and synthetic auxins; others restored responsiveness only to auxin precursors. We used positional information to determine that one ibr5 suppressor carried a mutation in PLEIOTROPIC DRUG RESISTANCE9 (PDR9/ABCG37/At3g53480), which encodes an ATP-binding cassette transporter previously implicated in cellular efflux of the synthetic auxin 2,4-dichlorophenoxyacetic acid.
DOI: 10.1091/mbc.e09-08-0672
2010
Cited 54 times
Interdependence of the Peroxisome-targeting Receptors in<i>Arabidopsis thaliana</i>: PEX7 Facilitates PEX5 Accumulation and Import of PTS1 Cargo into Peroxisomes
Peroxisomes compartmentalize certain metabolic reactions critical to plant and animal development. The import of proteins from the cytosol into the organelle matrix depends on more than a dozen peroxin (PEX) proteins, with PEX5 and PEX7 serving as receptors that shuttle proteins bearing one of two peroxisome-targeting signals (PTSs) into the organelle. PEX5 is the PTS1 receptor; PEX7 is the PTS2 receptor. In plants and mammals, PEX7 depends on PEX5 binding to deliver PTS2 cargo into the peroxisome. In this study, we characterized a pex7 missense mutation, pex7-2, that disrupts both PEX7 cargo binding and PEX7-PEX5 interactions in yeast, as well as PEX7 protein accumulation in plants. We examined localization of peroxisomally targeted green fluorescent protein derivatives in light-grown pex7 mutants and observed not only the expected defects in PTS2 protein import but also defects in PTS1 import. These PTS1 import defects were accompanied by reduced PEX5 accumulation in light-grown pex7 seedlings. Our data suggest that PEX5 and PTS1 import depend on the PTS2 receptor PEX7 in Arabidopsis and that the environment may influence this dependence. These data advance our understanding of the biogenesis of these essential organelles and provide a possible rationale for the retention of the PTS2 pathway in some organisms.
DOI: 10.1534/genetics.116.193169
2016
Cited 32 times
The Roles of β-Oxidation and Cofactor Homeostasis in Peroxisome Distribution and Function in <i>Arabidopsis thaliana</i>
Abstract Key steps of essential metabolic pathways are housed in plant peroxisomes. We conducted a microscopy-based screen for anomalous distribution of peroxisomally targeted fluorescence in Arabidopsis thaliana. This screen uncovered 34 novel alleles in 15 genes affecting oil body mobilization, fatty acid β-oxidation, the glyoxylate cycle, peroxisome fission, and pexophagy. Partial loss-of-function of lipid-mobilization enzymes conferred peroxisomes clustered around retained oil bodies without other notable defects, suggesting that this microscopy-based approach was sensitive to minor perturbations, and that fatty acid β-oxidation rates in wild type are higher than required for normal growth. We recovered three mutants defective in PECTIN METHYLESTERASE31, revealing an unanticipated role in lipid mobilization for this cytosolic enzyme. Whereas mutations reducing fatty acid import had peroxisomes of wild-type size, mutations impairing fatty acid β-oxidation displayed enlarged peroxisomes, possibly caused by excess fatty acid β-oxidation intermediates in the peroxisome. Several fatty acid β-oxidation mutants also displayed defects in peroxisomal matrix protein import. Impairing fatty acid import reduced the large size of peroxisomes in a mutant defective in the PEROXISOMAL NAD+ TRANSPORTER (PXN), supporting the hypothesis that fatty acid accumulation causes pxn peroxisome enlargement. The diverse mutants isolated in this screen will aid future investigations of the roles of β-oxidation and peroxisomal cofactor homeostasis in plant development.
DOI: 10.1073/pnas.2304870120
2023
Cited 5 times
The ubiquitin-protein ligase MIEL1 localizes to peroxisomes to promote seedling oleosin degradation and lipid droplet mobilization
Lipid droplets are organelles conserved across eukaryotes that store and release neutral lipids to regulate energy homeostasis. In oilseed plants, fats stored in seed lipid droplets provide fixed carbon for seedling growth before photosynthesis begins. As fatty acids released from lipid droplet triacylglycerol are catabolized in peroxisomes, lipid droplet coat proteins are ubiquitinated, extracted, and degraded. In Arabidopsis seeds, the predominant lipid droplet coat protein is OLEOSIN1 (OLE1). To identify genes modulating lipid droplet dynamics, we mutagenized a line expressing mNeonGreen-tagged OLE1 expressed from the OLE1 promoter and isolated mutants with delayed oleosin degradation. From this screen, we identified four miel1 mutant alleles. MIEL1 (MYB30-interacting E3 ligase 1) targets specific MYB transcription factors for degradation during hormone and pathogen responses [D. Marino et al ., Nat. Commun. 4 , 1476 (2013); H. G. Lee and P. J. Seo, Nat. Commun. 7 , 12525 (2016)] but had not been implicated in lipid droplet dynamics. OLE1 transcript levels were unchanged in miel1 mutants, indicating that MIEL1 modulates oleosin levels posttranscriptionally. When overexpressed, fluorescently tagged MIEL1 reduced oleosin levels, causing very large lipid droplets. Unexpectedly, fluorescently tagged MIEL1 localized to peroxisomes. Our data suggest that MIEL1 ubiquitinates peroxisome-proximal seed oleosins, targeting them for degradation during seedling lipid mobilization. The human MIEL1 homolog (PIRH2; p53-induced protein with a RING-H2 domain) targets p53 and other proteins for degradation and promotes tumorigenesis [A. Daks et al ., Cells 11 , 1515 (2022)]. When expressed in Arabidopsis , human PIRH2 also localized to peroxisomes, hinting at a previously unexplored role for PIRH2 in lipid catabolism and peroxisome biology in mammals.
DOI: 10.1016/s1534-5807(01)00077-6
2001
Cited 65 times
Auxin Signaling
Auxins are a class of phytohormones implicated in virtually every aspect of plant growth and development. Many early plant responses to auxin are apparently mediated by members of a family of Aux/IAA proteins that dimerize with and inhibit members of the auxin response factor (ARF) family of transcription factors. Aux/IAA proteins are unstable, and their degradation is triggered by a ubiquitin-protein ligase that is regulated by modification with a ubiquitin-related protein. Recent genetic and biochemical evidence indicates that auxin accelerates the degradation of the already short-lived Aux/IAA proteins to derepress transcription by ARF proteins. Several pieces of the auxin-signaling puzzle remain to be assembled, including the proteins that initially bind auxin, the proteins that convey this signal to the protein degradation machinery, and the targets of the transcriptional derepression.
DOI: 10.1038/nchembio0608-337
2008
Cited 50 times
A new path to auxin
DOI: 10.1007/s11103-011-9782-0
2011
Cited 36 times
Matrix proteins are inefficiently imported into Arabidopsis peroxisomes lacking the receptor-docking peroxin PEX14
Mutations in peroxisome biogenesis proteins (peroxins) can lead to developmental deficiencies in various eukaryotes. PEX14 and PEX13 are peroxins involved in docking cargo-receptor complexes at the peroxisomal membrane, thus aiding in the transport of the cargo into the peroxisomal matrix. Genetic screens have revealed numerous Arabidopsis thaliana peroxins acting in peroxisomal matrix protein import; the viable alleles isolated through these screens are generally partial loss-of-function alleles, whereas null mutations that disrupt delivery of matrix proteins to peroxisomes can confer embryonic lethality. In this study, we used forward and reverse genetics in Arabidopsis to isolate four pex14 alleles. We found that all four alleles conferred reduced PEX14 mRNA levels and displayed physiological and molecular defects suggesting reduced but not abolished peroxisomal matrix protein import. The least severe pex14 allele, pex14-3, accumulated low levels of a C-terminally truncated PEX14 product that retained partial function. Surprisingly, even the severe pex14-2 allele, which lacked detectable PEX14 mRNA and PEX14 protein, was viable, fertile, and displayed residual peroxisome matrix protein import. As pex14 plants matured, import improved. Together, our data indicate that PEX14 facilitates, but is not essential for peroxisomal matrix protein import in plants.
DOI: 10.1104/pp.114.247148
2014
Cited 33 times
Peroxisomal Ubiquitin-Protein Ligases Peroxin2 and Peroxin10 Have Distinct But Synergistic Roles in Matrix Protein Import and Peroxin5 Retrotranslocation in Arabidopsis
Peroxisomal matrix proteins carry peroxisomal targeting signals (PTSs), PTS1 or PTS2, and are imported into the organelle with the assistance of peroxin (PEX) proteins. From a microscopy-based screen to identify Arabidopsis (Arabidopsis thaliana) mutants defective in matrix protein degradation, we isolated unique mutations in PEX2 and PEX10, which encode ubiquitin-protein ligases anchored in the peroxisomal membrane. In yeast (Saccharomyces cerevisiae), PEX2, PEX10, and a third ligase, PEX12, ubiquitinate a peroxisome matrix protein receptor, PEX5, allowing the PEX1 and PEX6 ATP-hydrolyzing enzymes to retrotranslocate PEX5 out of the membrane after cargo delivery. We found that the pex2-1 and pex10-2 Arabidopsis mutants exhibited defects in peroxisomal physiology and matrix protein import. Moreover, the pex2-1 pex10-2 double mutant exhibited severely impaired growth and synergistic physiological defects, suggesting that PEX2 and PEX10 function cooperatively in the wild type. The pex2-1 lesion restored the unusually low PEX5 levels in the pex6-1 mutant, implicating PEX2 in PEX5 degradation when retrotranslocation is impaired. PEX5 overexpression altered pex10-2 but not pex2-1 defects, suggesting that PEX10 facilitates PEX5 retrotranslocation from the peroxisomal membrane. Although the pex2-1 pex10-2 double mutant displayed severe import defects of both PTS1 and PTS2 proteins into peroxisomes, both pex2-1 and pex10-2 single mutants exhibited clear import defects of PTS1 proteins but apparently normal PTS2 import. A similar PTS1-specific pattern was observed in the pex4-1 ubiquitin-conjugating enzyme mutant. Our results indicate that Arabidopsis PEX2 and PEX10 cooperate to support import of matrix proteins into plant peroxisomes and suggest that some PTS2 import can still occur when PEX5 retrotranslocation is slowed.
DOI: 10.1038/s41467-020-20099-y
2020
Cited 23 times
Peroxisomes form intralumenal vesicles with roles in fatty acid catabolism and protein compartmentalization in Arabidopsis
Abstract Peroxisomes are vital organelles that compartmentalize critical metabolic reactions, such as the breakdown of fats, in eukaryotic cells. Although peroxisomes typically are considered to consist of a single membrane enclosing a protein lumen, more complex peroxisomal membrane structure has occasionally been observed in yeast, mammals, and plants. However, technical challenges have limited the recognition and understanding of this complexity. Here we exploit the unusually large size of Arabidopsis peroxisomes to demonstrate that peroxisomes have extensive internal membranes. These internal vesicles accumulate over time, use ESCRT (endosomal sorting complexes required for transport) machinery for formation, and appear to derive from the outer peroxisomal membrane. Moreover, these vesicles can harbor distinct proteins and do not form normally when fatty acid β-oxidation, a core function of peroxisomes, is impaired. Our findings suggest a mechanism for lipid mobilization that circumvents challenges in processing insoluble metabolites. This revision of the classical view of peroxisomes as single-membrane organelles has implications for all aspects of peroxisome biogenesis and function and may help address fundamental questions in peroxisome evolution.
DOI: 10.1111/j.1600-0854.2010.01136.x
2010
Cited 33 times
Reducing <i>PEX13</i> Expression Ameliorates Physiological Defects of Late‐Acting Peroxin Mutants
Proteins are targeted to the peroxisome matrix via processes that are mechanistically distinct from those used by other organelles. Protein entry into peroxisomes requires peroxin (PEX) proteins, including early‐acting receptor (e.g. PEX5) and docking peroxins (e.g. PEX13 and PEX14) and late‐acting PEX5‐recycling peroxins (e.g. PEX4 and PEX6). We examined genetic interactions among Arabidopsis peroxin mutants and found that the weak pex13‐1 allele had deleterious effects when combined with pex5‐1 and pex14‐2 , which are defective in early‐acting peroxins, as shown by reduced matrix protein import and enhanced physiological defects. In contrast, combining pex13‐1 with pex4‐1 or pex6‐1 , which are defective in late‐acting peroxins, unexpectedly ameliorated mutant growth defects. Matrix protein import remained impaired in pex4‐1 pex13‐1 and pex6‐1 pex13‐1 , suggesting that the partial suppression of pex4‐1 and pex6‐1 physiological defects by a weak pex13 allele may result from restoring the balance between import and export of PEX5 or other proteins that are retrotranslocated from the peroxisome with the assistance of PEX4 and PEX6. Our results suggest that symptoms caused by pex mutants defective in late‐acting peroxins may result not only from defects in matrix protein import but also from inefficient removal of PEX5 from the peroxisomal membrane following cargo delivery.
DOI: 10.1046/j.1365-313x.2003.01826.x
2003
Cited 41 times
<i>ILR2</i>, a novel gene regulating IAA conjugate sensitivity and metal transport in <i>Arabidopsis thaliana</i>
Plants can regulate levels of the auxin indole-3-acetic acid (IAA) by conjugation to amino acids or sugars, and subsequent hydrolysis of these conjugates to release active IAA. These less active auxin conjugates constitute the majority of IAA in plants. We isolated the Arabidopsis ilr2-1 mutant as a recessive IAA-leucine resistant mutant that retains wild-type sensitivity to free IAA. ilr2-1 is also defective in lateral root formation and primary root elongation. In addition, ilr2-1 is resistant to manganese- and cobalt-mediated inhibition of root elongation, and microsomal preparations from the ilr2-1 mutant exhibit enhanced ATP-dependent manganese transport. We used a map-based positional approach to clone the ILR2 gene, which encodes a novel protein with no predicted membrane-spanning domains that is polymorphic among Arabidopsis accessions. Our results demonstrate that ILR2 modulates a metal transporter, providing a novel link between auxin conjugate metabolism and metal homeostasis.
DOI: 10.1104/pp.104.040519
2004
Cited 40 times
<i>IAR4</i>, a Gene Required for Auxin Conjugate Sensitivity in Arabidopsis, Encodes a Pyruvate Dehydrogenase E1<i>α</i> Homolog
The formation and hydrolysis of indole-3-acetic acid (IAA) conjugates represent a potentially important means for plants to regulate IAA levels and thereby auxin responses. The identification and characterization of mutants defective in these processes is advancing the understanding of auxin regulation and response. Here we report the isolation and characterization of the Arabidopsis iar4 mutant, which has reduced sensitivity to several IAA-amino acid conjugates. iar4 is less sensitive to a synthetic auxin and low concentrations of an ethylene precursor but responds to free IAA and other hormones tested similarly to wild type. The gene defective in iar4 encodes a homolog of the E1alpha-subunit of mitochondrial pyruvate dehydrogenase, which converts pyruvate to acetyl-coenzyme A. We did not detect glycolysis or Krebs-cycle-related defects in the iar4 mutant, and a T-DNA insertion in the IAR4 coding sequence conferred similar phenotypes as the originally identified missense allele. In contrast, we found that disruption of the previously described mitochondrial pyruvate dehydrogenase E1alpha-subunit does not alter IAA-Ala responsiveness or confer any obvious phenotypes. It is possible that IAR4 acts in the conversion of indole-3-pyruvate to indole-3-acetyl-coenzyme A, which is a potential precursor of IAA and IAA conjugates.
DOI: 10.1104/pp.107.100404
2007
Cited 32 times
Mutation of<i>E1-CONJUGATING ENZYME-RELATED1</i>Decreases RELATED TO UBIQUITIN Conjugation and Alters Auxin Response and Development
The ubiquitin-like protein RELATED TO UBIQUITIN (RUB) is conjugated to CULLIN (CUL) proteins to modulate the activity of Skp1-Cullin-F-box (SCF) ubiquitylation complexes. RUB conjugation to specific target proteins is necessary for the development of many organisms, including Arabidopsis (Arabidopsis thaliana). Here, we report the isolation and characterization of e1-conjugating enzyme-related1-1 (ecr1-1), an Arabidopsis mutant compromised in RUB conjugation. The ecr1-1 mutation causes a missense change located two amino acid residues from the catalytic site cysteine, which normally functions to form a thioester bond with activated RUB. A higher ratio of unmodified CUL1 relative to CUL1-RUB is present in ecr1-1 compared to wild type, suggesting that the mutation reduces ECR1 function. The ecr1-1 mutant is resistant to the auxin-like compound indole-3-propionic acid, produces fewer lateral roots than wild type, displays reduced adult height, and stabilizes a reporter fusion protein that is degraded in response to auxin, suggesting reduced auxin signaling in the mutant. In addition, ecr1-1 hypocotyls fail to elongate normally when seedlings are grown in darkness, a phenotype shared with certain other RUB conjugation mutants that is not general to auxin-response mutants. The suite of ecr1-1 molecular and morphological phenotypes reflects roles for RUB conjugation in many aspects of plant growth and development. Certain ecr1-1 elongation defects are restored by treatment with the ethylene-response inhibitor silver nitrate, suggesting that the short ecr1-1 root and hypocotyl result from aberrant ethylene accumulation. Further, silver nitrate supplementation in combination with various auxins and auxin-like compounds reveals that members of this growth regulator family may differentially rely on ethylene signaling to inhibit root growth.
DOI: 10.4161/auto.27565
2014
Cited 24 times
Mutation of the<i>Arabidopsis</i>LON2 peroxisomal protease enhances pexophagy
Peroxisomes are critical organelles housing various, often oxidative, reactions. Pexophagy, the process by which peroxisomes are selectively targeted for destruction via autophagy, is characterized in yeast and mammals but had not been reported in plants. In this article, we describe how the peroxisome-related aberrations of a mutant defective in the LON2 peroxisomal protease are suppressed when autophagy is prevented by mutating any of several key autophagy-related (ATG) genes. Our results reveal that plant peroxisomes can be degraded by selective autophagy and suggest that pexophagy is accelerated when the LON2 protease is disabled.
DOI: 10.1007/s11103-014-0223-8
2014
Cited 23 times
A viable Arabidopsis pex13 missense allele confers severe peroxisomal defects and decreases PEX5 association with peroxisomes
Peroxisomes are organelles that catabolize fatty acids and compartmentalize other oxidative metabolic processes in eukaryotes. Using a forward-genetic screen designed to recover severe peroxisome-defective mutants, we isolated a viable allele of the peroxisome biogenesis gene PEX13 with striking peroxisomal defects. The pex13-4 mutant requires an exogenous source of fixed carbon for pre-photosynthetic development and is resistant to the protoauxin indole-3-butyric acid. Delivery of peroxisome-targeted matrix proteins depends on the PEX5 receptor docking with PEX13 at the peroxisomal membrane, and we found severely reduced import of matrix proteins and less organelle-associated PEX5 in pex13-4 seedlings. Moreover, pex13-4 physiological and molecular defects were partially ameliorated when PEX5 was overexpressed, suggesting that PEX5 docking is partially compromised in this mutant and can be improved by increasing PEX5 levels. Because previously described Arabidopsis pex13 alleles either are lethal or confer only subtle defects, the pex13-4 mutant provides valuable insight into plant peroxisome receptor docking and matrix protein import.
DOI: 10.1186/s12870-015-0605-3
2015
Cited 20 times
Elevated growth temperature decreases levels of the PEX5 peroxisome-targeting signal receptor and ameliorates defects of Arabidopsis mutants with an impaired PEX4 ubiquitin-conjugating enzyme
Peroxisomes house critical metabolic reactions. For example, fatty acid β-oxidation enzymes, which are essential during early seedling development, are peroxisomal. Peroxins (PEX proteins) are needed to bring proteins into peroxisomes. Most matrix proteins are delivered to peroxisomes by PEX5, a receptor that forms transient pores to escort proteins across the peroxisomal membrane. After cargo delivery, a peroxisome-tethered ubiquitin-conjugating enzyme (PEX4) and peroxisomal ubiquitin-protein ligases mono- or polyubiquitinate PEX5 for recycling back to the cytosol or for degradation, respectively. Arabidopsis pex mutants β-oxidize fatty acids inefficiently and therefore fail to germinate or grow less vigorously. These defects can be partially alleviated by providing a fixed carbon source, such as sucrose, in the growth medium. Despite extensive characterization of peroxisome biogenesis in Arabidopsis grown in non-challenged conditions, the effects of environmental stressors on peroxisome function and pex mutant dysfunction are largely unexplored. We surveyed the impact of growth temperature on a panel of pex mutants and found that elevated temperature ameliorated dependence on external sucrose and reduced PEX5 levels in the pex4-1 mutant. Conversely, growth at low temperature exacerbated pex4-1 physiological defects and increased PEX5 levels. Overexpressing PEX5 also worsened pex4-1 defects, implying that PEX5 lingering on the peroxisomal membrane when recycling is impaired impedes peroxisome function. Growth at elevated temperature did not reduce the fraction of membrane-associated PEX5 in pex4-1, suggesting that elevated temperature did not restore PEX4 enzymatic function in the mutant. Moreover, preventing autophagy in pex4-1 did not restore PEX5 levels at high temperature. In contrast, MG132 treatment increased PEX5 levels, implicating the proteasome in degrading PEX5, especially at high temperature. We conclude that growth at elevated temperature increases proteasomal degradation of PEX5 to reduce overall PEX5 levels and ameliorate pex4-1 physiological defects. Our results support the hypothesis that efficient retrotranslocation of PEX5 after cargo delivery is needed not only to make PEX5 available for further rounds of cargo delivery, but also to prevent the peroxisome dysfunction that results from PEX5 lingering in the peroxisomal membrane.
DOI: 10.1104/pp.16.01211
2016
Cited 18 times
Genetic Interactions between PEROXIN12 and Other Peroxisome-Associated Ubiquitination Components
Most eukaryotic cells require peroxisomes, organelles housing fatty acid β-oxidation and other critical metabolic reactions. Peroxisomal matrix proteins carry peroxisome-targeting signals that are recognized by one of two receptors, PEX5 or PEX7, in the cytosol. After delivering the matrix proteins to the organelle, these receptors are removed from the peroxisomal membrane or matrix. Receptor retrotranslocation not only facilitates further rounds of matrix protein import but also prevents deleterious PEX5 retention in the membrane. Three peroxisome-associated ubiquitin-protein ligases in the Really Interesting New Gene (RING) family, PEX2, PEX10, and PEX12, facilitate PEX5 retrotranslocation. However, the detailed mechanism of receptor retrotranslocation remains unclear in plants. We identified an Arabidopsis (Arabidopsis thaliana) pex12 Glu-to-Lys missense allele that conferred severe peroxisomal defects, including impaired β-oxidation, inefficient matrix protein import, and decreased growth. We compared this pex12-1 mutant to other peroxisome-associated ubiquitination-related mutants and found that RING peroxin mutants displayed elevated PEX5 and PEX7 levels, supporting the involvement of RING peroxins in receptor ubiquitination in Arabidopsis. Also, we observed that disruption of any Arabidopsis RING peroxin led to decreased PEX10 levels, as seen in yeast and mammals. Peroxisomal defects were exacerbated in RING peroxin double mutants, suggesting distinct roles of individual RING peroxins. Finally, reducing function of the peroxisome-associated ubiquitin-conjugating enzyme PEX4 restored PEX10 levels and partially ameliorated the other molecular and physiological defects of the pex12-1 mutant. Future biochemical analyses will be needed to determine whether destabilization of the RING peroxin complex observed in pex12-1 stems from PEX4-dependent ubiquitination on the pex12-1 ectopic Lys residue.
DOI: 10.1104/pp.17.00548
2017
Cited 18 times
The PEX1 ATPase Stabilizes PEX6 and Plays Essential Roles in Peroxisome Biology
A variety of metabolic pathways are sequestered in peroxisomes, conserved organelles that are essential for human and plant survival. Peroxin (PEX) proteins generate and maintain peroxisomes. The PEX1 ATPase facilitates recycling of the peroxisome matrix protein receptor PEX5 and is the most commonly affected peroxin in human peroxisome biogenesis disorders. Here, we describe the isolation and characterization of, to our knowledge, the first Arabidopsis (Arabidopsis thaliana) pex1 missense alleles: pex1-2 and pex1-3pex1-2 displayed peroxisome-related defects accompanied by reduced PEX1 and PEX6 levels. These pex1-2 defects were exacerbated by growth at high temperature and ameliorated by growth at low temperature or by PEX6 overexpression, suggesting that PEX1 enhances PEX6 stability and vice versa. pex1-3 conferred embryo lethality when homozygous, confirming that PEX1, like several other Arabidopsis peroxins, is essential for embryogenesis. pex1-3 displayed symptoms of peroxisome dysfunction when heterozygous; this semidominance is consistent with PEX1 forming a heterooligomer with PEX6 that is poisoned by pex1-3 subunits. Blocking autophagy partially rescued PEX1/pex1-3 defects, including the restoration of normal peroxisome size, suggesting that increasing peroxisome abundance can compensate for the deficiencies caused by pex1-3 and that the enlarged peroxisomes visible in PEX1/pex1-3 may represent autophagy intermediates. Overexpressing PEX1 in wild-type plants impaired growth, suggesting that excessive PEX1 can be detrimental. Our genetic, molecular, and physiological data support the heterohexamer model of PEX1-PEX6 function in plants.
DOI: 10.1073/pnas.1721279115
2018
Cited 18 times
A <i>pex1</i> missense mutation improves peroxisome function in a subset of <i>Arabidopsis pex6</i> mutants without restoring PEX5 recycling
Significance ATPases have diverse cellular roles, including extracting proteins from membranes to maintain organellar function. The PEX1–PEX6 heterohexameric ATPases are thought to retrotranslocate PEX5 from the peroxisome membrane, and PEX1–PEX6 dysfunction impairs peroxisome biogenesis in humans and plants. We implemented a pex6 suppressor screen in Arabidopsis and recovered a compensatory pex1 allele that rescues several pex6 defects. Preventing autophagy also improved pex6 peroxisome function, and combining the pex1 and autophagy lesions delivered synergistic benefits. Surprisingly, these different alterations ameliorated pex6 symptoms without notably restoring the sole known function of PEX6, suggesting that PEX1–PEX6 has unexplored functions. Because the pex6 mutations ameliorated by pex1 are analogous to those in human pex6 patients, this study informs research on peroxisome dysfunction in other eukaryotes.
DOI: 10.1371/journal.pone.0148335
2016
Cited 16 times
The Early-Acting Peroxin PEX19 Is Redundantly Encoded, Farnesylated, and Essential for Viability in Arabidopsis thaliana
Peroxisomes are single-membrane bound organelles that are essential for normal development in plants and animals. In mammals and yeast, the peroxin (PEX) proteins PEX3 and PEX19 facilitate the early steps of peroxisome membrane protein (PMP) insertion and pre-peroxisome budding from the endoplasmic reticulum. The PEX3 membrane protein acts as a docking site for PEX19, a cytosolic chaperone for PMPs that delivers PMPs to the endoplasmic reticulum or peroxisomal membrane. PEX19 is farnesylated in yeast and mammals, and we used immunoblotting with prenylation mutants to show that PEX19 also is fully farnesylated in wild-type Arabidopsis thaliana plants. We examined insertional alleles disrupting either of the two Arabidopsis PEX19 isoforms, PEX19A or PEX19B, and detected similar levels of PEX19 protein in the pex19a-1 mutant and wild type; however, PEX19 protein was nearly undetectable in the pex19b-1 mutant. Despite the reduction in PEX19 levels in pex19b-1, both pex19a-1 and pex19b-1 single mutants lacked notable peroxisomal β-oxidation defects and displayed normal levels and localization of peroxisomal matrix and membrane proteins. The pex19a-1 pex19b-1 double mutant was embryo lethal, indicating a redundantly encoded critical role for PEX19 during embryogenesis. Expressing YFP-tagged versions of either PEX19 isoform rescued this lethality, confirming that PEX19A and PEX19B act redundantly in Arabidopsis. We observed that pex19b-1 enhanced peroxisome-related defects of a subset of peroxin-defective mutants, supporting a role for PEX19 in peroxisome function. Together, our data indicate that Arabidopsis PEX19 promotes peroxisome function and is essential for viability.
DOI: 10.1016/j.jbc.2024.106721
2024
Abstract 2398 Investigating the relationship between early-acting and receptor-recycling peroxins
Peroxisomes are conserved organelles involved in fatty acid beta-oxidation and the metabolism of reactive oxygen species; proteins called peroxins (PEX proteins) guide organelle formation and function. Peroxins can be grouped into three functional categories: early-acting peroxins (PEX3, PEX16, and PEX19) help insert peroxisomal membrane proteins, receptor/docking peroxins help transport cargo into the peroxisomal lumen, and receptor-recycling peroxins (e.g., PEX12) assist in recycling the cargo receptors back to the cytosol.
DOI: 10.1016/j.jbc.2024.106720
2024
Abstract 2397 Investigating the role of general autophagy inducers in pexophagy
Peroxisomes are critical organelles that house fatty acid ꞵ-oxidation and antioxidative enzymes that detoxify reactive oxygen and nitrogen species. Impairment of peroxisome function can impact many physiological processes in plants and humans. The primary mechanism for organelle turnover is autophagy, and stress-inducing conditions, including nutrient starvation and oxidative stress, and adaptor proteins, such as NBR1, promote general autophagy. The selective autophagy of peroxisomes is termed pexophagy.
DOI: 10.1016/j.jbc.2024.106723
2024
Abstract 2424 Peroxisomal quality control impacts lipid droplet dynamics
Peroxisomes are membrane-bound organelles in most eukaryotes carrying out fatty acid β-oxidation. Peroxisomes catabolize fatty acids derived from triacylglycerol stored in lipid droplets. This catabolism fuels oilseed germination but generates damaging reactive oxygen species. Peroxisomes contain antioxidant enzymes to decompose these reactive oxygen species, chaperones to refold and disaggregate misfolded and aggregated proteins, and proteases to degrade damaged proteins. In addition, obsolete and damaged peroxisomes can be degraded through pexophagy, a specialized form of autophagy.
DOI: 10.1111/tpj.13641
2017
Cited 16 times
Disparate peroxisome‐related defects in Arabidopsis <i>pex6</i> and <i>pex26</i> mutants link peroxisomal retrotranslocation and oil body utilization
Catabolism of fatty acids stored in oil bodies is essential for seed germination and seedling development in Arabidopsis. This fatty acid breakdown occurs in peroxisomes, organelles that sequester oxidative reactions. Import of peroxisomal enzymes is facilitated by peroxins including PEX5, a receptor that delivers cargo proteins from the cytosol to the peroxisomal matrix. After cargo delivery, a complex of the PEX1 and PEX6 ATPases and the PEX26 tail-anchored membrane protein removes ubiquitinated PEX5 from the peroxisomal membrane. We identified Arabidopsis pex6 and pex26 mutants by screening for inefficient seedling β-oxidation phenotypes. The mutants displayed distinct defects in growth, response to a peroxisomally metabolized auxin precursor, and peroxisomal protein import. The low PEX5 levels in these mutants were increased by treatment with a proteasome inhibitor or by combining pex26 with peroxisome-associated ubiquitination machinery mutants, suggesting that ubiquitinated PEX5 is degraded by the proteasome when the function of PEX6 or PEX26 is reduced. Combining pex26 with mutations that increase PEX5 levels either worsened or improved pex26 physiological and molecular defects, depending on the introduced lesion. Moreover, elevating PEX5 levels via a 35S:PEX5 transgene exacerbated pex26 defects and ameliorated the defects of only a subset of pex6 alleles, implying that decreased PEX5 is not the sole molecular deficiency in these mutants. We found peroxisomes clustered around persisting oil bodies in pex6 and pex26 seedlings, suggesting a role for peroxisomal retrotranslocation machinery in oil body utilization. The disparate phenotypes of these pex alleles may reflect unanticipated functions of the peroxisomal ATPase complex.
DOI: 10.1042/ebc20210059
2022
Cited 6 times
Plant peroxisome proteostasis—establishing, renovating, and dismantling the peroxisomal proteome
Plant peroxisomes host critical metabolic reactions and insulate the rest of the cell from reactive byproducts. The specialization of peroxisomal reactions is rooted in how the organelle modulates its proteome to be suitable for the tissue, environment, and developmental stage of the organism. The story of plant peroxisomal proteostasis begins with transcriptional regulation of peroxisomal protein genes and the synthesis, trafficking, import, and folding of peroxisomal proteins. The saga continues with assembly and disaggregation by chaperones and degradation via proteases or the proteasome. The story concludes with organelle recycling via autophagy. Some of these processes as well as the proteins that facilitate them are peroxisome-specific, while others are shared among organelles. Our understanding of translational regulation of plant peroxisomal protein transcripts and proteins necessary for pexophagy remain based in findings from other models. Recent strides to elucidate transcriptional control, membrane dynamics, protein trafficking, and conditions that induce peroxisome turnover have expanded our knowledge of plant peroxisomal proteostasis. Here we review our current understanding of the processes and proteins necessary for plant peroxisome proteostasis-the emergence, maintenance, and clearance of the peroxisomal proteome.
DOI: 10.1016/j.devcel.2024.04.016
2024
Hormonal control of underwater germination in rice
The ability to germinate, develop, and thrive underwater is key to efficient rice cultivation. In this issue of Developmental Cell, Wang et al. (2024) illuminate a hormone synthesis and inactivation cascade that promotes germination of submerged rice seeds and may allow improved germination in the field. The ability to germinate, develop, and thrive underwater is key to efficient rice cultivation. In this issue of Developmental Cell, Wang et al. (2024) illuminate a hormone synthesis and inactivation cascade that promotes germination of submerged rice seeds and may allow improved germination in the field. A peroxisomal cinnamate:CoA ligase-dependent phytohormone metabolic cascade in submerged rice germinationWang et al.Developmental CellApril 4, 2024In BriefWang et al. demonstrated a peroxisomal cinnamate:CoA ligase (CNL)-dependent phytohormone metabolic cascade in rice. Submerged imbibition-induced salicylic acid (SA) biosynthesis promotes anaerobic germination by inducing OsGH3-mediated indole-acetic acid (IAA)-amino acid conjugation, thus releasing IAA's inhibition of germination under water submergence conditions. Full-Text PDF
DOI: 10.1016/0092-8674(88)90435-7
1988
Cited 25 times
Hypersensitivity to heavy water: A new conditional phenotype
Wild-type strains of the yeast S. cerevisiae can grow on media containing 90% D20. Using chemical mutagenesis we obtained a number of strains that grow on H2O-containing media but not on otherwise identical media containing 90% D2O. The frequency of these D20-sensitive (ds) mutants is comparable to the frequency of conventional temperature-sensitive (ts) mutants in the same mutagenized sample, and the ds mutations are distributed over a large number of complementation groups. Furthermore, most ds mutants do not display other conditional phenotypes, such as heat, cold, or osmotic sensitivity. Conversely, of 17 cell division cycle is mutants tested, only 2 are also ds. Thus, the ds technique should be useful for producing conditional mutations in genes that are not amenable to the is and cs approaches, and also for generating alternative conditional (ds) alleles in many other genes. In addition, the ds technique should make it possible to generate conditional (ds) mutants in homeothermic animals, thereby extending the advantages of conditional phenotypes to mammalian and avian genetics.
DOI: 10.1021/ol800389q
2008
Cited 19 times
Trinorlupeol: A Major Nonsterol Triterpenoid in <i>Arabidopsis</i>
We report the structure determination of 20,29,30-trinorlup-18-en-3β-ol (trinorlupeol) and establish this novel C27 metabolite as a major nonsterol triterpenoid in Arabidopsis thaliana. Trinorlupeol was concentrated in cuticular waxes, notably in the plant stem, floral buds, and seedpods, but not in leaves. Based on expression data and functional characterization of A. thaliana oxidosqualene cyclases, we propose that LUP1 is the cyclase responsible for trinorlupeol biosynthesis. Also described are two oxidized trinorlupeols and additional biosynthetic insights.
DOI: 10.1104/pp.112.203208
2012
Cited 15 times
Focus on Ubiquitin in Plant Biology
Ubiquitin and ubiquitin-like modifiers are small proteins that are covalently joined via their C-terminal carboxyl group to substrate proteins, often on a Lys residue, thereby modifying the stability, localization, or function of the target. Although ubiquitin is present in all eukaryotes, plants
DOI: 10.3389/fcell.2022.838923
2022
Cited 5 times
The Structure of the Arabidopsis PEX4-PEX22 Peroxin Complex—Insights Into Ubiquitination at the Peroxisomal Membrane
Peroxisomes are eukaryotic organelles that sequester critical oxidative reactions and process the resulting reactive oxygen species into less toxic byproducts. Peroxisome function and formation are coordinated by peroxins (PEX proteins) that guide peroxisome biogenesis and division and shuttle proteins into the lumen and membrane of the organelle. Despite the importance of peroxins in plant metabolism and development, no plant peroxin structures have been reported. Here we report the X-ray crystal structure of the PEX4-PEX22 peroxin complex from the reference plant Arabidopsis thaliana. PEX4 is a ubiquitin-conjugating enzyme (UBC) that ubiquitinates proteins associated with the peroxisomal membrane, and PEX22 is a peroxisomal membrane protein that anchors PEX4 to the peroxisome and facilitates PEX4 activity. We co-expressed Arabidopsis PEX4 as a translational fusion with the soluble PEX4-interacting domain of PEX22 in E. coli. The fusion was linked via a protease recognition site, allowing us to separate PEX4 and PEX22 following purification and solve the structure of the complex. We compared the structure of the PEX4-PEX22 complex to the previously published structures of yeast orthologs. Arabidopsis PEX4 displays the typical UBC structure expected from its sequence. Although Arabidopsis PEX22 lacks notable sequence identity to yeast PEX22, it maintains a similar Rossmann fold-like structure. Several salt bridges are positioned to contribute to the specificity of PEX22 for PEX4 versus other Arabidopsis UBCs, and the long unstructured PEX22 tether would allow PEX4-mediated ubiquitination of distant peroxisomal membrane targets without dissociation from PEX22. The Arabidopsis PEX4-PEX22 structure also revealed that the residue altered in pex4-1 (P123L), a mutant previously isolated via a forward-genetic screen for peroxisomal dysfunction, is near the active site cysteine of PEX4. We demonstrated in vitro UBC activity for the PEX4-PEX22 complex and found that the pex4-1 enzyme has reduced in vitro ubiquitin-conjugating activity and altered specificity compared to PEX4. Our findings illuminate the role of PEX4 and PEX22 in peroxisome structure and function and provide tools for future exploration of ubiquitination at the peroxisome surface.
DOI: 10.1534/g3.112.004655
2013
Cited 11 times
Compensatory Mutations in Predicted Metal Transporters Modulate Auxin Conjugate Responsiveness in <i>Arabidopsis</i>
Levels of the phytohormone indole-3-acetic acid (IAA) can be altered by the formation and hydrolysis of IAA conjugates. The isolation and characterization of Arabidopsis thaliana mutants with reduced IAA-conjugate sensitivity and wild-type IAA responses is advancing the understanding of auxin homeostasis by uncovering the factors needed for conjugate metabolism. For example, the discovery that the IAA-Ala-resistant mutant iar1 is defective in a protein in the ZIP family of metal transporters uncovered a link between metal homeostasis and IAA-conjugate sensitivity. To uncover additional factors impacting auxin conjugate metabolism, we conducted a genetic modifier screen and isolated extragenic mutations that restored IAA-amino acid conjugate sensitivity to the iar1 mutant. One of these suppressor mutants is defective in a putative cation diffusion facilitator, MTP5 (At3g12100; formerly known as MTPc2). Loss of MTP5 function restored IAA conjugate sensitivity to iar1 but not to mutants defective in IAA-amino acid conjugate amidohydrolases. Our results are consistent with a model in which MTP5 and IAR1 transport metals in an antagonistic fashion to regulate metal homeostasis within the subcellular compartment in which the IAA-conjugate amidohydrolases reside, and support previous suggestions that the ion composition in this compartment influences hydrolase activity.
DOI: 10.1016/j.molcel.2015.06.004
2015
Cited 7 times
Proteaphagy—Selective Autophagy of Inactive Proteasomes
In this issue of Molecular Cell, Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar report that proteasomes, the ATP-dependent protease complexes that execute ubiquitin-dependent protein degradation in eukaryotes, can be degraded by a newly described form of selective autophagy, termed proteaphagy. In this issue of Molecular Cell, Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar report that proteasomes, the ATP-dependent protease complexes that execute ubiquitin-dependent protein degradation in eukaryotes, can be degraded by a newly described form of selective autophagy, termed proteaphagy. Autophagy and ubiquitin-dependent proteasomal degradation are the predominant pathways of protein degradation in eukaryotic cells. During autophagy (self-eating), cytoplasmic components are enveloped by a double-membrane structure that encircles condemned cargo to form an autophagosome. Autophagosomes fuse with vacuoles (in plants and yeast) or lysosomes (in animals), where the contents are degraded by resident hydrolases (reviewed in Li and Vierstra, 2012Li F. Vierstra R.D. Trends Plant Sci. 2012; 17: 526-537Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Ubiquitylation is a posttranslational process in which the ubiquitin protein is covalently conjugated to target proteins, usually on lysine residues, to alter the fate of the modified protein. Ubiquitin chains are formed when a lysine residue of ubiquitin itself is ubiquitylated. Proteins bearing particular ubiquitin chains can be degraded by the proteasome, a large (26S), complicated machine with subunits that recognize and deconstruct ubiquitin chains and that unfold and degrade substrate proteins (reviewed in Finley, 2009Finley D. Annu. Rev. Biochem. 2009; 78: 477-513Crossref PubMed Scopus (1275) Google Scholar). These two degradative processes were originally assumed to function independently, with ubiquitin-dependent proteasomal degradation targeting individual misfolded or short-lived regulatory proteins and autophagy destroying protein aggregates, damaged or superfluous organelles, and intracellular pathogens. However, intriguing connections between autophagy and the ubiquitin-proteasome system have long been apparent. For example, the ubiquitin-like ATG8 protein decorates the expanding autophagosome membrane after conjugation to phosphatidylethanolamine via a cascade of enzymes resembling ubiquitin-activating and -conjugating enzymes. Moreover, the discovery that ubiquitylation can not only target proteins for proteasomal degradation, but also target aggregates and organelles for autophagic destruction (reviewed in Kirkin et al., 2009Kirkin V. McEwan D.G. Novak I. Dikic I. Mol. Cell. 2009; 34: 259-269Abstract Full Text Full Text PDF PubMed Scopus (990) Google Scholar) suggests that autophagy provides a back-up for the proteasome when substrates are too large or entangled to be unfolded by the proteasome. In this issue of Molecular Cell, Vierstra and colleagues (Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar) reveal an additional point of cross-talk between these two degradative systems by demonstrating that inactivated Arabidopsis proteasomes are ubiquitylated and degraded via selective autophagy. The first indication that proteasomes were being degraded by autophagy came when Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar noted that proteasome subunits accumulated in a variety of Arabidopsis autophagy (atg) mutants, even when the plants were grown in nutrient-replete conditions that do not induce autophagy. The transcripts encoding the proteasome subunits were not elevated in atg mutants, so the observed protein accumulation suggested that proteasomes are being turned over by autophagy. Indeed, after inducing autophagy in wild-type plants via nitrogen starvation, GFP-tagged proteasomes were seen to accumulate in the vacuole, where they co-localized with mCherry-ATG8 puncta. Unlike nitrogen starvation, autophagy stimulants that induce proteotoxic stress—heat shock or treatment with tunicamycin or arsenite—were ineffective proteaphagy inducers, suggesting that the proteaphagy induction that follows nitrogen limitation was a general starvation response. Several lines of evidence support the conclusion that inactive or damaged proteasomes are particularly susceptible to autophagic destruction (Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). First, proteasome enzymatic activity was not elevated in atg mutants despite the increased abundance of proteasome subunits. Second, the proteasome subunit mutants rpt2a-2 and rpt24b-2, which display impaired proteasome assembly, also displayed heightened proteaphagy. Finally, treatment with the MG132 proteasome inhibitor, while not inducing general autophagy, induced proteaphagy even when nitrogen was abundant (Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). What targets these damaged proteasomes to the autophagy machinery? Selective autophagy typically employs bridging receptor proteins that bind both a protein on the condemned cargo and ATG8 on the expanding autophagosome membrane. For example, NBR1 binds both ubiquitin and ATG8 and is a selective autophagy receptor for ubiquitylated targets (reviewed in Kirkin et al., 2009Kirkin V. McEwan D.G. Novak I. Dikic I. Mol. Cell. 2009; 34: 259-269Abstract Full Text Full Text PDF PubMed Scopus (990) Google Scholar). Ubiquitylation of several proteasome subunits increases following inhibitor treatment (Book et al., 2010Book A.J. Gladman N.P. Lee S.S. Scalf M. Smith L.M. Vierstra R.D. J. Biol. Chem. 2010; 285: 25554-25569Crossref PubMed Scopus (96) Google Scholar, Kim et al., 2013Kim D.Y. Scalf M. Smith L.M. Vierstra R.D. Plant Cell. 2013; 25: 1523-1540Crossref PubMed Scopus (179) Google Scholar, Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar), making proteasome ubiquitylation a potential tag for autophagic destruction. Arabidopsis NBR1 is the only characterized autophagy receptor that is apparent in the Arabidopsis genome (Svenning et al., 2011Svenning S. Lamark T. Krause K. Johansen T. Autophagy. 2011; 7: 993-1010Crossref PubMed Scopus (228) Google Scholar, Zhou et al., 2013Zhou J. Wang J. Cheng Y. Chi Y.J. Fan B. Yu J.Q. Chen Z. PLoS Genet. 2013; 9: e1003196Crossref PubMed Scopus (231) Google Scholar). However, proteasome subunit levels were not elevated in an nbr1 mutant, and this nbr1 mutant was perfectly able to target damaged proteasomes for autophagy (Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar), indicating that NBR1 is not the proteaphagy receptor in plants. Unlike NBR1, Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar provide evidence that RPN10 serves as a proteaphagy receptor in Arabidopsis. RPN10 is a ubiquitin receptor and a component of the regulatory “lid” of the proteasome (reviewed in Finley, 2009Finley D. Annu. Rev. Biochem. 2009; 78: 477-513Crossref PubMed Scopus (1275) Google Scholar). RPN10 is an atypical proteasome subunit in that it assembles into proteasomes at substoichiometric levels and can exist in a free form that can shuttle ubiquitylated substrates to the proteasome (reviewed in Finley, 2009Finley D. Annu. Rev. Biochem. 2009; 78: 477-513Crossref PubMed Scopus (1275) Google Scholar). Interestingly, RPN10 association with the proteasome increased following proteasome inhibition and was reduced by treatment with a ubiquitin-conjugate hydrolase (Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar), suggesting that the increased RPN10-proteasome association seen following proteasome inhibition is via binding to ubiquitin moieties on the proteasome rather than increased incorporation into the proteasome lid (Figure 1). The authors demonstrate that Arabidopsis RPN10 binds not only ubiquitin, but also several isoforms of the Arabidopsis ubiquitin-like ATG8 protein (Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). The ATG8-RPN10 interaction requires one of the three C-terminal ubiquitin-interacting motifs (UIMs) of RPN10, and an rpn10 truncation mutant lacking the UIM region was unable to robustly target inhibitor-treated proteasomes for autophagy (Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar), consistent with the possibility that RPN10 acts as an autophagy receptor for the inactive, ubiquitylated proteasomes that accumulate following inhibitor treatment (Figure 1). Although ATG8 binding is conserved in the plant RPN10 lineage (Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar), the critical UIM is missing from Saccharomyces cerevisiae RPN10, so if yeast proteasomes are also targeted for autophagy upon inactivation, a different receptor may be involved. In summary, Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar show that upon proteasome inactivation by chemical or genetic means, proteasomes are ubiquitylated and targeted for autophagic destruction by RPN10, which binds both ATG8 and the ubiquitylated proteasome (Figure 1). Future research will undoubtedly fill in additional mechanistic details of this intriguing process. Which of the multitude of Arabidopsis ubiquitin-protein ligases are responsible for ubiquitylating proteasome subunits, and how do they sense that a proteasome is inactive? Do ubiquitylated proteasome substrates dangling from stalled proteasomes promote autophagic degradation by recruiting such ubiquitin-protein ligases or serve as RPN10 landing pads? How is the proteaphagy that occurs following nitrogen starvation executed (Figure 1)? This starvation-induced proteaphagy is not associated with increased proteasome ubiquitylation and does not require RPN10 (Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). The observation that proteasomes are not heavily ubiquitylated following nitrogen starvation implies that these proteasomes are not damaged but instead are being degraded to recover the nitrogen tied up in the amino acids of the proteasome subunits. Are proteasomes being swept up in non-specific autophagy, or is there a different selective autophagy receptor for these active, but presumably excessive, particles? Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Mol. Cell. 2015; 58 (this issue): 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar have added proteaphagy to the ever-growing list of selective autophagy pathways and provided a solid foundation for further elucidation of the fascinating and critical process of protein quality control. Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in ArabidopsisMarshall et al.Molecular CellMay 21, 2015In BriefMarshall et al. have revealed that the 26S proteasome is degraded by ATG8-mediated autophagy in Arabidopsis, a process stimulated separately by nitrogen starvation and chemical or genetic proteasome inhibition. Additionally, they showed that inhibited proteasomes become extensively ubiquitylated and that subsequent autophagic turnover is mediated by the ubiquitin receptor RPN10. Full-Text PDF Open Archive
DOI: 10.1007/978-3-7091-1788-0_14
2014
Cited 7 times
Protein Transport In and Out of Plant Peroxisomes