Silin Zhong

This paper reports the genome sequence of domesticated tomato, a major crop plant, and a draft sequence for its closest wild relative; comparative genomics reveal very little divergence between the two genomes but some important differences with the potato genome, another important food crop in the genus Solanum. Tomato (Solanum lycopersicum) is a major crop plant and a model system for fruit development. Solanum is one of the largest angiosperm genera1 and includes annual and perennial plants from diverse habitats. Here we present a high-quality genome sequence of domesticated tomato, a draft sequence of its closest wild relative, Solanum pimpinellifolium2, and compare them to each other and to the potato genome (Solanum tuberosum). The two tomato genomes show only 0.6% nucleotide divergence and signs of recent admixture, but show more than 8% divergence from potato, with nine large and several smaller inversions. In contrast to Arabidopsis, but similar to soybean, tomato and potato small RNAs map predominantly to gene-rich chromosomal regions, including gene promoters. The Solanum lineage has experienced two consecutive genome triplications: one that is ancient and shared with rosids, and a more recent one. These triplications set the stage for the neofunctionalization of genes controlling fruit characteristics, such as colour and fleshiness.

Watermelon, Citrullus lanatus, is an important cucurbit crop grown throughout the world. Here we report a high-quality draft genome sequence of the east Asia watermelon cultivar 97103 (2n = 2× = 22) containing 23,440 predicted protein-coding genes. Comparative genomics analysis provided an evolutionary scenario for the origin of the 11 watermelon chromosomes derived from a 7-chromosome paleohexaploid eudicot ancestor. Resequencing of 20 watermelon accessions representing three different C. lanatus subspecies produced numerous haplotypes and identified the extent of genetic diversity and population structure of watermelon germplasm. Genomic regions that were preferentially selected during domestication were identified. Many disease-resistance genes were also found to be lost during domestication. In addition, integrative genomic and transcriptomic analyses yielded important insights into aspects of phloem-based vascular signaling in common between watermelon and cucumber and identified genes crucial to valuable fruit-quality traits, including sugar accumulation and citrulline metabolism.

Ethylene regulates many aspects of the plant life cycle, including seed germination, root initiation, flower development, fruit ripening, senescence, and responses to biotic and abiotic stresses. It thus plays a key role in responses to the environment that have a direct bearing on a plant's fitness for adaptation and reproduction. In recent years, there have been major advances in our understanding of the molecular mechanisms regulating ethylene synthesis and action. Screening for mutants of the triple response phenotype of etiolated Arabidopsis seedlings, together with map-based cloning and candidate gene characterization of natural mutants from other plant species, has led to the identification of many new genes for ethylene biosynthesis, signal transduction, and response pathways. The simple chemical nature of ethylene contrasts with its regulatory complexity. This is illustrated by the multiplicity of genes encoding the key ethylene biosynthesis enzymes 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase, multiple ethylene receptors and signal transduction components, and the complexity of regulatory steps involving signalling relays and control of mRNA and protein synthesis and turnover. In addition, there are extensive interactions with other hormones. This review integrates knowledge from the model plant Arabidopsis and other plant species and focuses on key aspects of recent research on regulatory networks controlling ethylene synthesis and its role in flower development and fruit ripening.

Abstract N 6-Methyladenosine is a ubiquitous modification identified in the mRNA of numerous eukaryotes, where it is present within both coding and noncoding regions. However, this base modification does not alter the coding capacity, and its biological significance remains unclear. We show that Arabidopsis thaliana mRNA contains N 6-methyladenosine at levels similar to those previously reported for animal cells. We further show that inactivation of the Arabidopsis ortholog of the yeast and human mRNA adenosine methylase (MTA) results in failure of the developing embryo to progress past the globular stage. We also demonstrate that the arrested seeds are deficient in mRNAs containing N 6-methyladenosine. Expression of MTA is strongly associated with dividing tissues, particularly reproductive organs, shoot meristems, and emerging lateral roots. Finally, we show that MTA interacts in vitro and in vivo with At FIP37, a homolog of the Drosophila protein FEMALE LETHAL2D and of human WILMS' TUMOUR1-ASSOCIATING PROTEIN. The results reported here provide direct evidence for an essential function for N 6-methyladenosine in a multicellular eukaryote, and the interaction with At FIP37 suggests possible RNA processing events that might be regulated or altered by this base modification.

The kiwifruit (Actinidia chinensis) is an economically and nutritionally important fruit crop with remarkably high vitamin C content. Here we report the draft genome sequence of a heterozygous kiwifruit, assembled from ~140-fold next-generation sequencing data. The assembled genome has a total length of 616.1 Mb and contains 39,040 genes. Comparative genomic analysis reveals that the kiwifruit has undergone an ancient hexaploidization event (γ) shared by core eudicots and two more recent whole-genome duplication events. Both recent duplication events occurred after the divergence of kiwifruit from tomato and potato and have contributed to the neofunctionalization of genes involved in regulating important kiwifruit characteristics, such as fruit vitamin C, flavonoid and carotenoid metabolism. As the first sequenced species in the Ericales, the kiwifruit genome sequence provides a valuable resource not only for biological discovery and crop improvement but also for evolutionary and comparative genomics analysis, particularly in the asterid lineage.

INTRODUCTION Conventional Illumina RNA-Seq does not have the resolution to decode the complex eukaryote transcriptome due to the lack of RNA polarity information. Strand-specific RNA sequencing (ssRNA-Seq) can overcome these limitations and as such is better suited for genome annotation, de novo transcriptome assembly, and accurate digital gene expression analysis. This protocol describes a simple and robust method to generate ssRNA-Seq libraries for the Illumina sequencing platform. It has significantly increased the throughput to 96 libraries in a two-day preparation while simultaneously lowering the reagent costs to below ten dollars per library. It is compatible with both single-read and paired-end multiplex sequencing and, most importantly, its data can also be used with existing conventional RNA-Seq data. This is a significant advantage, because it enables researchers to switch to ssRNA-Seq even if a large amount of data has already been generated by the nonstrand specific methods.

Fruit has evolved myriad forms that facilitate seed dispersal in varied environmental and ecological contexts. Because fleshy fruits become attractive and nutritious to seed-dispersing animals, the transition from unripe to ripe fruit represents a dramatic shift in survival strategy—from protecting unripe fruit against damaging animals to making it appealing to those same animals once ripened. For optimal fitness, ripening therefore must be tightly controlled and coordinated with seed development. Fruits, like many vegetative tissues of plants that contribute to human diets, are also subject to decay, which is enhanced as a consequence of the ripening transition. As such, ripening control has enormous relevance for both plant biology and food security. Here, we review the complex interactions of hormones and transcription factors during fleshy-fruit ripening, with an emphasis on the recent discovery that epigenome dynamics are a critical and early regulator of the cascade of molecular events that ultimately contribute to fruit maturation and ripening.

Summary N 6‐adenosine methylation (m 6 A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m 6 A writer proteins in Arabidopsis thaliana . The components required for m 6 A in Arabidopsis included MTA , MTB , FIP 37, VIRILIZER and the E3 ubiquitin ligase HAKAI . Downregulation of these proteins led to reduced relative m 6 A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m 6 A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m 6 A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences.

The hormone jasmonate (JA), which functions in plant immunity, regulates resistance to pathogen infection and insect attack through triggering genome-wide transcriptional reprogramming in plants. We show that the basic helix-loop-helix transcription factor (TF) MYC2 in tomato (Solanum lycopersicum) acts downstream of the JA receptor to orchestrate JA-mediated activation of both the wounding and pathogen responses. Using chromatin immunoprecipitation sequencing (ChIP-seq) coupled with RNA sequencing (RNA-seq) assays, we identified 655 MYC2-targeted JA-responsive genes. These genes are highly enriched in Gene Ontology categories related to TFs and the early response to JA, indicating that MYC2 functions at a high hierarchical level to regulate JA-mediated gene transcription. We also identified a group of MYC2-targeted TFs (MTFs) that may directly regulate the JA-induced transcription of late defense genes. Our findings suggest that MYC2 and its downstream MTFs form a hierarchical transcriptional cascade during JA-mediated plant immunity that initiates and amplifies transcriptional output. As proof of concept, we showed that during plant resistance to the necrotrophic pathogen Botrytis cinerea, MYC2 and the MTF JA2-Like form a transcription module that preferentially regulates wounding-responsive genes, whereas MYC2 and the MTF ETHYLENE RESPONSE FACTOR.C3 form a transcription module that preferentially regulates pathogen-responsive genes.

Fleshy fruits using ethylene to regulate ripening have developed multiple times in the history of angiosperms, presenting a clear case of convergent evolution whose molecular basis remains largely unknown. Analysis of the fruitENCODE data consisting of 361 transcriptome, 71 accessible chromatin, 147 histone and 45 DNA methylation profiles reveals three types of transcriptional feedback circuits controlling ethylene-dependent fruit ripening. These circuits are evolved from senescence or floral organ identity pathways in the ancestral angiosperms either by neofunctionalisation or repurposing pre-existing genes. The epigenome, H3K27me3 in particular, has played a conserved role in restricting ripening genes and their orthologues in dry and ethylene-independent fleshy fruits. Our findings suggest that evolution of ripening is constrained by limited hormone molecules and genetic and epigenetic materials, and whole-genome duplications have provided opportunities for plants to successfully circumvent these limitations.

The spatial organization of the genome plays an important role in the regulation of gene expression. However, the core structural features of animal genomes, such as topologically associated domains (TADs) and chromatin loops, are not prominent in the extremely compact Arabidopsis genome. In this study, we examine the chromatin architecture, as well as their DNA methylation, histone modifications, accessible chromatin, and gene expression, of maize, tomato, sorghum, foxtail millet, and rice with genome sizes ranging from 0.4 to 2.4 Gb. We found that these plant genomes can be divided into mammalian-like A/B compartments. At higher resolution, the chromosomes of these plants can be further partitioned to local A/B compartments that reflect their euchromatin, heterochromatin, and polycomb status. Chromatins in all these plants are organized into domains that are not conserved across species. They show similarity to the Drosophila compartment domains, and are clustered into active, polycomb, repressive, and intermediate types based on their transcriptional activities and epigenetic signatures, with domain border overlaps with the local A/B compartment junctions. In the large maize and tomato genomes, we observed extensive chromatin loops. However, unlike the mammalian chromatin loops that are enriched at the TAD border, plant chromatin loops are often formed between gene islands outside the repressive domains and are closely associated with active compartments. Our study indicates that plants have complex and unique 3D chromatin architectures, which require further study to elucidate their biological functions.

We previously showed that the N6-methyladenosine (m(6)A) mRNA methylase is essential during Arabidopsis thaliana embryonic development. We also demonstrated that this modification is present at varying levels in all mature tissues. However, the requirement for the m(6)A in the mature plant was not tested. Here we show that a 90% reduction in m(6)A levels during later growth stages gives rise to plants with altered growth patterns and reduced apical dominance. The flowers of these plants commonly show defects in their floral organ number, size, and identity. The global analysis of gene expression from reduced m(6)A plants show that a significant number of down-regulated genes are involved in transport, or targeted transport, and most of the up-regulated genes are involved in stress and stimulus response processes. An analysis of m(6)A distribution in fragmented mRNA suggests that the m(6)A is predominantly positioned toward the 3' end of transcripts in a region 100-150 bp before the poly(A) tail. In addition to the analysis of the phenotypic changes in the low methylation Arabidopsis plants we will review the latest advances in the field of mRNA internal methylation.

Cassava is a major tropical food crop in the Euphorbiaceae family that has high carbohydrate production potential and adaptability to diverse environments. Here we present the draft genome sequences of a wild ancestor and a domesticated variety of cassava and comparative analyses with a partial inbred line. We identify 1,584 and 1,678 gene models specific to the wild and domesticated varieties, respectively, and discover high heterozygosity and millions of single-nucleotide variations. Our analyses reveal that genes involved in photosynthesis, starch accumulation and abiotic stresses have been positively selected, whereas those involved in cell wall biosynthesis and secondary metabolism, including cyanogenic glucoside formation, have been negatively selected in the cultivated varieties, reflecting the result of natural selection and domestication. Differences in microRNA genes and retrotransposon regulation could partly explain an increased carbon flux towards starch accumulation and reduced cyanogenic glucoside accumulation in domesticated cassava. These results may contribute to genetic improvement of cassava through better understanding of its biology.

Fruit ripening is the summation of changes rendering fleshy fruit tissues attractive and palatable to seed dispersing organisms. For example, sugar content is influenced by plastid numbers and photosynthetic activity in unripe fruit and later by starch and sugar catabolism during ripening. Tomato fruit are sinks of photosynthate, yet unripe green fruit contribute significantly to the sugars that ultimately accumulate in the ripe fruit. Plastid numbers and chlorophyll content are influenced by numerous environmental and genetic factors and are positively correlated with photosynthesis and photosynthate accumulation. GOLDEN2-LIKE (GLK) transcription factors regulate plastid and chlorophyll levels. Tomato (Solanum lycopersicum), like most plants, contains two GLKs (i.e., GLK1 and GLK2/UNIFORM). Mutant and transgene analysis demonstrated that these genes encode functionally similar peptides, though differential expression renders GLK1 more important in leaves, while GLK2 is predominant in fruit. A latitudinal gradient of GLK2 expression influences the typical uneven coloration of green and ripe wild-type fruit. Transcriptome profiling revealed a broader fruit gene expression gradient throughout development. The gradient influenced general ripening activities beyond plastid development and was consistent with the easily observed yet poorly studied ripening gradient present in tomato and many fleshy fruits.

Tomato is considered as the genetic model for climacteric fruits, in which three major players control the fruit ripening process: ethylene, ripening transcription factors, and DNA methylation. The fruitENCODE project has now shown that there are multiple transcriptional circuits regulating fruit ripening in different species, and H3K27me3, instead of DNA methylation, plays a conserved role in restricting these ripening pathways. In addition, the function of the core tomato ripening transcription factors is now being questioned. We have employed CRISPR/Cas9 genome editing to mutate the SBP-CNR and NAC-NOR transcription factors, both of which are considered as master regulators in the current tomato ripening model. These plants only displayed delayed or partial non-ripening phenotypes, distinct from the original mutant plants, which categorically failed to ripen, suggesting that they might be gain-of-function mutants. Besides increased DNA methylation genome-wide, the original mutants also have hyper-H3K27me3 in ripening gene loci such as ACS2, RIN, and TDR4. It is most likely that multiple genetic and epigenetic factors have contributed to their strong non-ripening phenotypes. Hence, we propose that the field should move beyond these linear and two-dimensional models and embrace the fact that important biological processes such as ripening are often regulated by highly redundant network with inputs from multiple levels.

Abstract The transcription regulatory network inside a eukaryotic cell is defined by the combinatorial actions of transcription factors (TFs). However, TF binding studies in plants are too few in number to produce a general picture of this complex network. In this study, we use large-scale ChIP-seq to reconstruct it in the maize leaf, and train machine-learning models to predict TF binding and co-localization. The resulting network covers 77% of the expressed genes, and shows a scale-free topology and functional modularity like a real-world network. TF binding sequence preferences are conserved within family, while co-binding could be key for their binding specificity. Cross-species comparison shows that core network nodes at the top of the transmission of information being more conserved than those at the bottom. This study reveals the complex and redundant nature of the plant transcription regulatory network, and sheds light on its architecture, organizing principle and evolutionary trajectory.

Baculoviruses are important insect pathogens that have been developed as protein expression vectors in insect cells and as transduction vectors for mammalian cells. They have large double-stranded DNA genomes containing approximately 156 tightly spaced genes, and they present significant challenges for transcriptome analysis. In this study, we report the first comprehensive analysis of AcMNPV transcription over the course of infection in Trichoplusia ni cells, by a combination of strand-specific RNA sequencing (RNA-Seq) and deep sequencing of 5' capped transcription start sites and 3' polyadenylation sites. We identified four clusters of genes associated with distinctive patterns of mRNA accumulation through the AcMNPV infection cycle. A total of 218 transcription start sites (TSS) and 120 polyadenylation sites (PAS) were mapped. Only 29 TSS were associated with a canonical TATA box, and 14 initiated within or near the previously identified CAGT initiator motif. The majority of viral transcripts (126) initiated within the baculovirus late promoter motif (TAAG), and late transcripts initiated precisely at the second position of the motif. Analysis of 3' ends showed that 92 (77%) of the 3' PAS were located within 30 nucleotides (nt) downstream of a consensus termination signal (AAUAAA or AUUAAA). A conserved U-rich region was found approximately 2 to 10 nt downstream of the PAS for 58 transcripts. Twelve splicing events and an unexpectedly large number of antisense RNAs were identified, revealing new details of possible regulatory mechanisms controlling AcMNPV gene expression. Combined, these data provide an emerging global picture of the organization and regulation of AcMNPV transcription through the infection cycle.

Cytosine methylation at CG sites ((m)CG) plays critical roles in development, epigenetic inheritance, and genome stability in mammals and plants. In the dicot model plant Arabidopsis thaliana, methyltransferase 1 (MET1), a principal CG methylase, functions to maintain (m)CG during DNA replication, with its null mutation resulting in global hypomethylation and pleiotropic developmental defects. Null mutation of a critical CG methylase has not been characterized at a whole-genome level in other higher eukaryotes, leaving the generality of the Arabidopsis findings largely speculative. Rice is a model plant of monocots, to which many of our important crops belong. Here we have characterized a null mutant of OsMet1-2, the major CG methylase in rice. We found that seeds homozygous for OsMet1-2 gene mutation (OsMET1-2(-/-)), which directly segregated from normal heterozygote plants (OsMET1-2(+/-)), were seriously maldeveloped, and all germinated seedlings underwent swift necrotic death. Compared with wild type, genome-wide loss of (m)CG occurred in the mutant methylome, which was accompanied by a plethora of quantitative molecular phenotypes including dysregulated expression of diverse protein-coding genes, activation and repression of transposable elements, and altered small RNA profiles. Our results have revealed conservation but also distinct functional differences in CG methylases between rice and Arabidopsis.

Microbe-associated molecular patterns, such as those present in bacterial flagellin, are powerful inducers of the innate immune response in plants. Successful pathogens deliver virulence proteins, termed effectors, into the plant cell where they can interfere with the immune response and promote disease. Engineering the plant immune system to enhance disease resistance requires a thorough understanding of its components.We describe a high-throughput screen, using RNA sequencing and virus-induced gene silencing, to identify tomato genes whose expression is enhanced by the flagellin microbe-associated molecular pattern flgII-28, but reduced by activities of the Pseudomonas syringae pv. tomato (Pst) type III effectors AvrPto and AvrPtoB. Gene ontology terms for this category of Flagellin-induced repressed by effectors (FIRE) genes showed enrichment for genes encoding certain subfamilies of protein kinases and transcription factors. At least 25 of the FIRE genes have been implicated previously in plant immunity. Of the 92 protein kinase-encoding FIRE genes, 33 were subjected to virus-induced gene silencing and their involvement in pattern-triggered immunity was tested with a leaf-based assay. Silencing of one FIRE gene, which encodes the cell wall-associated kinase SlWAK1, compromised the plant immune response resulting in increased growth of Pst and enhanced disease symptoms.Our transcriptomic approach identifies FIRE genes that represent a pathogen-defined core set of immune-related genes. The analysis of this set of candidate genes led to the discovery of a cell wall-associated kinase that participates in plant defense. The FIRE genes will be useful for further elucidation of the plant immune system.

Fruit set in angiosperms marks the transition from flowering to fruit production and a commitment to seed dispersal. Studies with Solanum lycopersicum (tomato) fruit have shown that pollination and subsequent fertilization induce the biosynthesis of several hormones, including auxin and gibberellins (GAs), which stimulate fruit set. Circumstantial evidence suggests that the gaseous hormone ethylene may also influence fruit set, but this has yet to be substantiated with molecular or mechanistic data. Here, we examined fruit set at the biochemical and genetic levels, using hormone and inhibitor treatments, and mutants that affect auxin or ethylene signaling. The expression of system-1 ethylene biosynthetic genes and the production of ethylene decreased during pollination-dependent fruit set in wild-type tomato and during pollination-independent fruit set in the auxin hypersensitive mutant iaa9-3. Blocking ethylene perception in emasculated flowers, using either the ethylene-insensitive Sletr1-1 mutation or 1-methylcyclopropene (1-MCP), resulted in elongated parthenocarpic fruit and increased cell expansion, whereas simultaneous treatment with the GA biosynthesis inhibitor paclobutrazol (PAC) inhibited parthenocarpy. Additionally, the application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to pollinated ovaries reduced fruit set. Furthermore, Sletr1-1 parthenocarpic fruits did not exhibit increased auxin accumulation, but rather had elevated levels of bioactive GAs, most likely reflecting an increase in transcripts encoding the GA-biosynthetic enzyme SlGA20ox3, as well as a reduction in the levels of transcripts encoding the GA-inactivating enzymes SlGA2ox4 and SlGA2ox5. Taken together, our results suggest that ethylene plays a role in tomato fruit set by suppressing GA metabolism.

Abstract Background Cultivated watermelon [ Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus ] is an important agriculture crop world-wide. The fruit of watermelon undergoes distinct stages of development with dramatic changes in its size, color, sweetness, texture and aroma. In order to better understand the genetic and molecular basis of these changes and significantly expand the watermelon transcript catalog, we have selected four critical stages of watermelon fruit development and used Roche/454 next-generation sequencing technology to generate a large expressed sequence tag (EST) dataset and a comprehensive transcriptome profile for watermelon fruit flesh tissues. Results We performed half Roche/454 GS-FLX run for each of the four watermelon fruit developmental stages (immature white, white-pink flesh, red flesh and over-ripe) and obtained 577,023 high quality ESTs with an average length of 302.8 bp. De novo assembly of these ESTs together with 11,786 watermelon ESTs collected from GenBank produced 75,068 unigenes with a total length of approximately 31.8 Mb. Overall 54.9% of the unigenes showed significant similarities to known sequences in GenBank non-redundant (nr) protein database and around two-thirds of them matched proteins of cucumber, the most closely-related species with a sequenced genome. The unigenes were further assigned with gene ontology (GO) terms and mapped to biochemical pathways. More than 5,000 SSRs were identified from the EST collection. Furthermore we carried out digital gene expression analysis of these ESTs and identified 3,023 genes that were differentially expressed during watermelon fruit development and ripening, which provided novel insights into watermelon fruit biology and a comprehensive resource of candidate genes for future functional analysis. We then generated profiles of several interesting metabolites that are important to fruit quality including pigmentation and sweetness. Integrative analysis of metabolite and digital gene expression profiles helped elucidating molecular mechanisms governing these important quality-related traits during watermelon fruit development. Conclusion We have generated a large collection of watermelon ESTs, which represents a significant expansion of the current transcript catalog of watermelon and a valuable resource for future studies on the genomics of watermelon and other closely-related species. Digital expression analysis of this EST collection allowed us to identify a large set of genes that were differentially expressed during watermelon fruit development and ripening, which provide a rich source of candidates for future functional analysis and represent a valuable increase in our knowledge base of watermelon fruit biology.

Background Grafting has been extensively used to enhance the performance of horticultural crops. Since Charles Darwin coined the term “graft hybrid” meaning that asexual combination of different plant species may generate products that are genetically distinct, highly discrepant opinions exist supporting or against the concept. Recent studies have documented that grafting enables exchanges of both RNA and DNA molecules between the grafting partners, thus providing a molecular basis for grafting-induced genetic variation. DNA methylation is known as prone to alterations as a result of perturbation of internal and external conditions. Given characteristics of grafting, it is interesting to test whether the process may cause an alteration of this epigenetic marker in the grafted organismal products. Methodology/Principal Findings We analyzed relative global DNA methylation levels and locus-specific methylation patterns by the MSAP marker and locus-specific bisulfite-sequencing in the seed plants (wild-type controls), self- and hetero-grafted scions/rootstocks, selfed progenies of scions and their seed-plant controls, involving three Solanaceae species. We quantified expression of putative genes involved in establishing and/or maintaining DNA methylation by q-(RT)-PCR. We found that (1) hetero-grafting caused extensive alteration of DNA methylation patterns in a locus-specific manner, especially in scions, although relative methylation levels remain largely unaltered; (2) the altered methylation patterns in the hetero-grafting-derived scions could be inherited to sexual progenies with some sites showing further alterations or revisions; (3) hetero-grafting caused dynamic changes in steady-state transcript abundance of genes encoding for a set of enzymes functionally relevant to DNA methylation. Conclusions/Significance Our results demonstrate that inter-species grafting in plants could produce extensive and heritable alterations in DNA methylation. We suggest that these readily altered, yet heritable, epigenetic modifications due to interspecies hetero-grafting may shed one facet of insight into the molecular underpinnings for the still contentious concept of graft hybrid.

Panax notoginseng (Burk.) F.H. Chen is one of the most highly valued medicinal plants in the world. The major bioactive molecules are triterpene saponins, which are also known as ginsenosides. However, its large genome size has hindered the assembly of a draft genome by whole genome sequencing. Hence, genomic and transcriptomic details about P. notoginseng, especially its biosynthetic pathways and gene expression in different parts of the plant, have remained largely unknown until now.In this study, RNA sequencing of three different P. notoginseng tissues was performed using next generation DNA sequencing. After assembling the high quality sequencing reads into 107,340 unigenes, biochemical pathways were predicted and 9,908 unigenes were assigned to 135 KEGG pathways. Among them, 270 unigenes were identified to be involved in triterpene saponin biosynthesis. In addition, 350 and 342 unigenes were predicted to encode cytochrome P450s and glycosyltransferases, respectively, based on the annotation results, some of which encode enzymes responsible for the conversion of the triterpene saponin backbone into different ginsenosides. In particular, one unigene predominantly expressed in the root was annotated as CYP716A53v2, which probably participates in the formation of protopanaxatriol from protopanaxadiol in P. notoginseng. The differential expression of this gene was further confirmed by real-time PCR.We have established a global transcriptome dataset for P. notoginseng and provided additional genetic information for further genome-wide research and analyses. Candidate genes involved in ginsenoside biosynthesis, including putative cytochrome P450s and glycosyltransferases were obtained. The transcriptomes in different plant tissues also provide invaluable resources for future study of the differences in physiological processes and secondary metabolites in different parts of P. notoginseng.

Chloroplast development and chlorophyll(Chl)metabolism in unripe tomato contribute to the growth and quality of the fruit, however these mechanisms are poorly understood. In this study, we initially investigated seven homeobox-containing transcription factors (TFs) with specific ripening-associated expression patterns using virus-induced gene silencing (VIGS) technology and found that inhibiting the expression of one of these TFs, BEL1-LIKE HOMEODOMAIN11 (SlBEL11), significantly increased Chl levels in unripe tomato fruit. This enhanced Chl accumulation was further validated by generating stable RNA interference (RNAi) transgenic lines. RNA sequencing (RNA-seq) of RNAi-SlBEL11 fruit at the mature green (MG) stage showed that 48 genes involved in Chl biosynthesis, photosynthesis and chloroplast development were significantly upregulated compared with the wild type (WT) fruit. Genomic global scanning for Homeobox TF binding sites combined with RNA-seq differential gene expression analysis showed that 22 of these 48 genes were potential target genes of SlBEL11 protein. These genes included Chl biosynthesis-related genes encoding for protochlorophyllide reductase (POR), magnesium chelatase H subunit (CHLH) and chlorophyllide a oxygenase (CAO), and chloroplast development-related genes encoding for chlorophyll a/b binding protein (CAB), homeobox protein knotted 2 (TKN2) and ARABIDOPSIS PSEUDO RESPONSE REGULATOR 2-LIKE (APRR2-like). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation quantitative polymerase chain reaction (PCR) (ChIP-qPCR) assays were employed to verify that SlBEL11 protein could bind to the promoters for TKN2, CAB and POR. Taken together, our findings demonstrated that SlBEL11 plays an important role in chloroplast development and Chl synthesis in tomato fruit.

Abstract High-temperature (HT) stress is a major environmental stress that limits plant growth and development. MAPK cascades play key roles in plant growth and stress signaling, but their involvement in the HT stress response is poorly understood. Here, we describe a 47-kD MBP-phosphorylated protein (p47-MBPK) activated in tomato (Solanum lycopersicum) leaves under HT and identify it as SlMPK1 by tandem mass spectrometry analysis. Silencing of SlMPK1 in transgenic tomato plants resulted in enhanced tolerance to HT, while overexpression resulted in reduced tolerance. Proteomic analysis identified a set of proteins involved in antioxidant defense that are significantly more abundant in RNA interference-SlMPK1 plants than nontransgenic plants under HT stress. RNA interference-SlMPK1 plants also showed changes in membrane lipid peroxidation and antioxidant enzyme activities. Furthermore, using yeast two-hybrid screening, we identified a serine-proline-rich protein homolog, SlSPRH1, which interacts with SlMPK1 in yeast, in plant cells, and in vitro. We demonstrate that SlMPK1 can directly phosphorylate SlSPRH1. Furthermore, the serine residue serine-44 of SlSPRH1 is a crucial phosphorylation site in the SlMPK1-mediated antioxidant defense mechanism activated during HT stress. We also demonstrate that heterologous expression of SlSPRH1 in Arabidopsis (Arabidopsis thaliana) led to a decrease in thermotolerance and lower antioxidant capacity. Taken together, our results suggest that SlMPK1 is a negative regulator of thermotolerance in tomato plants. SlMPK1 acts by regulating antioxidant defense, and its substrate SlSPRH1 is involved in this pathway.

Abstract The methyltransferase complex (m 6 A writer), which catalyzes the deposition of N 6 -methyladenosine (m 6 A) in mRNAs, is highly conserved across most eukaryotic organisms, but its components and interactions between them are still far from fully understood. Here, using in vivo interaction proteomics, two HAKAI-interacting zinc finger proteins, HIZ1 and HIZ2, are discovered as components of the Arabidopsis m 6 A writer complex. HAKAI is required for the interaction between HIZ1 and MTA (mRNA adenosine methylase A). Whilst HIZ1 knockout plants have normal levels of m 6 A, plants in which it is overexpressed show reduced methylation and decreased lateral root formation. Mutant plants lacking HIZ2 are viable but have an 85% reduction in m 6 A abundance and show severe developmental defects. Our findings suggest that HIZ2 is likely the plant equivalent of ZC3H13 (Flacc) of the metazoan m 6 A-METTL Associated Complex.

Abstract Nicotiana benthamiana is an invaluable model plant and biotechnology platform with a ~3 Gb allotetraploid genome. To further improve its usefulness and versatility, we have produced high-quality chromosome-level genome assemblies, coupled with transcriptome, epigenome, microRNA and transposable element datasets, for the ubiquitously used LAB strain and a related wild accession, QLD. In addition, single nucleotide polymorphism maps have been produced for a further two laboratory strains and four wild accessions. Despite the loss of five chromosomes from the ancestral tetraploid, expansion of intergenic regions, widespread segmental allopolyploidy, advanced diploidization and evidence of recent bursts of Copia pseudovirus (Copia) mobility not seen in other Nicotiana genomes, the two subgenomes of N. benthamiana show large regions of synteny across the Solanaceae. LAB and QLD have many genetic, metabolic and phenotypic differences, including disparate RNA interference responses, but are highly interfertile and amenable to genome editing and both transient and stable transformation. The LAB/QLD combination has the potential to be as useful as the Columbia-0/Landsberg errecta partnership, utilized from the early pioneering days of Arabidopsis genomics to today.

Abstract While considerable knowledge exists about the enzymes pivotal for C 4 photosynthesis, much less is known about the cis- regulation important for specifying their expression in distinct cell types. Here, we use single-cell-indexed ATAC-seq to identify cell-type-specific accessible chromatin regions (ACRs) associated with C 4 enzymes for five different grass species. This study spans four C 4 species, covering three distinct photosynthetic subtypes: Zea mays and Sorghum bicolor (NADP-ME), Panicum miliaceum (NAD-ME), Urochloa fusca (PEPCK), along with the C 3 outgroup Oryza sativa . We studied the cis- regulatory landscape of enzymes essential across all C 4 species and those unique to C 4 subtypes, measuring cell-type-specific biases for C 4 enzymes using chromatin accessibility data. Integrating these data with phylogenetics revealed diverse co-option of gene family members between species, showcasing the various paths of C 4 evolution. Besides promoter proximal ACRs, we found that, on average, C 4 genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C 4 evolution. Examining the evolutionary history of these cell-type-specific ACRs revealed a spectrum of conserved and novel ACRs, even among closely related species, indicating ongoing evolution of cis -regulation at these C 4 loci. This study illuminates the dynamic and complex nature of CRE evolution in C 4 photosynthesis, particularly highlighting the intricate cis- regulatory evolution of key loci. Our findings offer a valuable resource for future investigations, potentially aiding in the optimization of C 3 crop performance under changing climatic conditions.

In the model plant Arabidopsis, members of a family of two-component system His kinase-like ethylene receptors have direct protein-protein interactions with a single downstream Ser/Thr kinase CTR1. These components of the ethylene signalling network found in Arabidopsis are conserved in the climacteric fruit tomato, but both the ethylene receptors and CTR1-like proteins (LeCTRs) in tomato are encoded by multigene families. Here, using a yeast two-hybrid interaction assay, it is shown that the tomato receptors LeETR1, LeETR2, and NEVER-RIPE (NR) can interact with multiple LeCTRs. In vivo protein localization studies with fluorescent tagged proteins revealed that the ethylene receptor NR was targeted to the endoplasmic reticulum (ER) when transiently expressed in onion epidermal cells, whereas the four LeCTR proteins were found in the cytoplasm and nucleus. When co-expressed with NR, three LeCTRs (1, 3, and 4), but not LeCTR2, also adopted the same ER localization pattern in an NR receptor-dependent manner but not in the absence of NR. The receptor-CTR interactions were confirmed by biomolecular fluorescence complementation (BiFC) showing that NR could form a protein complex with LeCTR1, 3, and 4. This suggested that ethylene receptors recruit these LeCTR proteins to the ER membrane through direct protein-protein interaction. The receptor-CTR interactions and localization observed in the study reinforce the idea that ethylene receptors transmit the signal to the downstream CTRs and show that a single receptor can interact with multiple CTR proteins. It remains unclear whether the different LeCTRs are functionally redundant or have unique roles in ethylene signalling.

Identifying the target genes of transcription factors is important for unraveling regulatory networks in all types of organisms. Our interest was precisely to uncover the spectrum of loci regulated by a widespread plant transcription factor involved in physiological adaptation to drought, a type of stress that plants have encountered since the colonization of land habitats 400 MYA. The regulator under study, named ASR1, is exclusive to the plant kingdom (albeit absent in Arabidopsis) and known to alleviate the stress caused by restricted water availability. As its target genes are still unknown despite the original cloning of Asr1 cDNA 20 years ago, we examined the tomato genome for specific loci interacting in vivo with this conspicuous protein.We performed ChIP followed by high throughput DNA sequencing (ChIP-seq) on leaves from stressed tomato plants, using a high-quality anti-ASR1 antibody. In this way, we unraveled a novel repertoire of target genes, some of which are clearly involved in the response to drought stress. Many of the ASR1-enriched genomic loci we found encode enzymes involved in cell wall synthesis and remodeling as well as channels implicated in water and solute flux, such as aquaporins. In addition, we were able to determine a robust consensus ASR1-binding DNA motif.The finding of cell wall synthesis and aquaporin genes as targets of ASR1 is consistent with their suggested role in the physiological adaptation of plants to water loss. The results gain insight into the environmental stress-sensing pathways leading to plant tolerance of drought.

Litchi is an evergreen woody tree widely cultivated in subtropical and tropical regions. Defective flowering is a major challenge for litchi production in time of climate change and global warming. Previous studies have shown that high temperature conditions encourage the growth of rudimentary leaves in panicles and suppress litchi flowering, while reactive oxygen species (ROS) generated by methyl viologen dichloride hydrate (MV) promote flowering and abortion of rudimentary leaves. To understand the molecular function of the ROS-induced abortion of rudimentary leaves in litchi, we sequenced and de novo assembled the litchi transcriptome.Our assembly encompassed 82,036 unigenes with a mean size of 710 bp, and over 58% (47,596) of unigenes showed significant similarities to known sequences in GenBank non-redundant (nr) protein database. 5,865 unigenes were found to be differentially expressed between ROS-treated and un-treated rudimentary leaves, and genes encoding signaling components of plant hormones such as ABA and ethylene were significantly enriched.Our transcriptome data represents the comprehensive collection of expressed sequence tags (ESTs) of litchi leaves, which is a vital resource for future studies on the genomics of litchi and other closely related species. The identified differentially expressed genes also provided potential candidates for functional analysis of genes involved in litchi flowering underlying the control of rudimentary leaves in the panicles.

Productive infection of Trichoplusia ni cells by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) leads to expression of ~156 viral genes and results in dramatic cell remodeling. How the cell transcriptome responds to viral infection was unknown due to the lack of a reference genome and transcriptome for T. ni. We used an ~60-Gb RNA sequencing (RNA-seq) data set from infected and uninfected T. ni cells to generate and annotate a de novo transcriptome assembly of approximately 70,322 T. ni unigenes (assembled transcripts), representing the 48-h infection cycle. Using differential gene expression analysis, we found that the majority of host transcripts were downregulated after 6 h postinfection (p.i.) and throughout the remainder of the infection. In contrast, 5.7% (4,028) of the T. ni unigenes were upregulated during the early period (0 to 6 h p.i.), followed by a decrease through the remainder of the infection cycle. Also, a small subset of genes related to metabolism and stress response showed a significant elevation of transcript levels at 18 and 24 h p.i. but a decrease thereafter. We also examined the responses of genes belonging to a number of specific pathways of interest, including stress responses, apoptosis, immunity, and protein trafficking. We identified specific pathway members that were upregulated during the early phase of the infection. Combined with the parallel analysis of AcMNPV expression, these results provide both a broad and a detailed view of how baculovirus infection impacts the host cell transcriptome to evade cellular defensive responses, to modify cellular biosynthetic pathways, and to remodel cell structure.Baculoviruses are insect-specific DNA viruses that are highly pathogenic to their insect hosts. In addition to their use for biological control of certain insects, baculoviruses also serve as viral vectors for numerous biotechnological applications, such as mammalian cell transduction and protein expression for vaccine production. While there is considerable information regarding viral gene expression in infected cells, little is known regarding responses of the host cell to baculovirus infection. In these studies, we assembled a cell transcriptome from the host Trichoplusia ni and used that transcriptome to analyze changes in host cell gene expression throughout the infection cycle. The study was performed in parallel with a prior study of changes in viral gene expression. Combined, these studies provide an unprecedented new level of detail and an overview of events in the infection cycle, and they will stimulate new experimental approaches to understand, modify, and utilize baculoviruses for a variety of applications.

Papaya (Carica papaya L.) is a climacteric fleshy fruit that undergoes dramatic changes during ripening, most noticeably a severe pulp softening. However, little is known regarding the genetics of the cell wall metabolism in papayas. The present work describes the identification and characterization of genes related to pulp softening. We used gene expression profiling to analyze the correlations and co-expression networks of cell wall-related genes, and the results suggest that papaya pulp softening is accomplished by the interactions of multiple glycoside hydrolases. The polygalacturonase cpPG1 appeared to play a central role in the network and was further studied. The transient expression of cpPG1 in papaya results in pulp softening and leaf necrosis in the absence of ethylene action and confirms its role in papaya fruit ripening.

Naturally-occurring epimutants are rare and have mainly been described in plants. However how these mutants maintain their epigenetic marks and how they are inherited remain unknown. Here we report that CHROMOMETHYLASE3 (SlCMT3) and other methyltransferases are required for maintenance of a spontaneous epimutation and its cognate Colourless non-ripening (Cnr) phenotype in tomato. We screened a series of DNA methylation-related genes that could rescue the hypermethylated Cnr mutant. Silencing of the developmentally-regulated SlCMT3 gene results in increased expression of LeSPL-CNR, the gene encodes the SBP-box transcription factor residing at the Cnr locus and triggers Cnr fruits to ripen normally. Expression of other key ripening-genes was also up-regulated. Targeted and whole-genome bisulfite sequencing showed that the induced ripening of Cnr fruits is associated with reduction of methylation at CHG sites in a 286-bp region of the LeSPL-CNR promoter, and a decrease of DNA methylation in differentially-methylated regions associated with the LeMADS-RIN binding sites. Our results indicate that there is likely a concerted effect of different methyltransferases at the Cnr locus and the plant-specific SlCMT3 is essential for sustaining Cnr epi-allele. Maintenance of DNA methylation dynamics is critical for the somatic stability of Cnr epimutation and for the inheritance of tomato non-ripening phenotype.

Abstract Chromatins are not randomly packaged in the nucleus and their organization plays important roles in transcription regulation, which is best studied in the mammalian models. Using in situ Hi‐C, we have compared the 3D chromatin architectures of rice mesophyll and endosperm, foxtail millet bundle sheath and mesophyll, and maize bundle sheath, mesophyll and endosperm tissues. We found that their global A/B compartment partitions are stable across tissues, while local A/B compartment has tissue‐specific dynamic associated with differential gene expression. Plant domains are largely stable across tissues, while new domain border formations are often associated with transcriptional activation in the region. Genes inside plant domains are not conserved across species, and lack significant co‐expression behavior unlike those in mammalian TADs. Although we only observed chromatin loops between gene islands in the large genomes, the maize loop gene pairs’ syntenic orthologs have shorter physical distances in small genome monocots, suggesting that loops instead of domains might have conserved biological function. Our study showed that plants’ chromatin features might not have conserved biological functions as the mammalian ones.

The Dormancy-associated MADS-box (DAM) gene cluster in peach serves as a key regulatory hub on which the seasonal temperatures act and orchestrate dormancy onset and exit, chilling response and floral bud developmental pace. Yet, how different temperature regimes interact with and regulate the six linked DAM genes remains unclear. Here, we demonstrate that chilling downregulates DAM1 and DAM3-6 in dormant floral buds with distinct patterns and identify DAM4 as the most abundantly expressed one. We reveal multiple epigenetic events, with tri-methyl histone H3 lysine 27 (H3K27me3) induced by chilling specifically in DAM1 and DAM5, a 21-nt sRNA in DAM3 and a ncRNA induced in DAM4. Such induction is inversely correlated with downregulation of their cognate DAMs. We also show that the six DAMs were hypermethylated, associating with the production of 24-nt sRNAs. Hence, the chilling-responsive dynamic of the different epigenetic elements and their interactions likely define distinct expression abundance and downregulation pattern of each DAM. We further show that the expression of the five DAMs remains steadily unchanged or continuously downregulated at the ensuing warm temperature after chilling, and this state of regulation correlates with robust increase of sRNA expression, H3K27me3 and CHH methylation, which is particularly pronounced in DAM4. Such robust increase of repressive epigenetic marks may irreversibly reinforce the chilling-imposed repression of DAMs to ensure flower-developmental programming free from any residual DAM inhibition. Taken together, we reveal novel information about genetic and epigenetic regulation of the DAM cluster in peach, which will be of fundamental significance in understanding of the regulatory mechanisms underlying chilling requirement and dormancy release, and of practical application for improvement of plasticity of flower time and bud break in fruit trees to adapt changing climates.

Spontaneous mutations in fruit-specific carotenoid biosynthetic genes of tomato (Solanum lycopersicum) have led to improved understanding of ripening-associated carotenogenesis. Here, we confirm that ZDS is encoded by a single gene in tomato transcriptionally regulated by ripening transcription factors RIN, NOR and ethylene. Manipulation of ZDS was achieved through transgenic repression and heterologous over-expression in tomato. CaMV 35S-driven RNAi repression inhibited carotenoid biosynthesis in all aerial tissues examined resulting in elevated levels of ζ-carotene isomers and upstream carotenoids, while downstream all trans-lycopene and subsequent photoprotective carotenes and xanthophylls were diminished. Consequently, immature fruit displayed photo-bleaching consistent with reduced levels of the photoprotective carotenes and developmental phenotypes related to a reduction in the carotenoid-derived phytohormone abscisic acid (ABA). ZDS-repressed ripe fruit was devoid of the characteristic red carotenoid, all trans-lycopene and displayed brilliant yellow pigmentation due to elevated 9,9' di-cis-ζ-carotene. Over-expression of the Arabidopsis thaliana ZDS (AtZDS) gene bypassed endogenous co-suppression and revealed ZDS as an additional bottleneck in ripening-associated carotenogenesis of tomato. Quantitation of carotenoids in addition to multiple ripening parameters in ZDS-altered lines and ABA-deficient fruit-specific carotenoid mutants was used to separate phenotypic consequences of ABA from other effects of ZDS manipulation and reveal a unique and dynamic ζ-carotene isomer profile in ripe fruit.

Summary Tomato ( Solanum lycopersicum L.) plants are cold‐sensitive, and the fruit are susceptible to postharvest chilling injury when stored at low temperature. However, the mechanisms underlying cold stress responses in tomato are poorly understood. We demonstrate that SlGRAS4 , encoding a transcription factor induced by low temperature, promotes chilling tolerance in tomato leaves and fruit. Combined genome‐wide ChIP‐seq and RNA‐seq approaches identified among cold stress‐associated genes those being direct targets of SlGRAS4 and protein studies revealed that SlGRAS4 forms a homodimer to self‐activate its own promoter. SlGRAS4 can also directly bind tomato SlCBF promoters to activate their transcription without inducing any growth retardation. The study identifies the SlGRAS4‐regulon as a new cold response pathway conferring cold stress tolerance in tomato independently of the ICE1‐CBF pathway. This provides new track for breeding strategies aiming to improve chilling tolerance of cultivated tomatoes and to preserve sensory qualities of tomato fruit often deteriorated by storage at low temperatures.

Non-coding cis-regulatory variants in animal genomes are an important driving force in the evolution of transcription regulation and phenotype diversity. However, cistrome dynamics in plants remain largely underexplored. Here, we compare the binding of GOLDEN2-LIKE (GLK) transcription factors in tomato, tobacco, Arabidopsis, maize and rice. Although the function of GLKs is conserved, most of their binding sites are species-specific. Conserved binding sites are often found near photosynthetic genes dependent on GLK for expression, but sites near non-differentially expressed genes in the glk mutant are nevertheless under purifying selection. The binding sites' regulatory potential can be predicted by machine learning model using quantitative genome features and TF co-binding information. Our study show that genome cis-variation caused wide-spread TF binding divergence, and most of the TF binding sites are genetically redundant. This poses a major challenge for interpreting the effect of individual sites and highlights the importance of quantitatively measuring TF occupancy.

Summary Ethylene (ETH) controls climacteric fruit ripening and can be triggered by osmotic stress. However, the mechanism regulating ETH biosynthesis during fruit ripening and under osmotic stress is largely unknown in apple ( Malus domestica ). Here, we explored the roles of SnRK2 protein kinases in ETH biosynthesis related to fruit ripening and osmoregulation. We identified the substrates of MdSnRK2‐I using phosphorylation analysis techniques. Finally, we identified the MdSnRK2‐I‐mediated signaling pathway for ETH biosynthesis related to fruit ripening and osmoregulation. The activity of two MdSnRK2‐I members, MdSnRK2.4 and MdSnRK2.9, was significantly upregulated during ripening or following mannitol treatment. Overexpression of MdSnRK2‐I increased ETH biosynthesis under normal and osmotic conditions in apple fruit. MdSnRK2‐I phosphorylated the transcription factors MdHB1 and MdHB2 to enhance their protein stability and transcriptional activity on MdACO1 . MdSnRK2‐I also interacted with MdACS1 and increased its protein stability through two phosphorylation sites. The increased MdACO1 expression and MdACS1 protein stability resulted in higher ETH production in apple fruit. In addition, heterologous expression of MdSnRK2‐I or manipulation of SlSnRK2‐I expression in tomato ( Solanum lycopersicum ) fruit altered fruit ripening and ETH biosynthesis. We established that MdSnRK2‐I functions in fruit ripening and osmoregulation, and identified the MdSnRK2‐I‐mediated signaling pathway controlling ETH biosynthesis.

Summary Photosynthesis in eukaryotic cells takes place in chloroplasts that develop from undifferentiated plastids in response to light. Two families of transcription factors known as Golden2-like (GLK) and GATA regulate plant chloroplast development, and the MIR171-targeted SCARECROW-LIKE (SCL) GRAS transcription factors regulate chlorophyll biosynthesis. Additionally, the Elongated Hypocotyl 5 (HY5) transcription factor plays a critical role in photomorphogenesis. The extent to which these proteins carry out conserved roles in non-seed plants such as the liverworts is not known. Here we determine the degree of functional conservation of the GLK, GATA, SCL and HY5 proteins in controlling chloroplast development in the model liverwort Marchantia polymorpha . Our results indicate that GATA, SCL and HY5 have a minimal or undetectable role in chloroplast biogenesis but loss of GLK function leads to reduced chloroplast size, underdeveloped thylakoid membranes and lower chlorophyll accumulation. ChIP-seq and RNA-seq analyses revealed that GLK can directly regulate many photosynthetic and chloroplast development-related genes. But there is extensive divergence between its function in M. polymorpha and flowering plants. Collectively our findings suggest that the function of GATA as well as SCL in chloroplast development and the more specialised role of HY5 in photomorphogenesis, either evolved after the divergence of vascular plants from bryophytes, that were lost in M. polymorpha , or that functional redundancy is masking their roles. In contrast, and consistent with its presence in algae, GLK plays a conserved role in chloroplast biogenesis of liverworts and vascular plants.

The gaseous hormone ethylene is perceived by a family of ethylene receptors which interact with the Raf-like kinase CTR1. SlTPR1 encodes a novel TPR (tetratricopeptide repeat) protein from tomato that interacts with the ethylene receptors NR and LeETR1 in yeast two-hybrid and in vitro protein interaction assays. SlTPR1 protein with a GFP fluorescent tag was localized in the plasmalemma and nuclear membrane in Arabidopsis, and SlTPR1-CFP and NR-YFP fusion proteins were co-localized in the plasmalemma and nuclear membrane following co-bombardment of onion cells. Overexpression of SlTPR1 in tomato resulted in ethylene-related pleiotropic effects including reduced stature, delayed and reduced production of inflorescences, abnormal and infertile flowers with degenerate styles and pollen, epinasty, reduced apical dominance, inhibition of abscission, altered leaf morphology, and parthenocarpic fruit. Similar phenotypes were seen in Arabidopsis overexpressing SlTPR1. SlTPR1 overexpression did not increase ethylene production but caused enhanced accumulation of mRNA from the ethylene responsive gene ChitB and the auxin-responsive gene SlSAUR1-like, and reduced expression of the auxin early responsive gene LeIAA9, which is known to be inhibited by ethylene and to be associated with parthenocarpy. Cuttings from the SlTPR1-overexpressors produced fewer adventitious roots and were less responsive to indole butyric acid. It is suggested that SlTPR1 overexpression enhances a subset of ethylene and auxin responses by interacting with specific ethylene receptors. SlTPR1 shares features with human TTC1, which interacts with heterotrimeric G-proteins and Ras, and competes with Raf-1 for Ras binding. Models for SlTPR1 action are proposed involving modulation of ethylene signalling or receptor levels.

A method is described for the detection of certain nucleotide modifications adjacent to the 5' 7-methyl guanosine cap of mRNAs from individual genes. The method quantitatively measures the relative abundance of 2'-O-methyl and N(6),2'-O-dimethyladenosine, two of the most common modifications. In order to identify and quantitatify the amounts of N(6),2'-O-dimethyladenosine, a novel method for the synthesis of modified adenosine phosphoramidites was developed. This method is a one step synthesis and the product can directly be used for the production of N(6),2'-O-dimethyladenosine containing RNA oligonucleotides. The nature of the cap-adjacent nucleotides were shown to be characteristic for mRNAs from individual genes transcribed in liver and testis.

Deep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3' end adapter containing a 5',5'-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3' hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available.We demonstrate that DNA oligo with 5' phosphate and 3' amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum) using the T4 RNA ligase 1 adenylated adapter.We provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples.

Leaves of C4 crops usually have higher radiation, water and nitrogen use efficiencies compared to the C3 species. Engineering C4 traits into C3 crops has been proposed as one of the most promising ways to repeal the biomass yield ceiling. To better understand the function of C4 photosynthesis, and to identify candidate genes that are associated with the C4 pathways, a comparative transcription network analysis was conducted on leaf developmental gradients of three C4 species including maize, green foxtail and sorghum and one C3 species, rice. By combining the methods of gene co-expression and differentially co-expression networks, we identified a total of 128 C4 specific genes. Besides the classic C4 shuttle genes, a new set of genes associated with light reaction, starch and sucrose metabolism, metabolites transportation, as well as transcription regulation, were identified as involved in C4 photosynthesis. These findings will provide important insights into the differential gene regulation between C3 and C4 species, and a good genetic resource for establishing C4 pathways in C3 crops.

Background and AimsIn recent years, increasing numbers of long non-coding RNAs (lncRNAs) have been identified in humans, animals and plants, and several of them have been shown to play important roles in diverse biological processes. However, little work has been performed on the regulation mechanism of lncRNA biogenesis and expression, especially in plants. Compared with studies of tomato MADS-box transcription factor RIPENING INHIBITOR (RIN) target coding genes, there are few reports on its relationship to non-coding RNAs. The aim of the present study was to identify and explore the specific role of RIN target lncRNAs in tomato fruit development and ripening.

Biogenesis of the complex 3D architecture of plant thylakoids remains an unsolved problem. Here, we analyzed this process in chloroplasts of germinating Arabidopsis thaliana cotyledons using 3D electron microscopy and gene expression analyses of chloroplast proteins. Our study identified a linear developmental sequence with five assembly stages: tubulo-vesicular prothylakoids (24 h after imbibition [HAI]), sheet-like pregranal thylakoids that develop from the prothylakoids (36 HAI), proliferation of pro-grana stacks with wide tubular connections to the originating pregrana thylakoids (60 HAI), structural differentiation of pro-grana stacks and expanded stroma thylakoids (84 HAI), and conversion of the pro-grana stacks into mature grana stacks (120 HAI). Development of the planar pregranal thylakoids and the pro-grana membrane stacks coincides with the appearance of thylakoid-bound polysomes and photosystem II complex subunits at 36 HAI. ATP synthase, cytochrome b6f, and light-harvesting complex II proteins are detected at 60 HAI, while PSI proteins and the curvature-inducing CURT1A protein appear at 84 HAI. If stromal ribosome biogenesis is delayed, prothylakoids accumulate until stromal ribosomes are produced, and grana-forming thylakoids develop after polysomes bind to the thylakoid membranes. In fzo-like (fzl) mutants, in which thylakoid organization is perturbed, pro-grana stacks in cotyledons form discrete, spiral membrane compartments instead of organelle-wide membrane networks, suggesting that FZL is involved in fusing membrane compartments together. Our data demonstrate that the assembly of thylakoid protein complexes, CURT1 proteins, and FZL proteins mediate distinct and critical steps in thylakoid biogenesis.

Abstract Tomato (Solanum lycopersicum) is a model plant for studying fruit development and ripening. In this study, we found that down-regulation of a tomato bell-like homeodomain 4 (SlBL4) resulted in a slightly darker-green fruit phenotype and increased accumulation of starch, fructose, and glucose. Analysis of chlorophyll content and TEM observations was consistent with these phenotypes, indicating that SlBL4 was involved in chlorophyll accumulation and chloroplast formation. Ripened fruit of SlBL4-RNAi plants had noticeably decreased firmness, larger intercellular spaces, and thinner cell walls than the wild-type. RNA-seq identified differentially expressed genes involved in chlorophyll metabolism, chloroplast development, cell wall metabolism, and carotenoid metabolism. ChIP-seq identified (G/A) GCCCA (A/T/C) and (C/A/T) (C/A/T) AAAAA (G/A/T) (G/A) motifs. SlBL4 directly inhibited the expression of protoporphyrinogen oxidase (SlPPO), magnesium chelatase H subunit (SlCHLD), pectinesterase (SlPE), protochlorophyllide reductase (SlPOR), chlorophyll a/b binding protein 3B (SlCAB-3B), and homeobox protein knotted 2 (TKN2). In contrast, it positively regulated the expression of squamosa promoter binding protein-like colorless non-ripening (LeSPL-CNR). Our results indicate that SlBL4 is involved in chlorophyll accumulation, chloroplast development, cell wall metabolism, and the accumulation of carotenoids during tomato fruit ripening, and provide new insights for the transcriptional regulation mechanism of BELL-mediated fruit growth and ripening.

Abstract Melon exhibits substantial natural variation especially in fruit ripening physiology, including both climacteric (ethylene-producing) and non-climacteric types. However, genomic mechanisms underlying such variation are not yet fully understood. Here, we report an Oxford Nanopore-based high-grade genome reference in the semi-climacteric cultivar Harukei-3 (378 Mb + 33,829 protein-coding genes), with an update of tissue-wide RNA-seq atlas in the Melonet-DB database. Comparison between Harukei-3 and DHL92, the first published melon genome, enabled identification of 24,758 one-to-one orthologue gene pairs, whereas others were candidates of copy number variation or presence/absence polymorphisms (PAPs). Further comparison based on 10 melon genome assemblies identified genome-wide PAPs of 415 retrotransposon Gag-like sequences. Of these, 160 showed fruit ripening-inducible expression, with 59.4% of the neighboring genes showing similar expression patterns ( r > 0.8). Our results suggest that retrotransposons contributed to the modification of gene expression during diversification of melon genomes, and may affect fruit ripening-inducible gene expression.

Fruit ripening relies on the precise spatiotemporal control of RNA polymerase II (Pol II)-dependent gene transcription, and the evolutionarily conserved Mediator (MED) coactivator complex plays an essential role in this process. In tomato (Solanum lycopersicum), a model climacteric fruit, ripening is tightly coordinated by ethylene and several key transcription factors. However, the mechanism underlying the transmission of context-specific regulatory signals from these ripening-related transcription factors to the Pol II transcription machinery remains unknown. Here, we report the mechanistic function of MED25, a subunit of the plant Mediator transcriptional coactivator complex, in controlling the ethylene-mediated transcriptional program during fruit ripening. Multiple lines of evidence indicate that MED25 physically interacts with the master transcription factors of the ETHYLENE-INSENSITIVE 3 (EIN3)/EIN3-LIKE (EIL) family, thereby playing an essential role in pre-initiation complex formation during ethylene-induced gene transcription. We also show that MED25 forms a transcriptional module with EIL1 to regulate the expression of ripening-related regulatory as well as structural genes through promoter binding. Furthermore, the EIL1-MED25 module orchestrates both positive and negative feedback transcriptional circuits, along with its downstream regulators, to fine-tune ethylene homeostasis during fruit ripening.

Yellow-seed trait is a desirable breeding characteristic of rapeseed (Brassica napus) that could greatly improve seed oil yield and quality. However, the underlying mechanisms controlling this phenotype in B. napus plants are difficult to discern because of their complexity. Here, we assemble high-quality genomes of yellow-seeded (GH06) and black-seeded (ZY821). Combining in-depth fine mapping of a quantitative trait locus (QTL) for seed color with other omics data reveal BnA09MYB47a, encoding an R2R3-MYB-type transcription factor, as the causal gene of a major QTL controlling the yellow-seed trait. Functional studies show that sequence variation of BnA09MYB47a underlies the functional divergence between the yellow- and black-seeded B. napus. The black-seed allele BnA09MYB47aZY821, but not the yellow-seed allele BnA09MYB47aGH06, promotes flavonoid biosynthesis by directly activating the expression of BnTT18. Our discovery suggests a possible approach to breeding B. napus for improved commercial value and facilitates flavonoid biosynthesis studies in Brassica crops.

Fluorescent protein labelling technologies enable dynamic protein actions to be imaged in living cells and can also be used in conjunction with other methods such as Forster resonance energy transfer and biomolecular fluorescence complementation. In this report, we describe the generation of a series of 23 novel GATEWAY-compatible vectors based on pGreenII and pDH51 backbones with the latest fluorescent protein tags (Cerulean, EGFP and Venus) and the choice of three in planta selection markers. These vectors can be obtained from the Nottingham Arabidopsis Stock Centre (N9819-N9846) and should be a powerful tool box for transgenic research in plants.

Ovarian folliculogenesis is always of great interest in reproductive biology. However, the molecular mechanisms that control follicle development, particularly the early phase of follicle activation or recruitment, still remain poorly understood. In an attempt to decipher the gene networks and signaling pathways involved in such transition, we conducted a transcriptomic analysis (RNA-seq) on zebrafish primary growth (PG, stage I; inactive) and previtellogenic (PV, stage II; activated) follicles. A total of 118 unique microRNAs (miRNAs) (11 downregulated and 83 upregulated during PG/PV transition) and 56711 unique messenger RNAs (mRNAs) (1839 downregulated and 7243 upregulated during PG/PV transition) were identified. Real-time quantitative polymerase chain reaction analysis confirmed differential expression of 46 miRNAs from 66 candidates (66.67%). Among which, we chose to focus on 13 miRNAs (let-7a, -7b, -7c-5p, -7d-5p, -7h, -7i; miR-21, -23a-3p, -27c-3p, -107a-3p, -125b-5p, -145-3p, and -202-5p) that exhibited significant differential expression between PG and PV follicles (P ≤ 0.045*). With this 13-miRNA expression signature alone, PG follicles can be well differentiated from PV follicles by hierarchical clustering, suggesting their functional relevance during PG-to-PV transition. By overlaying predicted target genes and the differentially expressed mRNAs revealed by the RNA-seq analysis, especially those showing reciprocal miRNA-mRNA expression patterns, we shortlisted a panel of miRNA downstream targets for luciferase reporter validation. The reporter assay confirmed the interactions of let-7i:: atg4a (P = 0.01*), miR-202-5p::c23h20orf24 (P = 0.0004***), and miR-144-5p::ybx1 (P = 0.003**), implicating these potential miRNA-mRNA gene pairs in follicle activation during folliculogenesis. Our transcriptomic data analyses suggest that miRNA-mediated post-transcriptional control may represent an important mechanism underlying follicle activation.

Abstract Chromatin is the main carrier of genetic information and is non-randomly distributed within the nucleus. Next-generation sequence-based chromatin conformation capture technologies have enabled us to directly examine its three-dimensional organization at an unprecedented scale and resolution. In the best-studied mammalian models, chromatin folding can be broken down into three hierarchical levels, compartment, domains, and loops, which play important roles in transcriptional regulation. Although similar structures have now been identified in plants, they might not possess exactly the same functions as the mammalian ones. Here, we review recent Hi-C studies in plants, compare plant chromatin structures with their mammalian counterparts, and discuss the differences between plants with different genome sizes.

Impaired root development caused by aluminum (Al) toxicity is a major cause of grain yield reduction in crops cultivated on acid soils, which are widespread worldwide. In sorghum, the major Al-tolerance locus, AltSB , is due to the function of SbMATE, which is an Al-activated root citrate transporter. Here we performed a molecular and physiological characterization of various AltSB donors and near-isogenic lines harboring various AltSB alleles. We observed a partial transfer of Al tolerance from the parents to the near-isogenic lines that was consistent across donor alleles, emphasizing the occurrence of strong genetic background effects related to AltSB . This reduction in tolerance was variable, with a 20% reduction being observed when highly Al-tolerant lines were the AltSB donors, and a reduction as great as 70% when other AltSB alleles were introgressed. This reduction in Al tolerance was closely correlated with a reduction in SbMATE expression in near-isogenic lines, suggesting incomplete transfer of loci acting in trans on SbMATE. Nevertheless, AltSB alleles from the highly Al-tolerant sources SC283 and SC566 were found to retain high SbMATE expression, presumably via elements present within or near the AltSB locus, resulting in significant transfer of the Al-tolerance phenotype to the derived near-isogenic lines. Allelic effects could not be explained by coding region polymorphisms, although occasional mutations may affect Al tolerance. Finally, we report on the extensive occurrence of alternative splicing for SbMATE, which may be an important component regulating SbMATE expression in sorghum by means of the nonsense-mediated RNA decay pathway.

Abstract Interspecific hybridization is a driving force in evolution and speciation of higher plants. Interspecific hybridization often induces immediate and saltational changes in gene expression, a phenomenon collectively termed “transcriptome shock”. Although transcriptome shock has been reported in various plant and animal taxa, the extent and pattern of shock‐induced expression changes are often highly idiosyncratic, and hence entails additional investigations. Here, we produced a set of interspecific F1 triploid hybrid plants between Oryza sativa , ssp. japonica (2 n = 2 x = 24, genome AA) and the tetraploid form of O. punctata (2 n = 4 x = 48, genome, BBCC), and conducted RNA‐seq transcriptome profiling of the hybrids and their exact parental plants. We analyzed both homeolog expression bias and overall gene expression level difference in the hybrids relative to the in silico “hybrids” (parental mixtures). We found that approximately 16% (2,541) of the 16,112 expressed genes in leaf tissue of the F1 hybrids showed nonadditive expression, which were specifically enriched in photosynthesis‐related pathways. Interestingly, changes in the maternal homeolog expression, including non‐stochastic silencing, were the major causes for altered homeolog expression partitioning in the F1 hybrids. Our findings have provided further insights into the transcriptome response to interspecific hybridization and heterosis.

Ripe Cara Cara sweet orange contains 25 times as much carotenoids in flesh as Newhall sweet orange, due to high accumulation of carotenes, mainly phytoene, lycopene and phytofluene. Only yellow globular chromoplasts were observed in Newhall flesh. Distinct yellow globular and red elongated crystalline chromoplasts were found in Cara Cara but only one type of chromoplast was present in each cell. The red crystalline chromoplasts contained lycopene as a dominant carotenoid and were associated with characteristic carotenoid sequestering structures. The increased accumulation of linear carotenes in Cara Cara is not explained by differences in expression of all 18 carotenogenic genes or gene family members examined, or sequence or abundance of mRNAs from phytoene synthase (PSY) and chromoplast-specific lycopene β-cyclase (CYCB) alleles. 2-(4-Chlorophenylthio)-triethylamine hydrochloride (CPTA) enhanced lycopene accumulation and induced occurrence of red crystalline chromoplasts in cultured Newhall juice vesicles, indicating that carotenoid synthesis and accumulation can directly affect chromoplast differentiation and structure. Norflurazon (NFZ) treatment resulted in high accumulation of phytoene and phytofluene in both oranges, and the biosynthetic activity upstream of phytoene desaturase was similar in Newhall and Cara Cara. Possible mechanisms for high carotene accumulation and unique development of red crystalline chromoplasts in Cara Cara are discussed.

Abstract Temperature changes affect apple development and production. Phenylpropanoid metabolism and hormone signaling play a crucial role in regulating apple growth and development in response to temperature changes. Here, we found that McMYB4 is induced by treatment at 28 °C and 18 °C, and McMYB4 overexpression results in flavonol and lignin accumulation in apple leaves. Yeast one-hybrid (Y1H) assays and electrophoretic mobility shift assays (EMSAs) further revealed that McMYB4 targets the promoters of the flavonol biosynthesis genes CHS and FLS and the lignin biosynthesis genes CAD and F5H . McMYB4 expression resulted in higher levels of flavonol and lignin biosynthesis in apple during growth at 28 °C and 18 °C than during growth at 23 °C. At 28 °C and 18 °C, McMYB4 also binds to the AUX / ARF and BRI / BIN promoters to activate gene expression, resulting in acceleration of the auxin and brassinolide signaling pathways. Taken together, our results demonstrate that McMYB4 promotes flavonol biosynthesis and brassinolide signaling, which decreases ROS contents to improve plant resistance and promotes lignin biosynthesis and auxin signaling to regulate plant growth. This study suggests that McMYB4 participates in the abiotic resistance and growth of apple in response to temperature changes by regulating phenylpropanoid metabolism and hormone signaling.

C4 plants partition photosynthesis enzymes between the bundle sheath (BS) and the mesophyll (M) cells for the better delivery of CO2 to RuBisCO and to reduce photorespiration. To better understand how C4 photosynthesis is regulated at the transcriptional level, we performed RNA-seq, ATAC-seq, ChIP-seq and Bisulfite-seq (BS-seq) on BS and M cells isolated from maize leaves. By integrating differentially expressed genes with chromatin features, we found that chromatin accessibility coordinates with epigenetic features, especially H3K27me3 modification and CHH methylation, to regulate cell type-preferentially enriched gene expression. Not only the chromatin-accessible regions (ACRs) proximal to the genes (pACRs) but also the distal ACRs (dACRs) are determinants of cell type-preferentially enriched expression. We further identified cell type-preferentially enriched motifs, e.g. AAAG for BS cells and TGACC/T for M cells, and determined their corresponding transcription factors: DOFs and WRKYs. The complex interaction between cis and trans factors in the preferential expression of C4 genes was also observed. Interestingly, cell type-preferentially enriched gene expression can be fine-tuned by the coordination of multiple chromatin features. Such coordination may be critical in ensuring the cell type-specific function of key C4 genes. Based on the observed cell type-preferentially enriched expression pattern and coordinated chromatin features, we predicted a set of functionally unknown genes, e.g. Zm00001d042050 and Zm00001d040659, to be potential key C4 genes. Our findings provide deep insight into the architectures associated with C4 gene expression and could serve as a valuable resource to further identify the regulatory mechanisms present in C4 species.

Understanding how cis-regulatory elements facilitate gene expression is a key question in biology. Recent advances in single-cell genomics have led to the discovery of cell-specific chromatin landscapes that underlie transcription programs in animal models. However, the high equipment and reagent costs of commercial systems limit their applications for many laboratories. In this study, we developed a combinatorial index and dual PCR barcode strategy to profile the Arabidopsis thaliana root single-cell epigenome without any specialized equipment. We generated chromatin accessibility profiles for 13 576 root nuclei with an average of 12 784 unique Tn5 integrations per cell. Integration of the single-cell assay for transposase-accessible chromatin sequencing and RNA sequencing data sets enabled the identification of 24 cell clusters with unique transcription, chromatin, and cis-regulatory signatures. Comparison with single-cell data generated using the commercial microfluidic platform from 10X Genomics revealed that this low-cost combinatorial index method is capable of unbiased identification of cell-type-specific chromatin accessibility. We anticipate that, by removing cost, instrumentation, and other technical obstacles, this method will be a valuable tool for routine investigation of single-cell epigenomes and provide new insights into plant growth and development and plant interactions with the environment.

Leaf angle is an important agronomic trait determining maize (Zea mays) planting density and light penetration into the canopy and contributes to the yield gain in modern maize hybrids. However, little is known about the molecular mechanisms underlying leaf angle beyond the ZmLG1 (liguleless1) and ZmLG2 (Liguleless2) genes. In this study, we found that the transcription factor (TF) ZmBEH1 (BZR1/BES1 homolog gene 1) is targeted by ZmLG2 and regulates leaf angle formation by influencing sclerenchyma cell layers on the adaxial side. ZmBEH1 interacted with the TF ZmBZR1 (Brassinazole Resistant 1), whose gene expression was also directly activated by ZmLG2. Both ZmBEH1 and ZmBZR1 are bound to the promoter of ZmSCL28 (SCARECROW-LIKE 28), a third TF that influences leaf angle. Our study demonstrates regulatory modules controlling leaf angle and provides gene editing targets for creating optimal maize architecture suitable for dense planting.

Plant virus technology, in particular virus-induced gene silencing, is a widely used reverse- and forward-genetics tool in plant functional genomics. However the potential of virus technology to express genes to induce phenotypes or to complement mutants in order to understand the function of plant genes is not well documented. Here we exploit Potato virus X as a tool for virus-induced gene complementation (VIGC). Using VIGC in tomato, we demonstrated that ectopic viral expression of LeMADS-RIN, which encodes a MADS-box transcription factor (TF), resulted in functional complementation of the non-ripening rin mutant phenotype and caused fruits to ripen. Comparative gene expression analysis indicated that LeMADS-RIN up-regulated expression of the SBP-box (SQUAMOSA promoter binding protein-like) gene LeSPL-CNR, but down-regulated the expression of LeHB-1, an HD-Zip homeobox TF gene. Our data support the hypothesis that a transcriptional network may exist among key TFs in the modulation of fruit ripening in tomato.

Vivipary, wherein seeds germinate prior to dispersal while still associated with the maternal plant, is an adaptation to extreme environments. It is normally inhibited by the establishment of dormancy. The genetic framework of vivipary has been well studied; however, the role of epigenetics in vivipary remains unknown. Here, we report that silencing of METHYLTRANSFERASE1 (SlMET1) promoted precocious seed germination and seedling growth within the tomato (Solanum lycopersicum) epimutant Colorless non-ripening (Cnr) fruits. This was associated with decreases in abscisic acid concentration and levels of mRNA encoding 9-cis-epoxycarotenoid-dioxygenase (SlNCED), which is involved in abscisic acid biosynthesis. Differentially methylated regions were identified in promoters of differentially expressed genes, including SlNCED. SlNCED knockdown also induced viviparous seedling growth in Cnr fruits. Strikingly, Cnr ripening reversion suppressed vivipary. Moreover, neither SlMET1/SlNCED-virus-induced gene silencing nor transgenic SlMET1-RNA interference produced vivipary in wild-type tomatoes; the latter affected leaf architecture, arrested flowering, and repressed seed development. Thus, a dual pathway in ripening and SlMET1-mediated epigenetics coordinates the blockage of seed vivipary.

Membrane trafficking is essential for plant growth and responses to external signals. The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex (endosomal sorting complex required for transport). FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body (MVB), vacuolar protein transport and vacuole biogenesis, and autophagic degradation. FREE1 knockout plants show defective MVB formation, abnormal vacuolar transport, fragmented vacuoles, accumulated autophagosomes, and seedling lethality. To further uncover the underlying mechanisms of FREE1 function in plants, we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants. The obtained mutants are termed as suppressors of free1 (sof). To date, 229 putative sof mutants have been identified. Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors. Also 145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants. Through next-generation sequencing (NGS) of bulked DNA from F2 mapping population of two RNAi-related sof mutants, FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly. These FREE1- and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis, as well as for uncovering the new components involved in the regulation of silencing pathways in plants.

Artificial light at night (ALAN) is a major driver of firefly population declines, but its physiological effects are not well understood. To investigate the impact of ALAN on firefly development, we exposed larval Aquatica ficta fireflies to ALAN for two weeks. High larval mortality was observed in the periods of 1–68 days and 106–134 days post-treatment, which may represent the short- and long-term impacts of ALAN. We then profiled the transcriptome of larval Aquatica ficta fireflies following two weeks of ALAN exposure. A total of 1262 (1.67% out of 75777 unigenes) were differentially expressed in the treatment group: 1157 were down-regulated, and 105 were up-regulated. Up-regulated unigenes were related to regulation of hormone levels, ecdysteroid metabolic process, and response to stimulus; down-regulated unigenes were related to negative regulation of insulin receptor signaling, germ cell development, oogenesis, spermatid development, and regulation of neuron differentiation. Transcriptome results suggest that the endocrine, reproductive, and neural development of firefly larvae could be impaired by even relatively brief period of ALAN exposure. This report contributes a much-needed molecular perspective to the growing body of research documenting the fitness impacts of ALAN on bioluminescent fireflies.

Abstract Cis -regulatory elements (CREs) are critical in regulating gene expression, and yet our understanding of CRE evolution remains a challenge. Here, we constructed a comprehensive single-cell atlas of chromatin accessibility in Oryza sativa , integrating data from 104,029 nuclei representing 128 discrete cell states across nine distinct organs. We used comparative genomics to compare cell-type resolved chromatin accessibility between O. sativa and 57,552 nuclei from four additional grass species ( Zea mays, Sorghum bicolor, Panicum miliaceum , and Urochloa fusca ). Accessible chromatin regions (ACRs) had different levels of conservation depending on the degree of cell-type specificity. We found a complex relationship between ACRs with conserved noncoding sequences, cell-type specificity, conservation, and tissue-specific switching. Additionally, we found that epidermal ACRs were less conserved compared to other cell types, potentially indicating that more rapid regulatory evolution has occurred in the L1 epidermal layer of these species. Finally, we identified and characterized a conserved subset of ACRs that overlapped the repressive histone modification H3K27me3, implicating them as potentially critical silencer CREs maintained by evolution. Collectively, this comparative genomics approach highlights the dynamics of cell-type-specific CRE evolution in plants.

In this article, we comprehensively reviewed Argentina’s Vaca Muerta formation, which encompasses a geological overview, advances in extraction technologies, the potential environmental impact, and economic analysis. Detailed geological analysis is discussed, emphasizing the stratigraphy, lithology, and depositional environments of the formation, which is crucial for understanding the distribution and quality of hydrocarbon resources. The latest advancements in hydraulic fracturing and horizontal drilling are explored, which have significantly improved efficiency and increased recoverable resources. The environmental implications of these extraction methods are critically examined. This includes a discussion of the necessity of sustainable practices in hydrocarbon extraction, highlighting the balance between resource development and environmental stewardship. The economic viability of the Vaca Muerta formation is analyzed, with a focus on cost-effectiveness, market trends, and investment patterns. This section assesses the formation’s potential as a profitable venture and its impact on the global energy market. Finally, the review anticipates future technological and policy developments. The strategic importance of the Vaca Muerta formation in the global energy sector is underscored, and its potential role in shaping future hydrocarbon exploration and production strategies is examined. In short, this essay not only presents data and findings, but also contextualizes them within the broader scope of energy production, environmental sustainability, and economic viability. This comprehensive approach provides a multi-faceted understanding of the Vaca Muerta formation’s significance in the global energy landscape.

DNA cytosine methylation is one of the most abundant epigenetic marks found in the plant nuclear genome. Bisulfite sequencing (BS-Seq) is the method of choice for profiling DNA cytosine methylation genome-wide at a single nucleotide resolution. The basis of this technique is that the unmethylated cytosine can be deaminated to uracil by sodium bisulfite, while the methylated cytosine is resistant to the treatment. By deep sequencing of the bisulfite converted genomic DNA, the methylation level of each mappable cytosine position in the genome could be measured. In this chapter, we present a detailed 2-day protocol for performing a BS-Seq experiment and a simple bioinformatic workflow for wet lab biologists to visualize the methylation data.

Litchi is an important subtropical fruit tree that requires an appropriately low temperature to trigger floral initiation. Our previous studies have shown that reactive oxygen species (ROS) are involved in litchi flowering. To identify oxidative stress-induced flowering related genes in leaves, ‘Nuomici’ potted trees were grown at medium low-temperature conditions (18/13 °C for day/night, medium-temperature). The trees were treated with the ROS generator methyl viologen dichloride hydrate (MV) as the MV-generated ROS treatment (MM, medium-temperature plus MV) and water as the control treatment (M, medium-temperature plus water). Sixteen RNA-sequencing libraries were constructed, and each library generated more than 5,000,000 clean reads. A total of 517 differentially expressed genes (DEGs) were obtained. Among those DEGs, plant hormone biosynthesis and signal transduction genes, ROS-specific transcription factors, such as AP2/ERF and WRKY genes, stress response genes, and flowering-related genes FLOWERING LOCUS T1 (FT1) and FLOWERING LOCUS T2 (FT2) were significantly enriched. Then, as a confirmatory experiment, the potted trees were uniformly sprayed with MV, N,N’-dimethylthiourea (DMTU, ROS scavenger) plus MV, and water at medium-temperature. The results showed that the MV-generated ROS promoted flowering and changed related gene expression, but these effects were repressed by DMTU treatment. The results of our studies indicate that ROS could promote flowering and partly bypass chilling for litchi flowering.

Whole-genome bisulfite sequencing (WGBS) allows single-base resolution and genome-wide profiling of DNA methylation in plants and animals. This technology provides a powerful tool to identify genes that are potentially controlled by dynamic changes of DNA methylation and demethylation. However, naturally occurring epimutants are rare and genes under epigenetic regulation as well as their biological relevances are often difficult to define. In tomato, fruit development and ripening are a complex process that involves epigenetic control. We have taken the advantage of the tomato epimutant Colourless non-ripening (Cnr) and performed comparative mining of the WGBS datasets for the Cnr and SlCMT3-silenced Cnr fruits. We compared DNA methylation profiles for the promoter sequences of approximately 5,000 bp immediately upstream of the coding region of a list of 20 genes. Differentially methylated regions were found for some of these genes. Virus-induced gene silencing (VIGS) of differentially methylated gene SlDET1 or SlPDS resulted in unusual brown pigmentation in Cnr fruits. These results suggest that comparative WGBS coupled with VIGS can be used to identify genes that may contribute to the colourless unripe phenotype of fruit in the Cnr epimutant.

Abstract Rice ( Oryza sativa ) is a staple crop that supports half the world's population and an important monocot model system. Monocot leaf matures in a basipetal manner and has a well-defined developmental gradient along the longitudinal axis. However, little is known about its transcriptional dynamics after leaf maturation. In this study, we have reconstructed a high spatial resolution transcriptome for the matured rice leaf by sectioning the leaf into seven 3-cm fragments. We have performed strand-specific Illumina sequencing to generate gene expression profiles for each fragment. We found that the matured leaf contains a longitudinal gene expression gradient, with 6.97% (2,603) of the expressed genes showing differentially expression along the seven sections. The leaf transcriptome showed a gradual transition from accumulating transcripts related to primary cell wall and basic cellular metabolism at the base to those involved in photosynthesis and energy production in the middle and catabolic metabolism process toward the tip.

Abstract Advances in genome sequencing and annotation have eased the difficulty of identifying new gene sequences. Predicting the functions of these newly identified genes remains challenging. Genes descended from a common ancestral sequence are likely to have common functions. As a result, homology is widely used for gene function prediction. This means functional annotation errors also propagate from one species to another. Several approaches based on machine learning classification algorithms were evaluated for their ability to accurately predict gene function from non‐homology gene features. Among the eight supervised classification algorithms evaluated, random‐forest‐based prediction consistently provided the most accurate gene function prediction. Non‐homology‐based functional annotation provides complementary strengths to homology‐based annotation, with higher average performance in Biological Process GO terms, the domain where homology‐based functional annotation performs the worst, and weaker performance in Molecular Function GO terms, the domain where the accuracy of homology‐based functional annotation is highest. GO prediction models trained with homology‐based annotations were able to successfully predict annotations from a manually curated “gold standard” GO annotation set. Non‐homology‐based functional annotation based on machine learning may ultimately prove useful both as a method to assign predicted functions to orphan genes which lack functionally characterized homologs, and to identify and correct functional annotation errors which were propagated through homology‐based functional annotations.

Maize ear development determines the crop yield, and many important transcription factors (TFs) have been identified to function in this process. However, their transcriptional regulation mechanisms are still elusive. In this study, we generated the genome-wide DNA binding sites for 8 TFs which are known to function or highly expressed in inflorescence by applying the tsCUT&Tag method in maize leaf protoplast. We exposed a regulatory grid of 4 WUSCHEL-related homeobox (WOX) genes and revealed their potential regulatory mechanisms. In addition, a hierarchical regulation network for the determinacy and specification of maize inflorescence meristems were also constructed using the binding profiles of a floral development gene INDETERMINATE FLORAL APEX1 (IFA1) and 3 MADS-box genes. Our study provides an in-depth understanding and new insights of the regulatory mechanisms during maize inflorescence development.

Plant growth and immunity are tightly interconnected. Oligogalacturonic acids (OGs) are pectic fragments and have been well investigated in plant immunity as a damage-associated molecular pattern. However, little is known regarding how OGs affect plant growth. Here, we reveal that OGs inhibit the growth of intact etiolated seedling by using the horticultural crop tomato as a model. This inhibitory effect is partially suppressed by the action of ethylene biosynthesis inhibitors, or the gene silencing of SlACS2, an essential rate-limiting enzyme for ethylene biosynthesis, suggesting that SlACS2-mediated ethylene production promotes OG-induced growth inhibition. Furthermore, OGs treatment elevates the SlACS2 protein phosphorylation, and its decrease by the kinase inhibitor K252a partially rescue OG-induced growth inhibition, indicating that SlACS2 phosphorylation involves in OG-induced growth inhibition. Moreover, the mitogen-activated protein kinase SlMPK3 could be activated by OGs treatment and can directly phosphorylate SlACS2 in vitro, and the bimolecular fluorescence complementation combining with the yeast two-hybrid assay shows that SlMPK3 interacts with SlACS2, indicating that SlMPK3 may participate in modulating the OG-induced SlACS2 phosphorylation and growth inhibition. Our results reveal a regulatory mechanism at both the transcriptional and post-transcriptional levels by which OGs inhibit the growth of intact plant seedlings.

Seedless chestnut rose (Rosa sterilis S. D. Shi), a promising fruit with a high content of vitamin C, characterizes in bearing seedless fruit due to the severe male sterility, which is scarce among the Rosa species. To better understand the period and cause of the complete male sterility of seedless chestnut rose, the pollen mother cell meiosis, microspore development, callose dynamics, and pollen morphological characteristics of this plant species were investigated in comparison with those of with congener, chestnut rose (Rosa roxburghii Tratt). Cytologically, seedless chestnut rose showed abnormal behaviors of chromosomes during meiosis I, including the formation of chromosome bridges, lagging chromosomes, fragmented chromosomes, asymmetric chromosomal arrangement, etc. which were further ascribed to the structure mutation of chromosomes. During the formation of tetrads, anomalous tetrads were also observed. In the early mononuclear stage, the callose disintegration was delayed in comparison with that of chestnut rose, partially leading to the underdevelopment of pollen grains. Subsequently nucleus appeared to disintegrate during the binuclear stage, giving rise to the termination of microspore development. Microstructural and ultrastructural observations of microspore formation and development demonstrated that the pollen abortion of seedless chestnut rose started in meiosis of the pollen mother cell, followed by anomalies of the tetrad, afterwards, with the delayed disintegration of callose in the early mononucleate stage, as well as by nucleolysis in the binucleate stage, led to the malformation of pollen grain. Therefore, abnormal behavior of meiotic chromosomes was the predominant cause for complete male sterility of seedless chestnut rose, which may be helpful for the further elucidation of the mechanism of male sterility in the genus Rosa the formation of male sterility in the genus Rosa.

Litchi (Litchi chinensis Sonn.) is an important tropical and subtropical evergreen woody fruit tree, and it has been shown that nitric oxide (NO) could promote litchi flowering. NO responsive genes of litchi (cv. Nuomici) primordia were identified through a suppression subtractive hybridization (SSH) library screen. We obtained 1 563 expressed sequences tags (ESTs) that were enriched in the NO treated inflorescence primordia. We then used a reverse Northern analysis to identify 728 true NO responsive ESTs, the sequences of which have been further analyzed. They represent 70 litchi unique genes that could be classified into 9 categories: 14 % of them were involved in transport facilitation, 7 % in transcription regulation, 9 % in stress response, 7 % in sugar metabolism, 9 % in secondary metabolism, 10 % in intracellular signalling, and 44 % in other metabolism, whereas 11 % were genes with unknown functions, and 7 % were genes with no hit found. Next, we performed a real-time quantitative polymerase chain reaction (RT-qPCR) to determine the expression of selected candidate genes during a time-course of NO treatment and of normal floral tissue development.

Fruit ripening is a developmental process evolved to foster animal-mediated seed dispersal, and considerable progress have been made through studies of tomato (Solanum lycopersicum), which is an important vegetable crop as well as the model plant for the Solanaceae family. The timing of tomato fruit ripening is tightly regulated and dependent upon the plant hormone ethylene. However, ethylene is involved in many other aspects of plant growth and development, and is unable to induce ripening in immature fruit when the seeds are not viable. It has long been hypothesized that an epigenetic regulatory mechanism is present to oversee this critical transition to ripening competency and ability to respond to ethylene. Recent genome-wide DNA methylation analysis revealed that the tomato genome is heavily methylated and contains primarily asymmetrical CHH methylation. Small RNAs are enriched in euchromatic regions and form tight clusters in gene promoters coincident with peaks of DNA methylation and repetitive regions. During tomato fruit development, extensive epigenome reprogramming targets the promoter of genes in hormone and metabolic pathways. Most importantly, epigenetic changes also take place in the binding sites of key transcription factor regulating fruit development. These binding sites are hypermethylated in immature fruits, but become substantially demethylated in association with fruit maturation. Hence, the epigenome could have been selected to regulate plant growth and development through altering tissue-specific hormone signalling, metabolite biosynthesis and transcription factor binding. In the case of tomato fruit, DNA methylation might have been exploited by evolution to ensure fidelity and constraint of an important developmental process that directly impacts the effectiveness of seed dispersal. The tomato genome-wide DNA methylation changes also provides a novel perspective towards understanding how plant development can be regulated, and highlights the importance of establishing future crop improvement strategies that consider both genetic and epigenetic variations.

The environmental residue/sublethal doses of neonicotinoid insecticides are believed to generate a negative impact on pollinators, including honey bees. Here we report our recent investigation on how imidacloprid, one of the major neonicotinoids, affects worker bees by profiling the transcriptomes of various ages of bees exposed to different doses of imidacloprid during the larval stage. The results show that imidacloprid treatments during the larval stage severely altered the gene expression profiles and may induce precocious foraging. Differential expression of foraging regulators was found in 14-day-old treated adults. A high transcriptome similarity between larvae-treated 14-day-old adults and 20-day-old controls was also observed, and the similarity was positively correlated with the dose of imidacloprid. One parts per billion (ppb) of imidacloprid was sufficient to generate a long-term impact on the bee’s gene expression as severe as with 50 ppb imidacloprid. The disappearance of nurse bees may be driven not only by the hive member constitution but also by the neonicotinoid-induced precocious foraging behavior.

Litchi is an important Sapindaceae fruit tree. Flowering in litchi is triggered by low temperatures in autumn and winter. It can be divided into early-, medium-, and late-flowering phenotypes according to the time for floral induction. Early-flowering varieties need low chilling accumulation level for floral induction, whereas the late-flowering varieties require high chilling accumulation level. In the present study, RNA-Seq of 87 accessions was performed and transcriptome-based genome-wide association studies (GWAS) was used to identify candidate genes involved in chilling accumulation underlying the time for floral induction. A total of 98,155 high-quality single-nucleotide polymorphism (SNP) sites were obtained. A total of 1,411 significantly associated SNPs and 1,115 associated genes (AGs) were identified, of which 31 were flowering-related, 23 were hormone synthesis-related, and 27 were hormone signal transduction-related. Association analysis between the gene expression of the AGs and the flowering phenotypic data was carried out, and differentially expressed genes (DEGs) in a temperature-controlled experiment were obtained. As a result, 15 flowering-related candidate AGs (CAGs), 13 hormone synthesis-related CAGs, and 11 hormone signal transduction-related CAGs were further screened. The expression levels of the CAGs in the early-flowering accessions were different from those in the late-flowering ones, and also between the flowering trees and non-flowering trees. In a gradient chilling treatment, flowering rates of the trees and the CAGs expression were affected by the treatment. Our present work for the first time provided candidate genes for genetic regulation of flowering in litchi using transcriptome-based GWAS.

Abstract The transcription regulatory network underlying essential and complex functionalities inside a eukaryotic cell is defined by the combinatorial actions of transcription factors (TFs). However, TF binding studies in plants are too few in number to produce a general picture of this complex regulatory netowrk. Here, we used ChIP-seq to determine the binding profiles of 104 TF expressed in the maize leaf. With this large dataset, we could reconstruct a transcription regulatory network that covers over 77% of the expressed genes, and reveal its scale-free topology and functional modularity like a real-world network. We found that TF binding occurs in clusters covering ∼2% of the genome, and shows enrichment for sequence variations associated with eQTLs and GWAS hits of complex agronomic traits. Machine-learning analyses were used to identify TF sequence preferences, and showed that co-binding is key for TF specificity. The trained models were used to predict and compare the regulatory networks in other species and showed that the core network is evolutionarily conserved. This study provided an extensive description of the architecture, organizing principle and evolution of the transcription regulatory network inside the plant leaf.

Light plays an important role in many plant biological processes such as photosynthesis and photomorphogenesis.In this study, we applied RNA-seq, ATAC-seq and ChIP-seq to study transcriptional regulation at multiple levels in Brachypodium distachyon (B.distachyon) under controlled light and extended darkness conditions.We have identified 8,400 differentially expressed genes (DEGs), and they are enriched in photosynthesis Gene Ontology terms.ATAC-seq signal decreased after extended darkness, indicating that light has a genome-wide impact on chromatin accessibility.We also found that differential H3K4me3 and H3K9ac modifications are enriched in gene loci associated with photosynthesis and other light-dependent reactions.Finally, ChIP-seq revealed that a HY5-Like transcription factor could directly bind to the promoter open chromatin regions of these light reaction genes.Taken together, our results indicated the dynamic transcriptional reprograming associated with light-dark changes in Brachypodium could be resulted from the combinatorial action of multiple epigenetic features. Hosted

Abstract Fleshy fruit evolved independently multiple times during angiosperm history. Many climacteric fruits utilize the hormone ethylene to regulate ripening. The fruitENCODE project shows there are multiple evolutionary origins of the regulatory circuits that govern climacteric fruit ripening. Eudicot climacteric fruits with recent whole-genome duplications (WGDs) evolved their ripening regulatory systems using the duplicated floral identity genes, while others without WGD utilised carpel senescence genes. The monocot banana uses both leaf senescence and duplicated floral-identity genes, forming two interconnected regulatory circuits. H3K27me3 plays a conserved role in restricting the expression of key ripening regulators and their direct orthologs in both the ancestral dry fruit and non-climacteric fleshy fruit species. Our findings suggest that evolution of climacteric ripening was constrained by limited availability of signalling molecules and genetic and epigenetic materials, and WGD provided new resources for plants to circumvent this limit. Understanding these different ripening mechanisms makes it possible to design tailor-made ripening traits to improve quality, yield and minimize postharvest losses. One Sentence Summary The fruitENCODE project discovered three evolutionary origins of the regulatory circuits that govern climacteric fruit ripening.

Warm winters and hot springs may promote panicle leaf growing and repress floral development. To identify genes potentially involved in litchi panicle leaf senescence, eight RNA-sequencing (RNA-Seq) libraries of the senescing panicle leaves under low temperature (LT) conditions and the developing panicle leaves under high temperature (HT) conditions were constructed. For each library, 4.78⁻8.99 × 10⁶ clean reads were generated. Digital expression of the genes was compared between the senescing and developing panicle leaves. A total of 6477 upregulated differentially expressed genes (DEGs) (from developing leaves to senescing leaves), and 6318 downregulated DEGs were identified, 158 abscisic acid (ABA)-, 68 ethylene-, 107 indole-3-acetic acid (IAA)-, 27 gibberellic acid (GA)-, 68 cytokinin (CTK)-, 37 salicylic acid (SA)-, and 23 brassinolide (BR)-related DEGs. Confirmation of the RNA-Seq data by quantitative real-time PCR (qRT-PCR) analysis suggested that expression trends of the 10 candidate genes using qRT-PCR were similar to those revealed by RNA-Seq, and a significantly positive correlation between the obtained data from qRT-PCR and RNA-Seq were found, indicating the reliability of our RNA-Seq data. The present studies provided potential genes for the future molecular breeding of new cultivars that can induce panicle leaf senescence and reduce floral abortion under warm climates.

Abstract Advances in genome sequencing and annotation have eased the difficulty of identifying new gene sequences. Predicting the functions of these newly identified genes remains challenging. Genes descended from a common ancestral sequence are likely to have common functions. As a result homology is widely used for gene function prediction. This means functional annotation errors also propagate from one species to another. Several approaches based on machine learning classification algorithms were evaluated for their ability to accurately predict gene function from non-homology gene features. Among the eight supervised classification algorithms evaluated, random forest-based prediction consistently provided the most accurate gene function prediction. Non-homology-based functional annotation provides complementary strengths to homology-based annotation, with higher average performance in Biological Process GO terms, the domain where homology-based functional annotation performs the worst, and weaker performance in Molecular Function GO terms, the domain where the accuracy of homology-based functional annotation is highest. Non-homology-based functional annotation based on machine learning may ultimately prove useful both as a method to assign predicted functions to orphan genes which lack functionally characterized homologs, and to identify and correct functional annotation errors which were propagated through homology-based functional annotations.

Abstract A striking feature of micro-RNAs is that they are often clustered in the genomes of animals. The functional and evolutionary consequences of this clustering remain obscure. Here, we investigated a micro-RNA cluster miR-6/5/4/286/3/309 that is conserved across drosophilid lineages. Small RNA sequencing revealed expression of this micro-RNA cluster in Drosophila melanogaster leg discs, and conditional overexpression of the whole cluster resulted in leg appendage shortening. Transgenic overexpression lines expressing different combinations of micro-RNA cluster members were also constructed. Expression of individual micro-RNAs from the cluster resulted in a normal wild-type phenotype, but either the expression of several ancient micro-RNAs together (miR-5/4/286/3/309) or more recently evolved clustered micro-RNAs (miR-6-1/2/3) can recapitulate the phenotypes generated by the whole-cluster overexpression. Screening of transgenic fly lines revealed downregulation of leg-patterning gene cassettes in generation of the leg-shortening phenotype. Furthermore, cell transfection with different combinations of micro-RNA cluster members revealed a suite of downstream genes targeted by all cluster members, as well as complements of targets that are unique for distinct micro-RNAs. Considered together, the micro-RNA targets and the evolutionary ages of each micro-RNA in the cluster demonstrate the importance of micro-RNA clustering, where new members can reinforce and modify the selection forces on both the cluster regulation and the gene regulatory network of existing micro-RNAs. Key words: micro-RNA, cluster, evolution.

Wheat TaWRKY10 is an uncharacterized group II member of the WRKY super family transcription factor and is regulated by high salinity and PEG. In order to study the diverse biological functions of TaWRKY10, we cloned a 783 bp cDNA fragment of this gene from the wheat accession Yangmai 158 ('Triticum aestivem' L.). Subsequent sequence analysis revealed that the cloned fragment contains UTRs and coding region and shows 96% sequence identity to theTaWRKY10gene in Genbank. The amino acid sequences analysis identified15 amino acid differences, mostly at the C-terminus of this protein of 222 amino acids, compared with the TaWRKY10, suggesting that it is likely to be a novel allele of TaWRKY10. Hence we designated it as TaWRKY10-1. RT-PCR results showed thatTaWRKY10-1gene was up-regulated by NaCl and PEG in wheat. To further elucidate its function, we constructed achemical-inducible TaWRKY10-1over-expression plasmid, pKIGW-TaWRKY10-1, via Gateway cloning technology and transformed it into Arabidopsis plants. Over expression of TaWRKY10-1gene increases the sensitivity of transgenic plants to NaCl, PEG and mannitol, indicating TaWRKY10-1 mightact as a negative regulator in response to the salinity and osmostresses. This work provides the key basis for further functional analysis of wheat TaWRKY10-1 gene.

Fleshy fruit evolved independently multiple times during flowering plant (angiosperm) history yet many utilize the hormone ethylene to regulate ripening. The fruitENCODE project highlights three evolutionary origins of ripening regulatory circuits. Eudicots that experienced recent whole-genome duplications (WGDs) evolved their regulatory systems using duplicated floral identity genes, while others without WGD utilised carpel senescence genes. The monocot banana uses both duplicated floral-identity and leaf senescence genes, forming two interconnected regulatory circuits. Epigenetic marks restricting the expression of key ripening regulators could be tracked to their direct ancestral orthologs. Our findings suggest that evolution of climacteric fruit was constrained by limited optimal signalling molecules and genetic and epigenetic materials, and WGD provided new resources for plants to circumvent this limit. Understanding these mechanisms could improve quality, yield and minimize postharvest losses.

Abstract Chromatins are not randomly packaged in the nucleus and their organization plays important roles in transcription regulation. Using in situ Hi-C, we have compared the 3D chromatin architectures of rice mesophyll and endosperm, foxtail millet bundle sheath and mesophyll, and maize bundle sheath, mesophyll and endosperm tissues. We have also profiled their DNA methylation, open chromatin, histone modification and gene expression to investigate whether chromatin structural dynamics are associated with epigenome features changes. We found that plant global A/B compartment partitions are stable across tissues, while local A/B compartment has tissue-specific dynamic that is associated with differential gene expression. Plant domains are largely stable across tissues, while rare domain border changes are often associated with gene activation. Genes inside plant domains are not conserved across species, and lack significant co-expression behavior unlike those in mammalian cells. When comparing synteny gene pairs, we found those maize genes involved in gene island chromatin loops have shorter genomic distances in smaller genomes without gene island loops such as rice and foxtail millet, suggesting that they have conserved functions. Our study revealed that the 3D configuration of the plant chromatin is also complex and dynamic with unique features that need to be further examined.

In the mammalian nucleus, 3D organization of the chromatin plays an important role in the regulation of gene expression. Similar chromatin structures such as A/B compartments, domains, and loops have now been found in plants, yet their biological function remained to be further elucidated. In this chapter, we present a detailed protocol for examining genome-wide chromatin interaction using in situ Hi-C optimized for plant tissues. We also provide a step-by-step bioinformatic workflow for Hi-C sequencing data alignment, and subsequent compartment, domain, and chromatin loop identification.

ABSTRACT Understanding how cis- regulatory elements facilitate gene expression is a key question in biology. Recent advances in single-cell genomics have led to the discovery of cell-specific chromatin landscapes that underlie transcription programs. However, the high equipment and reagent costs of commercial systems limit their applications for many laboratories. In this study, we profiled the Arabidopsis root single-cell epigenome using a combinatorial index and dual PCR barcode strategy without the need of any specialized equipment. We generated chromatin accessibility profiles for 13,576 Arabidopsis thaliana root nuclei with an average of 12,784 unique Tn5 integrations per cell and 85% of the Tn5 insertions localizing to discrete accessible chromatin regions. Comparison with data generated from a commercial microfluidic platform revealed that our method is capable of unbiased identification of cell type-specific chromatin accessibility with improved throughput, quality, and efficiency. We anticipate that by removing cost, instrument, and other technical obstacles, this combinatorial indexing method will be a valuable tool for routine investigation of single-cell epigenomes and usher new insight into plant growth, development and their interactions with the environment.

We isolated the coding sequence a putative SbCO gene from Sorghum cultivar, Roma, by in silico sequence analysis and subsequently, reverse-transcription PCR. The DNA sequencing data revealed 92% similarity between SbCO and the Arabidopsis flowering time regulator CONSTANS. To investigate the biological function of SbCO in sorghum, we have constructed an inducible expression vector for SbCO based on Gateway cloning technology. This study lays a solid foundation for future analysis of flowering time control in Sorghum.

：Objective To examine therole of comprehensive percutaneous permeation of radix salviae miltiorrhizae in treatingevent-related potentials( P300 )of vascular dementia patients. Methods A total of 60patients were randomly classified into two groups: the treatment group( percutaneouspermeation of radix salviae miltiorrhizae ) and the control group. The two groups weremeasured by means of Hasegawa s Dementia scale(HDS),activity of daily living (ADL) andevent-related potentials (P300 ) by physical therapist before and after treatment. ResultsCompared with the control group, the treatment group manifested shorter latency and higheramplitude of Target P3 wave of P300 after treatment ( P＜0.01), and there was alsosignificant difference in HDS and P300 within the treatment group before and aftertreatment (P ＜0.01).Conclusion Comprehensive percutaneous permeation of radix salviae miltiorrhizae iseffective in improving the cognitive ability of vascular dementia patients, and it's alsoan assistant therapy method and one of the preventive measures for vascular dementiapatients.. Key words: Percutaneous medicament permeation of radix salviaemiltiorrhizae;  Vascular dementia; P300