Elucidating the callus-to-shoot-forming mechanism in Capsicum annuum 'Dempsey' through comparative transcriptome analyses.

BACKGROUND: The formation of shoots plays a pivotal role in plant organogenesis and productivity. Despite its significance, the underlying molecular mechanism of de novo regeneration has not been extensively elucidated in Capsicum annuum 'Dempsey', a bell pepper cultivar. To address this, we performed a comparative transcriptome analysis focusing on the differential expression in C. annuum 'Dempsey' shoot, callus, and leaf tissue. We further investigated phytohormone-related biological processes and their interacting genes in the C. annuum 'Dempsey' transcriptome based on comparative transcriptomic analysis across five species. RESULTS: We provided a comprehensive view of the gene networks regulating shoot formation on the callus, revealing a strong involvement of hypoxia responses and oxidative stress. Our comparative transcriptome analysis revealed a significant conservation in the increase of gene expression patterns related to auxin and defense mechanisms in both callus and shoot tissues. Consequently, hypoxia response and defense mechanism emerged as critical regulators in callus and shoot formation in C. annuum 'Dempsey'. Current transcriptome data also indicated a substantial decline in gene expression linked to photosynthesis within regenerative tissues, implying a deactivation of the regulatory system governing photosynthesis in C. annuum 'Dempsey'. CONCLUSION: Coupled with defense mechanisms, we thus considered spatial redistribution of auxin to play a critical role in the shoot morphogenesis via primordia outgrowth. Our findings shed light on shoot formation mechanisms in C. annuum 'Dempsey' explants, important information for regeneration programs, and have broader implications for precise molecular breeding in recalcitrant crops.

The CARBON CATABOLITE REPRESSION 4A-mediated RNA deadenylation pathway acts on the transposon RNAs that are not regulated by small RNAs.

Transposable elements (TEs) are mobile genetic elements that can impair the host genome stability and integrity. It has been well documented that activated transposons in plants are suppressed by small interfering (si) RNAs. However, transposon repression by the cytoplasmic RNA surveillance system is unknown. Here, we show that mRNA deadenylation is critical for controlling transposons in Arabidopsis. Trimming of poly(A) tail is a rate-limiting step that precedes the RNA decay and is primarily mediated by the CARBON CATABOLITE REPRESSION 4 (CCR4)-NEGATIVE ON TATA-LESS (NOT) complex. We found that the loss of CCR4a leads to strong derepression and mobilization of TEs in Arabidopsis. Intriguingly, CCR4a regulates a largely distinct set of TEs from those controlled by RNA-dependent RNA Polymerase 6 (RDR6), a key enzyme that produces cytoplasmic siRNAs. This indicates that the cytoplasmic RNA quality control mechanism targets the TEs that are poorly recognized by the previously well-characterized RDR6-mediated pathway, and thereby augments the host genome stability. Our study suggests a hitherto unknown mechanism for transposon repression mediated by RNA deadenylation and unveils a complex nature of the host's strategy to maintain the genome integrity.

Comprehensive Analysis of Rice Seedling Transcriptome during Dehydration and Rehydration.

Drought is a harmful abiotic stress that threatens the growth, development, and yield of rice plants. To cope with drought stress, plants have evolved their diverse and sophisticated stress-tolerance mechanisms by regulating gene expression. Previous genome-wide studies have revealed many rice drought stress-responsive genes that are involved in various forms of metabolism, hormone biosynthesis, and signaling pathways, and transcriptional regulation. However, little is known about the regulation of drought-responsive genes during rehydration after dehydration. In this study, we examined the dynamic gene expression patterns in rice seedling shoots during dehydration and rehydration using RNA-seq analysis. To investigate the transcriptome-wide rice gene expression patterns during dehydration and rehydration, RNA-seq libraries were sequenced and analyzed to identify differentially expressed genes (DEGs). DEGs were classified into five clusters based on their gene expression patterns. The clusters included drought-responsive DEGs that were either rapidly or slowly recovered to control levels by rehydration treatment. Representative DEGs were selected and validated using qRT-PCR. In addition, we performed a detailed analysis of DEGs involved in nitrogen metabolism, phytohormone signaling, and transcriptional regulation. In this study, we revealed that drought-responsive genes were dynamically regulated during rehydration. Moreover, our data showed the potential role of nitrogen metabolism and jasmonic acid signaling during the drought stress response. The transcriptome data in this study could be a useful resource for understanding drought stress responses in rice and provide a valuable gene list for developing drought-resistant crop plants.

Reciprocal inhibition of expression between RAV1 and BES1 modulates plant growth and development in Arabidopsis.

RAV1 (Related to ABI3/VP1) is a plant-specific B3 and AP2 domain-containing transcription factor that acts as a negative regulator of growth in many plant species. The expression of RAV1 is downregulated by brassinosteroids (BRs); large-scale transcriptome analyses have shown that the expression of RAV1 was previously targeted by BRI1-EMS-SUPPRESOR1 (BES1) and BRASSINAZOLE-RESISTANT1 (BZR1), which are critical transcription factors for the BR-signaling process. Using RAV1-overexpressing transgenic plants, we showed that RAV1 overexpression reduced the BR signaling capacity, resulting in the downregulation of BR biosynthetic genes and BES1 expression. Furthermore, we demonstrated that BES1, not BZR1, is directly bound to the RAV1 promoter and repressed RAV1 expression, and vice versa; RAV1 is also bound to the BES1 promoter and repressed BES1 expression. This mutual inhibition was specific to RAV1 and BES1 because RAV1 exhibited binding activity to the BZR1 promoter but did not repress BZR1 expression. We observed that constitutively activated BR signaling phenotypes in bes1-D were attenuated by the repression of endogenous BES1 expression in transgenic bes1-D plants overexpressing RAV1. RNA-sequencing analysis of RAV1-overexpressing transgenic plants and bes1-D mutant plants revealed differentially expressed genes by RAV1 and BES1 and genes that were oppositely co-regulated by RAV1 and BES1. RAV1 and BES1 regulated different transcriptomes but co-regulated a specific set of genes responsible for the balance between growth and defense. These results suggested that the mutual inhibitory transcriptional activities of RAV1 and BES1 provide fine regulatory mechanisms for plant growth and development.

PhenGenVar: A User-Friendly Genetic Variant Detection and Visualization Tool for Precision Medicine.

Precision medicine has been revolutionized by the advent of high-throughput next-generation sequencing (NGS) technology and development of various bioinformatic analysis tools for large-scale NGS big data. At the population level, biomedical studies have identified human diseases and phenotype-associated genetic variations using NGS technology, such as whole-genome sequencing, exome sequencing, and gene panel sequencing. Furthermore, patients' genetic variations related to a specific phenotype can also be identified by analyzing their genomic information. These breakthroughs paved the way for the clinical diagnosis and precise treatment of patients' diseases. Although many bioinformatics tools have been developed to analyze the genetic variations from the individual patient's NGS data, it is still challenging to develop user-friendly programs for clinical physicians who do not have bioinformatics programing skills to diagnose a patient's disease using the genomic data. In response to this demand, we developed a Phenotype to Genotype Variation program (PhenGenVar), which is a user-friendly interface for monitoring the variations in a gene of interest for molecular diagnosis. This allows for flexible filtering and browsing of variants of the disease and phenotype-associated genes. To test this program, we analyzed the whole-genome sequencing data of an anonymous person from the 1000 human genome project data. As a result, we were able to identify several genomic variations, including single-nucleotide polymorphism, insertions, and deletions in specific gene regions. Therefore, PhenGenVar can be used to diagnose a patient's disease. PhenGenVar is freely accessible and is available at our website.

Narrow lpa1 Metaxylems Enhance Drought Tolerance and Optimize Water Use for Grain Filling in Dwarf Rice.

Rice cultivation needs extensive amounts of water. Moreover, increased frequency of droughts and water scarcity has become a global concern for rice cultivation. Hence, optimization of water use is crucial for sustainable agriculture. Here, we characterized Loose Plant Architecture 1 (LPA1) in vasculature development, water transport, drought resistance, and grain yield. We performed genetic combination of lpa1 with semi-dwarf mutant to offer the optimum rice architecture for more efficient water use. LPA1 expressed in pre-vascular cells of leaf primordia regulates genes associated with carbohydrate metabolism and cell enlargement. Thus, it plays a role in metaxylem enlargement of the aerial organs. Narrow metaxylem of lpa1 exhibit leaves curling on sunny day and convey drought tolerance but reduce grain yield in mature plants. However, the genetic combination of lpa1 with semi-dwarf mutant (dep1-ko or d2) offer optimal water supply and drought resistance without impacting grain-filling rates. Our results show that water use, and transports can be genetically controlled by optimizing metaxylem vessel size and plant height, which may be utilized for enhancing drought tolerance and offers the potential solution to face the more frequent harsh climate condition in the future.

Small regulatory RNAs in rice epigenetic regulation.

Plant small RNAs (sRNAs) are short non-coding RNAs that are implicated in various regulatory processes involving post-transcriptional gene silencing and epigenetic gene regulation. In epigenetic regulation, sRNAs are primarily involved in RNA-directed DNA methylation (RdDM) pathways. sRNAs in the RdDM pathways play a role not only in the suppression of transposable element (TE) activity but also in gene expression regulation. Although the major components of the RdDM pathways have been well studied in Arabidopsis, recent studies have revealed that the RdDM pathways in rice have important biological functions in stress response and developmental processes. In this review, we summarize and discuss recent literature on sRNA-mediated epigenetic regulation in rice. First, we describe the RdDM mechanisms in plants. We then introduce recent discoveries on the biological roles of rice genes involved in the RdDM pathway and TE-derived sRNAs working at specific genomic loci for epigenetic control in rice.

Global Analysis of the Human RNA Degradome Reveals Widespread Decapped and Endonucleolytic Cleaved Transcripts.

RNA decay is an important regulatory mechanism for gene expression at the posttranscriptional level. Although the main pathways and major enzymes that facilitate this process are well defined, global analysis of RNA turnover remains under-investigated. Recent advances in the application of next-generation sequencing technology enable its use in order to examine various RNA decay patterns at the genome-wide scale. In this study, we investigated human RNA decay patterns using parallel analysis of RNA end-sequencing (PARE-seq) data from XRN1-knockdown HeLa cell lines, followed by a comparison of steady state and degraded mRNA levels from RNA-seq and PARE-seq data, respectively. The results revealed 1103 and 1347 transcripts classified as stable and unstable candidates, respectively. Of the unstable candidates, we found that a subset of the replication-dependent histone transcripts was polyadenylated and rapidly degraded. Additionally, we identified 380 endonucleolytically cleaved candidates by analyzing the most abundant PARE sequence on a transcript. Of these, 41.4% of genes were classified as unstable genes, which implied that their endonucleolytic cleavage might affect their mRNA stability. Furthermore, we identified 1877 decapped candidates, including HSP90B1 and SWI5, having the most abundant PARE sequences at the 5'-end positions of the transcripts. These results provide a useful resource for further analysis of RNA decay patterns in human cells.

Genome-wide analysis of brassinosteroid responsive small RNAs in Arabidopsis thaliana.

BACKGROUND: Brassinosteroids (BRs) are a class of phytohormones with important roles in regulating physiological and developmental processes. Small RNAs, including small interfering RNAs and microRNAs (miRNAs), are non-protein coding RNAs that regulate gene expression at the transcriptional and post-transcriptional levels. However, the roles of small RNAs in BR response have not been studied well. OBJECTIVE: In this study, we aimed to identify BR-responsive small RNA clusters and miRNAs in Arabidopsis. In addition, the effect of BR-responsive small RNAs on their transcripts and target genes were examined. METHODS: Small RNA libraries were constructed from control and epibrassinolide-treated seedlings expressing wild-type BRI1-Flag protein under its native promoter in the bri1-5 mutant. After sequencing the small RNA libraries, differentially expressed small RNA clusters were identified by examining the expression levels of small RNAs in 100-nt bins of the Arabidopsis genome. To identify the BR-responsive miRNAs, the expression levels of all the annotated mature miRNAs, registered in miRBase, were analyzed. Previously published RNA-seq data were utilized to monitor the BR-responsive expression patterns of differentially expressed small RNA clusters and miRNA target genes. RESULTS: In results, 38 BR-responsive small RNA clusters, including 30 down-regulated and eight up-regulated clusters, were identified. These differentially expressed small RNA clusters were from miRNA loci, transposons, protein-coding genes, pseudogenes and others. Of these, a transgene, BRI1, accumulates small RNAs, which are not found in the wild type. Small RNAs in this transgene are up-regulated by BRs while BRI1 mRNA is down-regulated by BRs. By analyzing the expression patterns of mature miRNAs, we have identified BR-repressed miR398a-5p and BR-induced miR156g. Although miR398a-5p is down-regulated by BRs, its predicted targets were not responsive to BRs. However, SPL3, a target of BR-inducible miR156g, is down-regulated by BRs. CONCLUSION: BR-responsive small RNAs and miRNAs identified in this study will provide an insight into the role of small RNAs in BR responses in plants. Especially, we suggest that miR156g/SPL3 module might play a role in BR-mediated growth and development in Arabidopsis.

Analysis of Brachypodium miRNA targets: evidence for diverse control during stress and conservation in bioenergy crops.

BACKGROUND: Since the proposal of Brachypodium distachyon as a model for the grasses, over 500 Bdi-miRNAs have been annotated in miRBase making Brachypodium second in number only to rice. Other monocots, such as switchgrass, are completely absent from the miRBase database. While a significant number of miRNAs have been identified which are highly conserved across plants, little research has been done with respect to the conservation of miRNA targets. Plant responses to abiotic stresses are regulated by diverse pathways many of which involve miRNAs; however, it can be difficult to identify miRNA guided gene regulation when the miRNA is not the primary regulator of the target mRNA. RESULTS: To investigate miRNA target conservation and stress response involvement, a set of PARE (Parallel Analysis of RNA Ends) libraries totaling over two billion reads was constructed and sequenced from Brachypodium, switchgrass, and sorghum representing the first report of RNA degradome data from the latter two species. Analysis of this data provided not only PARE evidence for miRNA guided cleavage of over 7000 predicted target mRNAs in Brachypodium, but also evidence for miRNA guided cleavage of over 1000 homologous transcripts in sorghum and switchgrass. A pipeline was constructed to compare RNA-seq and PARE data made from Brachypodium plants exposed to various abiotic stress conditions. This resulted in the identification of 44 miRNA targets which exhibit stress regulated cleavage. Time course experiments were performed to reveal the relationship between miR393ab, miR169a, miR394ab, and their respective targets throughout the first 36 h of the cold stress response in Brachypodium. CONCLUSIONS: Knowledge gained from this study provides considerable insight into the RNA degradomes and the breadth of miRNA target conservation among these three species. Additionally, associations of a number of miRNAs and target mRNAs with the stress responses have been revealed which could aid in the development of stress tolerant transgenic crops.

Transcriptome profiling of drought responsive noncoding RNAs and their target genes in rice.

BACKGROUND: Plant transcriptome profiling has provided a tool for understanding the mechanisms by which plants respond to stress conditions. Analysis of genome-wide transcriptome will provides a useful dataset of drought responsive noncoding RNAs and their candidate target genes that may be involved in drought stress responses. RESULTS: Here RNA-seq analyses of leaves from drought stressed rice plants was performed, producing differential expression profiles of noncoding RNAs. We found that the transcript levels of 66 miRNAs changed significantly in response to drought conditions and that they were negatively correlated with putative target genes during the treatments. The negative correlations were further validated by qRT-PCR using total RNAs from both drought-treated leaves and various tissues at different developmental stages. The drought responsive miRNA/target pairs were confirmed by the presence of decay intermediates generated by miRNA-guided cleavages in Parallel Analysis of RNA Ends (PARE) libraries. We observed that the precursor miR171f produced two different mature miRNAs, miR171f-5p and miR171f-3p with 4 candidate target genes, the former of which was responsive to drought conditions. We found that the expression levels of the miR171f precursor negatively correlated with those of one candidate target gene, but not with the others, suggesting that miR171f-5p was drought-responsive, with Os03g0828701-00 being a likely target. Pre-miRNA expression profiling indicated that miR171f is involved in the progression of rice root development and growth, as well as the response to drought stress. Ninety-eight lncRNAs were also identified, together with their corresponding antisense transcripts, some of which were responsive to drought conditions. CONCLUSIONS: We identified rice noncoding RNAs (66 miRNAs and 98 lncRNAs), whose expression was highly regulated by drought stress conditions, and whose transcript levels negatively correlated with putative target genes.

Identification of SMG6 cleavage sites and a preferred RNA cleavage motif by global analysis of endogenous NMD targets in human cells.

In metazoans, cleavage by the endoribonuclease SMG6 is often the first degradative event in non-sense-mediated mRNA decay (NMD). However, the exact sites of SMG6 cleavage have yet to be determined for any endogenous targets, and most evidence as to the identity of SMG6 substrates is indirect. Here, we use Parallel Analysis of RNA Ends to specifically identify the 5' termini of decay intermediates whose production is dependent on SMG6 and the universal NMD factor UPF1. In this manner, the SMG6 cleavage sites in hundreds of endogenous NMD targets in human cells have been mapped at high resolution. In addition, a preferred sequence motif spanning most SMG6 cleavage sites has been discovered and validated by mutational analysis. For many SMG6 substrates, depletion of SMG6 resulted in the accumulation of decapped transcripts, an effect indicative of competition between SMG6-dependent and SMG6-independent NMD pathways. These findings provide key insights into the mechanisms by which mRNAs targeted by NMD are degraded.

Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon.

BACKGROUND: The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using parallel analysis of RNA ends (PARE) data is lacking in B. distachyon. RESULTS: B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset for analyzing small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. CONCLUSIONS: B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants.

Comprehensive investigation of microRNAs enhanced by analysis of sequence variants, expression patterns, ARGONAUTE loading, and target cleavage.

MicroRNAs (miRNAs) are a class of small RNAs that typically function by guiding the cleavage of target messenger RNAs. They have been shown to play major roles in a variety of plant processes, including development, and responses to pathogens and environmental stresses. To identify new miRNAs and regulation in Arabidopsis (Arabidopsis thaliana), 27 small RNA libraries were constructed and sequenced from various tissues, stresses, and small RNA biogenesis mutants, resulting in 95 million genome-matched sequences. The use of rdr2 to enrich the miRNA population greatly enhanced this analysis and led to the discovery of new miRNAs arising from both known and new precursors, increasing the total number of Arabidopsis miRNAs by about 10%. Parallel Analysis of RNA Ends data provide evidence that the majority guide target cleavage. Many libraries represented novel stress/tissue conditions, such as submergence-stressed flowers, which enabled the identification of new stress regulation of both miRNAs and their targets, all of which were validated in wild-type plants. By combining small RNA expression analysis with ARGONAUTE immunoprecipitation data and global target cleavage data from Parallel Analysis of RNA Ends, a much more complete picture of Arabidopsis miRNAs was obtained. In particular, the discovery of ARGONAUTE loading and target cleavage biases gave important insights into tissue-specific expression patterns, pathogen responses, and the role of sequence variation among closely related miRNA family members that would not be evident without this combinatorial approach.

Methods for validation of miRNA sequence variants and the cleavage of their targets.

MicroRNA (miRNA) variants that share the sequences with other closely related miRNAs have been identified by deep sequencing and have been implicated in the diverse regulation of their target genes. The miRNA variants that originate from the same miRNA precursor are among the most common and have been termed "isomiRs." IsomiRs can be generated by several mechanisms such as differential processing by DICER, RNA degradation, or RNA editing. Members of the same miRNA family that have distinct sequences also contribute to the diversity of miRNA variants. Although many miRNA variants are lowly expressed and may function redundantly with their reference miRNAs, some miRNA variants are highly and/or differentially expressed. In addition, slight differences in sequence among miRNA variants can affect their specificity in target selection. Here, we describe two methods for detecting or validating miRNA variants and the target events they mediate.

Roles of DCL4 and DCL3b in rice phased small RNA biogenesis.

Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5' rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased small RNA biogenesis pathway in rice.

MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs.

Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those that are deleterious or pathogenic. Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide (nt) "phased" intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for phased siRNAs (phasiRNAs) found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt microRNA (miRNA) families that target conserved domains in these NB-LRRs and trigger the production of trans-acting siRNAs. High levels of small RNAs were matched to >60% of all ∼540 encoded Medicago NB-LRRs; in the potato, a model for mycorrhizal interactions, phasiRNAs were also produced from NB-LRRs. DCL2 and SGS3 transcripts were also cleaved by these 22-nt miRNAs, generating phasiRNAs, suggesting synchronization between silencing and pathogen defense pathways. In addition, a new example of apparent "two-hit" phasiRNA processing was identified. Our data reveal complex tasiRNA-based regulation of NB-LRRs that potentially evolved to facilitate symbiotic interactions and demonstrate miRNAs as master regulators of a large gene family via the targeting of highly conserved, protein-coding motifs, a new paradigm for miRNA function.

Massive analysis of rice small RNAs: mechanistic implications of regulated microRNAs and variants for differential target RNA cleavage.

Small RNAs have a variety of important roles in plant development, stress responses, and other processes. They exert their influence by guiding mRNA cleavage, translational repression, and chromatin modification. To identify previously unknown rice (Oryza sativa) microRNAs (miRNAs) and those regulated by environmental stress, 62 small RNA libraries were constructed from rice plants and used for deep sequencing with Illumina technology. The libraries represent several tissues from control plants and plants subjected to different environmental stress treatments. More than 94 million genome-matched reads were obtained, resulting in more than 16 million distinct small RNA sequences. This allowed an evaluation of ~400 annotated miRNAs with current criteria and the finding that among these, ~150 had small interfering RNA-like characteristics. Seventy-six new miRNAs were found, and miRNAs regulated in response to water stress, nutrient stress, or temperature stress were identified. Among the new examples of miRNA regulation were members of the same miRNA family that were differentially regulated in different organs and had distinct sequences Some of these distinct family members result in differential target cleavage and provide new insight about how an agriculturally important rice phenotype could be regulated in the panicle. This high-resolution analysis of rice miRNAs should be relevant to plant miRNAs in general, particularly in the Poaceae.

OsCOL4 is a constitutive flowering repressor upstream of Ehd1 and downstream of OsphyB.

Plants recognize environmental factors to determine flowering time. CONSTANS (CO) plays a central role in the photoperiod flowering pathway of Arabidopsis, and CO protein stability is modulated by photoreceptors. In rice, Hd1, an ortholog of CO, acts as a flowering promoter, and phytochromes repress Hd1 expression. Here, we investigated the functioning of OsCOL4, a member of the CONSTANS-like (COL) family in rice. OsCOL4 null mutants flowered early under short or long days. In contrast, OsCOL4 activation-tagging mutants (OsCOL4-D) flowered late in either environment. Transcripts of Ehd1, Hd3a, and RFT1 were increased in the oscol4 mutants, but reduced in the OsCOL4-D mutants. This finding indicates that OsCOL4 is a constitutive repressor functioning upstream of Ehd1. By comparison, levels of Hd1, OsID1, OsMADS50, OsMADS51, and OsMADS56 transcripts were not significantly changed in oscol4 or OsCOL4-D, suggesting that OsCOL4 functions independently from previously reported flowering pathways. In osphyB mutants, OsCOL4 expression was decreased and osphyB oscol4 double mutants flowered at the same time as the osphyB single mutants, indicating OsCOL4 functions downstream of OsphyB. We also present evidence for two independent pathways through which OsPhyB controls flowering time. These pathways are: (i) night break-sensitive, which does not need OsCOL4; and (ii) night break-insensitive, in which OsCOL4 functions between OsphyB and Ehd1.

Prediction of novel miRNAs and associated target genes in Glycine max.

BACKGROUND: Small non-coding RNAs (21 to 24 nucleotides) regulate a number of developmental processes in plants and animals by silencing genes using multiple mechanisms. Among these, the most conserved classes are microRNAs (miRNAs) and small interfering RNAs (siRNAs), both of which are produced by RNase III-like enzymes called Dicers. Many plant miRNAs play critical roles in nutrient homeostasis, developmental processes, abiotic stress and pathogen responses. Currently, only 70 miRNA have been identified in soybean. METHODS: We utilized Illumina's SBS sequencing technology to generate high-quality small RNA (sRNA) data from four soybean (Glycine max) tissues, including root, seed, flower, and nodules, to expand the collection of currently known soybean miRNAs. We developed a bioinformatics pipeline using in-house scripts and publicly available structure prediction tools to differentiate the authentic mature miRNA sequences from other sRNAs and short RNA fragments represented in the public sequencing data. RESULTS: The combined sequencing and bioinformatics analyses identified 129 miRNAs based on hairpin secondary structure features in the predicted precursors. Out of these, 42 miRNAs matched known miRNAs in soybean or other species, while 87 novel miRNAs were identified. We also predicted the putative target genes of all identified miRNAs with computational methods and verified the predicted cleavage sites in vivo for a subset of these targets using the 5' RACE method. Finally, we also studied the relationship between the abundance of miRNA and that of the respective target genes by comparison to Solexa cDNA sequencing data. CONCLUSION: Our study significantly increased the number of miRNAs known to be expressed in soybean. The bioinformatics analysis provided insight on regulation patterns between the miRNAs and their predicted target genes expression. We also deposited the data in a soybean genome browser based on the UCSC Genome Browser architecture. Using the browser, we annotated the soybean data with miRNA sequences from four tissues and cDNA sequencing data. Overlaying these two datasets in the browser allows researchers to analyze the miRNA expression levels relative to that of the associated target genes. The browser can be accessed at http://digbio.missouri.edu/soybean_mirna/.