Long Mao

Novel microRNAs uncovered by deep sequencing of small RNA transcriptomes in bread wheat (Triticum aestivum L.) and Brachypodium distachyon (L.) Beauv

The small RNA transcriptomes of bread wheat and its emerging model Brachypodium distachyon were obtained by using deep sequencing technology. Small RNA compositions were analyzed in these two species. In addition to 70 conserved microRNAs (miRNAs) from 25 families, 23 novel wheat miRNAs were identified. For Brachypodium, 12 putative miRNAs were predicted from a limited number of expressed sequence tags, of which one was a potential novel miRNA. Also, 94 conserved miRNAs from 28 families were identified in this species. Expression validation was performed for several novel wheat miRNAs. RNA ligase-mediated 5′ rapid amplification of complementary DNA ends experiments demonstrated their capability to cleave predicted target genes including three disease-resistant gene analogs. Differential expression of miRNAs was observed between Brachypodium vegetative and reproductive tissues, suggesting their different roles at the two growth stages. Our work significantly increases the novel miRNA numbers in wheat and provides the first set of small RNAs in B. distachyon.

mRNA and Small RNA Transcriptomes Reveal Insights into Dynamic Homoeolog Regulation of Allopolyploid Heterosis in Nascent Hexaploid Wheat

Newly synthesized allohexaploid wheat lines, which exhibit hybrid vigor or heterosis, may recapitulate the initial genetic state of common wheat. This work shows that, in addition to nonadditive expression, parental expression-level dominance of subgenomes in nascent allohexaploid wheat may contribute distinctively to heterosis through dynamic small RNA–mediated homoeolog regulations. Nascent allohexaploid wheat may represent the initial genetic state of common wheat (Triticum aestivum), which arose as a hybrid between Triticum turgidum (AABB) and Aegilops tauschii (DD) and by chromosome doubling and outcompeted its parents in growth vigor and adaptability. To better understand the molecular basis for this success, we performed mRNA and small RNA transcriptome analyses in nascent allohexaploid wheat and its following generations, their progenitors, and the natural allohexaploid cultivar Chinese Spring, with the assistance of recently published A and D genome sequences. We found that nonadditively expressed protein-coding genes were rare but relevant to growth vigor. Moreover, a high proportion of protein-coding genes exhibited parental expression level dominance, with genes for which the total homoeolog expression level in the progeny was similar to that in T. turgidum potentially participating in development and those with similar expression to that in Ae. tauschii involved in adaptation. In addition, a high proportion of microRNAs showed nonadditive expression upon polyploidization, potentially leading to differential expression of important target genes. Furthermore, increased small interfering RNA density was observed for transposable element–associated D homoeologs in the allohexaploid progeny, which may account for biased repression of D homoeologs. Together, our data provide insights into small RNA–mediated dynamic homoeolog regulation mechanisms that may contribute to heterosis in nascent hexaploid wheat.

Evolutionary and functional study of the CDPK gene family in wheat (Triticum aestivum L.)

Calcium-dependent protein kinases (CDPKs) are crucial sensors of calcium concentration changes in plant cells under diverse endogenous and environmental stimuli. We identified 20 CDPK genes from bread wheat and performed a comprehensive study on their structural, functional and evolutionary characteristics. Full-length cDNA sequences of 14 CDPKs were obtained using various approaches. Wheat CDPKs were found to be similar to their counterparts in rice in genomic structure, GC content, subcellular localization, and subgroup classification. Divergence time estimation of wheat CDPK gene pairs and wheat–rice orthologs suggested that most duplicated genes already existed in the common ancestor of wheat and rice. The number of CDPKs in diploid wheat genome was estimated to be at least 26, a number close to that in rice, Arabidopsis, and poplar. However, polymorphism among EST sequences uncovered transcripts of all three homoeologous alleles for 13 out of 20 CDPKs. Thus, the hexaploid wheat should have 2–3 fold more CDPK genes expressing in their cells than the diploid species. Wheat CDPK genes were found to respond to various biotic and abiotic stimuli, including cold, hydrogen peroxide (H2O2), salt, drought, powdery mildew (Blumeria graminis tritici, Bgt), as well as phytohormones abscisic acid (ABA) and gibberellic acid (GA). Each CDPK gene often responded to multiple treatments, suggesting that wheat CDPKs are converging points for multiple signal transduction pathways. The current work represents the first comprehensive study of CDPK genes in bread wheat and provides a foundation for further functional study of this important gene family in Triticeae.

Roles of DICER-LIKE and ARGONAUTE Proteins in TAS-Derived Small Interfering RNA-Triggered DNA Methylation

Trans-acting small interfering RNAs (ta-siRNAs; TAS) emerge as a class of plant-specific small RNAs that are initiated from microRNA-mediated cleavage of TAS gene transcripts. It has been revealed that ta-siRNAs are generated by the sequential activities of SUPPRESSOR OF GENE SILENCING3 (SGS3), RNA-DEPENDENT RNA POLYMERASE6 (RDR6), and DICER-LIKE4 (DCL4), and loaded into ARGONAUTE1 (AGO1) proteins to posttranscriptionally regulate several target genes by messenger RNA cleavage in trans. Here, we showed a high cytosine DNA methylation status at ta-siRNA-generating loci in Arabidopsis (Arabidopsis thaliana), which is dependent on RDR6, SGS3, and DNA-DIRECTED RNA POLYMERASE V (PolV). More important, we found that DCL1 is the only DCL protein that is required for TAS3 loci DNA methylation, and all four DCLs exert combinatory functions in the methylation of TAS1 loci, suggesting a previously unknown role for DCL1 in directly processing TAS gene transcripts. Furthermore, we demonstrated that AGO4/6 complexes rather than AGO1 are responsible for TAS siRNA-guided DNA methylation. Based upon these findings, we propose a novel ta-siRNA pathway that acts at both the messenger RNA and chromatin level.

Regulation of FLOWERING LOCUS T by a MicroRNA in Brachypodium distachyon

This work identifies a Pooideae-specific microRNA that posttranscriptionally regulates the florigen gene FT under different daylength conditions in Brachypodium distachyon, revealing one mechanism in the complex but precise genetic regulatory pathways for flowering time control in plants. The highly conserved florigen gene FLOWERING LOCUS T (FT) functions at the core of the flowering pathways. Extensive studies have examined the transcriptional regulation of FT; however, other layers of FT regulation remain unclear. Here, we identified miR5200 a Pooideae-specific microRNA that is expressed in leaves and targets Brachypodium distachyon FT orthologs for mRNA cleavage. miR5200 was abundantly expressed in plants grown under short-day (SD) conditions but was dramatically repressed in plants transferred to long-day (LD) conditions. We also found that the epigenetic chromatin status, specifically the levels of histone methylation marks, at miR5200 precursor loci changed in response to daylength. Moreover, artificial interruption of miR5200 activity by target mimicry in B. distachyon altered flowering time in SD but not in LD conditions, suggesting that miR5200 functions in photoperiod-mediated flowering time regulation. Together, these findings illustrate a posttranscriptional regulation mechanism of FT and provide insights into understanding of the multiple concerted pathways for flowering time control in plants.

The SEPALLATA MADS‐box protein SLMBP21 forms protein complexes with JOINTLESS and MACROCALYX as a transcription activator for development of the tomato flower abscission zone

Organ abscission is a key step in a plants life cycle and is one of the most important agronomic traits for crops. In tomato, two MADS-box genes, JOINTLESS (J) and MACROCAYLYX (MC), have been shown to be implicated in development of the flower abscission zone (AZ), but the molecular mechanisms underlying this process are not well known. We report here that the SEPALLATA (SEP) MADS-box gene SLMBP21 acts as an additional factor for development of the AZ in tomato. We show that knockdown of SLMBP21 abolishes development of the flower AZ, while overexpression of SLMBP21 produces small cells at the proximal section of the pedicel and the peduncle. Bimolecular fluorescence complementation analysis confirms that SLMBP21 interacts with J and MC, and co-immunoprecipitation assays further demonstrates that these three proteins may form higher-order protein complexes. In situ hybridization shows that SLMBP21, J, and MC transcripts accumulate in distinct regions, but overlap at the AZ vasculature. In addition, transactivation assays in yeast show that, of the three interacting proteins, only SLMBP21 can activate reporter gene transcription. RNA-seq analysis furthermore reveals that loss of function of SLMBP21, J, or MC affects a common subset of meristem activity genes including LeWUS and LATERAL SUPPRESSOR that were specifically expressed in the AZ on the tomato flower pedicel. Since SLMBP21 belongs to the FBP9/23 subclade of the SEP gene family, which is absent in Arabidopsis, the SLMBP21-J-MC complex may represent a distinct mechanism for development of the AZ in plants.

Identification of novel MiRNAs and MiRNA expression profiling during grain development in indica rice.

BackgroundMicroRNAs (miRNAs) modulate gene expression in different tissues and at diverse developmental stages, including grain development in japonica rice. To identify novel miRNAs in indica rice and to study their expression patterns during the entire grain filling process, small RNAs from all stages of grain development were sequenced and their expression patterns were studied using customized miRNA chips.ResultsA total of 21 conserved and 91 non-conserved miRNA families were found in developing indica grains. We also discovered 11 potential novel miRNAs based on the presence of their miRNA*s. Expression patterns of these identified miRNAs were analyzed using customized miRNA chips. The results showed that during the filling phase about half of the detected miRNAs were up-regulated, whereas the remainder were down-regulated. Predicted targets of differentially expressed miRNAs may participate in carbohydrate metabolism, hormone signaling and pathways associated with seed maturity, suggesting potentially important roles in rice grain development.ConclusionsThis study is the first genome-wide investigation of miRNAs during the grain-filling phase of an indica variety of rice. The novel miRNAs identified might be involved in new miRNA regulatory pathways for grain development. The complexity of these miRNAs and their targets and interactions require further study to obtain a better understanding of the molecular mechanisms underlying grain development.

Transcriptome analysis of grain-filling caryopses reveals involvement of multiple regulatory pathways in chalky grain formation in rice

BackgroundGrain endosperm chalkiness of rice is a varietal characteristic that negatively affects not only the appearance and milling properties but also the cooking texture and palatability of cooked rice. However, grain chalkiness is a complex quantitative genetic trait and the molecular mechanisms underlying its formation are poorly understood.ResultsA near-isogenic line CSSL50-1 with high chalkiness was compared with its normal parental line Asominori for grain endosperm chalkiness. Physico-biochemical analyses of ripened grains showed that, compared with Asominori, CSSL50-1 contains higher levels of amylose and 8 DP (degree of polymerization) short-chain amylopectin, but lower medium length 12 DP amylopectin. Transcriptome analysis of 15 DAF (day after flowering) caryopses of the isogenic lines identified 623 differential expressed genes (P < 0.01), among which 324 genes are up-regulated and 299 down-regulated. These genes were classified into 18 major categories, with 65.3% of them belong to six major functional groups: signal transduction, cell rescue/defense, transcription, protein degradation, carbohydrate metabolism and redox homeostasis. Detailed pathway dissection demonstrated that genes involved in sucrose and starch synthesis are up-regulated, whereas those involved in non-starch polysaccharides are down regulated. Several genes involved in oxidoreductive homeostasis were found to have higher expression levels in CSSL50-1 as well, suggesting potential roles of ROS in grain chalkiness formation.ConclusionExtensive gene expression changes were detected during rice grain chalkiness formation. Over half of these differentially expressed genes are implicated in several important categories of genes, including signal transduction, transcription, carbohydrate metabolism and redox homeostasis, suggesting that chalkiness formation involves multiple metabolic and regulatory pathways.

Grass MicroRNA Gene Paleohistory Unveils New Insights into Gene Dosage Balance in Subgenome Partitioning after Whole-Genome Duplication

Reconstruction of the grass genome paleohistory revealed subgenome partitioning of microRNA (miRNA) genes during post-whole-genome duplication diploidization. The evolutionary scenario of miRNAs from the ancestral founder pool to the modern complements displayed dosage balance constrictions on the deletion/retention of miRNAs and associated target genes wherein transposable elements may play a major role in miRNA gene synteny disruption. The recent availability of plant genome sequences, combined with a robust evolutionary scenario of the modern monocot and eudicot karyotypes from their diploid ancestors, offers an opportunity to gain insights into microRNA (miRNA) gene paleohistory in plants. Characterization and comparison of miRNAs and associated protein-coding targets in plants allowed us to unravel (1) contrasted genome conservation patterns of miRNAs in monocots and eudicots after whole-genome duplication (WGD), (2) an ancestral miRNA founder pool in the monocot genomes dating back to 100 million years ago, (3) miRNA subgenome dominance during the post-WGD diploidization process with selective miRNA deletion complemented with possible transposable element–mediated return flows, and (4) the miRNA/target interaction-directed differential loss/retention of miRNAs following the gene dosage balance rule. Together, our data suggest that overretained miRNAs in grass genomes may be implicated in connected gene regulations for stress responses, which is essential for plant adaptation and useful for crop variety innovation.

Genome-Wide Analysis of the MADS-Box Gene Family in Brachypodium distachyon

MADS-box genes are important transcription factors for plant development, especially floral organogenesis. Brachypodium distachyon is a model for biofuel plants and temperate grasses such as wheat and barley, but a comprehensive analysis of MADS-box family proteins in Brachypodium is still missing. We report here a genome-wide analysis of the MADS-box gene family in Brachypodium distachyon. We identified 57 MADS-box genes and classified them into 32 MIKCc-type, 7 MIKC*-type, 9 Mα, 7 Mβ and 2 Mγ MADS-box genes according to their phylogenetic relationships to the Arabidopsis and rice MADS-box genes. Detailed gene structure and motif distribution were then studied. Investigation of their chromosomal localizations revealed that Brachypodium MADS-box genes distributed evenly across five chromosomes. In addition, five pairs of type II MADS-box genes were found on synteny blocks derived from whole genome duplication blocks. We then performed a systematic expression analysis of Brachypodium MADS-box genes in various tissues, particular floral organs. Further detection under salt, drought, and low-temperature conditions showed that some MADS-box genes may also be involved in abiotic stress responses, including type I genes. Comparative studies of MADS-box genes among Brachypodium, rice and Arabidopsis showed that Brachypodium had fewer gene duplication events. Taken together, this work provides useful data for further functional studies of MADS-box genes in Brachypodium distachyon.