OsNAC103, a NAC Transcription Factor, Positively Regulates Leaf Senescence and Plant Architecture in Rice.

Leaf senescence, the last stage of leaf development, is essential for crop yield by promoting nutrition relocation from senescence leaves to new leaves and seeds. NAC (NAM/ATAF1/ATAF2/CUC2) proteins, one of the plant-specific transcription factors, widely distribute in plants and play important roles in plant growth and development. Here, we identified a new NAC member OsNAC103 and found that it plays critical roles in leaf senescence and plant architecture in rice. OsNAC103 mRNA levels were dramatically induced by leaf senescence as well as different phytohormones such as ABA, MeJA and ACC and abiotic stresses including dark, drought and high salinity. OsNAC103 acts as a transcription factor with nuclear localization signals at the N terminal and a transcriptional activation signal at the C terminal. Overexpression of OsNAC103 promoted leaf senescence while osnac103 mutants delayed leaf senescence under natural condition and dark-induced condition, meanwhile, senescence-associated genes (SAGs) were up-regulated in OsNAC103 overexpression (OsNAC103-OE) lines, indicating that OsNAC103 positively regulates leaf senescence in rice. Moreover, OsNAC103-OE lines exhibited loose plant architecture with larger tiller angles while tiller angles of osnac103 mutants decreased during the vegetative and reproductive growth stages due to the response of shoot gravitropism, suggesting that OsNAC103 can regulate the plant architecture in rice. Taken together, our results reveal that OsNAC103 plays crucial roles in the regulation of leaf senescence and plant architecture in rice.

OsFTL12, a member of FT-like family, modulates the heading date and plant architecture by florigen repression complex in rice.

FLOWERING LOCUS T (FT), a florigen in Arabidopsis, plays critical roles in floral transition. Among 13 FT-like members in rice, OsFTL2 (Hd3a) and OsFTL3 (RFT1), two rice homologues of FT, have been well characterized to act as florigens to induce flowering under short-day (SD) and long-day (LD) conditions, respectively, but the functions of other rice FT-like members remain largely unclear. Here, we show that OsFTL12 plays an antagonistic function against Hd3a and RFT1 to modulate the heading date and plant architecture in rice. Unlike Hd3a and RFT1, OsFTL12 is not regulated by daylength and highly expressed in both SD and LD conditions, and delays the heading date under either SD or LD conditions. We further demonstrate that OsFTL12 interacts with GF14b and OsFD1, two key components of the florigen activation complex (FAC), to form the florigen repression complex (FRC) by competing with Hd3a for binding GF14b. Notably, OsFTL12-FRC can bind to the promoters of the floral identity genes OsMADS14 and OsMADS15 and suppress their expression. The osmads14 osmads15 double mutants could not develop panicles and showed erect leaves. Taken together, our results reveal that different FT-like members can fine-tune heading date and plant architecture by regulating the balance of FAC and FRC in rice.

SDG712, a Putative H3K9-Specific Methyltransferase Encoding Gene, Delays Flowering through Repressing the Expression of Florigen Genes in Rice.

SET domain group (SDG) proteins have been identified to be involved in histone modification and participate in diverse biological processes. Rice contains 41 SDG genes, however, most of which have not been functionally characterized. Here, we report the identification and functional investigation of rice SDG712 gene. Phylogenic analysis revealed that SDG712 belongs to the H3K9-specific SDG subclade. SDG712 is highly expressed in leaves during reproductive growth stage with obvious circadian rhythmic pattern. Mutation of SDG712 promotes rice flowering, while overexpression of SDG712 delays rice flowering. Gene expression analysis suggested that SDG712 acts downstream of Hd1, while acts upstream of Ehd1, Hd3a and RFT1. Subcellular localization assay demonstrated that SDG712 is localized in the nucleus. Chromatin immunoprecipitation (ChIP) assay showed that the H3K9me2 levels at Hd3a and RFT1 loci were increased in SDG712 overexpression transgenic plants, indicating that SDG712 may mediate the H3K9 di-methylation on these loci to repress rice flowering. Taken together, our findings demonstrated that SDG712 is a negative flowering regulatory gene in rice, and it delays flowering through repressing key flowering regulator gene Ehd1 and the florigen genes Hd3a and RFT1.

TarDB: an online database for plant miRNA targets and miRNA-triggered phased siRNAs.

BACKGROUND: In plants, microRNAs (miRNAs) are pivotal regulators of plant development and stress responses. Different computational tools and web servers have been developed for plant miRNA target prediction; however, in silico prediction normally contains false positive results. In addition, many plant miRNA target prediction servers lack information for miRNA-triggered phased small interfering RNAs (phasiRNAs). Creating a comprehensive and relatively high-confidence plant miRNA target database is much needed. RESULTS: Here, we report TarDB, an online database that collects three categories of relatively high-confidence plant miRNA targets: (i) cross-species conserved miRNA targets; (ii) degradome/PARE (Parallel Analysis of RNA Ends) sequencing supported miRNA targets; (iii) miRNA-triggered phasiRNA loci. TarDB provides a user-friendly interface that enables users to easily search, browse and retrieve miRNA targets and miRNA initiated phasiRNAs in a broad variety of plants. TarDB has a comprehensive collection of reliable plant miRNA targets containing previously unreported miRNA targets and miRNA-triggered phasiRNAs even in the well-studied model species. Most of these novel miRNA targets are relevant to lineage-specific or species-specific miRNAs. TarDB data is freely available at http://www.biosequencing.cn/TarDB . CONCLUSIONS: In summary, TarDB serves as a useful web resource for exploring relatively high-confidence miRNA targets and miRNA-triggered phasiRNAs in plants.

Extensive profiling of the expressions of tRNAs and tRNA-derived fragments (tRFs) reveals the complexities of tRNA and tRF populations in plants.

Evidence is emerging that tRNA-derived fragments (tRFs) are regulatory molecules. Studies of tRFs in plants have been based on conventional small RNA sequencing, and focused on profiling of tRF-5 and tRF-3 species. A more comprehensive and quantitative analysis of the entire tRF population is highly necessary. Here, we employ tRNA-seq and YAMAT-seq, and develop a bioinformatics tool to comprehensively profile the expressions of tRNAs and tRFs in plants. We show that in Arabidopsis, approximately half of tRNA genes are extremely weakly expressed, accounting for only 1% of total tRNA abundance, while ~12% of tRNA genes contribute to ~80% of tRNA abundance. Our tRNA sequencings in various plants reveal that tRNA expression profiles exhibit a cross-species conserved pattern. By characterizing the composition of a highly heterogeneous tRF population, we show that tRNA halves and previously unnoticed 10-16-nt tiny tRFs represent substantial portions. The highly accumulated 13-nt and 16-nt tiny tRFs in Arabidopsis indicate that tiny tRFs are not random tRNA degradation products. Finally, we provide a user-friendly database for displaying the dynamic spatiotemporal expressions of tRNAs and tRFs in the model plants Arabidopsis and rice.

PRMdb: A Repository of Predicted RNA Modifications in Plants.

Evidence is mounting that RNA modifications play essential roles in posttranscriptional regulation of gene expression. So far, over 150 RNA modifications catalyzed by distinct enzymes have been documented. In plants, genome-wide identification of RNA modifications is largely limited to the model species Arabidopsis thaliana, while lacking in diverse non-model plants. Here, we present PRMdb, a plant RNA modification database, based on the analysis of thousands of RNA-seq, degradome-seq and small RNA-seq data from a wide range of plant species using the well-documented tool HAMR (high-throughput analysis of modified ribonucleotide). PRMdb provides a user-friendly interface that enables easy browsing and searching of the tRNA and mRNA modification data. We show that PRMdb collects high-confidence RNA modifications including novel RNA modification sites that can be validated by genomic PCR and reverse transcription PCR. In summary, PRMdb provides a valuable web resource for deciphering the epitranscriptomes in diverse plant species and will facilitate functional studies of RNA modifications in plants. RPMdb is available via http://www.biosequencing.cn/PRMdb/.

Overexpression of OsARD1 Improves Submergence, Drought, and Salt Tolerances of Seedling Through the Enhancement of Ethylene Synthesis in Rice.

Acireductone dioxygenase (ARD) is a metal-binding metalloenzyme and involved in the methionine salvage pathway. In rice, OsARD1 binds Fe2+ and catalyzes the formation of 2-keto-4-methylthiobutyrate (KMTB) to produce methionine, which is an initial substrate in ethylene synthesis pathway. Here, we report that overexpression of OsARD1 elevates the endogenous ethylene release rate, enhances the tolerance to submergence stress, and reduces the sensitivity to drought, salt, and osmotic stresses in rice. OsARD1 is strongly induced by submergence, drought, salinity, PEG6000, and mechanical damage stresses and exhibits high expression level in senescent leaves. Transgenic plants overexpressing OsARD1 (OsARD1-OE) display fast elongation growth to escape submergence stress. The ethylene content is significantly maximized in OsARD1-OE plants compared with the wide type. OsARD1-OE plants display increased shoot elongation and inhibition of root elongation under the submergence stress and grow in dark due to increase of ethylene. The elongation of coleoptile under anaerobic germination is also significantly promoted in OsARD1-OE lines due to the increase of ethylene content. The sensitivity to drought and salt stresses is reduced in OsARD1-OE transgenic lines. Water holding capacity is enhanced, and the stomata and trichomes on leaves increase in OsARD1-OE lines. Drought and salt tolerance and ethylene synthesis-related genes are upregulated in OsARD1-OE plants. Subcellular localization shows that OsARD1 displays strong localization signal in cell nucleus, suggesting OsARD1 may interact with the transcription factors. Taken together, the results provide the understanding of the function of OsARD1 in ethylene synthesis and abiotic stress response in rice.

Overexpression of OsHox32 Results in Pleiotropic Effects on Plant Type Architecture and Leaf Development in Rice.

BACKGROUND: The Class III homeodomain Leu zipper (HD-Zip III) gene family plays important roles in plant growth and development. Here, we analyze the function of OsHox32, an HD-Zip III family member, and show that it exhibits pleiotropic effects on regulating plant type architecture and leaf development in rice. RESULTS: Transgenic lines overexpressing OsHox32 (OsHox32-OV) produce narrow leaves that roll towards the adaxial side. Histological analysis revealed a decreased number of bulliform cells in OsHox32-OV lines. In addition, the angle between the leaf and culm was reduced, resulting in an erect plant phenotype. The height of the plants was reduced, resulting in a semi-dwarf phenotype. In addition, the chlorophyll level was reduced, resulting in a decrease in the photosynthetic rate, but water use efficiency was significantly improved, presumably due to the rolled leaf phenotype. OsHox32 exhibited constitutive expression in different organs, with higher mRNA levels in the stem, leaf sheath, shoot apical meristems and young roots, suggesting a role in plant-type and leaf development. Moreover, OsHox32 mRNA levels were higher in light and lower in the dark under both long-day and short-day conditions, indicating that OsHox32 may be associated with light regulation. Photosynthesis-associated and chlorophyll biosynthesis-associated genes were down-regulated to result in the reduction of photosynthetic capacity in OsHox32-OV lines. mRNA level of six rice YABBY genes is up-regulated or down-regulated by OsHox32, suggesting that OsHox32 may regulate the architecture of plant type and leaf development by controlling the expression of YABBY genes in rice. In addition, OsHox32 mRNA level was induced by the phytohormones, indicating that OsHox32 may be involved in phytohormones regulatory pathways. CONCLUSIONS: OsHox32, an HD-Zip III family member, plays pleiotropic effects on plant type architecture and leaf development in rice.

Knockdown of OsHox33, a member of the class III homeodomain-leucine zipper gene family, accelerates leaf senescence in rice.

The class III homeodomain-leucine zipper (HD-Zip III) gene family plays important roles in plant growth and development, including regulation of apical embryo patterning, embryonic shoot meristem formation, leaf polarity, vascular development, and meristem function, with a particularly crucial function in leaf development. Although HD-Zip III members are highly conserved in land plants, previous studies, such as genetic analyses based on multiple mutants in Arabidopsis and other plants, suggest that various HD-Zip III family genes have evolved with distinct functions and pleiotropic effects on plant growth and development. In this study, we analyzed a HD-Zip III member, OsHox33, and demonstrated that it plays an important role in age-dependent leaf senescence in rice. We constructed two specific RNAi vectors derived from the 5'-end region and 3'-UTR of OsHox33 to knockdown its expression. Transgenic plants harboring either RNAi construct displayed similar phenotypes of precocious leaf senescence symptoms, suggesting that knockdown of OsHox33 accelerates leaf senescence in rice. pOsHox33::GUS fusion expression and RT-PCR revealed that OsHox33 is highly expressed in young organs, especially in young meristems such as shoot apical meristems, intercalary meristems, and young callus. In addition, real-time PCR indicated that OsHox33 was more highly expressed in young leaves than in old leaves. To further investigate OsHox33 function, we analyzed chloroplast ultrastructure in different-aged leaves of RNAi plants, and found that OsHox33 knockdown accelerated chloroplast degradation, which is consistent with RNAi phenotypes. Finally, real-time PCR studies showed that OsHox33 can regulate the expression of GS1 and GS2, two senescence-associated genes. Taken together, the work presented here provides new insights into the function of HD-Zip III members in plants.

Interaction between temperature and photoperiod in regulation of flowering time in rice.

Photoperiod and temperature are two pivotal regulatory factors of plant flowering. The floral transition of plants depends on accurate measurement of changes in photoperiod and temperature. The flowering time of rice (Oryza sativa) as a facultative short-day (SD) plant is delayed under long-day (LD) and/or low temperature conditions. To elucidate the regulatory functions of photoperiod and temperature on flowering time in rice, we systematically analyzed the expression and regulation of several key genes (Hd3a, RFT1, Ehd1, Ghd7, RID1/Ehd2/OsId1, Se5) involved in the photoperiodic flowering regulatory pathway under different temperature and photoperiod treatments using a photoperiod-insensitive mutant and wild type plants. Our results indicate that the Ehd1-Hd3a/RFT1 pathway is common to and conserved in both the photoperiodic and temperature flowering regulatory pathways. Expression of Ehd1, Hd3a and RFT1 is dramatically reduced at low temperature (23°C), suggesting that suppression of Ehd1, Hd3a and RFT1 transcription is an essential cause of delayed flowering under low temperature condition. Under LD condition, Ghd7 mRNA levels are promoted at low temperature (23°C) compared with normal temperature condition (28°C), suggesting low temperature and LD treatment have a synergistic role in the expression of Ghd7. Therefore, upregulation of Ghd7 might be a crucial cause of delayed flowering under low temperature condition. We also analyzed Hd1 regulatory relationships in the photoperiodic flowering pathway, and found that Hd1 can negatively regulate Ehd1 transcription under LD condition. In addition, Hd1 can also positively regulate Ghd7 transcription under LD condition, suggesting that the heading-date of rice under LD condition is also regulated by the Hd1-Ghd7-Ehd1-RFT1 pathway.

OsCD1 encodes a putative member of the cellulose synthase-like D sub-family and is essential for rice plant architecture and growth.

The cell wall plays important roles in plant architecture and morphogenesis. The cellulose synthase-like super-families were reported to contain glycosyltransferases motif and are required for the biosynthesis of cell wall polysaccharides. Here, we describe a curled leaf and dwarf mutant, cd1, in rice, which exhibits multiple phenotypic traits such as the reduction of plant height and leaf width, curled leaf morphology and a decrease in the number of grains and in the panicle length. Map-based cloning indicates that a member of the cellulose synthase-like D (CSLD) group is a candidate for OsCD1. RNAi transgenic plants with the candidate CSLD gene display a similar phenotype to the cd1 mutant, suggesting that OsCD1 is a member of the CSLD sub-family. Furthermore, sequence analysis indicates that OsCD1 contains the common D,D,D,QXXRW motif, which is a feature of the cellulose synthase-like super-family. Analysis of OsCD1 promoter with GUS fusion expression shows that OsCD1 exhibits higher expression in young meristem tissues such as fresh roots, young panicle and stem apical meristem. Cell wall composition analysis reveals that cellulose content and the level of xylose are significantly reduced in mature culm owing to loss of OsCD1 function. Take together, the work presented here is useful for expanding the understanding of cell wall biosynthesis.

Identification and characterization of HTD2: a novel gene negatively regulating tiller bud outgrowth in rice.

Tiller number is highly regulated by controlling the formation of tiller bud and its subsequent outgrowth in response to endogenous and environmental signals. Here, we identified a rice mutant htd2 from one of the 15,000 transgenic rice lines, which is characterized by a high tillering and dwarf phenotype. Phenotypic analysis of the mutant showed that the mutation did not affect formation of tiller bud, but promoted the subsequent outgrowth of tiller bud. To isolate the htd2 gene, a map-based cloning strategy was employed and 17 new insertions-deletions (InDels) markers were developed. A high-resolution physical map of the chromosomal region around the htd2 gene was made using the F(2) and F(3) population. Finally, the gene was mapped in 12.8 kb region between marker HT41 and marker HT52 within the BAC clone OSJNBa0009J13. Cloning and sequencing of the target region from the mutant showed that the T-DNA insertion caused a 463 bp deletion between the promoter and first exon of an esterase/lipase/thioesterase family gene in the 12.8 kb region. Furthermore, transgenic rice with reduced expression level of the gene exhibited an enhanced tillering and dwarf phenotype. Accordingly, the esterase/lipase/thioesterase family gene (TIGR locus Os03g10620) was identified as the HTD2 gene. HTD2 transcripts were expressed mainly in leaf. Loss of function of HTD2 resulted in a significantly increased expression of HTD1, D10 and D3, which were involved in the strigolactone biosynthetic pathway. The results suggest that the HTD2 gene could negatively regulate tiller bud outgrowth by the strigolactone pathway.

The effect of the crosstalk between photoperiod and temperature on the heading-date in rice.

Photoperiod and temperature are two important environmental factors that influence the heading-date of rice. Although the influence of the photoperiod on heading has been extensively reported in rice, the molecular mechanism for the temperature control of heading remains unknown. This study reports an early heading mutant derived from tissue culture lines of rice and investigates the heading-date of wild type and mutant in different photoperiod and temperature treatments. The linkage analysis showed that the mutant phenotype cosegregated with the Hd1 locus. Sequencing analysis found that the mutant contained two insertions and several single-base substitutions that caused a dramatic reduction in Hd1mRNA levels compared with wild type. The expression patterns of Hd1 and Hd3a were also analyzed in different photoperiod and temperature conditions, revealing that Hd1 mRNA levels displayed similar expression patterns for different photoperiod and temperature treatments, with high expression levels at night and reduced levels in the daytime. In addition, Hd1 displayed a slightly higher expression level under long-day and low temperature conditions. Hd3a mRNA was present at a very low level under low temperature conditions regardless of the day-length. This result suggests that suppression of Hd3a expression is a principle cause of late heading under low temperature and long-day conditions.

Genetic structure and diversity of cultivated soybean (Glycine max (L.) Merr.) landraces in China.

The Chinese genebank contains 23,587 soybean landraces collected from 29 provinces. In this study, a representative collection of 1,863 landraces were assessed for genetic diversity and genetic differentiation in order to provide useful information for effective management and utilization. A total of 1,160 SSR alleles at 59 SSR loci were detected including 97 unique and 485 low-frequency alleles, which indicated great richness and uniqueness of genetic variation in this core collection. Seven clusters were inferred by STRUCTURE analysis, which is in good agreement with a neighbor-joining tree. The cluster subdivision was also supported by highly significant pairwise Fst values and was generally in accordance with differences in planting area and sowing season. The cluster HSuM, which contains accessions collected from the region between 32.0 and 40.5 degrees N, 105.4 and 122.2 degrees E along the central and downstream parts of the Yellow River, was the most genetically diverse of the seven clusters. This provides the first molecular evidence for the hypotheses that the origin of cultivated soybean is the Yellow River region. A high proportion (95.1%) of pairs of alleles from different loci was in LD in the complete dataset. This was mostly due to overall population structure, since the number of locus pairs in LD was reduced sharply within each of the clusters compared to the complete dataset. This shows that population structure needs to be accounted for in association studies conducted within this collection. The low value of LD within the clusters can be seen as evidence that much of the recombination events in the past have been maintained in soybean, fixed in homozygous self-fertilizing landraces.

An efficient field screening procedure for identifying transposants for constructing an Ac/Ds-based insertional-mutant library of rice.

An efficient system was developed, and several variables tested, for generating a large-scale insertional-mutagenesis population of rice. The most important feature in this improved Ac/Ds tagging system is that one can conveniently carry out large-scale screening in the field and select transposants at the seedling stage. Rice was transformed with a plasmid that includes a Basta-resistance gene (bar). After the Ds element is excised during transposition, bar becomes adjacent to the ubiquitin promoter, and the rice plant becomes resistant to the herbicide Basta. In principle, one can plant up to one million plants in the field and select those plants that survive after spraying with Basta. To test the utility of this system, 4 Ds starter lines were crossed with 14 different Ac plants, and many transposants were successfully identified after planting 134,285 F2 plants in the field. Over 2,800 of these transposants were randomly chosen for PCR analysis, and the results fully confirmed the reliability of the field screening procedure.

[Representativeness of Northeast China spring soybeans and their genetic diversity at SSR loci].

A total of 283 accessions were selected from the total 3 226 Northeast China spring soybeans, which represented > 80% of the whole based on their qualitative and quantitative traits. The representative samples were analyzed by 61 SSR loci, and a total of 534 alleles were detected, ranging 2 - 16 alleles per locus, with an average of 8.75 alleles per locus. Among the accessions, the Simpson diversity index (SDI) for each locus ranged from 0.406 to 0.886, with a mean of 0.704, which was relatively lower since there were dominant alleles at most of loci in the representative samples. 35 accessions had specific alleles, which distributed among 29 loci. The differentiation coefficient was lower than 9.27% in 61 loci among the three provinces of Northeast China, which might be caused by lots of common alleles shared among these provinces. The genetic diversity in Jilin and Liaoning provinces was nearly equal, but was higher than that in Heilongjiang Province. The landraces of the representative samples at 61 loci had a higher genetic diversity than that of the released cultivars. The genetic diversity appeared within and between the provinces could be used to broaden the genetic base of modern cultivars, and also, the landraces were the major source for soybean breeding because of their high genetic diversity.

[Ac/Ds tagging system and functional genomics in rice].

With the completion of the rice genome sequence in 2002, the study on functional genomics in rice has become a major task. Establishment of rice mutant library is an essential approach for rice functional genomics study. At present, utilizing maize transposable element Ac/Ds (Activator/Dissociation) is a promising method to construct insertional mutagenesis library of rice. Ac/Ds tagging system has received extensive application in rice during the past several years, but it is still confronted with practical problems. In this paper, constructing rice insertional mutagenesis library using Ac/Ds-tagging system and transpositional behaviors of an Ac/Ds-tagging system, difficulties and advantages are reviewed. The research advances and challenge in rice functional genomics study using Ac/Ds tagging system are also discussed and summarized in the paper.