Functional characterization of D-type cyclins involved in cell division in rice.

BACKGROUND: D-type cyclins (CYCD) regulate the cell cycle G1/S transition and are thus closely involved in cell cycle progression. However, little is known about their functions in rice. RESULTS: We identified 14 CYCD genes in the rice genome and confirmed the presence of characteristic cyclin domains in each. The expression of the OsCYCD genes in different tissues was investigated. Most OsCYCD genes were expressed at least in one of the analyzed tissues, with varying degrees of expression. Ten OsCYCD proteins could interact with both retinoblastoma-related protein (RBR) and A-type cyclin-dependent kinases (CDKA) forming holistic complexes, while OsCYCD3;1, OsCYCD6;1, and OsCYCD7;1 bound only one component, and OsCYCD4;2 bound to neither protein. Interestingly, all OsCYCD genes except OsCYCD7;1, were able to induce tobacco pavement cells to re-enter mitosis with different efficiencies. Transgenic rice plants overexpressing OsCYCD2;2, OsCYCD6;1, and OsCYCD7;1 (which induced cell division in tobacco with high-, low-, and zero-efficiency, respectively) were created. Higher levels of cell division were observed in both the stomatal lineage and epidermal cells of the OsCYCD2;2- and OsCYCD6;1-overexpressing plants, with lower levels seen in OsCYCD7;1-overexpressing plants. CONCLUSIONS: The distinct expression patterns and varying effects on the cell cycle suggest different functions for the various OsCYCD proteins. Our findings will enhance understanding of the CYCD family in rice and provide a preliminary foundation for the future functional verification of these genes.

Stacking Multiple Genes Improves Resistance to Chilo suppressalis, Magnaporthe oryzae, and Nilaparvata lugens in Transgenic Rice.

The ability of various pests and diseases to adapt to a single plant resistance gene over time leads to loss of resistance in transgenic rice. Therefore, introduction of different pest and disease resistance genes is critical for successful cultivation of transgenic rice strains with broad-spectrum resistance to multiple pathogens. Here, we produced resistance rice lines with multiple, stacked resistance genes by stacking breeding and comprehensively evaluated their resistance to Chilo suppressalis (striped rice stemborer), Magnaporthe oryzae (rice blast), and Nilaparvata lugens (brown planthopper) in a pesticide-free environment. CRY1C and CRY2A are exogenous genes from Bacillus thuringiensis. Pib, Pikm, and Bph29 are natural genes in rice. CH121TJH was introduced into CRY 1C, Pib, Pikm, and Bph29. CH891TJH and R205XTJH were introduced into CRY 2A, Pib, Pikm, and Bph29. Compared with those observed in their recurrent parents, CH121TJH significantly increased the mortality of borers. The other two lines CH891TJH and R205XTJH are the same result. Three lines introduction of Pib and Pikm significantly reduced the area of rice blast lesions, and introduction of Bph29 significantly reduced seedling mortality from N. lugens. Introduction of the exogenous genes had relatively few effects on agronomic and yield traits of the original parents. These findings suggest that stacking of rice resistance genes through molecular marker-assisted backcross breeding can confer broad spectrum and multiple resistance in differently genetic backgrounds.

Analysis of the Genetic Stability of Insect and Herbicide Resistance Genes in Transgenic Rice Lines: A Laboratory and Field Experiment.

A lack of stability in the expression of Bacillus thuringiensis genes (CRY) and the dialaninophosphate resistance gene (BAR) in transgenic rice plants can lead to the loss of important characters. The genetic stability of transgenic expression in high-generation lines is thus critically important for ensuring the success of molecular breeding efforts. Here, we studied the genetic stability of resistance to insect pests and herbicides in transgenic rice lines at the molecular and phenotypic levels in a pesticide-free environment. Southern blot analysis, real-time polymerase chain reaction, and enzyme-linked immunosorbent assays revealed high stability in the copy numbers and expression levels of CRY1C, CRY2A, and BAR in transgenic lines across different generations, and gene expression levels were highly correlated with protein expression levels. The insecticide resistance of the transgenic rice lines was high. The larval mortality of Chilo suppressalis was 50.25% to 68.36% higher in transgenic lines than in non-transgenic control lines. Percent dead hearts and percent white spikelets were 16.66% to 22.15% and 27.07% to 33.47% lower in transgenic lines than in non-transgenic control lines, respectively. The herbicide resistance of the transgenic rice lines was also high. The bud length and root length ranged were 2.53 cm to 4.20 cm and 0.28 cm to 0.73 cm higher in transgenic lines than in non-transgenic control lines in the budding stage, respectively. Following application of the herbicide Basta, the chlorophyll content of the transgenic lines began to recover 2 d later in the seedling and tillering stages and 3 d later in the booting and heading stages, by contrast, the chlorophyll content of the non-transgenic lines did not recover and continued to decrease. These findings revealed high genetic stability of the resistance to insect pests and herbicides across several generations of transgenic rice regardless of the genetic background.

A base substitution in OsphyC disturbs its Interaction with OsphyB and affects flowering time and chlorophyll synthesis in rice.

BACKGROUND: Phytochromes are important photoreceptors in plants, and play essential roles in photomorphogenesis. The functions of PhyA and PhyB in plants have been fully analyzed, while those of PhyC in plant are not well understood. RESULTS: A rice mutant, late heading date 3 (lhd3), was characterized, and the gene LHD3 was identified with a map-based cloning strategy. LHD3 encodes phytochrome C in rice. Animo acid substitution in OsphyC disrupted its interaction with OsphyB or itself, restraining functional forms of homodimer or heterodimer formation. Compared with wild-type plants, the lhd3 mutant exhibited delayed flowering under both LD (long-day) and SD (short-day) conditions, and delayed flowering time was positively associated with the day length via the Ehd1 pathway. In addition, lhd3 showed a pale-green-leaf phenotype and a slower chlorophyll synthesis rate during the greening process. The transcription patterns of many key genes involved in photoperiod-mediated flowering and chlorophyll synthesis were altered in lhd3. CONCLUSION: The dimerization of OsPhyC is important for its functions in the regulation of chlorophyll synthesis and heading. Our findings will facilitate efforts to further elucidate the function and mechanism of OsphyC and during light signal transduction in rice.

Rice microtubule-associated protein OsMAP65-3.1, but not OsMAP65-3.2, plays a critical role in phragmoplast microtubule organization in cytokinesis.

In plants, MAP65 preferentially cross-links the anti-parallel microtubules (MTs) and plays an important role for cytokinesis. However, the functions of MAP65 isoforms in rice (Oryza sativa. L) are largely unknown. Here, we identified two MAP65-3 homologs in rice, OsMAP65-3.1 and OsMAP65-3.2. We found that both OsMAP65-3.1 and OsMAP65-3.2 were similar in dimerization and location to AtMAP65-3, and the expression of either rice genes driven by the AtMAP65-3 promoter suppressed the cytokinesis failure and growth defect of atmap65-3. However, OsMAP65-3.1 with native promoter also recovered the atmap65-3, but OsMAP65-3.2 with its own promoter had no effects. OsMAP65-3.1 but not OsMAP65-3.2 was actively expressed in tissues enriched with dividing cells. R1R2R3-Myb (MYB3R) transcription factors directly bound to the OsMAP65-3.1 promoter but not that of OsMAP65-3.2. Furthermore, osmap65-3.2 had no obvious phenotype, while either osmap65-3.1 or osmap65-3.1(+/-) was lethal. The eminent MTs around the daughter nuclei and cytokinesis defects were frequently observed in OsMAP65-3.1-defective plants. Taken together, our findings suggest that OsMAP65-3.1, rather than OsMAP65-3.2, plays essential roles in rice cytokinesis resulting from their differential expression which were passably directly regulated by OsMYB3Rs.

The genetic editing of GS3 via CRISPR/Cas9 accelerates the breeding of three-line hybrid rice with superior yield and grain quality.

Grain size is one of the major traits that determine rice grain yield and quality. The GS3 gene is the first major quantitative trait locus (QTL) that was identified in regulating rice grain length and weight. It was reported that the gs3 allele with a mutation in the organ size regulation (OSR) domain of the GS3 protein produced longer grains. In this study, we used the CRISPR/Cas9 gene editing technology to introduce an edited gs3 allele into our indica maintainer line, Mei1B, to enhance its grain yield and quality. Through molecular analysis and sequencing, a homologous edited-gs3 mutant line without any transgene was obtained in the T1 generation and was named Mei2B. A superior male sterile line Mei2A was generated by backcrossing the cytoplasmic male sterile (CMS) line Mei1A with Mei2B. Mei2B had a higher grain quality and yield compared to its wild-type Mei1B. Its grain length increased by 7.9%, its length/width ratio increased from 3.89 to 4.19, TGW increased by 6.7%, and grain yield per plant increased by 14.9%. In addition, genetic improvement of other quality traits including brown rice length (6.83 mm), brown rice grain length/width ratio (3.61), matched the appearance standards set for traditional Simiao (silk seedling) type cultivars. Two restorer lines were outcrossed to both Mei1A and Mei2A to produce hybrid rice. Compared to two hybrids of Mei1A, the hybrids of Mei2A had longer grains, higher length/width ratio, TGW, and yield per plant. In addition, the hybrids of Mei2A showed a better grain appearance including better translucency, a lower chalky rice rate, and degree of chalkiness than the hybrids of Mei1A. These results demonstrated that the introduction of an elite gs3 allele into Mei1A via CRISPR/Cas9 gene editing technology led to significant genetic improvement of the rice grain. The resultant CMS line Mei2A(gs3) displayed much higher grain quality and yield than the original Mei1A. Therefore, our study demonstrated that the targeted genetic improvement via gene editing technology can enhance rice breeding, especially the breeding of three-line hybrid rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01290-z.

Comparison of the Phenotypic Performance, Molecular Diversity, and Proteomics in Transgenic Rice.

The extent of molecular diversity and differentially expressed proteins (DEPs) in transgenic lines provide valuable information to understand the phenotypic performance of transgenic crops compared with their parents. Here, we compared the differences in the phenotypic variation of twelve agronomic and end-use quality traits, the extent of microsatellite diversity, and DEPs of a recurrent parent line with three transgenic rice restorer lines carrying either CRY1C gene on chromosome 11 or CRY2A gene on chromosome 12 or both genes. The three transgenic lines had significantly smaller stem borer infestation than the recurrent parent without showing significant differences among most agronomic traits, yield components, and end-use quality traits. Using 512 microsatellite markers, the three transgenic lines inherited 2.9-4.3% of the Minghui 63 donor genome and 96.3-97.1% of the CH891 recurrent parent genome. As compared with the recurrent parent, the number of upregulated and down-regulated proteins in the three transgenic lines varied from 169 to 239 and from 131 to 199, respectively. Most DEPs were associated with the secondary metabolites biosynthesis transport and catabolism, carbohydrate transport and metabolism, post-translational modification, and signal transduction mechanisms. Although several differentially expressed proteins were observed between transgenic rice and its recurrent parent, the differences may not have been associated with grain yield and most other phenotypic traits in transgenic rice.

Detection of QTLs for panicle-related traits using an indica × japonica recombinant inbred line population in rice.

BACKGROUND: The panicle is the most important organ in rice, and all the panicle-related traits are correlated with rice grain yield. Understanding the underlying genetic mechanisms controlling panicle development is very important for improving rice production. METHODS: Nine panicle-related traits including heading date, panicle length, number of primary branches, number of secondary branches, number of grains per panicle, number of panicles per plant, number of filled grains per plant, seed-setting rate, and grain yield per plant were investigated. To map the quantitative trait loci (QTLs) for the nine panicle-related traits, a PCR-based genetic map with 208 markers (including 121 simple sequence repeats and 87 InDels) and a high-density linkage map with 18,194 single nucleotide polymorphism (SNP) markers were both used. RESULTS: Using a recombinant inbred line population derived from an indica variety Huanghuazhan and a japonica line Jizi 1560, a total of 110 and 112 QTLs were detected for panicle-related traits by PCR-based genetic map and by high-density linkage map, respectively. Most of the QTLs were clustered on chromosomes 1, 2, 3, 6, and 7 while no QTLs were detected on chromosome 10. Almost all the QTLs with LOD values of more than 5.0 were repeatedly detected, indicating the accuracy of the two methods and the stability of the QTL effects. No genes for panicle-related traits have been previously reported in most of these regions. QTLs found in JD1006-JD1007 and RM1148-RM5556 with high LOD and additive values deserved further research. The results of this study are beneficial for marker-assisted breeding and provide research foundation for further fine-mapping and cloning of these QTLs for panicle-related traits.

Fine Mapping of a Novel Major Quantitative Trait Locus, qPAA7, That Controls Panicle Apical Abortion in Rice.

The panicle apical abortion (PAA) causes severe yield losses in rice production, but details about its development and molecular basis remain elusive. Here, we detected PAA quantitative trait loci (QTLs) in three environments using a set of chromosome segment substitution lines (CSSLs) that was constructed with indica Changhui121 as the recurrent parent and japonica Koshihikari as the donor parent. First, we identified a novel major effector quantitative trait locus, qPAA7, and selected a severe PAA line, CSSL176, which had the highest PAA rate among CSSLs having Koshihikari segments at this locus. Next, an F2 population was constructed from a cross between CSS176 and CH121. Using F2 to make recombinantion analysis, qPAA7 was mapped to an 73.8-kb interval in chromosome 7. Among nine candidate genes within this interval, there isn't any known genes affecting PAA. According to the gene annotation, gene expression profile and alignment of genomic DNA, LOC_Os07g41220 and LOC_Os07g41280 were predicted as putative candidate genes of qPAA7. Our study provides a foundation for cloning and functional characterization of the target gene from this locus.

qTGW12a, a naturally varying QTL, regulates grain weight in rice.

KEY MESSAGE: A stable QTL associated with rice grain type with a large effect value was found in multiple environments, and its candidate genes were verified by genetic transformation. Rice (Oryza sativa L.) grain size is critical to both yield and appearance quality. Therefore, the discovery and identification of rice grain size genes can provide pathways for the cultivation of high-yielding varieties. In the present work, 45,607 SNP markers were used to construct a high-density genetic map of rice recombinant inbred lines, and hence a total of 14 quantitative trait loci (QTLs) were detected based on the phenotypic data of grain weight, grain length and grain width under four different environments. qTGW12a and qGL12 are newly detected QTLs related to grain weight, and are located between 22.43 Mb and 22.45 Mb on chromosome 12. Gene annotation shows that the QTL region contains the LOC_Os12g36660 annotated gene, which encodes the multidrug and toxic compound extrusion (MATE) transporter. Mutations in exons and the splice site were responsible for the changes in grain type and weight. Gene knockout experiments were used to verify these results. Hence, these results provide a basis for the cloning of qTGW12a. This discovery provides new insights for studying the genetic mechanism of rice grain morphology, and reveals a promising gene to ultimately increase rice yield.

Combined proteomics, metabolomics and physiological analyses of rice growth and grain yield with heavy nitrogen application before and after drought.

BACKGROUND: Nitrogen application can effectively mitigate the damage to crop growth and yield caused by drought. However, the efficiency of heavy nitrogen application before drought (NBD) and heavy nitrogen application after drought (NAD) to regulate rice response to drought stress remains controversial. In this study, we profiled physiology, proteomics and metabolomics in rice variety Wufengyou 286 of two nitrogen management modes (NBD and NAD) to investigate their yield formation and the mechanism of nitrogen regulation for drought resistance. RESULTS: Results revealed that the yield of NBD and NAD decreased significantly when it was subjected to drought stress at the stage of young panicle differentiation, while the yield of NBD was 33.85 and 36.33% higher than that of NAD in 2017 and 2018, reaching significant levels. Under drought conditions, NBD increased chlorophyll content and net photosynthetic rate in leaves, significantly improved the activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase and catalase, and decreased malondialdehyde (MDA) content compared with NAD. NBD promoted nitrogen assimilation in leaves, which was characterized by increased activities of nitrate reductase (NR) and glutamine synthetase (GS). In addition, NBD significantly increased the contents of osmotic regulatory substances such as soluble sugar, soluble protein and free proline. Gene ontology and KEGG enrichment analysis of 234 differentially expressed proteins and 518 differential metabolites showed that different nitrogen management induced strong changes in photosynthesis pathway, energy metabolism pathway, nitrogen metabolism and oxidation-reduction pathways. CONCLUSION: Different nitrogen management methods have significant differences in drought resistance of rice. These results suggest that heavy nitrogen application before drought may be an important pathway to improve the yield and stress resistance of rice, and provide a new ecological perspective on nitrogen regulation in rice.

Ribosome profiling reveals the effects of nitrogen application translational regulation of yield recovery after abrupt drought-flood alternation in rice.

Abrupt drought-flood alternation is a frequent meteorological disaster during the summer in Southern China. The study of physiological and translation mechanisms of rice yield recovery after abrupt drought-flood alternation has great potential benefits in field production. Our results showed that yield recovery upon nitrogen (N) application after abrupt drought-flood alternation was due to the increase in effective panicle numbers per plant. The N application resulted in the regulation of physiological and biochemical as well as growth development processes, which led to a rapid growth recovery effect after abrupt drought-flood alternation stress in rice. Using ribosome profiling combined with RNA sequencing (RNA-seq) technology, the interactions between transcription and translation for N application after abrupt drought-flood alternation were analyzed. It was found that a small proportion of response genes were shared at the transcriptional and translational levels, that is, 14% of the expressed genes were upregulated and 6.6% downregulated. Further analysis revealed that the translation efficiency (TE) of the genes was influenced by their sequence characteristics, including their GC content, coding sequence length and normalized minimal free energy. Compared with the number of untranslated upstream open reading frames (uORFs), the increased number of translated uORFs promoted the improvement of TE. The TE of the uORFs for N application was lower than the control without N application after abrupt drought-flood alternation. This study characterizes the translational regulatory pattern in response to N application after abrupt drought-flood alternation stress.

OsPLS4 Is Involved in Cuticular Wax Biosynthesis and Affects Leaf Senescence in Rice.

Leaf senescence is one of the most common factors that affects the growth and yield of rice. Although numerous genes affecting leaf senescence have been identified, few involved in cuticular wax synthesis have been described for rice premature leaf senescence. Here, we cloned and characterized Premature Leaf Senescence 4 (PLS4) in rice (Oryza sativa), which encodes a putative 3-oxoacyl-reductase in the fatty acid biosynthetic pathway. Subcellular localization of OsPLS4 was observed in the chloroplast. A single nucleotide substitution in OsPLS4 reduced leaf cuticular wax, and the expression levels of most wax biosynthesis-associated genes were downregulated. TEM showed chloroplast development were defective in the pls4 mutant. Further investigation revealed that the chlorophyll (Chl) content was reduced in the pls4 mutant compared with the WT and that the photosynthesis rate was lower, which caused ROS dramatic accumulation at the heading stage. These results confirmed premature leaf senescence in pls4 plants. Cold treatment indicated that the mutant was more sensitive than the WT was to cold stress. Together, all the above results indicate that the OsPLS4 mutation affects cuticular wax biosynthesis and chloroplast development in rice, causing reduced cuticular wax and premature leaf senescence.

Comprehensive metabolomic, proteomic and physiological analyses of grain yield reduction in rice under abrupt drought-flood alternation stress.

Abrupt drought-flood alternation (T1) is a meteorological disaster that frequently occurs during summer in southern China and the Yangtze river basin, often causing a significant loss of rice production. In this study, the response mechanism of yield decline under abrupt drought-flood alternation stress at the panicle differentiation stage was analyzed by looking at the metabolome, proteome as well as yield and physiological and biochemical indexes. The results showed that drought and flood stress caused a decrease in the yield of rice at the panicle differentiation stage, and abrupt drought-flood alternation stress created a synergistic effect for the reduction of yield. The main reason for the decrease of yield per plant under abrupt drought-flood alternation was the decrease of seed setting rate. Compared with CK0 (no drought and no flood), the net photosynthetic rate and soluble sugar content of T1 decreased significantly and its hydrogen peroxidase, superoxide dismutase, peroxidase activity increased significantly. The identified differential metabolites and differentially expressed proteins indicated that photosynthesis metabolism, energy metabolism pathway and reactive oxygen species response have changed strongly under abrupt drought-flood alteration stress, which are factors that leads to the rice grain yield reduction.

Genetic analysis for the grain number heterosis of a super-hybrid rice WFYT025 combination using RNA-Seq.

BACKGROUND: Despite the great contributions of utilizing heterosis to crop productivity worldwide, the molecular mechanism of heterosis remains largely unexplored. Thus, the present research is focused on the grain number heterosis of a widely used late-cropping indica super hybrid rice combination in China using a high-throughput next-generation RNA-seq strategy. RESULTS: Here, we obtained 872 million clean reads, and at least one read could maps 27,917 transcripts out of 35,679 annotations. Transcript differential expression analysis revealed a total of 5910 differentially expressed genes (DGHP) between super-hybrid rice Wufengyou T025 (WFYT025) and its parents were identified in the young panicles. Out of the 5910 DGHP, 63.1% had a genetic action mode of over-dominance, 17.3% had a complete-dominance action, 15.6% had a partial-dominance action and 4.0% had an additive action. DGHP were significantly enriched in carotenoid biosynthesis, diterpenoid biosynthesis and plant hormone signal transduction pathways, with the key genes involved in the three pathways being up-regulated in the hybrid. By comparing the DGHP enriched in the KEGG pathway with QTLs associated with grain number, several DGHP were located on the same chromosomal segment with some of these grain number QTLs. CONCLUSION: Through young panicle development transcriptome analysis, we conclude that the over-dominant effect is probably the major contributor to the grain number heterosis of WFYT025. The DGHP sharing the same location with grain number QTLs could be considered a candidate gene and provide valuable targets for the cloning and functional analysis of these grain number QTLs.

Genome-wide analysis of long non-coding RNAs affecting roots development at an early stage in the rice response to cadmium stress.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been found to play a vital role in several gene regulatory networks involved in the various biological processes in plants related to stress response. However, systematic analyses of lncRNAs expressed in rice Cadmium (Cd) stress are seldom studied. Thus, we presented the characterization and expression of lncRNAs in rice root development at an early stage in response to Cd stress. RESULTS: The lncRNA deep sequencing revealed differentially expressed lncRNAs among Cd stress and normal condition. In the Cd stress group, 69 lncRNAs were up-regulated and 75 lncRNAs were down-regulated. Furthermore, 386 matched lncRNA-mRNA pairs were detected for 120 differentially expressed lncRNAs and 362 differentially expressed genes in cis, and target gene-related pathway analyses exhibited significant variations in cysteine and methionine metabolism pathway-related genes. For the genes in trans, overall, 28,276 interaction relationships for 144 lncRNAs and differentially expressed protein-coding genes were detected. The pathway analyses found that secondary metabolites, such as phenylpropanoids and phenylalanine, and photosynthesis pathway-related genes were significantly altered by Cd stress. All of these results indicate that lncRNAs may regulate genes of cysteine-rich peptide metabolism in cis, as well as secondary metabolites and photosynthesis in trans, to activate various physiological and biochemical reactions to respond to excessive Cd. CONCLUSION: The present study could provide a valuable resource for lncRNA studies in response to Cd treatment in rice. It also expands our knowledge about lncRNA biological function and contributes to the annotation of the rice genome.

Mapping QTL for Seed Germinability under Low Temperature Using a New High-Density Genetic Map of Rice.

Mapping major quantitative trait loci (QTL) responsible for rice seed germinability under low temperature (GULT) can provide valuable genetic source for improving cold tolerance in rice breeding. In this study, 124 rice backcross recombinant inbred lines (BRILs) derived from a cross indica cv. Changhui 891 and japonica cv. 02428 were genotyped through re-sequencing technology. A bin map was generated which includes 3057 bins covering distance of 1266.5 cM with an average of 0.41 cM between markers. On the basis of newly constructed high-density genetic map, six QTL were detected ranging from 40 to 140 kb on Nipponbare genome. Among these, two QTL qCGR8 and qGRR11 alleles shared by 02428 could increase GULT and seed germination recovery rate after cold stress, respectively. However, qNGR1 and qNGR4 may be two major QTL affecting indica Changhui 891germination under normal condition. QTL qGRR1 and qGRR8 affected the seed germination recovery rate after cold stress and the alleles with increasing effects were shared by the Changhui 891 could improve seed germination rate after cold stress dramatically. These QTL could be a highly valuable genetic factors for cold tolerance improvement in rice lines. Moreover, the BRILs developed in this study will serve as an appropriate choice for mapping and studying genetic basis of rice complex traits.

Genome-wide identification and phylogenetic analysis of the chalcone synthase gene family in rice.

The enzymes of the chalcone synthase family are also known as type III polyketide synthases (PKS), and produce a series of secondary metabolites in bacteria, fungi and plants. In a number of plants, genes encoding PKS comprise a large multigene family. Currently, detailed reports on rice (Oryza sativa) PKS (OsPKS) family genes and tissue expression profiling are limited. Here, 27 candidate OsPKS genes were identified in the rice genome,and 23 gene structures were confirmed by EST and cDNA sequencing; phylogenetic analysis has indicated that these 23 OsPKS members could be clustered into three groups (I-III). Comparative analysis has shown OsPKS08 and OsPKS26 could be classified with the CHS genes of other species. Two members OsPKS10 and OsPKS21 were grouped into anther specific chalcone synthase-like (ASCL) clade. Intron/exon structure analysis revealed that nearly all of the OsPKS members contained one phase-1 intron at a conserved Cys. Analysis of chromosomal localization and genome distribution showed that some of the members were distributed on a chromosome as a cluster. Expression data exhibited widespread distribution of the rice OsPKS gene family within plant tissues, suggesting functional diversification of the OsPKS genes. Our results will contribute to future study of the complexity of the OsPKS gene family in rice.

Using RNA-seq to Profile Gene Expression of Spikelet Development in Response to Temperature and Nitrogen during Meiosis in Rice (Oryza sativa L.).

Rice reproductive development is sensitive to high temperature and soil nitrogen supply, both of which are predicted to be increased threats to rice crop yield. Rice spikelet development is a critical process that determines yield, yet little is known about the transcriptional regulation of rice spikelet development in response to the combination of heat stress and low nitrogen availability. Here, we profiled gene expression of rice spikelet development during meiosis under heat stress and different nitrogen levels using RNA-seq. We subjected plants to four treatments: 1) NN: normal nitrogen level (165 kg ha-1) with normal temperature (30°C); 2) HH: high nitrogen level (264 kg ha-1) with high temperature (37°C); 3) NH: normal nitrogen level and high temperature; and 4) HN: high nitrogen level and normal temperature. The de novo transcriptome assembly resulted in 52,250,482 clean reads aligned with 76,103 unigenes, which were then used to compare differentially expressed genes (DEGs) in the different treatments. Comparing gene expression in samples with the same nitrogen levels but different temperatures, we identified 70 temperature-responsive DEGs in normal nitrogen levels (NN vs NH) and 135 DEGs in high nitrogen levels (HN vs HH), with 27 overlapping DEGs. We identified 17 and seven nitrogen-responsive DEGs by comparing changes in nitrogen levels in lower temperature (NN vs HN) and higher temperature (NH vs HH), with one common DEG. The temperature-responsive genes were principally associated with cytochrome, heat shock protein, peroxidase, and ubiquitin, while the nitrogen-responsive genes were mainly involved in glutamine synthetase, amino acid transporter, pollen development, and plant hormone. Rice spikelet fertility was significantly reduced under high temperature, but less reduced under high-nitrogen treatment. In the high temperature treatments, we observed downregulation of genes involved in spikelet development, such as pollen tube growth, pollen maturation, especially sporopollenin biosynthetic process, and pollen exine formation. Moreover, we observed higher expression levels of the co-expressed DEGs in HN vs HH compared to NN vs NH. These included the six downregulated genes (one pollen maturation and five pollen exine formation genes), as well as the four upregulated DEGs in response to heat. This suggests that high-nitrogen treatment may enhance the gene expression levels to mitigate aspects of heat-stress. The spikelet genes identified in this study may play important roles in response to the combined effects of high temperature and high nitrogen, and may serve as candidates for crop improvement.

RNA-seq reveals differentially expressed genes of rice (Oryza sativa) spikelet in response to temperature interacting with nitrogen at meiosis stage.

BACKGROUND: Rice (Oryza sativa) is one of the most important cereal crops, providing food for more than half of the world's population. However, grain yields are challenged by various abiotic stresses such as drought, fertilizer, heat, and their interaction. Rice at reproductive stage is much more sensitive to environmental temperatures, and little is known about molecular mechanisms of rice spikelet in response to high temperature interacting with nitrogen (N). RESULTS: Here we reported the transcriptional profiling analysis of rice spikelet at meiosis stage using RNA sequencing (RNA-seq) as an attempt to gain insights into molecular events associated with temperature and nitrogen. This study received four treatments: 1) NN: normal nitrogen level (165 kg ha(-1)) with natural temperature (30 °C); 2) HH: high nitrogen level (330 kg ha(-1)) with high temperature (37 °C); 3) NH: normal nitrogen level and high temperature; and 4) HN: high nitrogen level and natural temperature, respectively. The de novo assembly generated 52,553,536 clean reads aligned with 72,667 unigenes. About 10 M reads were identified from each treatment. In these differentially expressed genes (DEGs), we found 151 and 323 temperature-responsive DEGs in NN-vs-NH and HN-vs-HH, and 114 DEGs were co-expressed. Meanwhile, 203 and 144 nitrogen-responsive DEGs were focused in NN-vs-HN and NH-vs-HH, and 111 DEGs were co-expressed. The temperature-responsive genes were principally associated with calcium-dependent protein, cytochrome, flavonoid, heat shock protein, peroxidase, ubiquitin, and transcription factor while the nitrogen-responsive genes were mainly involved in glutamine synthetase, transcription factor, anthocyanin, amino acid transporter, leucine zipper protein, and hormone. It is noted that, rice spikelet fertility was significantly decreased under high temperature, but it was more reduced under higher nitrogen. Accordingly, numerous spikelet genes involved in pollen development, pollen tube growth, pollen germination, especially sporopollenin biosynthetic process, and pollen exine formation were mainly down-regulated under high temperature. Moreover, the expression levels of co-expressed DEGs including 5 sporopollenin biosynthetic process and 7 pollen exine formation genes of NN-vs-NH were lower than that of HN-vs-HH. Therefore, these spikelet genes may play important roles in response to high temperature with high nitrogen and may be good candidates for crop improvement. CONCLUSIONS: This RNA-seq study will help elucidate the molecular mechanisms of rice spikelet defense response to high temperature interacting with high nitrogen level.