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.

Alternative polyadenylation profiles of susceptible and resistant rice (Oryza sativa L.) in response to bacterial leaf blight using RNA-seq.

BACKGROUND: Alternative polyadenylation (APA) is an important pattern of post-transcriptional regulation of genes widely existing in eukaryotes, involving plant physiological and pathological processes. However, there is a dearth of studies investigating the role of APA profile in rice leaf blight. RESULTS: In this study, we compared the APA profile of leaf blight-susceptible varieties (CT 9737-613P-M) and resistant varieties (NSIC RC154) following bacterial blight infection. Through gene enrichment analysis, we found that the genes of two varieties typically exhibited distal poly(A) (PA) sites that play different roles in two kinds of rice, indicating differential APA regulatory mechanisms. In this process, many disease-resistance genes displayed multiple transcripts via APA. Moreover, we also found five polyadenylation factors of similar expression patterns of rice, highlighting the critical roles of these five factors in rice response to leaf blight about PA locus diversity. CONCLUSION: Notably, the present study provides the first dynamic changes of APA in rice in early response to biotic stresses and proposes a possible functional conjecture of APA in plant immune response, which lays the theoretical foundation for in-depth determination of the role of APA events in plant stress response and other life processes.

Metabolomics revealed metabolite biomarkers of antioxidant properties and flavonoid metabolite accumulation in purple rice after grain filling.

The correlation between flavonoids, phenolic metabolites and the total antioxidant capacity is well established. However, specific biomarkers of metabolites with antioxidant properties in purple rice grains remain unidentified. This study integrated nontargeted metabolomics, quantitative detection of flavonoids and phenolic compounds, and physiological and biochemical data to identify metabolite biomarkers of the antioxidant properties of purple rice grains after filling. The findings demonstrated a significant enhancement in the biosynthesis of flavonoids during the middle and late filling stages in purple rice grains. Additionally, the pathways involved in anthocyanin and flavonoid biosynthesis were significantly enriched. Catalase (CAT), phenylalanine ammonia-lyase (PAL), total phenols (TP), flavonoids (FD), and oligomeric proanthocyanidin (OPC) were significantly correlated with philorizin, myricetin 3-galactoside, and trilobatin. Phlorizin, myricetin 3-galactoside, and trilobatin were metabolite biomarkers of antioxidant properties in purple rice grains. This study provides new ideas for the cultivation of high-quality coloured rice varieties with high antioxidant activity.

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 anthocyanins and total flavonoids content in functional rice and its recombination inbred lines.

Anthocyanin is one of the flavonoids, which has strong antioxidant properties. Functional rice rich in anthocyanins can not only improve immunity, but also anti-radiation, beauty, anti-aging effect, very popular in the market. In this study, we used Zibaoxiangnuo 1 (ZBXN 1), a functional rice variety which is rich in total flavonoids and anthocyanins, as the experimental material to construct Recombination Inbred Lines (RILs) with Minghui63 (MH63), a variety without anthocyanins. The contents of anthocyanins and total flavonoids of RILs and two parents were determined for three consecutive generations. The average anthocyanin content of parent ZBXN 1 was 319.31 mg/kg, and the anthocyanin inheritance of RIL population was relatively stable, with 10 samples higher than ZBXN 1. In addition, there was no significant difference in the total flavonoids content between the two parents, the total flavonoids content of Z25 in RIL population was 0.33%. Based on these studies, we believe that ZBXN 1 has abundant and stable anthocyanins, which can be used as an intermediate breeding material for breeding high-quality varieties with high anthocyanins, and lay a foundation for breeding more anthocyanin-rich rice varieties.

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.

Processing affects (decreases or increases) metabolites, flavonoids, black rice pigment, and total antioxidant capacity of purple glutinous rice grains.

The objective of this study was to understand the effects of processing on metabolites, flavonoids, black rice pigments and total antioxidant capacity of purple grains. The biochemical indicators and metabolites were determined before and after processing of purple grains. The results showed that the total antioxidant capacity, total phenol (TP), flavonoid (PD), oligomeric proanthocyanidin (OPC), ascorbic acid (AsA), cyanidin-3-O-glucoside (C3OG), peonidin 3-glucoside (P3G) contents of purple grains were greatly decreased after brown rice grains were processed into polished rice grains. The TP, PD, OPC, AsA, C3OG, and P3G of Yangzinuo No.1 brown rice (YZN1_B) or polished rice grains (YZN1_H) were higher than those of Yangzinuo No.2 brown rice (YZN2_B) or polished rice grains (YZN2_H). 154 differential metabolites (DMs) were identified between YZN1_B and YZN1_H. 52 DMs were identified between YZN2_B and YZN2_H. Citric acid and isocyanate are key metabolites affected during processing and have good correlations with various biochemical indicators.

Transcriptome and Metabolome Analysis Provides Insights into the Heterosis of Yield and Quality Traits in Two Hybrid Rice Varieties (Oryza sativa L.).

Heterosis is a common biological phenomenon that is useful for breeding superior lines. Using heterosis to increase the yield and quality of crops is one of the main achievements of modern agricultural science. In this study, we analysed the transcriptome and metabolome of two three-line hybrid rice varieties, Taiyou 871 (TY871), and Taiyou 398 (TY398) and the parental grain endosperm using RNA-seq (three biological repeats per variety) and untargeted metabolomic (six biological repeats per variety) methods. TY871 and TY398 showed specific heterosis in yield and quality. Transcriptome analysis of the hybrids revealed 638 to 4059 differentially expressed genes in the grain when compared to the parents. Metabolome analysis of the hybrids revealed 657 to 3714 differential grain metabolites when compared to the parents. The honeydew1 and grey60 module core genes Os04g0350700 and Os05g0154700 are involved in the regulation of awn development, grain size, and grain number, as well as the regulation of grain length and plant height, respectively. Rice grain length may be an important indicator for improving the quality of three-line hybrid rice. In addition, the rice quality-related metabolite NEG_M341T662 was highly connected to the module core genes Os06g0254300 and Os03g0168100. The functions of Os06g0254300 and Os03g0168100 are EF-hand calcium binding protein and late embroideries absolute protein repeat containing protein, respectively. These genes may play a role in the formation of rice quality. We constructed a gene and metabolite coexpression network, which provides a scientific basis for the utilization of heterosis in producing high-yield and high-quality hybrid 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.

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.

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.

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.

[Cloning and sequence analysis of full-length trypsin cDNA of Culex pipiens pallens].

The expression difference of trypsin gene between deltamethrin-resistant strain and -susceptible strain of Culex pipiens pallens was further investigated, and the results showed that the expression of trypsin gene were respectively 4.3 and 3.9 fold more in the resistant strain than in the susceptible strain, by reverse Northern blot and Northern blot assays. A full-length trypsin cDNA of 909 base pairs (GenBank/NCBI AY034060) with an open reading frame of 786 base pairs was cloned from the constructed cDNA library by the rapid amplification of cDNA ends, and the deduced protein of 261 amino acids was 55% homologous with Anopheles gambiae trypsin.