Zhonghua 11 Research Articles

Exogenous MgH2-derived hydrogen alleviates cadmium toxicity through m6A RNA methylation in rice

Cadmium (Cd) contamination poses a substantial threat to crop yields and human health. While magnesium hydride (MgH2) has been reported as a hydrogen (H2) donor that promotes plant growth under heavy metal contamination, its role in rice remains elusive. Herein, seedlings of Oryza sativa L. Japonica variety Zhonghua 11 (ZH11) were selected and exposed to 20 µL of 1-mol/L cadmium chloride (CdCl2) solution via hydroponics to simulate Cd stress. Meanwhile, 0.1 mg of MgH2 was used to slow-release H2 to the experimental group to explore its potential effects on rice over a 2-week period. The results indicated that Cd exposure severely inhibited the growth and development of ZH11 rice seedlings. However, the exogenous slow-release of H2 from MgH2 effectively mitigated this inhibitory effect by restoring the balance of reactive oxygen species (ROS), maintaining endogenous H2 homeostasis, and supporting the photosynthetic system. High-performance liquid chromatography analysis revealed that exogenous H2 reduces m6A RNA methylation levels in mRNA under Cd stress. Consequently, MeRIP-seq was conducted to investigate the effect of Cd exposure in rice in the presence and absence of H2. The m6A modifications were enriched at the start codon, stop codon, and 3′ UTR. By integrating RNA-seq data, 118 transcripts were identified as differentially methylated and expressed genes under Cd stress. These gene annotations were associated with ROS, biological stress, and hormonal responses. Notably, 297 differentially methylated and expressed genes were identified under Cd stress in the presence of H2, linked to heavy metals, protein kinases, and calcium signaling regulation. Cd strongly activates the MAPK pathway in response to stress. Exogenous H2 reduces Cd accumulation as well as enhances plant tolerance and homeostasis by lowering m6A levels, thereby decreasing the mRNA stability of these genes. Our findings indicate that MgH2, by supplying H2, regulates gene expression through m6A RNA methylation and confers Cd tolerance in rice. This study provides potential candidate genes for studying the remediation of heavy metal pollution in plants.

Controlling diurnal flower-opening time by manipulating the jasmonate pathway accelerates development of indica-japonica hybrid rice breeding.

Inter-subspecific indica-japonica hybrid rice (Oryza sativa) has the potential for increased yields over traditional indica intra-subspecies hybrid rice, but limited yield of F1 hybrid seed production (FHSP) hinders the development of indica-japonica hybrid rice breeding. Diurnal flower-opening time (DFOT) divergence between indica and japonica rice has been a major contributing factor to this issue, but few DFOT genes have been cloned. Here, we found that manipulating the expression of jasmonate (JA) pathway genes can effectively modulate DFOT to improve the yield of FHSP in rice. Treating japonica cultivar Zhonghua 11 (ZH11) with methyl jasmonate (MeJA) substantially advanced DFOT. Furthermore, overexpressing the JA biosynthesis gene OPDA REDUCTASE 7 (OsOPR7) and knocking out the JA inactivation gene CHILLING TOLERANCE 1 (OsHAN1) in ZH11 advanced DFOT by 1- and 2-h respectively; and knockout of the JA signal suppressor genes JASMONATE ZIM-DOMAIN PROTEIN 7 (OsJAZ7) and OsJAZ9 resulted in 50-min and 1.5-h earlier DFOT respectively. The yields of FHSP using japonica male-sterile lines GAZS with manipulated JA pathway genes were significantly higher than that of GAZS wildtype. Transcriptome analysis, cytological observations, measurements of elastic modulus and determination of cell wall components indicated that the JA pathway could affect the loosening of the lodicule cell walls by regulating their composition through controlling sugar metabolism, which in turn influences DFOT. This research has vital implications for breeding japonica rice cultivars with early DFOT to facilitate indica-japonica hybrid rice breeding.

Overexpression of ATP Synthase Subunit Beta (Atp2) Confers Enhanced Blast Disease Resistance in Transgenic Rice.

Previous research has shown that the pathogenicity and appressorium development of Magnaporthe oryzae can be inhibited by the ATP synthase subunit beta (Atp2) present in the photosynthetic bacterium Rhodopseudomonas palustris. In the present study, transgenic plants overexpressing the ATP2 gene were generated via genetic transformation in the Zhonghua11 (ZH11) genetic background. We compared the blast resistance and immune response of ATP2-overexpressing lines and wild-type plants. The expression of the Atp2 protein and the physiology, biochemistry, and growth traits of the mutant plants were also examined. The results showed that, compared with the wild-type plant ZH11, transgenic rice plants heterologously expressing ATP2 had no significant defects in agronomic traits, but the disease lesions caused by the rice blast fungus were significantly reduced. When infected by the rice blast fungus, the transgenic rice plants exhibited stronger antioxidant enzyme activity and a greater ratio of chlorophyll a to chlorophyll b. Furthermore, the immune response was triggered stronger in transgenic rice, especially the increase in reactive oxygen species (ROS), was more strongly triggered in plants. In summary, the expression of ATP2 as an antifungal protein in rice could improve the ability of rice to resist rice blast.

Effects of stacking breeding on the methylome and transcriptome profile of transgenic rice with glyphosate tolerance.

Transcriptomics and methylomics were used to identify the potential effects resulting from GM rice breeding stacks, which provided scientific data for the safety assessment strategy of stacked GM crops in China. Gene interaction is one of the main concerns for stacked genetically modified crop safety. With the development of technology, the combination of omics and bioinformatics has become a useful tool to evaluate the unintended effects of genetically modified crops. In this study, transcriptomics and methylomics were used as molecular profiling techniques to identify the potential effects of stack through breeding. Stacked transgenic rice En-12 × Ec-26 was used as material, which was obtained through hybridization using parents En-12 and Ec-26, in which the foreign protein can form functional EPSPS protein by intein-mediated trans-splitting. Differentially methylated region (DMR) analysis showed that the effect of stacking breeding on methylation was less than that of genetic transformation at the methylome level. Differentially expressed gene (DEG) analysis showed that the DEGs between En-12 × Ec-26 and its parents were far fewer than those between transgenic rice and Zhonghua 11 (ZH11), and no unintended new genes were found in En-12 × Ec-26. Statistical analysis of gene expression and methylation involved in shikimic acid metabolism showed that there was no difference in gene expression, although there were 16 and 10 DMR genes between En-12 × Ec-26 and its parents (En and Ec) in methylation, respectively. The results indicated that the effect of stacking breeding on gene expression and DNA methylation was less than the effect of genetic transformation. This study provides scientific data supporting safety assessments of stacked GM crops in China.

Transcriptome and Proteome Analysis Revealed That Hormone and Reactive Oxygen Species Synergetically Regulate Dormancy of Introgression Line in Rice (Oryza sativa L.)

Dormancy is a complex agronomy phenotype controlled by multiple signaling and a key trait repressing pre-harvest sprouting (PHS). However, the signaling network of dormancy remains unclear. In this study, we used Zhonghua11 (ZH11) with a weak dormancy, and Introgression line (IL) with a strong dormancy to study the mechanism of hormones and reactive oxygen species (ROS) crosstalk regulating rice dormancy. The germination experiment showed that the germination rate of ZH11 was 76.86%, while that of IL was only 1.25%. Transcriptome analysis showed that there were 1658 differentially expressed genes (DEGs) between IL and ZH11, of which 577 were up-regulated and 1081 were down-regulated. Additionally, DEGs were mainly enriched in oxidoreductase activity, cell periphery, and plant hormone signal transduction pathways. Tandem mass tags (TMT) quantitative proteomics analysis showed 275 differentially expressed proteins (DEPs) between IL and ZH11, of which 176 proteins were up-regulated, 99 were down-regulated, and the DEPs were mainly enriched in the metabolic process and oxidation-reduction process. The comprehensive transcriptome and proteome analysis showed that their correlation was very low, and only 56 genes were co-expressed. Hormone content detection showed that IL had significantly lower abscisic acid (ABA) contents than the ZH11 while having significantly higher jasmonic acid (JA) contents than the ZH11. ROS content measurement showed that the hydrogen peroxide (H2O2) content of IL was significantly lower than the ZH11, while the production rate of superoxide anion (O2.-) was significantly higher than the ZH11. These results indicate that hormones and ROS crosstalk to regulate rice dormancy. In particular, this study has deepened our mechanism of ROS and JA crosstalk regulating rice dormancy and is conducive to our precise inhibition of PHS.

OsGRF4AA compromises heat tolerance of developing pollen grains in rice.

Extreme high temperature at the meiosis stage causes a severe decrease in spikelet fertility and grain yield in rice. The rice variety grain size on chromosome 2 (GS2) contains sequence variations of OsGRF4 (Oryza sativa growth-regulating factor 4; OsGRF4AA ), escaping the microRNA miR396-mediated degradation of this gene at the mRNA level. Accumulation of OsGRF4 enhances nitrogen usage and metabolism, and increases grain size and grain yield. In this study, we found that pollen viability and seed-setting rate under heat stress (HS) decreased more seriously in GS2 than in its comparator, Zhonghua 11 (ZH11). Transcriptomic analysis revealed that, following HS, genes related to carbohydrate metabolic processes were expressed and regulated differentially in the anthers of GS2 and ZH11. Moreover, the expression of genes involved in chloroplast development and photosynthesis, lipid metabolism, and key transcription factors, including eight male sterile genes, were inhibited by HS to a greater extent in GS2 than in ZH11. Interestingly, pre-mRNAs of OsGRF4, and a group of essential genes involved in development and fertilization, were differentially spliced in the anthers of GS2 and ZH11. Taken together, our results suggest that variation in OsGRF4 affects proper transcriptional and splicing regulation of genes under HS, and that this can be mediated by, and also feed back to, carbohydrate and nitrogen metabolism, resulting in a reduction in the heat tolerance of rice anthers.

Overexpression of a beta-1,6-glucanase gene GluM in transgenic rice confers high resistance to rice blast, sheath blight and false smut.

Frequent fungal diseases tend to lead to severe losses in rice production. As a main component of the fungal cell wall, glucan plays an important role in the growth and development of fungi. Glucanase can inhibit the growth of fungi by breaking glycosidic bonds, and may be a promising target for developing rice varieties with broad-spectrum disease resistance. We transferred a codon-optimized β-1,6-glucanase gene (GluM) from myxobacteria into the japonica rice variety Zhonghua11 (ZH11), and obtained a large number of individual transgenic plants with GluM overexpression. Based on molecular analysis, three single-copy homozygous lines with GluM overexpression were selected for assessment of fungal disease resistance at the T3 generation. Compared with that of the recipient cultivar ZH11, the area of rice blast lesion in transgenic rice was reduced by 82.71%; that of sheath blight lesion was decreased by 35.76%-43.67%; the sheath blight resistance in the field was enhanced by an average of 0.75 grade over 3 years; and the incidence of diseased panicles due to rice false smut was decreased by 65.79%. More importantly, there was no obvious loss of yield (without a significant effect on agronomic traits). Furthermore, plants overexpressing a β-1,6-glucanase gene showed higher disease resistance than rice plants overexpressing a β-1,3-glucanase gene derived from tobacco. The β-1,6-glucanase gene GluM can confer broad-spectrum disease resistance to rice, providing an environmentally friendly alternative way to effectively manage fungal pathogens in rice production. © 2023 Society of Chemical Industry.

Seed priming with calcium chloride enhances stress tolerance in rice seedlings

Calcium is a crucial second messenger in plant cells and contributes to plant resistance against biotic and abiotic stress. Plant defense priming with natural or synthetic compounds leads to quicker and stronger resistance responses. However, whether pretreatment of plant seeds with calcium could improve their resistance to stress remains poorly understood. In this study, we showed that rice seedlings grown from calcium chloride (CaCl2)-pretreated seeds displayed enhanced resistance to the rice blast fungus Magnaporthe oryzae and the rice bacterial pathogen Xanthomonas oryzae pv. Oryzae (Xoo). Seed priming with CaCl2 also led to enhanced rice tolerance to salt and cold. Furthermore, the reactive oxygen species (ROS) burst increased significantly upon immunity activation in the leaves of rice seedlings grown from CaCl2-pretreated seeds. Additionally, we analyzed the rice calmodulin-binding protein 60 (OsCBP60) family and found that there were 19 OsCBP60s in rice cultivar Zhonghua 11 (ZH11). The transcripts of several OsCBP60s were chitin- and M. oryzae-inducible, suggesting that they may contribute to rice resistance. Taken together, these data indicate that seed priming with CaCl2 can effectively enhance rice tolerance to multiple stresses, perhaps by boosting the burst of ROS, and OsCBP60 family members may also play an essential role in this process.

Mechanism of drought and salt tolerance of <italic>OsLPL2/PIR </italic>gene in rice

<p id="C3">Drought threatens global agricultural production and limits the prospects for sustainable agricultural development. Plant leaf epidermis plays a vital role in the process of growth, development, and resistance to adversity stress, and water and gas exchange with the external environment. In this study, compared with the wild-type Zhonghua 11 (ZH11), we found that mutants <italic>less pronounced lobe epidermal cell 2-1</italic> (<italic>lpl2-1</italic>) and<italic> less pronounced lobe epidermal cell 2-2</italic> (<italic>lpl2-2</italic>) were more sensitive to drought and salt stress response, and the survival rate after rewatering was extremely significantly reduced, which was less than half of the control. Compared with ZH11, <italic>lpl2-1 </italic>and<italic> lpl2-2</italic> had shorter plant height, shorter root length, significantly increased stomatal density and stomatal openings in the same phyllodes, and the serrated lobe of the epidermal cell margin becomes smoother, and the epidermal cell nesting was not tight, which might result in faster and more water loss of <italic>lpl2-1</italic> and <italic>lpl2-2</italic> than ZH11. The water loss experiment of separated leaves also proved that the water loss rate of <italic>lpl2-1</italic> and <italic>lpl2-2</italic> leaves was higher than that of the ZH11 in equal time. Overexpression of <italic>OsLPL2</italic> was transferred into <italic>lpl2-1</italic>, and the <italic>OE-OsLPL2</italic>/<italic>lpl2-1</italic> transgenic positive plants recovered the smooth epidermis of <italic>lpl2-1</italic> and the sensitive phenotype to drought and salt stress. These results showed that <italic>OsLPL2</italic> gene not only controlled the microfilament synthesis and morphogenesis of rice epidermal cells, but also played a key role in response to plant stress by regulating stomatal density, stomatal conductance, and root growth and development. This study provides a theoretical basis for revealing the molecular regulation mechanism of <italic>OsLPL2</italic> in response to drought stress in rice.

The GzMYB-7D1 gene of Guizimai No.1 wheat is essential for seed anthocyanins accumulation and yield regulation

Anthocyanins are antioxidants with important benefits for human health. Therefore, they have caught the interest of plant breeding programs. In this study, GzMYB-7D1, the key gene responsible for anthocyanin synthesis regulation in the purple Guizimai No.1 wheat, was transferred into Zhonghua 11 (ZH11) rice. Compared to wild-type ZH11, anthocyanin accumulated in the seeds of GzMYB-7D1 overexpressing lines. Furthermore, anthocyanin content kept increasing in the growing panicle of GzMYB-7D1 overexpressing lines, accumulating mostly in the rice glumes and grains during maturation, along with a concomitant steady decrease in chlorophyll. Genes related to anthocyanin synthesis, including OsPAL4, Os4CL3, OsCHS, OsDFR, OsANS, and Os3GT, exhibited much higher expression in the panicles of GzMYB-7D1 overexpressing lines than in those of wild-type ZH11. Interestingly, the grain yield per plant was significantly improved in GzMYB-7D1 overexpressing lines, as indicated by a higher tiller number per plant and branching of the secondary panicle, together with a significantly higher content of total amino acids. In conclusion, the GzMYB-7D1 gene of Guizimai No.1 wheat is essential for regulating seed anthocyanin levels and grain yield in rice, and could be applied to attain rice varieties with better nutritional value and improved yields.

An Integration of MicroRNA and Transcriptome Sequencing Analysis Reveal Regulatory Roles of miRNAs in Response to Chilling Stress in Wild Rice.

A chromosome single segment substitution line (CSSL) DC90, which was generated by introgressing CTS-12, a locus derived from common wild rice (Oryza rufipogon Griff.), into the 9311 (Oryza sativa L. ssp. indica) background, exhibits a chilling tolerance phenotype under chilling stress. Here, an integration of microRNA (miRNA) deep sequencing and transcriptomic sequencing analysis was performed to explore the expression profiles of miRNAs and their target genes mediated by CTS-12 under chilling stress, and to reveal the possible regulatory mechanisms of miRNAs that are involved in chilling tolerance. Integration analysis revealed that a number of differentially expressed miRNAs (DEMs) and putative target genes with different expression patterns and levels were identified in 9311 and DC90 under chilling stress. KEGG enrichment analysis revealed that the target genes that are regulated by chilling-induced miRNAs are involved in the regulation of various biological processes/pathways, including protein biosynthesis, redox process, photosynthetic process, and chloroplast development in two genotypes. CRISPR/Cas9 editing of the target genes of the key DEMs in a chilling tolerant rice variety Zhonghua 11 (ZH11) found that LOC_Os11g48020 (OsGL1-11), one of the putative target genes of osa-miR1846a/b-5p and encoding a wax synthesis protein, is correlated with a chilling stress tolerance phenotype, implying osa-miR1846a/b-5p/OsGL1-11 plays an important role in CTS-12-mediated chilling stress tolerance regulatory pathway(s). Therefore, we speculate that the CTS-12 may regulate the key miRNA target genes in response to chilling stress by differential regulation of miRNAs in wild rice, thereby resulting in the variation of chilling tolerance phenotype between 9311 and DC90.

Transcription factor OsMADS25 improves rice tolerance to cold stress.

Cold stress is the limiting factor of rice growth and production, and it is important to clone cold stress tolerant genes and cultivate cold tolerance rice varieties. The MADS transcription factors play an important role in abiotic stress signaling in rice. This study showed that OsMADS25 was up-regulated by low temperature and abscisic acid (ABA), suggesting that OsMADS25 may be involved in ABA-dependent signaling. The OsMADS25 overexpression vector, pCambia1300-221-OsMADS25-Flag, was constructed and introduced into the rice variety Zhonghua 11 (ZH11) through Agrobacterium tumefacian-mediated genetic transformation. Two homozygous lines with high expression levels were selected for phenotypic identification. OsMADS25 overexpression lines show significantly improved cold stress tolerance and the sensitivity to ABA at the seedling stage of rice. Reactive oxygen species (ROS) was detected by diaminobenzidine (DAB) staining and nitroblue tetrazolium (NBT) staining. After treatment with cold stress, little ROS accumulation was observed in OsMADS25 overexpression lines compared to wild-type ZH11. In conclusion, OsMADS25 plays a role in scavenging reactive oxygen species (ROS) and could improve rice tolerance to cold stress involved in ABA-dependent pathway.

OsPRR37 Alternatively Promotes Heading Date Through Suppressing the Expression of Ghd7 in the Japonica Variety Zhonghua 11 under Natural Long-Day Conditions

Heading date is an important agronomic trait of rice (Oryza sativa L.) and is regulated by numerous genes, some of which exhibit functional divergence in a genetic background-dependent manner. Here, we identified a late heading date 7 (lhd7) mutant that flowered later than wild-type Zhonghua 11 (ZH11) under natural long-day (NLD) conditions. Map-based cloning facilitated by the MutMap strategy revealed that LHD7 was on the same locus as OsPRR37 but exhibited a novel function as a promoter of heading date. A single-nucleotide mutation of G-to-A in the coding region caused a substitution of aspartic acid for glycine at site 159 within the pseudo-receiver (PR) domain of OsPRR37. Transcriptional analysis revealed that OsPRR37 suppressed Ghd7 expression in both ZH11 background under NLD conditions and the Zhenshan 97 background under natural short-day conditions. Consistently, the expression of Ehd1, Hd3a and RFT1 was enhanced by OsPRR37 in the ZH11 background. Genetic analysis indicated that the promotion of heading date and reduction in grain yield by OsPRR37 were partially dependent on Ghd7. Further investigation showed that the alternative function of OsPRR37 required an intact Ghd7-related regulatory pathway involving not only its upstream regulators OsGI and PhyB but also its interacting partner Hd1. Our study revealed the distinct role of OsPRR37 in the ZH11 background, which provides a more comprehensive understanding of OsPRR37 function and enriches the theoretical bases for improvement of rice heading date in the future.

Rice SST Variation Shapes the Rhizosphere Bacterial Community, Conferring Tolerance to Salt Stress through Regulating Soil Metabolites.

Some plant-specific resistance genes could affect rhizosphere microorganisms by regulating the release of root exudates. In a previous study, the SST (seedling salt tolerant) gene in rice (Oryza sativa) was identified, and loss of SST function resulted in better plant adaptation to salt stress. However, whether the rice SST variation could alleviate salt stress via regulating soil metabolites and microbiota in the rhizosphere is still unknown. Here, we used transgenic plants with SST edited in the Huanghuazhan (HHZ) and Zhonghua 11 (ZH11) cultivars by the CRISPR/Cas9 system and found that loss of SST function increased the accumulation of potassium and reduced the accumulation of sodium ions in rice plants. Using 16S rRNA gene amplicon high-throughput sequencing, we found that the mutant material shifted the rhizobacterial assembly under salt-free stress. Importantly, under salt stress, the sst, HHZcas, and ZH11cas plants significantly changed the assembly of the rhizobacteria. Furthermore, the rice SST gene also affected the soil metabolites, which were closely related to the dynamics of rhizosphere microbial communities, and we further determined the relationship between the rhizosphere microbiota and soil metabolites. Overall, our results show the effects of the rice SST gene on the response to salt stress associated with the soil microbiota and metabolites in the rhizosphere. This study reveals a helpful linkage among the rice SST gene, soil metabolites, and rhizobacterial community assembly and also provides a theoretical basis for improving crop adaptation through soil microbial management practices.IMPORTANCE Soil salinization is one of the major environmental stresses limiting crop productivity. Crops in agricultural ecosystems have developed various strategies to adapt to salt stress. We used rice mutant and CRISPR-edited lines to investigate the relationships among the Squamosa promoter Binding Protein box (SBP box) family gene (SST/OsSPL10), soil metabolites, and the rhizosphere bacterial community. We found that during salt stress, there are significant differences in the rhizosphere bacterial community and soil metabolites between the plants with the SST gene and those without it. Our findings provide a useful paradigm for revealing the roles of key genes of plants in shaping rhizosphere microbiomes and their relationships with soil metabolites and offer new insights into strategies to enhance rice tolerance to high salt levels from microbial and ecological perspectives.

Identification of FERONIA-like receptor genes involved in rice-Magnaporthe oryzae interaction

The receptor-like kinase (RLK) FERONIA functions in immunity in Arabidopsis. Here, we systemically screened rice RLK genes encoding FERONIA-like receptor (FLRs) that may be involved in rice-Magnaporthe oryzae interaction. The expression of 16 FLR genes was examined in response to the infection of M. oryzae in different rice varieties. For each FLR gene, at least two independent mutants were generated by CRISPR/Cas9 gene-editing technology in rice variety Zhonghua 11 (ZH11). Blast disease assay identified that the mutants of FLR1 and FLR13 showed increased susceptibility, whereas the mutants of FLR2 and FLR11 displayed enhanced resistance. Consistently, the mutant of FLR1 enhanced, but the mutant of FLR2 delayed the M. oryzae infection progress, which might be associated with the altered expression of defense-related genes. Together, these data indicate that at least 4 FLR genes are involved in rice-M. oryzae interaction and thus are potentially valuable in blast disease resistance.

OsNAC45 plays complex roles by mediating POD activity and the expression of development-related genes under various abiotic stresses in rice root

OsNAC45 belongs to the NAC family of (for NAM/ATAF1/2/CUC2) transcription factors, which play an important role in plant development and abiotic stress responses. Transgenic rice plants overexpressing OsNAC45 have exhibited improved tolerance to salt and drought stress. This study aimed to investigate the mechanism underlying the effects of OsNAC45 on root development under abiotic stress in rice Zhonghua11 (ZH11) (Oryza sativa L.). The OsNAC45 GUS expression was strong in roots of the adult stage, weak in the seedling stage, and induced by low-temperature stress in the seedling stage. Under NaCl and low temperature stress, the OsNAC45 overexpression (OE) lines exhibited maintenance of root structure and normal growth of the seedling root, but the RNAi lines of OsNAC45 were more sensitive. The roots of the OE lines accumulated less superoxide anion under low temperature stress and more hydrogen peroxide under normal conditions than WT. After NaCl treatment, the Peroxidase (POD) assay showed POD activity of the roots in OE lines were elevated, but were decreased in RNAi lines and WT; additionally, the increment of lignin content in OE lines was significantly higher than RNAi lines and WT in roots. Furthermore, expression levels of some rice root development-related genes were altered in the transgenic plants. Under cold and salt stresses, expression of NCED4, a key enzyme in abscisic acid synthesis, was decreased in OE lines and raised in RNAi lines compared to WT. The data suggests that OsNAC45 may participate in the response of cold/salt stresses by lessening the accumulation of reactive oxygen species and increasing lignin in rice roots thus alleviating the restraint to the growth of seedling root. Overexpression of OsNAC45 weakened the high temperature tolerance; however, inhibiting its expression could improve the tolerance. Together, these results indicate that OsNAC45 plays an important role in the coordination, development, and response of rice plants under abiotic stresses.

OsJAZ1 Attenuates Drought Resistance by Regulating JA and ABA Signaling in Rice.

Jasmonates (JAs) and abscisic acid (ABA) are phytohormones known play important roles in plant response and adaptation to various abiotic stresses including salinity, drought, wounding, and cold. JAZ (JASMONATE ZIM-domain) proteins have been reported to play negative roles in JA signaling. However, direct evidence is still lacking that JAZ proteins regulate drought resistance. In this study, OsJAZ1 was investigated for its role in drought resistance in rice. Expression of OsJAZ1 was strongly responsive to JA treatment, and it was slightly responsive to ABA, salicylic acid, and abiotic stresses including drought, salinity, and cold. The OsJAZ1-overexpression rice plants were more sensitive to drought stress treatment than the wild-type (WT) rice Zhonghua 11 (ZH11) at both the seedling and reproductive stages, while the jaz1 T-DNA insertion mutant plants showed increased drought tolerance compared to the WT plants. The OsJAZ1-overexpression plants were hyposensitive to MeJA and ABA, whereas the jaz1 mutant plants were hypersensitive to MeJA and ABA. In addition, there were significant differences in shoot and root length between the OsJAZ1 transgenic and WT plants under the MeJA and ABA treatments. A subcellular localization assay indicated that OsJAZ1 was localized in both the nucleus and cytoplasm. Transcriptome profiling analysis by RNA-seq revealed that the expression levels of many genes in the ABA and JA signaling pathways exhibited significant differences between the OsJAZ1-overexpression plants and WT ZH11 under drought stress treatment. Quantitative real-time PCR confirmed the expression profiles of some of the differentially expressed genes, including OsNCED4, OsLEA3, RAB21, OsbHLH006, OsbHLH148, OsDREB1A, OsDREB1B, SNAC1, and OsCCD1. These results together suggest that OsJAZ1 plays a role in regulating the drought resistance of rice partially via the ABA and JA pathways.

Single-point Mutation of an Histidine-aspartic Domain-containing Gene involving in Chloroplast Ribosome Biogenesis Leads to White Fine Stripe Leaf in Rice

Plant leaves are a crucial organ associated closely with chloroplast development, photosynthesis rate and crop productivity. In this study, a white fine stripe leaf 1 (wfsl1) mutant was isolated and characterized from the japonica rice Zhonghua11 (ZH11) after ethyl methanesulfonate mutagenesis. The wfsl1 displayed white fine stripe leaves since tillering stage and abnormal chloroplast structure. Map-based cloning and Bioinformatic analysis indicated that WFSL1 on chromosome 1 contains an “A” to “T” substitution in protein coding region, and encodes a putative metal-dependent phosphohydrolase with HD domain at the N-terminus. WFSL1 was targeted to the chloroplasts and had higher expression in mature leaves and sheaths. RNA-seq analysis revealed that chloroplast development and photosynthesis genes were significantly affected in wfsl1 plants. Levels of WFSL1 and chloroplast encoded proteins were decreased in wfsl1 mutants via western blot analysis. Compared with WT, wfsl1 exhibits lower Chl content and defective in biogenesis of chloroplast ribosomes, which resulted in reduced grain yield. Taken together, our results show that WFSL1 is critical for chloroplast development, ribosome biogenesis, and light energy utilization, finally affects grain yield.

Development and Event-specific Detection of Transgenic Glyphosate-resistant Rice Expressing the G2-EPSPS Gene.

Glyphosate is a widely used herbicide, due to its broad spectrum, low cost, low toxicity, high efficiency, and non-selective characteristics. Rice farmers rarely use glyphosate as a herbicide, because the crop is sensitive to this chemical. The development of transgenic glyphosate-tolerant rice could greatly improve the economics of rice production. Here, we transformed the Pseudomonas fluorescens G2 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene G2-EPSPS, which conferred tolerance to glyphosate herbicide into a widely used japonica rice cultivar, Zhonghua 11 (ZH11), to develop two highly glyphosate-tolerant transgenic rice lines, G2-6 and G2-7, with one exogenous gene integration. Seed germination tests and glyphosate-tolerance assays of plants grown in a greenhouse showed that the two transgenic lines could greatly improve glyphosate-tolerance compared with the wild-type; The glyphosate-tolerance field test indicated that both transgenic lines could grow at concentrations of 20,000 ppm glyphosate, which is more than 20-times the recommended concentration in the field. Isolation of the flanking sequence of transgenic rice G2-6 indicated that the 5′-terminal of T-DNA was inserted into chromosome 8 of the rice genome. An event-specific PCR test system was established and the limit of detection of the primers reached five copies. Overall, the G2-EPSPS gene significantly improved glyphosate-tolerance in transgenic rice; furthermore, it is a useful candidate gene for the future development of commercial transgenic rice.

Plant architecture and grain yield are regulated by the novel DHHC-type zinc finger protein genes in rice (Oryza sativa L.)

In many plants, architecture and grain yield are affected by both the environment and genetics. In rice, the tiller is a vital factor impacting plant architecture and regulated by many genes. In this study, we cloned a novel DHHC-type zinc finger protein gene Os02g0819100 and its alternative splice variant OsDHHC1 from the cDNA of rice (Oryza sativa L.), which regulate plant architecture by altering the tiller in rice. The tillers increased by about 40% when this type of DHHC-type zinc finger protein gene was over-expressed in Zhong Hua 11 (ZH11) rice plants. Moreover, the grain yield of transgenic rice increased approximately by 10% compared with wild-type ZH11. These findings provide an important genetic engineering approach for increasing rice yields.