The OsTIL1 lipocalin protects cell membranes from reactive oxygen species damage and maintains the 18:3-containing glycerolipid biosynthesis under cold stress in rice.

Lipocalins constitute a conserved protein family that binds to and transports a variety of lipids while fatty acid desaturases (FADs) are required for maintaining the cell membrane fluidity under cold stress. Nevertheless, it remains unclear whether plant lipocalins promote FADs for the cell membrane integrity under cold stress. Here, we identified the role of OsTIL1 lipocalin in FADs-mediated glycerolipid remodeling under cold stress. Overexpression and CRISPR/Cas9 mediated gene edition experiments demonstrated that OsTIL1 positively regulated cold stress tolerance by protecting the cell membrane integrity from reactive oxygen species damage and enhancing the activities of peroxidase and ascorbate peroxidase, which was confirmed by combined cold stress with a membrane rigidifier dimethyl sulfoxide or a H2 O2 scavenger dimethyl thiourea. OsTIL1 overexpression induced higher 18:3 content, and higher 18:3/18:2 and (18:2 + 18:3)/18:1 ratios than the wild type under cold stress whereas the gene edition mutant showed the opposite. Furthermore, the lipidomic analysis showed that OsTIL1 overexpression led to higher contents of 18:3-mediated glycerolipids, including galactolipids (monoglactosyldiacylglycerol and digalactosyldiacylglycerol) and phospholipids (phosphatidyl glycerol, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and phosphatidyl inositol) under cold stress. RNA-seq and enzyme linked immunosorbent assay analyses indicated that OsTIL1 overexpression enhanced the transcription and enzyme abundance of four ω-3 FADs (OsFAD3-1/3-2, 7, and 8) under cold stress. These results reveal an important role of OsTIL1 in maintaining the cell membrane integrity from oxidative damage under cold stress, providing a good candidate gene for improving cold tolerance in rice.

OsFBN7-OsKAS I module promotes formation of plastoglobules clusters in rice chloroplasts.

Plastoglobules (PGs) contiguous with the outer leaflets of thylakoid membranes regulate lipid metabolism, plastid developmental transitions, and responses to environmental stimuli. However, the function of OsFBN7, a PG-core fibrillin gene in rice, has not been elucidated. Using molecular genetics and physiobiochemical approaches, we observed that OsFBN7 overexpression promoted PG clustering in rice chloroplasts. OsFBN7 interacted with two KAS I enzymes, namely OsKAS Ia and OsKAS Ib, in rice chloroplasts. Lipidomic analysis of chloroplast subcompartments, including PGs in the OsFBN7 overexpression lines, confirmed that levels of diacylglycerol (DAG), a chloroplast lipid precursor and monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), the main chloroplast membrane lipids, were increased in PGs and chloroplasts. Furthermore, OsFBN7 enhanced the abundances of OsKAS Ia/Ib in planta and their stability under oxidative and heat stresses. In addition, RNA sequencing and real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses showed that the expression of the DAG synthetase gene PAP1 and MGDG synthase gene MDG2 was upregulated by OsFBN7. In conclusion, this study proposes a new model in which OsFBN7 binds to OsKAS Ia/Ib in chloroplast and enhances their abundance and stability, thereby regulating the chloroplast and PG membrane lipids involved in the formation of PG clusters.

Global identification and integrated analysis of heat-responsive long non-coding RNAs in contrasting rice cultivars.

KEY MESSAGE: Identified 2743 rice lncRNAs LncRNAs in response to heat stress Function prediction of HRLs Network among HRLs, genes and miRNAs co-localization of HRLs with QTLs Significant motifs in HRL sequences Long non-coding RNAs (lncRNAs) play vital roles in plant responses to environmental challenges. A better understanding of the gene regulation mediated by lncRNAs and their systematic identification would provide great benefits for modern agriculture. In this study, we performed strand-specific RNA sequencing for two rice varieties, heat-tolerant ZS97B and heat-susceptible SYD2 under heat stress. In total, 2743 putative lncRNAs were identified, and their expression profiles in response to heat treatments were established. We identified 231 differentially expressed lncRNAs (DELs) under heat stress, including 31 DELs common to both varieties and 103 and 97 specific to ZS97B and SYD2, respectively, all defined as heat-responsive lncRNAs (HRLs). The target-coding genes of HRLs were predicted, and GO and KEGG annotations of HRL targets revealed functions in which HRLs might be involved. The interaction network between HRLs, target genes and relevant miRNAs was constructed. The HRLs and their targets were compared with publicly available QTLs for rice seedling growth under heat stimulus. Ten HRLs and twelve target genes were linked with five heat stress-relevant QTLs. Sequence analysis revealed several motifs significantly enriched within the 231 HRL sequences. Our findings provide a valuable resource for further characterization of lncRNAs in terms of heat response and plant heat tolerance improvement.

Co-overexpression of the Constitutively Active Form of OsbZIP46 and ABA-Activated Protein Kinase SAPK6 Improves Drought and Temperature Stress Resistance in Rice.

Drought is one of the major abiotic stresses threatening rice (Oryza sativa) production worldwide. Drought resistance is controlled by multiple genes, and therefore, a multi-gene genetic engineering strategy is theoretically useful for improving drought resistance. However, the experimental evidence for such a strategy is still lacking. In this study, a few drought-responsive genes from rice were assembled by a multiple-round site-specific assembly system, and the constructs were introduced into the rice cultivar KY131 via Agrobacterium-mediated transformation. The transgenic lines of the multi-gene and corresponding single-gene constructs were pre-evaluated for drought resistance. We found that the co-overexpression of two genes, encoding a constitutively active form of a bZIP transcription factor (OsbZIP46CA1) and a protein kinase (SAPK6) involved in the abscisic acid signaling pathway, showed significantly enhanced drought resistance compared with the single-gene transgenic lines and the negative transgenic plants. Single-copy lines of this bi-gene combination (named XL22) and the corresponding single-gene lines were further evaluated for drought resistance in the field using agronomical traits. The results showed that XL22 exhibited greater yield, biomass, spikelet number, and grain number under moderate drought stress conditions. The seedling survival rate of XL22 and the single-gene overexpressors after drought stress treatment also supported the drought resistance results. Furthermore, expression profiling by RNA-Seq revealed that many genes involved in the stress response were specifically up-regulated in the drought-treated XL22 lines and some of the stress-related genes activated in CA1-OE and SAPK6-OE were distinct, which could partially explain the different performances of these lines with respect to drought resistance. In addition, the XL22 seedlings showed improved tolerance to heat and cold stresses. Our results demonstrate that the multi-gene assembly in an appropriate combination may be a promising approach in the genetic improvement of drought resistance.

Comparative transcriptome analysis highlights the crucial roles of photosynthetic system in drought stress adaptation in upland rice.

Drought stress is one of the major adverse environmental factors reducing plant growth. With the aim to elucidate the underlying molecular basis of rice response to drought stress, comparative transcriptome analysis was conducted between drought susceptible rice cultivar Zhenshan97 and tolerant cultivar IRAT109 at the seedling stage. 436 genes showed differential expression and mainly enriched in the Gene Ontology (GO) terms of stress defence. A large number of variations exist between these two genotypes including 2564 high-quality insertion and deletions (INDELs) and 70,264 single nucleotide polymorphism (SNPs). 1041 orthologous gene pairs show the ratio of nonsynonymous nucleotide substitution rate to synonymous nucleotide substitutions rate (Ka/Ks) larger than 1.5, indicating the rapid adaptation to different environments during domestication. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of positive selection genes suggested that photosynthesis represents the most significant category. The collocation of positively selected genes with the QTLs of photosynthesis and the different photosynthesis performance of these two cultivars further illuminate the crucial function of photosynthesis in rice adaptation to drought stress. Our results also provide fruitful functional markers and candidate genes for future genetic research and improvement of drought tolerance in rice.

Identification of a cluster of PR4-like genes involved in stress responses in rice.

PR4 proteins constitute a pathogenesis-related (PR) protein family with a conserved BARWIN domain. In this study, we analyzed PR4-homologous genes in rice (Oryza sativa L.) and identified five putative PR4 genes designated as OsPR4a-e. The five PR4 genes are located in tandem on chromosome 11 and constitute a gene cluster with high sequence similarity to each other. The OsPR4 proteins have high sequence similarity to reported PR4 proteins from monocotyledonous species and are predicted to be class II PR4 proteins. Distinct diversification of plant PR4 proteins exists between monocotyledonous and dicotyledonous plants. Except for OsPR4e, which was not detected with any transcript, the other four OsPR4 genes showed diverse temporal-spatial expression patterns, and their expressions are responsive to Magnaporthe grisea infection. Interestingly, the OsPR4 genes are also responsive to abiotic stresses. Their expression levels were strongly induced by at least one of the stress treatments including drought, salt, cold, wounding, heat shock, and ultraviolet. The transcript levels of OsPR4 genes were also induced by some phytohormones such as abscisic acid and jasmonic acid. Transgenic rice with overexpression of OsPR4a showed enhanced tolerance to drought at both seedling and reproductive stages. We conclude that rice PR4 genes are also involved in abiotic stress responses and tolerance in addition to their responsiveness to pathogen attacks.

Evaluation of seven function-known candidate genes for their effects on improving drought resistance of transgenic rice under field conditions.

Many stress responsive genes have been reported with an effect on improving stress resistance in model plants under greenhouse conditions. Towards identification of genes for drought resistance breeding, seven well documented genes (CBF3, SOS2, NCED2, NPK1, LOS5, ZAT10, and NHX1) in stress resistance were selected in this study and transformed into rice cultivar Zhonghua 11 under the control of constitutive promoter Actin1 and stress-inducible promoter of a rice HVA22 homolog, and transgenic rice were tested for drought resistance under field conditions. A total of 1598 independent transgenic T0 plants were generated. The percentages of single copy and expression of the transgenes were 36.7% and 57.6%, respectively. For each gene construct, 30 T1 families with expression of transgene were selected for drought resistance testing at the reproductive stage in field, and 10 of them were tested in PVC pipes with a defined stress protocol at the same stage. Relative yield and relative spikelet fertility were used as two major criteria to evaluate drought resistance performance because significantly decreased yield was observed in the T1 generation. Transgenic families of eight constructs (HVA22P:CBF3, HVA22P:NPK1, Actin1:LOS5, HVA22P:LOS5, Actin1:ZAT10, HVA22P:ZAT10, Actin1:NHX1, and HVA22P:NHX1) showed significantly higher RY than wild-type (WT) under both drought stress field and PVC tube conditions. Transgenic families of 9 constructs (HVA22P:SOS2 and CBF3, LOS5, ZAT10, and NHX1 by both promoters) showed significantly higher relative spikelet fertility than WT in the field or PVC pipes. In the field drought resistance testing of T2 families derived from the T1 families with relatively lower yield decrease, transgenic families of seven constructs (HVA22P:CBF3, Actin1:NPK1, HVA22P:NPK1, Actin1:LOS5, HVA22P:LOS5, Actin1:ZAT10, and HVA22P:ZAT10) showed significantly higher yield per plant than WT, and families of nine constructs (Actin1:CBF3, HVA22P:CBF3, HVA22P:SOS2, HVA22P:NPK1, Actin1:LOS5, HVA22P:LOS5, Actin1:ZAT10, HVA22P:ZAT10, and Actin1:NHX1) had higher spikelet fertility than WT. In general, LOS5 and ZAT10 showed relatively better effect than the other five genes in improving drought resistance of transgenic rice under field conditions. The results and experience obtained from this study could be a useful reference for drought resistance engineering in rice.

Characterization of a stress responsive proteinase inhibitor gene with positive effect in improving drought resistance in rice.

A full-length cDNA gene, designated Oryza sativa chymotrypsin inhibitor-like 1 (OCPI1), was characterized in rice. The predicted protein of OCPI1 shows very high sequence identity to reported chymotrypsin inhibitors from various plant species. Northern-blot analysis showed that the expression of OCPI1 was strongly induced by dehydration stresses and abscisic acid (ABA). The expression of beta-glucuronidase (GUS) reporter gene under the control of OCPI1 promoter transformed into rice was strongly induced by drought and salt stresses. Interestingly, strong dehydration stress-induced GUS activity was also detected in the transgenic rice containing the reverse sequence of OCPI1 promoter fused to GUS gene, suggesting of a bidirectional transcriptional activity in the OCPI1 promoter. OCPI1 gene was over-expressed in japonica cv. Zhonghua 11 and transgenic plants containing single copy of transgene were tested for drought resistance at reproductive stage. The positive transgenic plants (OCPI1 was over-expressed) had significantly higher grain yield and seed setting rate than the wild type and the negative transgenic control (no over-expression of the transgene) under the severe drought stress conditions, whereas the potential yield of transgenic plants under normal growth conditions was not affected. Chymotrypsin-inhibitor activity assay showed that the crude protein of the positive transgenic plants had stronger inhibitory activity than the negative control. Transgenic plants had less decrease of total proteins than the wild type under drought stress. Taken together, these data indicate that OCPI1 might potentially be useful in the genetic improvement of drought resistance in rice.

Over-expression of a LEA gene in rice improves drought resistance under the field conditions.

Late embryogenesis abundant (LEA) proteins have been implicated in many stress responses of plants. In this report, a LEA protein gene OsLEA3-1 was identified and over-expressed in rice to test the drought resistance of transgenic lines under the field conditions. OsLEA3-1 is induced by drought, salt and abscisic acid (ABA), but not by cold stress. The promoter of OsLEA3-1 isolated from the upland rice IRAT109 exhibits strong activity under drought- and salt-stress conditions. Three expression constructs consisting of the full-length cDNA driven by the drought-inducible promoter of OsLEA3-1 (OsLEA3-H), the CaMV 35S promoter (OsLEA3-S), and the rice Actin1 promoter (OsLEA3-A) were transformed into the drought-sensitive japonica rice Zhonghua 11. Drought resistance pre-screening of T(1) families at anthesis stage revealed that the over-expressing families with OsLEA3-S and OsLEA3-H constructs had significantly higher relative yield (yield under drought stress treatment/yield under normal growth conditions) than the wild type under drought stress conditions, although a yield penalty existed in T(1) families under normal growth conditions. Nine homozygous families, exhibiting over-expression of a single-copy of the transgene and relatively low yield penalty in the T(1) generation, were tested in the field for drought resistance in the T(2) and T(3) generations and in the PVC pipes for drought tolerance in the T(2) generation. Except for two families (transformed with OsLEA3-A), all the other families (transformed with OsLEA3-S and OsLEA3-H constructs) had higher grain yield than the wild type under drought stress in both the field and the PVC pipes conditions. No significant yield penalty was detected for these T(2 )and T(3) families. These results indicate that transgenic rice with significantly enhanced drought resistance and without yield penalty can be generated by over-expressing OsLEA3-1 gene with appropriate promoters and following a bipartite (stress and non-stress) in-field screening protocol.

Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice.

Drought and salinity are major abiotic stresses to crop production. Here, we show that overexpression of stress responsive gene SNAC1 (STRESS-RESPONSIVE NAC 1) significantly enhances drought resistance in transgenic rice (22-34% higher seed setting than control) in the field under severe drought stress conditions at the reproductive stage while showing no phenotypic changes or yield penalty. The transgenic rice also shows significantly improved drought resistance and salt tolerance at the vegetative stage. Compared with WT, the transgenic rice are more sensitive to abscisic acid and lose water more slowly by closing more stomatal pores, yet display no significant difference in the rate of photosynthesis. SNAC1 is induced predominantly in guard cells by drought and encodes a NAM, ATAF, and CUC (NAC) transcription factor with transactivation activity. DNA chip analysis revealed that a large number of stress-related genes were up-regulated in the SNAC1-overexpressing rice plants. Our data suggest that SNAC1 holds promising utility in improving drought and salinity tolerance in rice.