Jacalin-related lectin 45 (OsJRL45) isolated from 'sea rice 86' enhances rice salt tolerance at the seedling and reproductive stages.

BACKGROUND: Rice (Oryza sativa L.) is one of the most widely cultivated grain crops in the world that meets the caloric needs of more than half the world's population. Salt stress seriously affects rice production and threatens food security. Therefore, mining salt tolerance genes in salt-tolerant germplasm and elucidating their molecular mechanisms in rice are necessary for the breeding of salt tolerant cultivars. RESULTS: In this study, a salt stress-responsive jacalin-related lectin (JRL) family gene, OsJRL45, was identified in the salt-tolerant rice variety 'sea rice 86' (SR86). OsJRL45 showed high expression level in leaves, and the corresponding protein mainly localized to the endoplasmic reticulum. The knockout mutant and overexpression lines of OsJRL45 revealed that OsJRL45 positively regulates the salt tolerance of rice plants at all growth stages. Compared with the wild type (WT), the OsJRL45 overexpression lines showed greater salt tolerance at the reproductive stage, and significantly higher seed setting rate and 1,000-grain weight. Moreover, OsJRL45 expression significantly improved the salt-resistant ability and yield of a salt-sensitive indica cultivar, L6-23. Furthermore, OsJRL45 enhanced the antioxidant capacity of rice plants and facilitated the maintenance of Na+-K+ homeostasis under salt stress conditions. Five proteins associated with OsJRL45 were screened by transcriptome and interaction network analysis, of which one, the transmembrane transporter Os10g0210500 affects the salt tolerance of rice by regulating ion transport-, salt stress-, and hormone-responsive proteins. CONCLUSIONS: The OsJRL45 gene isolated from SR86 positively regulated the salt tolerance of rice plants at all growth stages, and significantly increased the yield of salt-sensitive rice cultivar under NaCl treatment. OsJRL45 increased the activity of antioxidant enzyme of rice and regulated Na+/K+ dynamic equilibrium under salinity conditions. Our data suggest that OsJRL45 may improve the salt tolerance of rice by mediating the expression of ion transport-, salt stress response-, and hormone response-related genes.

OsJRL40, a Jacalin-Related Lectin Gene, Promotes Salt Stress Tolerance in Rice.

High salinity is a major stress factor affecting the quality and productivity of rice (Oryza sativa L.). Although numerous salt tolerance-related genes have been identified in rice, their molecular mechanisms remain unknown. Here, we report that OsJRL40, a jacalin-related lectin gene, confers remarkable salt tolerance in rice. The loss of function of OsJRL40 increased sensitivity to salt stress in rice, whereas its overexpression enhanced salt tolerance at the seedling stage and during reproductive growth. ß-glucuronidase (GUS) reporter assays indicated that OsJRL40 is expressed to higher levels in roots and internodes than in other tissues, and subcellular localization analysis revealed that the OsJRL40 protein localizes to the cytoplasm. Further molecular analyses showed that OsJRL40 enhances antioxidant enzyme activities and regulates Na+-K+ homeostasis under salt stress. RNA-seq analysis revealed that OsJRL40 regulates salt tolerance in rice by controlling the expression of genes encoding Na+/K+ transporters, salt-responsive transcription factors, and other salt response-related proteins. Overall, this study provides a scientific basis for an in-depth investigation of the salt tolerance mechanism in rice and could guide the breeding of salt-tolerant rice cultivars.

SiNCED1, a 9-cis-epoxycarotenoid dioxygenase gene in Setaria italica, is involved in drought tolerance and seed germination in transgenic Arabidopsis.

Foxtail millet (Setaria italica L.) is a vital cereal food crop with promising development and utilization potential because of its outstanding ability to resist drought stress. However, the molecular mechanisms underlying its drought stress resistance remain unclear. In this study, we aimed to elucidate the molecular function of a 9-cis-epoxycarotenoid dioxygenase gene, SiNCED1, in the drought stress response of foxtail millet. Expression pattern analysis showed that SiNCED1 expression was significantly induced by abscisic acid (ABA), osmotic stress, and salt stress. Furthermore, ectopic overexpression of SiNCED1 could enhance drought stress resistance by elevating endogenous ABA levels and promoting stomatal closure. Transcript analysis indicated that SiNCED1 modulated ABA-related stress responsive gene expression. In addition, we found that ectopic expression of SiNCED1 delayed seed germination under normal and abiotic stress conditions. Taken together, our results show that SiNCED1 plays a positive role in the drought tolerance and seed dormancy of foxtail millet by modulating ABA biosynthesis. In conclusion, this study revealed that SiNCED1 is an important candidate gene for the improvement of drought stress tolerance in foxtail millet and could be beneficial in the breeding and investigation of drought tolerance in other agronomic crops.

Identification of Salt Tolerance Related Candidate Genes in 'Sea Rice 86' at the Seedling and Reproductive Stages Using QTL-Seq and BSA-Seq.

Salt stress seriously affects plant growth and development and reduces the yield of rice. Therefore, the development of salt-tolerant high-yielding rice cultivars through quantitative trait locus (QTL) identification and bulked segregant analysis (BSA) is the main focus of molecular breeding projects. In this study, sea rice (SR86) showed greater salt tolerance than conventional rice. Under salt stress, the cell membrane and chlorophyll were more stable and the antioxidant enzyme activity was higher in SR86 than in conventional rice. Thirty extremely salt-tolerant plants and thirty extremely salt-sensitive plants were selected from the F2 progenies of SR86 × Nipponbare (Nip) and SR86 × 9311 crosses during the whole vegetative and reproductive growth period and mixed bulks were generated. Eleven salt tolerance related candidate genes were located using QTL-seq together with BSA. Real time quantitative PCR (RT-qPCR) analysis showed that LOC_Os04g03320.1 and BGIOSGA019540 were expressed at higher levels in the SR86 plants than in Nip and 9311 plants, suggesting that these genes are critical for the salt tolerance of SR86. The QTLs identified using this method could be effectively utilized in future salt tolerance breeding programs, providing important theoretical significance and application value for rice salt tolerance breeding.

Association between sequence variants in cadmium-related genes and the cadmium accumulation trait in thermo-sensitive genic male sterile rice.

Although cultivation of hybrid rice varieties has been increasing, there are risks that high levels of cadmium (Cd) will accumulate in grain when such rice is grown in Cd-polluted environments. To produce Cd-safe hybrid rice, one practical approach is the generation of low Cd-accumulating parental lines. In two-line hybrid breeding, thermosensitive genic male sterile (TGMS) lines function as female parents to yield hybrid seeds. Recently, Cd accumulation-related genes have been identified; however, the effect of these genes on Cd accumulation in the grains of TGMS lines has yet to be reported. Here, 174 TGMS lines were selected for Cd accumulation phenotyping, and 30 TGMS lines, including 15 stable low-Cd and 15 high-Cd lines, were selected for single-nucleotide polymorphism (SNP) genotyping and association analysis. Association studies were conducted to identify the relationship between Cd accumulation and variable sites within seven candidate Cd-associated genes using logistic models. Nine sequence variant sites in four of the candidate genes were found to be significantly associated with Cd accumulation, two of which in OsNRAMP1 and OsNRAMP5 are low-Cd favorable variants, explaining 46.4% and 22.6% of the phenotypic variation, respectively. These loci could be developed as new molecular markers for identification of Cd accumulation characteristics and low-Cd marker-assisted breeding.

OsNCED5, a 9-cis-epoxycarotenoid dioxygenase gene, regulates salt and water stress tolerance and leaf senescence in rice.

9-cis-epoxycarotenoid dioxygenase (NCED) is a rate-limiting enzyme for abscisic acid (ABA) biosynthesis. However, the molecular mechanisms of NCED5 that modulate plant development and abiotic stress tolerance are still unclear, particular in rice. Here, we demonstrate that a rice NCED gene, OsNCED5, was expressed in all tissues we tested, and was induced by exposure to salt stress, water stress, and darkness. Mutational analysis showed that nced5 mutants reduced ABA level and decreased tolerance to salt and water stress and delayed leaf senescence. However, OsNCED5 overexpression increased ABA level, enhanced tolerance to the stresses, and accelerated leaf senescence. Transcript analysis showed that OsNCED5 regulated ABA-dependent abiotic stress and senescence-related gene expression. Additionally, ectopic expression of OsNCED5 tested in Arabidopsis thaliana altered plant size and leaf morphology and delayed seed germination and flowering time. Thus, OsNCED5 may regulate plant development and stress resistance through control of ABA biosynthesis. These findings contribute to our understanding of the molecular mechanisms by which NCED regulates plant development and responses to abiotic stress in different crop species.

9-cis-Epoxycarotenoid Dioxygenase 3 Regulates Plant Growth and Enhances Multi-Abiotic Stress Tolerance in Rice.

Although abscisic acid (ABA) is an important hormone that regulates seed dormancy, stomatal closure, plant development, as well as responses to environmental stimuli, the physiological mechanisms of ABA response to multiple stress in rice remain poorly understood. In the ABA biosynthetic pathway, 9-cis-epoxycarotenoid dioxygenase (NCED) is the key rate-limiting enzyme. Here, we report important functions of OsNCED3 in multi-abiotic stress tolerance in rice. The OsNCED3 is constitutively expressed in various tissues under normal condition, Its expression is highly induced by NaCl, PEG, and H2O2 stress, suggesting the roles for OsNCED3 in response to the multi-abiotic stress tolerance in rice. Compared with wild-type plants, nced3 mutants had earlier seed germination, longer post-germination seedling growth, increased sensitivity to water stress and H2O2 stress and increased stomata aperture under water stress and delayed leaf senescence. Further analysis found that nced3 mutants contained lower ABA content compared with wild-type plants, overexpression of OsNCED3 in transgenic plants could enhance water stress tolerance, promote leaf senescence and increase ABA content. We conclude that OsNCED3 mediates seed dormancy, plant growth, abiotic stress tolerance, and leaf senescence by regulating ABA biosynthesis in rice; and may provide a new strategy for improving the quality of crop.

Exploring the genetic characteristics of 93-11 and Nipponbare recombination inbred lines based on the GoldenGate SNP assay.

Understanding genetic characteristics in rice populations will facilitate exploring evolutionary mechanisms and gene cloning. Numerous molecular markers have been utilized in linkage map construction and quantitative trait locus (QTL) mappings. However, segregation-distorted markers were rarely considered, which prevented understanding genetic characteristics in many populations. In this study, we designed a 384-marker GoldenGate SNP array to genotype 283 recombination inbred lines (RILs) derived from 93-11 and Nipponbare Oryza sativa crosses. Using 294 markers that were highly polymorphic between parents, a linkage map with a total genetic distance of 1,583.2 cM was constructed, including 231 segregation-distorted markers. This linkage map was consistent with maps generated by other methods in previous studies. In total, 85 significant quantitative trait loci (QTLs) with phenotypic variation explained (PVE) values≥5% were identified. Among them, 34 QTLs were overlapped with reported genes/QTLs relevant to corresponding traits, and 17 QTLs were overlapped with reported sterility-related genes/QTLs. Our study provides evidence that segregation-distorted markers can be used in linkage map construction and QTL mapping. Moreover, genetic information resulting from this study will help us to understand recombination events and segregation distortion. Furthermore, this study will facilitate gene cloning and understanding mechanism of inter-subspecies hybrid sterility and correlations with important agronomic traits in rice.

Mitochondrial DNA genetic polymorphism in thirteen rice cytoplasmic male sterile lines.

KEY MESSAGE: Thirteen rice CMS lines derived from different cytoplasms were classified into eight groups by PCR amplification on mtDNA. The orf79 gene, which causes Boro II CMS, possibly results in Dian1-CMS. Thirteen rice cytoplasmic male sterile (CMS) lines derived from different cytoplasms are widely used for hybrid rice breeding. Based on 27 loci on mitochondrial DNA, including single nucleotide polymorphisms and segmental sequence variations between typical indica and japonica as well as high-polymorphism segmental sequence variations and single nucleotide polymorphisms among rice CMS lines, the 13 rice CMS lines were classified into eight groups: (I) wild-abortive CMS, Indonesian Shuitiangu CMS, K-CMS, Gang CMS, D-CMS and dwarf abortive CMS; (II) Maxie-CMS; (III) Honglian CMS; (IV) Boro II CMS; (V) Dian1-CMS; (VI) Liao-CMS; (VII) Lead CMS; and (VIII) Chinese wild rice CMS. According to their pollen abortion phenotypes, groups I and II (including 7 CMS lines) were classified as sporophytic CMS lines, the cytoplasmic genetic relationships among which were very close. They could have originated from similar, or even the same, cytoplasm donors. Groups III-VIII (including 6 CMS lines) were categorized as gametophytic CMS lines, the cytoplasms of which differed from one another, with some having relatively far genetic relationships. Dian1-CMS was found to harbor the orf79 gene, which causes Boro II CMS, whereas Liao-CMS had an orf79 structure that does not result in Lead CMS. Therefore, we speculated that orf79 is associated with Dian1-CMS but not with Liao-CMS. The atp6-orf79 structure related to sterility was also found to experience multiple evolutionary turnovers. All sporophytic CMS lines were indica-like. Except the Honglian CMS line, which was indica-like, all gametophytic CMS lines were japonica-like.

Expression analysis of miRNAs and highly-expressed small RNAs in two rice subspecies and their reciprocal hybrids.

Heterosis, or hybrid vigor, is the phenomenon whereby progeny of two inbred lines exhibit superior agronomic performance compared with either parent. We analyzed the expression of miRNAs and highly expressed small RNAs (defined according to Solexa sequencing results) in two rice (Oryza sativa) subspecies (japonica cv. Nipponbare and indica cv. 93-11) and their reciprocal hybrids using microarrays. We found that of all the 1141 small RNAs tested, 140 (12%, 140 of 1141) and 157 (13%, 157 of 1141) were identified being significantly differentially expressed in two reciprocal hybrids, respectively. All possible modes of action, including additive, high- and low- parent, above high- and below low-parent modes were exhibited. Both F1 hybrids showed non-additive expression patterns, with downregulation predominating. Interestingly, 15 miRNAs displayed stark opposite expression trends relative to mid-parent in reciprocal hybrids. Computational prediction of targets of differentially expressed miRNAs showed that they participated in multifaceted developmental pathways, and were not distinguishable from the targets of non-differentially expressed miRNAs. Together, our findings reveal that small RNAs play roles in heterosis and add a new layer in the understanding and exploitation of molecular mechanisms of heterosis.

Global epigenetic and transcriptional trends among two rice subspecies and their reciprocal hybrids.

The behavior of transcriptomes and epigenomes in hybrids of heterotic parents is of fundamental interest. Here, we report highly integrated maps of the epigenome, mRNA, and small RNA transcriptomes of two rice (Oryza sativa) subspecies and their reciprocal hybrids. We found that gene activity was correlated with DNA methylation and both active and repressive histone modifications in transcribed regions. Differential epigenetic modifications correlated with changes in transcript levels among hybrids and parental lines. Distinct patterns in gene expression and epigenetic modifications in reciprocal hybrids were observed. Through analyses of single nucleotide polymorphisms from our sequence data, we observed a high correlation of allelic bias of epigenetic modifications or gene expression in reciprocal hybrids with their differences in the parental lines. The abundance of distinct small RNA size classes differed between the parents, and more small RNAs were downregulated than upregulated in the reciprocal hybrids. Together, our data reveal a comprehensive overview of transcriptional and epigenetic trends in heterotic rice crosses and provide a useful resource for the rice community.

A genome-wide transcription analysis reveals a close correlation of promoter INDEL polymorphism and heterotic gene expression in rice hybrids.

Heterosis, or hybrid vigor, refers to the phenomenon in which hybrid progeny of two inbred varieties exhibits enhanced growth or agronomic performance. Although a century-long history of research has generated several hypotheses regarding the genetic basis of heterosis, the molecular mechanisms underlying heterosis and heterotic gene expression remain elusive. Here, we report a genome-wide gene expression analysis of two heterotic crosses in rice, taking advantage of its fully sequenced genomes. Approximately 7-9% of the genes were differentially expressed in the seedling shoots from two sets of heterotic crosses, including many transcription factor genes, and exhibited multiple modes of gene action. Comparison of the putative promoter regions of the ortholog genes between inbred parents revealed extensive sequence variation, particularly small insertions/deletions (INDELs), many of which result in the formation/disruption of putative cis-regulatory elements. Together, these results suggest that a combinatorial interplay between expression of transcription factors and polymorphic promoter cis-regulatory elements in the hybrids is one plausible molecular mechanism underlying heterotic gene action and thus heterosis in rice.

[Multiplication technique of rice T(P) GMS line with double low critical temperature values using cold water irrigation].

In this paper, cold-water irrigation multiplication technique was used to solve the difficulty of multiplying rice TGMS line 96-5-2S(Oryza sative L.) with double low critical temperature values under natural air temperature condition. The results indicated that irrigating rice TGMS line 96-5-2S with different temperature underground cold water from the stage of pistil and stamen primordia differentiation to the stage of pollen filling for 15 days, and keeping the water depth 18-22 cm, the seed set rate of 96-5-2S decreased with increasing irrigating water temperature. When the average temperature of irrigation water was 18.5-19.8 degrees C, the seed set rate ranged from 40.5% to 57.6%, and the yield ranged from 3.30 t.hm-2 to 4.35 t.hm-2. While the temperature was 20.5-21.3 degrees C, the seed set rate and yield sharply decreased to 2.5-10.4% and 0.21-0.90 t.hm-2, respectively. When the average temperature was 22.3-23.5 degrees C, both the seed set rate and yield were zero. Under the same irrigation water temperature (average 19.8 degrees C) and the same irrigating period (phase IV to phase VII), the seed set rate and yield of 96-5-2S with deep water irrigation (18-22 cm) was very significantly higher than those of 96-5-2S with shallow water irrigation (7-10 cm). Under the conditions of same irrigation water temperature (average 19.8 degrees C) and same water depth (18-22 cm) but different irrigating periods (15 days from phase IV to phase VII, 20 days from phase III to phase VII, and 25 days from phase II to phase VII), the difference of seed set rate and yield of 96-5-2S between 15-day treatment and 20-25-day treatment was not significantly different. It is confident that 96-5-2S could multiply with underground cold water irrigation. The main technique index was that irrigating water temperature should be 18-20 degrees C, irrigating period should be from phase IV to phase VII, and irrigating water depth should be 18-22 cm.