Knocking out isopropylmalate synthase simultaneously improves grain appearance and nutritional quality in rice.

Grain appearance and nutritional quality are critical traits for rice marketing. However, how to simultaneously improve grain appearance (slender grain and low chalkiness) and nutritional quality (improved protein and amino acid contents) in rice remains a major challenge. Here, we show that knocking out rice isopropylmalate synthase genes OsIPMS1 and OsIPMS2 can improve both grain appearance and nutritional quality. We find that OsIPMS1 directly interacts with OsIPMS2 to form heterodimers. Meanwhile, we observe that OsIPMS1 and OsIPMS2 influence the expression of genes previously reported to be involved in the determination of grain size and nutritional quality in the developing panicles and grains. Furthermore, we show that Osipms1/2 double mutants exhibit significantly improved grain appearance and nutritional quality in polished rice in both the japonica (Wuyungeng 23) and indica (Huanghuazhan) varieties. Our findings indicate that OsIPMS is a useful target gene for breeding of rice varieties appealing for marketing and with health-benefiting properties.

Rice seed germination priming by salicylic acid and the emerging role of phytohormones in anaerobic germination.

This Commentary examines a recent study that identified peroxisomal cinnamate: CoA ligases as key enzymes for salicylic acid biosynthesis, which promotes submerged germination by releasing the inhibition of rice germination by indole-acetic acid. This study thus provides important information for developing rice varieties suitable for direct seeding.

OsNAC3 regulates seed germination involving abscisic acid pathway and cell elongation in rice.

Seed germination is a critical trait for the success of direct seeding in rice cultivation. However, the underlying mechanism determining seed germination is largely unknown in rice. Here, we report that NAC transcription factor OsNAC3 positively regulates seed germination of rice. OsNAC3 regulates seed germination involving abscisic acid (ABA) pathway and cell elongation. OsNAC3 can directly bind to the promoter of ABA catabolic gene OsABA8ox1 and cell expansion gene OsEXP4, which consequently activates their expressions during seed germination. We also find that the expression of OsEXP4 is reduced by ABA during seed germination in rice. OsNAC3 regulates seed germination by influencing cell elongation of the embryo through directly affecting OsEXP4 expression and indirectly ABA-medicated OsEXP4 expression. The OsNAC3 elite haplotype is useful for genetic improvement of seed germination, and overexpression of OsNAC3 can significantly increase seed germination. We therefore propose that OsNAC3 is a potential target in breeding of rice varieties with high seed germination for direct seeding cultivation.

UDP-glucosyltransferase OsUGT75A promotes submergence tolerance during rice seed germination.

Submergence stress represents a major obstacle limiting the application of direct seeding in rice cultivation. Under flooding conditions, coleoptile elongation can function as an escape strategy that contributes to submergence tolerance during seed germination in rice; however, the underlying molecular bases have yet to be fully determined. Herein, we report that natural variation of rice coleoptile length subjected to submergence is determined by the glucosyltransferase encoding gene OsUGT75A. OsUGT75A regulates coleoptile length via decreasing free abscisic acid (ABA) and jasmonic acid (JA) levels by promoting glycosylation of these two phytohormones under submergence. Moreover, we find that OsUGT75A accelerates coleoptile length through mediating the interactions between JASMONATE ZIMDOMAIN (OsJAZ) and ABSCISIC ACID-INSENSITIVE (OsABI) proteins. Last, we reveal the origin of the haplotype that contributes to coleoptile length in response to submergence and transferring this haplotype to indica rice can enhance coleoptile length in submergence conditions. Thus, we propose that OsUGT75A is a useful target in breeding of rice varieties suitable for direct seeding cultivation.

Comparative Transcriptome Analysis of the Heterosis of Salt Tolerance in Inter-Subspecific Hybrid Rice.

Soil salinity is one of the major abiotic stresses limiting rice growth. Hybrids outperform their parents in salt tolerance in rice, while its mechanism is not completely understood. In this study, a higher seedling survival was observed after salt treatment in an inter-subspecific hybrid rice, Zhegengyou1578 (ZGY1578), compared with its maternal japonica Zhegeng7A (ZG7A) and paternal indica Zhehui1578 (ZH1578). A total of 2584 and 3061 differentially expressed genes (DEGs) with at least twofold changes were identified between ZGY1578 and ZG7A and between ZGY1578 and ZH1578, respectively, in roots under salt stress using the RNA sequencing (RNA-Seq) approach. The expressions of a larger number of DEGs in hybrid were lower or higher than those of both parents. The DEGs associated with transcription factors, hormones, and reactive oxygen species (ROS)-related genes might be involved in the heterosis of salt tolerance. The expressions of the majority of transcription factors and ethylene-, auxin-, and gibberellin-related genes, as well as peroxidase genes, were significantly higher in the hybrid ZGY1578 compared with those of both parents. The identified genes provide valuable clues to elucidate the heterosis of salt tolerance in inter-subspecific hybrid rice.

Isopropylmalate synthase NtIPMS as a potential molecular marker for seed vigor in tobacco.

Seed vigor is an important trait for tobacco production. However, the evaluation of seed vigor using molecular biomarkers is scarcely reported in tobacco. In this study, the development of molecular marker isopropylmalate synthase NtIPMS was conducted to detect seed ageing degree and seed priming effect in tobacco. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression of NtIPMS was significantly induced at the initial imbibition stage during seed germination. The NtIPMS expression was positively correlated with the degree of seed ageing in non-pelleted and pelleted seeds. The mRNA level of NtIPMS was gradually increased with the increasing degree of seed ageing. The early best effect of gibberellin priming was observed in 30-h primed seeds, and the highest expression of NtIPMS was observed in 12-h primed seeds. The best stop time-point of seed priming is likely at the time 18 h after the relatively higher NtIPMS expression occurred during seed priming process. The NtIPMS mRNA detection has the potential usage as a potential molecular marker for the evaluation of seed vigor in tobacco.

Dynamic RNA-Seq Study Reveals the Potential Regulators of Seed Germination in Paris polyphylla var. yunnanensis.

Paris polyphylla var. yunnanensis is an important traditional Chinese medicine, but poor seed germination limits its large-scale artificial cultivation. Thus, it is crucial to understand the regulators of seed germination to obtain clues about how to improve the artificial cultivation of Paris polyphylla. In this study, the seeds at three germination stages, including ungerminated seeds (stage 1), germinated seeds with a 0.5 cm radicel length (stage 2), and germinated seeds with a 2.0 cm radicel length (stage 3) after warm stratification (20 °C) for 90 days were used for RNA sequencing. Approximately 220 million clean reads and 447,314 annotated unigenes were obtained during seed germination, of which a total of 4454, 5150, and 1770 differentially expressed genes (DEGs) were identified at stage 1 to stage 2, stage 1 to stage 3, and stage 2 to stage 3, respectively. Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEGs were significantly enriched in carbohydrate metabolism, lipid metabolism, signal transduction, and translation. Of them, several genes encoding the glutamate decarboxylase, glutamine synthetase, alpha-galactosidase, auxin-responsive protein IAA30, abscisic-acid-responsive element binding factor, mitogen-activated protein kinase kinase 9/18, and small and large subunit ribosomal proteins were identified as potentially involved in seed germination. The identified genes provide a valuable resource to study the molecular basis of seed germination in Paris polyphylla var. yunnanensis.

Characteristics of Seed Vigor in Rice Varieties with Different Globulin Accumulations.

Seed vigor of rice is an important trait for direct seeding. The objective of this study was to reveal the relationship between globulin and seed vigor, and then to explore a method for evaluating seed vigor. Several rice varieties with different levels of 52 kDa globulin accumulation were used to compare seed vigor under normal and aged conditions. Results showed that varieties with high globulin accumulation obtained significantly higher seed vigor, measured by germination percentage and germination index, compared with those varieties with low globulin accumulation under normal and aged conditions. Meanwhile, a significantly higher accumulation of reactive oxygen species (ROS) was observed in the early germinating seeds of varieties with high globulin accumulation compared to those varieties with low globulin accumulation under normal and aged conditions. Collectively, the globulin content could be applied in the evaluation of seed vigor, which contributes to the selection of rice varieties for direct seeding.

Leaf Mutant 7 Encoding Heat Shock Protein OsHSP40 Regulates Leaf Size in Rice.

Leaf size is an important agronomic trait directly affecting yield in rice, and thus understanding the genes determining leaf size is important in breeding. In this study, one Leaf Mutant 7 (lm7) with small leaf size was isolated using ethyl methane sulphonate (EMS) mutagenesis from the japonica Zhenggeng 1925. MutMap by whole genome resequencing of phenotypic bulks revealed that LM7 is likely located in the 133 kb region on chromosome 7 using F2 population from a cross between lm7 and wild-type (WT) Zhenggeng 1925. The candidate gene encoding heat shock protein OsHSP40 for LM7 was functionally validated. Disruption of this gene in Oshsp40 mutants significantly reduced the leaf size compared with that of WT in rice. Microscopic examination showed that OsHSP40 modulated leaf size via regulating the veins formation and cell size/cell number. Nucleotide diversity analysis indicated that a single nucleotide polymorphism (SNP) variation of C to T in the coding region of OsHSP40 may cause small leaves among rice accessions. Therefore, the natural variation of OsHSP40 contributing to leaf size might be useful for rice breeding.

Advances in the Understanding of Reactive Oxygen Species-Dependent Regulation on Seed Dormancy, Germination, and Deterioration in Crops.

Reactive oxygen species (ROS) play an essential role in the regulation of seed dormancy, germination, and deterioration in plants. The low level of ROS as signaling particles promotes dormancy release and triggers seed germination. Excessive ROS accumulation causes seed deterioration during seed storage. Maintaining ROS homeostasis plays a central role in the regulation of seed dormancy, germination, and deterioration in crops. This study highlights the current advances in the regulation of ROS homeostasis in dry and hydrated seeds of crops. The research progress in the crosstalk between ROS and hormones involved in the regulation of seed dormancy and germination in crops is mainly summarized. The current understandings of ROS-induced seed deterioration are reviewed. These understandings of ROS-dependent regulation on seed dormancy, germination, and deterioration contribute to the improvement of seed quality of crops in the future.

Genome-wide association study reveals that the cupin domain protein OsCDP3.10 regulates seed vigour in rice.

Seed vigour is an imperative trait for the direct seeding of rice. In this study, we examined the genetic regulation of seedling percentage at the early germination using a genome-wide association study in rice. One major quantitative trait loci qSP3 for seedling percentage was identified, and the candidate gene was validated as qSP3, encoding a cupin domain protein OsCDP3.10 for the synthesis of 52 kDa globulin. Disruption of this gene in Oscdp3.10 mutants reduced the seed vigour, including the germination potential and seedling percentage, at the early germination in rice. The lacking accumulation of 52 kDa globulin was observed in the mature grains of the Oscdp3.10 mutants. The significantly lower amino acid contents were observed in the mature grains and the early germinating seeds of the Oscdp3.10 mutants compared with those of wild-type. Rice OsCDP3.10 regulated seed vigour mainly via modulating the amino acids e.g. Met, Glu, His, and Tyr that contribute to hydrogen peroxide (H2 O2 ) accumulation in the germinating seeds. These results provide important insights into the application of seed priming with the amino acids and the selection of OsCDP3.10 to improve seed vigour in rice.

A genome-wide association study reveals that the cytochrome b5 involved in seed reserve mobilization during seed germination in rice.

KEY MESSAGE: A candidate gene cytochrome b5 for the major QTL qSRMP9 for rice seed reserve mobilization was validated during seed germination using a genome-wide association study approach. Seed reserve mobilization plays important roles in the early seedling growth in rice. However, the genetic basis underlying this process is poorly understood. In this study, the genetic architecture of variation in seed reserve mobilization during seed germination was studied using a genome-wide association study approach in rice. Three quantitative trait loci (QTL) including qSRMP6, qSRMP9, and qSRMP12 for seed reserve mobilization percentage were identified. In which, the candidate gene cytochrome b5 (OsCyb5) for the major QTL qSRMP9 was validated. Disruption of this gene in Oscyb5 mutants reduced the seed reserve mobilization and seedling growth compared with wild-type (WT) in rice. There were no significant differences of grain size, starch, protein and total soluble sugar content in the mature grains between Oscyb5 mutants and WT. However, the α-amylase activity in the germinating seeds of Oscyb5 mutants was significantly decreased compared to that of WT, and then, the starch and sugar mobilization and the glucose accumulation during seed germination were significantly decreased in Oscyb5 mutants. Two elite haplotypes of OsCyb5 associated with the higher seed reserve mobilization percentage and its elite single nucleotide polymorphism variations were mainly existed in the INDICA and AUS accessions. The natural variation of OsCyb5 contributing to seed reserve mobilization might be useful for the future rice breeding.

Advances in the Identification of Quantitative Trait Loci and Genes Involved in Seed Vigor in Rice.

Seed vigor is a complex trait, including the seed germination, seedling emergence, and growth, as well as seed storability and stress tolerance, which is important for direct seeding in rice. Seed vigor is established during seed development, and its level is decreased during seed storage. Seed vigor is influenced by genetic and environmental factors during seed development, storage, and germination stages. A lot of factors, such as nutrient reserves, seed dying, seed dormancy, seed deterioration, stress conditions, and seed treatments, will influence seed vigor during seed development to germination stages. This review highlights the current advances on the identification of quantitative trait loci (QTLs) and regulatory genes involved in seed vigor at seed development, storage, and germination stages in rice. These identified QTLs and regulatory genes will contribute to the improvement of seed vigor by breeding, biotechnological, and treatment approaches.

A genome-wide association study reveals that the 2-oxoglutarate/malate translocator mediates seed vigor in rice.

Seed vigor is an important trait for the direct seeding of rice (Oryza sativa L.). In this study, we examined the genetic architecture of variation in the germination rate using a diverse panel of rice accessions. Four quantitative trait loci for germination rate were identified using a genome-wide association study during early germination. One candidate gene, encoding the 2-oxoglutarate/malate translocator (OsOMT), was validated for qGR11. Disruption of this gene (Osomt mutants) reduced seed vigor, including seed germination and seedling growth, in rice. Functional analysis revealed that OsOMT influences seed vigor mainly by modulating amino acid levels and glycolysis and tricarboxylic acid cycle processes. The levels of most amino acids, including the Glu family (Glu, Pro, Arg, and GABA), Asp family (Asp, Thr, Lys, Ile, and Met), Ser family (Ser, Gly, and Cys), and others (His, Ala, Leu, and Val), were significantly reduced in the mature grains and the early germinating seeds of Osomt mutants compared to wild type (WT). The glucose and soluble sugar contents, as well as adenosine triphosphate levels, were significantly decreased in germinating seeds of Osomt mutants compared to WT. These results provide important insights into the role of OsOMT in seed vigor in rice.

The Rice Small Auxin-Up RNA Gene OsSAUR33 Regulates Seed Vigor via Sugar Pathway during Early Seed Germination.

Seed vigor affects seed germination and seedling emergence, and therefore is an important agronomic trait in rice. Small auxin-up RNAs (SAURs) function in a range of developmental processes, but their role in seed vigor remains unclear. Here, we observed that disruption of OsSAUR33 resulted in reduced germination rates and low seed uniformity in early germination. Expression of OsSAUR33 was higher in mature grains and early germinating seeds. RNA-seq analysis revealed that OsSAUR33 modulated seed vigor by affecting the mobilization of stored reserves during germination. Disruption of OsSAUR33 increased the soluble sugar content in dry mature grains and seeds during early germination. OsSAUR33 interacted with the sucrose non-fermenting-1-related protein kinase OsSnRK1A, a regulator of the sugar signaling pathway, which influences the expression of sugar signaling-related genes during germination. Disruption of OsSAUR33 increased sugar-sensitive phenotypes in early germination, suggesting OsSAUR33 likely affects seed vigor through the sugar pathway. One elite haplotype of OsSAUR33 associated with higher seed vigor was identified mainly in indica accessions. This study provides insight into the effects of OsSAUR33 on seed vigor in rice.

A genome-wide association study reveals that the glucosyltransferase OsIAGLU regulates root growth in rice.

Good root growth in the early post-germination stages is an important trait for direct seeding in rice, but its genetic control is poorly understood. In this study, we examined the genetic architecture of variation in primary root length using a diverse panel of 178 accessions. Four QTLs for root length (qRL3, qRL6, qRL7, and qRL11) were identified using genome-wide association studies. One candidate gene was validated for the major QTL qRL11, namely the glucosyltransferase OsIAGLU. Disruption of this gene in Osiaglu mutants reduced the primary root length and the numbers of lateral and crown roots. The natural allelic variations of OsIAGLU contributing to root growth were identified. Functional analysis revealed that OsIAGLU regulates root growth mainly via modulating multiple hormones in the roots, including levels of auxin, jasmonic acid, abscisic acid, and cytokinin. OsIAGLU also influences the expression of multiple hormone-related genes associated with root growth. The regulation of root growth through multiple hormone pathways by OsIAGLU makes it a potential target for future rice breeding for crop improvement.

Identification of candidate tolerance genes to low-temperature during maize germination by GWAS and RNA-seqapproaches.

BACKGROUND: Maize (Zea mays L.) is one of the main agricultural crops with the largest yield and acreage in the world. However, maize germplasm is very sensitive to low temperatures, mainly during germination, and low temperatures significantly affect plant growth and crop yield. Therefore, the identification of genes capable of increasing tolerance to low temperature has become necessary. RESULTS: In this study, fourteen phenotypic traits related to seed germination were used to assess the genetic diversity of maize through genome-wide association study (GWAS). A total of 30 single-nucleotide polymorphisms (SNPs) linked to low-temperature tolerance were detected (-log10(P) > 4), fourteen candidate genes were found to be directly related to the SNPs, further additional 68 genes were identified when the screen was extended to include a linkage disequilibrium (LD) decay distance of r2 ≥ 0.2 from the SNPs. RNA-sequencing (RNA-seq) analysis was then used to confirm the linkage between the candidate gene and low-temperature tolerance. A total of ten differentially expressed genes (DEGs) (|log2 fold change (FC)| ≥ 0.585, P < 0.05) were found within the set distance of LD decay (r2 ≥ 0.2). Among these genes, the expression of six DEGs was verified using qRT-PCR. Zm00001d039219 and Zm00001d034319 were putatively involved in 'mitogen activated protein kinase (MAPK) signal transduction' and 'fatty acid metabolic process', respectively, based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Thus, these genes appeared to be related to low-temperature signal transduction and cell membrane fluidity. CONCLUSION: Overall, by integrating the results of our GWAS and DEG analysis of low-temperature tolerance during germination in maize, we were able to identify a total of 30 SNPs and 82 related candidate genes, including 10 DEGs, two of which were involved in the response to tolerance to low temperature. Functional analysis will provide valuable information for understanding the genetic mechanism of low-temperature tolerance during germination in maize.

Indole-3-acetate beta-glucosyltransferase OsIAGLU regulates seed vigour through mediating crosstalk between auxin and abscisic acid in rice.

Seed vigour is an important trait for direct seeding in rice. In this study, indole-3-acetate beta-glucosyltransferase OsIAGLU was cloned in rice, and its roles on seed vigour were mainly investigated. Disruption of OsIAGLU resulted in low seed vigour in rice. Quantitative RT-PCR analysis showed that the expressions of OsIAGLU were relatively higher in the late developing and the early germinating seeds and were significantly induced by indole-3-acetic acid (IAA) and abscisic acid (ABA). Transcriptome analysis revealed that the IAA- and ABA-related genes were involved in the OsIAGLU regulation of seed vigour in rice. The higher levels of free IAA and ABA were identified in germinating seeds of osiaglu mutants compared to wild-type (WT) plants. When treated with exogenous IAA and ABA, the osiaglu mutants and WT plants showed sensitivity to ABA while not IAA, but the exogenous IAA amplified ABA-induced reduction of seed vigour in rice. The continuously higher expressions of ABA-INSENSITIVE 3 (OsABI3) and OsABI5 occurred in germinating seeds of osiaglu mutants compared to WT plants. The regulation of seed vigour by OsIAGLU might be through modulating IAA and ABA levels to alert OsABIs expression in germinating seeds in rice. Based on analysis of single-nucleotide polymorphism data of rice accessions, two haplotypes of OsIAGLU that positively correlated with seed vigour were identified in indica accessions. This study provides important insights into the roles of OsIAGLU on seed vigour and facilitates the practical use of OsIAGLU in rice breeding.

A quantitative trait locus, qSE3, promotes seed germination and seedling establishment under salinity stress in rice.

Seed germination is a complex trait determined by both quantitative trait loci (QTLs) and environmental factors and also their interactions. In this study, we mapped one major QTLqSE3 for seed germination and seedling establishment under salinity stress in rice. To understand the molecular basis of this QTL, we isolated qSE3 by map-based cloning and found that it encodes a K+ transporter gene, OsHAK21. The expression of qSE3 was significantly upregulated by salinity stress in germinating seeds. Physiological analysis suggested that qSE3 significantly increased K+ and Na+ uptake in germinating seeds under salinity stress, resulting in increased abscisic acid (ABA) biosynthesis and activated ABA signaling responses. Furthermore, qSE3 significantly decreased the H2 O2 level in germinating seeds under salinity stress. All of these seed physiological changes modulated by qSE3 might contribute to seed germination and seedling establishment under salinity stress. Based on analysis of single-nucleotide polymorphism data of rice accessions, we identified a HAP3 haplotype of qSE3 that was positively correlated with seed germination under salinity stress. This study provides important insights into the roles of qSE3 in seed germination and seedling establishment under salinity stress and facilitates the practical use of qSE3 in rice breeding.