Identification of hub genes involved in GA-regulated coleoptile elongation under submerged germinations in rice.

Submergence stress hinders the direct seeding in rice cultivation. Rapid elongation of rice seed coleoptiles to reach the water surface enables rice to survive submergence stress. Gibberellin (GA) positively influences rice growth. However, the molecular mechanisms underlying GA-regulated coleoptile elongation under submergence conditions remain unclear. Here, we performed a WGCNA analysis to preliminarily investigate the mechanisms. Our results identify four key modules with a high correlation to the GA regulation of rice submergence tolerance. The genes within these modules are mainly involved in Golgi apparatus and carbohydrate metabolic pathways, suggesting involvement of these biological processes in enhancing rice submergence tolerance. Moreover, natural variation analysis reveals that the hub genes, specifically, Os03g0337900, Os03g0355600, and Os07g0638400, exhibited a strong correlation with the subspecies divergence of the coleoptile elongation phenotype. Mutation of Os07g0638400 results in a lower germination potential and a stronger inhibition of coleoptile elongation under submergence conditions in rice, indicating the reliability of the analyses. The hub genes identified in this study provide deep insights into understanding the molecular mechanisms underlying GA-dependent submergence stress tolerance in rice and provide a theoretical basis for innovating rice germplasm for direct seeding application.

Disruption of the rice ALS1 localized in chloroplast causes seedling-lethal albino phenotype.

Chloroplasts are the organelles responsible for photosynthesis and regulate normal plant growth. Although translation elongation factors play important roles in chloroplast development, functional studies of chloroplast translation elongation factors in higher plants remain very sparse. Here, we obtained a rice mutant exhibiting seedling-lethal albino phenotype and named it albino and lethal seedling 1 (als1). Consistently, low content of photosynthetic pigments, malformed chloroplasts and defective photosynthesis were observed in als1 mutant leaves. Map-based cloning experiment showed that als1 mutant had a T base insertion in Os02g0595700, causing a frame shift and premature stop codon. ALS1 encoded a GTP-binding protein EF-Tu, which acts as a translation elongation factor in chloroplast protein translation. ALS1 was found to be expressed throughout plant with highest expression level in young leaves. Moreover, ALS1 was located in chloroplast, whereas the truncated als1 could not normally be located in chloroplast. Additionally, the ALS1 mutation significantly influenced the expression of downstream genes, such as genes relevant to chlorophyll biosynthesis, photosynthesis as well as chloroplast development. These results show that ALS1 acts as a key regulator of chloroplast development and plant growth.

Identification of hub genes that variate the qCSS12-mediated cold tolerance between indica and japonica rice using WGCNA.

KEY MESSAGE: Cold-tolerant QTL qCSS12-regulated 14 hub genes are involved in the chloroplastic biological processes and in the protein synthesis and degradation processes in japonica rice. Low temperature is a main constraint factor for rice growth and production. To better understand the regulatory mechanisms underlying the cold tolerance phenotype in rice, here, we selected a cold-sensitive nearly isogenic line (NIL) NIL(qcss12) as materials to identify hub genes that are mediated by the cold-tolerant locus qCSS12 through weighted gene co-expression network analysis (WGCNA). Fourteen cold-responsive genes were identified, of which, 6 are involved in regulating biological processes in chloroplasts, including the reported EF-Tu, Prk, and ChlD, and 8 are involved in the protein synthesis and degradation processes. Differential expression of these genes between NIL(qcss12) and its controls under cold stress may be responsible for qCSS12-mediated cold tolerance in japonica rice. Moreover, natural variations in 12 of these hub genes are highly correlated with the cold tolerance divergence in two rice subspecies. The results provide deep insights into a better understanding of the molecular basis of cold adaptation in rice and provide a theoretical basis for molecular breeding.

Improvement of resistance to rice blast and bacterial leaf streak by CRISPR/Cas9-mediated mutagenesis of Pi21 and OsSULTR3;6 in rice (Oryza sativa L.).

Rice (Oryza sativa L.) is a staple food in many countries around the world, particularly in China. The production of rice is seriously affected by the bacterial leaf streak and rice blast, which can reduce rice yield or even cause it to fail to be harvested. In this study, susceptible material 58B was edited by CRISPR/Cas9, targeting a target of the Pi21 gene and a target of the effector-binding element (EBE) of the OsSULTR3;6 gene, and the mutants 58b were obtained by Agrobacterium-mediated method. The editing efficiency of the two targets in the T0 generation was higher than 90.09%, the homozygous mutants were successfully selected in the T0 generation, and the homozygous mutation rate of each target was higher than 26.67%. The expression of the edited pi21 and EBE of Ossultr3;6 was significantly reduced, and the expression of defense responsive genes was significantly upregulated after infected with rice blast. The lesion areas of rice blast and bacterial leaf streak were significantly reduced in 58b, and the resistance of both was effectively improved. Furthermore, the gene editing events did not affect the agronomic traits of rice. In this study, the resistance of 58b to rice blast and bacterial leaf streak was improved simultaneously. This study provides a reference for using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) to accelerate the improvement of rice varieties and the development of new materials for rice breeding.

Recent Advances in Molecular Mechanism and Breeding Utilization of Brown Planthopper Resistance Genes in Rice: An Integrated Review.

Over half of the world's population relies on rice as their staple food. The brown planthopper (Nilaparvata lugens Stål, BPH) is a significant insect pest that leads to global reductions in rice yields. Breeding rice varieties that are resistant to BPH has been acknowledged as the most cost-effective and efficient strategy to mitigate BPH infestation. Consequently, the exploration of BPH-resistant genes in rice and the development of resistant rice varieties have become focal points of interest and research for breeders. In this review, we summarized the latest advancements in the localization, cloning, molecular mechanisms, and breeding of BPH-resistant rice. Currently, a total of 70 BPH-resistant gene loci have been identified in rice, 64 out of 70 genes/QTLs were mapped on chromosomes 1, 2, 3, 4, 6, 8, 10, 11, and 12, respectively, with 17 of them successfully cloned. These genes primarily encode five types of proteins: lectin receptor kinase (LecRK), coiled-coil-nucleotide-binding-leucine-rich repeat (CC-NB-LRR), B3-DNA binding domain, leucine-rich repeat domain (LRD), and short consensus repeat (SCR). Through mediating plant hormone signaling, calcium ion signaling, protein kinase cascade activation of cell proliferation, transcription factors, and miRNA signaling pathways, these genes induce the deposition of callose and cell wall thickening in rice tissues, ultimately leading to the inhibition of BPH feeding and the formation of resistance mechanisms against BPH damage. Furthermore, we discussed the applications of these resistance genes in the genetic improvement and breeding of rice. Functional studies of these insect-resistant genes and the elucidation of their network mechanisms establish a strong theoretical foundation for investigating the interaction between rice and BPH. Furthermore, they provide ample genetic resources and technical support for achieving sustainable BPH control and developing innovative insect resistance strategies.

Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of SRL1 and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis.

Leaf rolling is a crucial agronomic trait to consider in rice (Oryza sativa L.) breeding as it keeps the leaves upright, reducing interleaf shading and improving photosynthetic efficiency. The SEMI-ROLLED LEAF 1 (SRL1) gene plays a key role in regulating leaf rolling, as it encodes a glycosylphosphatidylinositol-anchored protein located on the plasma membrane. In this study, we used CRISPR/Cas9 to target the second and third exons of the SRL1 gene in the indica rice line GXU103, which resulted in the generation of 14 T0 transgenic plants with a double-target mutation rate of 21.4%. After screening 120 T1 generation plants, we identified 26 T-DNA-free homozygous double-target mutation plants. We designated the resulting SRL1 homozygous double-target knockout as srl1-103. This line exhibited defects in leaf development, leaf rolling in the mature upright leaves, and a compact nature of the fully grown plants. Compared with the wild type (WT), the T2 generation of srl1-103 varied in two key aspects: the width of flag leaf (12.6% reduction compared with WT) and the leaf rolling index (48.77% increase compared with WT). In order to gain a deeper understanding of the involvement of SRL1 in the regulatory network associated with rice leaf development, we performed a transcriptome analysis for the T2 generation of srl1-103. A comparison of srl1-103 with WT revealed 459 differentially expressed genes (DEGs), including 388 upregulated genes and 71 downregulated genes. In terms of the function of the DEGs, there seemed to be a significant enrichment of genes associated with cell wall synthesis (LOC_Os08g01670, LOC_Os05g46510, LOC_Os04g51450, LOC_Os10g28080, LOC_Os04g39814, LOC_Os01g71474, LOC_Os01g71350, and LOC_Os11g47600) and vacuole-related genes (LOC_Os09g23300), which may partially explain the increased leaf rolling in srl1-103. Furthermore, the significant downregulation of BAHD acyltransferase-like protein gene (LOC_Os08g44840) could be the main reason for the decreased leaf angle and the compact nature of the mutant plants. In summary, this study successfully elucidated the gene regulatory network in which SRL1 participates, providing theoretical support for targeting this gene in rice breeding programs to promote variety improvement.

Discovery of aphid-transmitted Rice tiller inhibition virus from native plants through metagenomic sequencing.

A major threat to rice production is the disease epidemics caused by insect-borne viruses that emerge and re-emerge with undefined origins. It is well known that some human viruses have zoonotic origins from wild animals. However, it remains unknown whether native plants host uncharacterized endemic viruses with spillover potential to rice (Oryza sativa) as emerging pathogens. Here, we discovered rice tiller inhibition virus (RTIV), a novel RNA virus species, from colonies of Asian wild rice (O. rufipogon) in a genetic reserve by metagenomic sequencing. We identified the specific aphid vector that is able to transmit RTIV and found that RTIV would cause low-tillering disease in rice cultivar after transmission. We further demonstrated that an infectious molecular clone of RTIV initiated systemic infection and causes low-tillering disease in an elite rice variety after Agrobacterium-mediated inoculation or stable plant transformation, and RTIV can also be transmitted from transgenic rice plant through its aphid vector to cause disease. Finally, global transcriptome analysis indicated that RTIV may disturb defense and tillering pathway to cause low tillering disease in rice cultivar. Thus, our results show that new rice viral pathogens can emerge from native habitats, and RTIV, a rare aphid-transmitted rice viral pathogen from native wild rice, can threaten the production of rice cultivar after spillover.

Detection of QTLs Regulating Six Agronomic Traits of Rice Based on Chromosome Segment Substitution Lines of Common Wild Rice (Oryza rufipogon Griff.) and Mapping of qPH1.1 and qLMC6.1.

Wild rice is a primary source of genes that can be utilized to generate rice cultivars with advantageous traits. Chromosome segment substitution lines (CSSLs) are consisting of a set of consecutive and overlapping donor chromosome segments in a recipient's genetic background. CSSLs are an ideal genetic population for mapping quantitative traits loci (QTLs). In this study, 59 CSSLs from the common wild rice (Oryza rufipogon Griff.) accession DP15 under the indica rice cultivar (O. sativa L. ssp. indica) variety 93-11 background were constructed through multiple backcrosses and marker-assisted selection (MAS). Through high-throughput whole genome re-sequencing (WGRS) of parental lines, 12,565 mapped InDels were identified and designed for polymorphic molecular markers. The 59 CSSLs library covered 91.72% of the genome of common wild rice accession DP15. The DP15-CSSLs displayed variation in six economic traits including grain length (GL), grain width (GW), thousand-grain weight (TGW), grain length-width ratio (GLWR), plant height (PH), and leaf margin color (LMC), which were finally attributed to 22 QTLs. A homozygous CSSL line and a purple leave margin CSSL line were selected to construct two secondary genetic populations for the QTLs mapping. Thus, the PH-controlling QTL qPH1.1 was mapped to a region of 4.31-Mb on chromosome 1, and the LMC-controlling QTL qLMC6.1 was mapped to a region of 370-kb on chromosome 6. Taken together, these identified novel QTLs/genes from common wild rice can potentially promote theoretical knowledge and genetic applications to rice breeders worldwide.

Development of Soft Rice Lines by Regulating Amylose Content via Editing the 5'UTR of the Wx Gene.

The type of soft rice with low amylose content (AC) is more and more favored by consumers for its better eating and cooking quality, as people's quality of life continuously improves in China. The Wx gene regulates the AC of rice grains, thus affecting the degree of softness of the rice. Mei Meng B (MMB), Tian Kang B (TKB), and DR462 are three indica rice maintained lines with good morphological characters, but also with undesirably high AC. Therefore, CRISPR/Cas9 technology was used to edit the Wx gene of these lines to create a batch of soft rice breeding materials. New gene-edited lines MMB-10-2, TKB-21-12, and DR462-9-9, derived from the above parental lines, respectively, were selected in the T2 generations, with an AC of 17.2%, 16.8%, and 17.8%, and gel consistency (GC) of 78.6 mm, 77.4 mm, and 79.6 mm, respectively. The rapid viscosity analysis (RVA) spectrum showed that the three edited lines had a better eating quality as compared to the corresponding wild type, and showing new characteristics, different from the high-quality soft rice popular in the market. There was no significant difference in the main agronomic traits in the three edited lines compared to the corresponding wild types. Moreover, the chalkiness of DR462-9-9 was reduced, resulting in an improved appearance of its polished rice. The present study created soft rice germplasms for breeding improved quality hybrid rice, without changing the excellent traits of their corresponding wild type varieties.

CRISPR/Cas9-Induced Mutagenesis of TMS5 Confers Thermosensitive Genic Male Sterility by Influencing Protein Expression in Rice (Oryza sativa L.).

The development of thermosensitive genic male sterile (TGMS) lines is the key to breeding two-line hybrid rice, which has been widely applied in China to increase grain yield. CRISPR/Cas9 has been widely used in genome editing to create novel mutants in rice. In the present study, a super grain quality line, GXU 47, was used to generate a new TGMS line with specific mutations in a major TGMS gene tms5 generated with CRISPR/Cas9-mediated genome editing in order to improve the rice quality of two-line hybrids. A mutagenesis efficiency level of 75% was achieved, and three homozygous T-DNA-free mutant lines were screened out. The mutants exhibited excellent thermosensitive male fertility transformation characteristics with complete male sterility at ≥24 °C and desirable male fertility at around 21 °C. Proteomic analysis based on isobaric tags for relative and absolute quantification (iTRAQ) was performed to unveil the subsequent proteomic changes. A total of 192 differentially expressed proteins (DEPs), including 35 upregulated and 157 downregulated, were found. Gene ontology (GO) analysis revealed that the DEPs were involved in a single-organism biosynthetic process, a single-organism metabolic process, oxidoreductase activity, and catalytic activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEPs were involved in ubiquinone and other terpenoid quinone biosynthesis, the biosynthesis of secondary metabolites, metabolic pathways, and phenylpropanoid biosynthesis. Our study shows that high mutation efficiency was achieved in both target sites, and T-DNA-free mutant lines were obtained in the T1 generation. The present study results prove that it is feasible and efficient to generate an excellent mutant line with CRISPR/Cas9, which provides a novel molecular mechanism of male sterility caused by the mutation of tms5.

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.

Transcriptome Analysis in Response to Infection of Xanthomonas oryzae pv. oryzicola Strains with Different Pathogenicity.

Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important quarantine diseases in the world. Breeding disease-resistant varieties can solve the problem of prevention and treatment of BLS from the source. The discovery of the molecular mechanism of resistance is an important driving force for breeding resistant varieties. In this study, a BLS-resistant near isogenic line NIL-bls2 was used as the material. Guangxi Xoc strain gx01 (abbreviated as WT) and its mutant strain (abbreviated as MT) with a knockout type III effectors (T3Es) gene were used to infect rice material NIL-bls2. The molecular interaction mechanism of rice resist near isogenic lines in response to infection by different pathogenic strains was analyzed by transcriptome sequencing. The results showed that there were 415, 134 and 150 differentially expressed genes (DEGs) between the WT group and the MT group at 12, 24 and 48 h of post inoculation (hpi). Through GO and KEGG enrichment analysis, it was found that, compared with non-pathogenic strains, the T3Es secreted by pathogenic strains inhibited the signal transduction pathway mediated by ethylene (ET), jasmonic acid (JA) and salicylic acid (SA), and the MAPKK (MAPK kinase) and MAPKKK (MAPK kinase kinase) in the MAPK (mitogen-activated protein kinase) cascade reaction, which prevented plants from sensing extracellular stimuli in time and starting the intracellular immune defense mechanism; and inhibited the synthesis of lignin and diterpenoid phytochemicals to prevent plants from establishing their own physical barriers to resist the invasion of pathogenic bacteria. The inhibitory effect was the strongest at 12 h, and gradually weakened at 24 h and 48 h. To cope with the invasion of pathogenic bacteria, rice NIL-bls2 material can promote wound healing by promoting the synthesis of traumatic acid at 12 h; at 24 h, hydrogen peroxide was degraded by dioxygenase, which reduced and eliminated the attack of reactive oxygen species on plant membrane lipids; and at 48 h, rice NIL-bls2 material can resist the invasion of pathogenic bacteria by promoting the synthesis of lignin, disease-resistant proteins, monoterpene antibacterial substances, indole alkaloids and other substances. Through transcriptome sequencing analysis, the molecular interaction mechanism of rice resistance near isogenic lines in response to infection by different pathogenic strains was expounded, and 5 genes, Os01g0719300, Os02g0513100, Os03g0122300, Os04g0301500, and Os10g0575100 closely related to BLS, were screened. Our work provides new data resources and a theoretical basis for exploring the infection mechanism of Xoc strain gx01 and the resistance mechanism of resistance gene bls2.

Comparative transcriptome-wide identification and differential expression of genes and lncRNAs in rice near-isogenic line (KW-Bph36-NIL) in response to BPH feeding.

Brown planthopper (BPH) is the most devastating pest of rice in Asia, causing substantial yield losses and has become a challenging task to be controlled under field conditions. Although extensive measures have been taken over the past decades, which resulted in the evolution of new resistant BPH strains. Therefore, besides other possible approaches, equipping host plants with resistant genes is the most effective and environment-friendly technique for BPH control. Here, we systematically analyzed transcriptome changes in the susceptible rice variety Kangwenqingzhan (KW) and the resistant near-isogenic line (NIL) KW-Bph36-NIL, through RNA-seq, depicting the differential expression profiles of mRNAs and long non-coding RNAs (lncRNAs) in rice before and after BPH feeding. We observed a proportion of genes (1.48%) and (2.74%) were altered in KW and NIL, respectively, indicating different responses of rice strains against BPH feeding. Nevertheless, we characterized 384 differentially expressed long non-coding RNAs (DELs) that can be impacted by the two strains by alternatively changing the expression patterns of the respective coding genes, suggesting their certain involvement in response to BPH feeding. In BPH invasion, KW and NIL responded differently by modifying the synthesis, storage, and transformation of intracellular substances, adjusting the nutrient accumulation and utilization inside and outside the cells. In addition, NIL expressed stronger resistance by acutely up-regulating genes and other transcription factors related to stress resistance and plant immunity. Altogether, our study elaborates valuable insights into the genome-wide DEGs and DELs expression profiles of rice under BPH invasion by high throughput sequencing and further suggests that NILs can be utilized in BPH resistance breeding programs in developing high-resistance rice lines.

Identification and characterization of An-4, a potential quantitative trait locus for awn development in rice.

BACKGROUND: Awn of rice is an important domestication trait closely associated with yield traits. Therefore, the identification of genes for awn development is of great significance for the elucidation of molecular mechanism of awn development and the genetic improvement of yield traits in rice. RESULTS: In this study, using chromosome segment substitution lines (CSSLs) derived from a long-awned Guangxi common wild rice (GXCWR, Oryza rufipogon Griff.) and a short-awned indica cultivar 9311, we identified An-4, a potential quantitative trait locus (QTL) for awn development. Then, An-4 was fine mapped into a 56-kb region of chromosome 2, which contained four annotated genes. Among these four annotated genes, Os02g0594800 was concluded to be the potential candidate gene for An-4. An-4 exhibited pleiotropic effects on awn development and several yield traits. Scanning electron microscopy (SEM) analysis showed that An-4 significantly promoted awn development at Sp7 and Sp8 stage of spikelet development. Transcriptome analysis suggested that An-4 might influence the development of awn by regulating the expression of genes related to growth, developmental process, channel regulation and extracellular region. By contrast to those of 9311, the expression level of OsRR5 in CSSL128 was significantly down-regulated, whereas the expression levels of OsCKX2 and OsGA2ox5 in CSSL128 were significantly up-regulated. In addition, our study showed that An-4 had additive effects with other genes for awn development, such as An-1, An-2/LABA1 and An-3/GAD1/RAE2. CONCLUSIONS: The identification of An-4 lays a foundation for cloning of An-4 and further elucidation of the molecular mechanism of awn development. Moreover, the identification of favorable allelic variation of An-4 from 9311 will be useful to improve rice yield traits.

CRISPR/Cas9 Guided Mutagenesis of Grain Size 3 Confers Increased Rice (Oryza sativa L.) Grain Length by Regulating Cysteine Proteinase Inhibitor and Ubiquitin-Related Proteins.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas9)-mediated genome editing has become an important way for molecular breeding in crop plants. To promote rice breeding, we edited the Grain Size 3 (GS3) gene for obtaining valuable and stable long-grain rice mutants. Furthermore, isobaric tags for the relative and absolute quantitation (iTRAQ)-based proteomic method were applied to determine the proteome-wide changes in the GS3 mutants compared with wild type (WT). Two target sites were designed to construct the vector, and the Agrobacterium-mediated method was used for rice transformation. Specific mutations were successfully introduced, and the grain length (GL) and 1000-grain weight (GWT) of the mutants were increased by 31.39% and 27.15%, respectively, compared with WT. The iTRAQ-based proteomic analysis revealed that a total of 31 proteins were differentially expressed in the GS3 mutants, including 20 up-regulated and 11 down-regulated proteins. Results showed that differentially expressed proteins (DEPs) were mainly related to cysteine synthase, cysteine proteinase inhibitor, vacuolar protein sorting-associated, ubiquitin, and DNA ligase. Furthermore, functional analysis revealed that DEPs were mostly enriched in cellular process, metabolic process, binding, transmembrane, structural, and catalytic activities. Pathway enrichment analysis revealed that DEPs were mainly involved in lipid metabolism and oxylipin biosynthesis. The protein-to-protein interaction (PPI) network found that proteins related to DNA damage-binding, ubiquitin-40S ribosomal, and cysteine proteinase inhibitor showed a higher degree of interaction. The homozygous mutant lines featured by stable inheritance and long-grain phenotype were obtained using the CRISPR/Cas9 system. This study provides a convenient and effective way of improving grain yield, which could significantly accelerate the breeding process of long-grain japonica parents and promote the development of high-yielding rice.

The Wild Rice Locus CTS-12 Mediates ABA-Dependent Stomatal Opening Modulation to Limit Water Loss Under Severe Chilling Stress.

A near-isogenic line (NIL) DC90 which was generated by introgressing a wild rice (Oryza rufipogon Griff.) locus CTS-12 into the 9311(Oryza sativa L. ssp. indica) background confers chilling tolerance phenotype. Here, our pilot trials showed that chilling tolerance was positively correlated with abscisic acid (ABA) biosynthesis. To understand how CTS-12 mediated the ABA-dependent multi-levels of regulation, the integration of transcriptomic and metabolomic profiling using the two-way orthogonal projections to latent structures (O2PLS) and discriminant analysis (OPLS-DA) modeling was performed to investigate the mechanisms underlying chilling tolerance. Our results revealed that metabolic shifts, including the activation of stachyose biosynthesis, amino acid metabolism pathways, phenylpropanoid/flavonoid biosynthesis, ABA biosynthesis, and perturbation of glycolysis, occurred under chilling treatment; in the recovery period, glutamate-related pathways, ß-alanine biosynthesis and degradation, and serotonin biosynthesis pathways were differentiated between 9311 and DC90. Particularly, the differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs), including galactinol, ß-alanine, glutamate, naringenin, serotonin, ABA, and LOC_Os03g44380 (9-cis-epoxycarotenoid dioxygenase 3, OsNCED3), might be involved in the chilling tolerance variation of 9311 and DC90. CRISPR/Cas9-edited OsNCED3 resulted in chilling sensitive of japonica rice ZH11, demonstrating the involvement of ABA pathway in chilling stress response. In addition, chilling tolerance of rice was associated with the balance of water uptake and loss that was modulated by stomatal movement under chilling stress. Therefore, we speculated that the CTS-12-mediated ABA signaling pathway leads to transcriptional regulation of chilling-responsive genes and, in turn, triggers metabolic shifts to coordinately regulate the stomatal movement of guard cells. The results of this study improve our understanding of the multilevel regulation of wild rice in response to chilling stress.

Correction to: Weighted gene coexpression network analysis-based identification of key modules and hub genes associated with drought sensitivity in rice.

An amendment to this paper has been published and can be accessed via the original article.

Precise Editing of the OsPYL9 Gene by RNA-Guided Cas9 Nuclease Confers Enhanced Drought Tolerance and Grain Yield in Rice (Oryza sativa L.) by Regulating Circadian Rhythm and Abiotic Stress Responsive Proteins.

Abscisic acid (ABA) is involved in regulating drought tolerance, and pyrabactin resistance-like (PYL) proteins are known as ABA receptors. To elucidate the role of one of the ABA receptors in rice, OsPYL9 was mutagenized through CRISPR/Cas9 in rice. Homozygous and heterozygous mutant plants lacking any off-targets and T-DNA were screened based on site-specific sequencing and used for morpho-physiological, molecular, and proteomic analysis. Mutant lines appear to accumulate higher ABA, antioxidant activities, chlorophyll content, leaf cuticular wax, and survival rate, whereas a lower malondialdehyde level, stomatal conductance, transpiration rate, and vascular bundles occur under stress conditions. Proteomic analysis found a total of 324 differentially expressed proteins (DEPs), out of which 184 and 140 were up and downregulated, respectively. The OsPYL9 mutants showed an increase in grain yield under both drought and well watered field conditions. Most of the DEPs related to circadian clock rhythm, drought response, and reactive oxygen species were upregulated in the mutant plants. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEPs were only involved in circadian rhythm and Gene Ontology (GO) analysis showed that most of the DEPs were involved in response to abiotic stimulus, and abscisic acid-activated signaling pathways. Protein GIGANTEA, Adagio-like, and Pseudo-response regulator proteins showed higher interaction in protein-protein interaction (PPI) network. Thus, the overall results showed that CRISPR/Cas9-generated OsPYL9 mutants have potential to improve both drought tolerance and the yield of rice. Furthermore, global proteome analysis provides new potential biomarkers and understandings of the molecular mechanism of rice drought tolerance.

Weighted gene coexpression network analysis-based identification of key modules and hub genes associated with drought sensitivity in rice.

BACKGROUND: Drought stress is an adverse factor with deleterious effects on several aspects of rice growth. However, the mechanism underlying drought resistance in rice remains unclear. To understand the molecular mechanism of the drought response in rice, drought-sensitive CSSL (Chromosome Single-substitution Segment Line) PY6 was used to map QTLs of sensitive phenotypes and to reveal the impact of the QTLs on transcriptional profiling. RESULTS: The QTL dss-1 was mapped onto the short arm of chromosome 1 of rice. According to transcriptomic analysis, the identified differentially expressed genes (DEGs) exhibited a downregulated pattern and were mainly enriched in photosynthesis-related GO terms, indicating that photosynthesis was greatly inhibited under drought. Further, according to weighted gene coexpression network analysis (WGCNA), specific gene modules (designating a group of genes with a similar expression pattern) were strongly correlated with H2O2 (4 modules) and MDA (3 modules), respectively. Likewise, GO analysis revealed that the photosynthesis-related GO terms were consistently overrepresented in H2O2-correlated modules. Functional annotation of the differentially expressed hub genes (DEHGs) in the H2O2 and MDA-correlated modules revealed cross-talk between abiotic and biotic stress responses for these genes, which were annotated as encoding WRKYs and PR family proteins, were notably differentially expressed between PY6 and PR403. CONCLUSIONS: We speculated that drought-induced photosynthetic inhibition leads to H2O2 and MDA accumulation, which can then trigger the reprogramming of the rice transcriptome, including the hub genes involved in ROS scavenging, to prevent oxidative stress damage. Our results shed light on and provide deep insight into the drought resistance mechanism in rice.

The Molecular Regulatory Pathways and Metabolic Adaptation in the Seed Germination and Early Seedling Growth of Rice in Response to Low O2 Stress.

As sessile organisms, flooding/submergence is one of the major abiotic stresses for higher plants, with deleterious effects on their growth and survival. Therefore, flooding/submergence is a large challenge for agriculture in lowland areas worldwide. Long-term flooding/submergence can cause severe hypoxia stress to crop plants and can result in substantial yield loss. Rice has evolved distinct adaptive strategies in response to low oxygen (O2) stress caused by flooding/submergence circumstances. Recently, direct seeding practice has been increasing in popularity due to its advantages of reducing cultivation cost and labor. However, establishment and growth of the seedlings from seed germination under the submergence condition are large obstacles for rice in direct seeding practice. The physiological and molecular regulatory mechanisms underlying tolerant and sensitive phenotypes in rice have been extensively investigated. Here, this review focuses on the progress of recent advances in the studies of the molecular mechanisms and metabolic adaptions underlying anaerobic germination (AG) and coleoptile elongation. Further, we highlight the prospect of introducing quantitative trait loci (QTL) for AG into rice mega varieties to ensure the compatibility of flooding/submergence tolerance traits and yield stability, thereby advancing the direct seeding practice and facilitating future breeding improvement.