Rice Genes Research Articles

OsAlR3 regulates aluminum tolerance through promoting the secretion of organic acids and the expression of antioxidant genes in rice

In acidic soils, aluminum (Al) toxicity inhibits the growth and development of plant roots and affects nutrient and water absorption, leading to reduced yield and quality. Therefore, it is crucial to investigate and identify candidate genes for Al tolerance and elucidate their physiological and molecular mechanisms under Al stress. In this study, we identified a new gene OsAlR3 regulating Al tolerance, and analyzed its mechanism from physiological, transcriptional and metabolic levels. Compared with the WT, malondialdehyde (MDA) and hydrogen peroxide (H2O2) content were significantly increased, superoxide dismutase (SOD) activity and citric acid (CA) content were significantly decreased in the osalr3 mutant lines when exposed to Al stress. Under Al stress, the osalr3 exhibited decreased expression of antioxidant-related genes and lower organic acid content compared with WT. Integrated transcriptome and metabolome analysis showed the phenylpropanoid biosynthetic pathway plays an important role in OsAlR3-mediated Al tolerance. Exogenous CA and oxalic acid (OA) could increase total root length and enhance the antioxidant capacity in the mutant lines under Al stress. Conclusively, we found a new gene OsAlR3 that positively regulates Al tolerance by promoting the chelation of Al ions through the secretion of organic acids, and increasing the expression of antioxidant genes.

Mapping and Candidate Gene Analysis of the Low-Temperature-Sensitive Albino Gene OsLTSA8 in Rice Seedlings.

Chloroplasts are organelles responsible for photosynthesis in plants, providing energy for growth and development. However, the genetic regulatory mechanisms underlying early chloroplast development in rice remain incompletely understood. In this study, we identified a rice seedling thermosensitive chlorophyll-deficient mutant, osltsa8, and the genetic analysis of two F2 populations suggested that this trait may be controlled by more than one pair of alleles. Through reciprocal F2 populations and QTL-seq technology, OsLTSA8 was mapped to the interval of 24,280,402-25,920,942 bp on rice chromosome 8, representing a novel albino gene in rice. Within the candidate gene region of OsLTSA8, there were 258 predicted genes, among which LOC_Os08g39050, LOC_Os08g39130, and LOC_Os08g40870 encode pentatricopeptide repeat (PPR) proteins. RNA-seq identified 18 DEGs (differentially expressed genes) within the candidate interval, with LOC_Os08g39420 showing homology to the pigment biosynthesis-related genes Zm00001d017656 and Sb01g000470; LOC_Os08g39430 and LOC_Os08g39850 were implicated in chlorophyll precursor synthesis. RT-qPCR was employed to assess the expression levels of LOC_Os08g39050, LOC_Os08g39130, LOC_Os08g40870, LOC_Os08g39420, LOC_Os08g39430, and LOC_Os08g39850 in the wild-type and mutant plants. Among them, the differences in the expression levels of LOC_Os08g39050 and LOC_Os08g39430 were the most significant. This study will contribute to further elucidating the molecular mechanisms of rice chloroplast development.

Genetic Analysis and Fine Mapping of a New Rice Mutant, Leaf Tip Senescence 2.

Premature leaf senescence significantly reduces rice yields. Despite identifying numerous factors influencing these processes, the intricate genetic regulatory networks governing leaf senescence demand further exploration. We report the characterization of a stably inherited, ethyl methanesulfonate(EMS)-induced rice mutant with wilted leaf tips from seedling till harvesting, designated lts2. This mutant exhibits dwarfism and early senescence at the leaf tips and margins from the seedling stage when compared to the wild type. Furthermore, lts2 displays a substantial decline in both photosynthetic activity and chlorophyll content. Transmission electron microscopy revealed the presence of numerous osmiophilic granules in chloroplast cells near the senescent leaf tips, indicative of advanced cellular senescence. There was also a significant accumulation of H2O2, alongside the up-regulation of senescence-associated genes within the leaf tissues. Genetic mapping situated lts2 between SSR markers Q1 and L12, covering a physical distance of approximately 212 kb in chr.1. No similar genes controlling a premature senescence leaf phenotype have been identified in the region, and subsequent DNA and bulk segregant analysis (BSA) sequencing analyses only identified a single nucleotide substitution (C-T) in the exon of LOC_Os01g35860. These findings position the lts2 mutant as a valuable genetic model for elucidating chlorophyll metabolism and for further functional analysis of the gene in rice.

A CRISPR/Cas9-mediated mutant library of seed-preferred genes in rice.

A CRISPR/Cas9-mediated mutant library of seed-preferred genes in rice.

Rice Big Grain1 improves grain yield in ectopically expressing rice and heterologously expressing tobacco plants.

Functional genomics through transgenesis has provided faster and more reliable methods for identifying, characterizing, and utilizing genes or quantitative trait loci linked to agronomic traits to target yield. The present study explored the role of Big Grain1 (BG1) gene of rice (Oryza sativa L.) in yield improvement of crop plants. We aimed to identify the genetic variation of OsBG1 in various indica rice cultivars by studying the allelic polymorphism of the gene, while also investigating the gene's potential to increase crop yield through the transgenic approach. Our study reports the presence of an extra 393bp sequence having two 6bp enhancer elements in the 3' regulatory sequence of OsBG1 in the large-grain cultivar IR64 but not in the small-grain cultivar Badshahbhog. A single copy of the OsBG1 gene in both the cultivars and a 4.1-fold higher expression of OsBG1 in IR64 than in Badshahbhog imply that the grain size is positively correlated with the level of OsBG1 expression in rice. The ectopic expression of OsBG1 under the endosperm-specific glutelin C promoter in Badshahbhog enhanced the flag leaf length, panicle weight, and panicle length by an average of 33.2%, 33.7%, and 30.5%, respectively. The length of anthers, spikelet fertility, and grain yield per plant increased in transgenic rice lines by an average of 27.5%, 8.3%, and 54.4%, respectively. Heterologous expression of OsBG1 under the constitutive 2xCaMV35S promoter improved the number of seed pods per plant and seed yield per plant in transgenic tobacco lines by an average of 2.2-fold and 2.6-fold, respectively. Improving crop yield is crucial to ensure food security and socio-economic stability, and identifying suitable genetic factor is the essential step towards this endeavor. Our findings suggest that the OsBG1 gene is a promising candidate for improving the grain yield of monocot and dicot plant systems by molecular breeding and genetic engineering.

The potential of genome editing to create novel alleles of resistance genes in rice.

Rice, a staple food for a significant portion of the global population, faces persistent threats from various pathogens and pests, necessitating the development of resilient crop varieties. Deployment of resistance genes in rice is the best practice to manage diseases and reduce environmental damage by reducing the application of agro-chemicals. Genome editing technologies, such as CRISPR-Cas, have revolutionized the field of molecular biology, offering precise and efficient tools for targeted modifications within the rice genome. This study delves into the application of these tools to engineer novel alleles of resistance genes in rice, aiming to enhance the plant's innate ability to combat evolving threats. By harnessing the power of genome editing, researchers can introduce tailored genetic modifications that bolster the plant's defense mechanisms without compromising its essential characteristics. In this study, we synthesize recent advancements in genome editing methodologies applicable to rice and discuss the ethical considerations and regulatory frameworks surrounding the creation of genetically modified crops. Additionally, it explores potential challenges and future prospects for deploying edited rice varieties in agricultural landscapes. In summary, this study highlights the promise of genome editing in reshaping the genetic landscape of rice to confront emerging challenges, contributing to global food security and sustainable agriculture practices.

Quantitative profiling of m6A at single base resolution across the life cycle of rice and Arabidopsis

N6-methyladenosine (m6A) plays critical roles in regulating mRNA metabolism. However, comprehensive m6A methylomes in different plant tissues with single-base precision have yet to be reported. Here, we present transcriptome-wide m6A maps at single-base resolution in different tissues of rice and Arabidopsis using m6A-SAC-seq. Our analysis uncovers a total of 205,691 m6A sites distributed across 22,574 genes in rice, and 188,282 m6A sites across 19,984 genes in Arabidopsis. The evolutionarily conserved m6A sites in rice and Arabidopsis ortholog gene pairs are involved in controlling tissue development, photosynthesis and stress response. We observe an overall mRNA stabilization effect by 3’ UTR m6A sites in certain plant tissues. Like in mammals, a positive correlation between the m6A level and the length of internal exons is also observed in plant mRNA, except for the last exon. Our data suggest an active m6A deposition process occurring near the stop codon in plant mRNA. In addition, the MTA-installed plant mRNA m6A sites correlate with both translation promotion and translation suppression, depicting a more complicated regulatory picture. Our results therefore provide in-depth resources for relating single-base resolution m6A sites with functions in plants and uncover a suppression-activation model controlling m6A biogenesis across species.

Genome-wide association study reveals that JASMONATE ZIM-DOMAIN 5 regulates seed germination in rice

Seed germination is a complex trait regulated by multiple genes in rice. However, the regulators of rice seed germination have yet to be sufficiently determined. Here, a quantitative trait locus (QTL) for rice seed germination was identified in a genome-wide association study. The candidate gene JASMONATE ZIM-DOMAIN 5 (OsJAZ5) of the QTL was verified that positively regulates seed germination. OsJAZ5 regulation of seed germination involves an OsABI3-mediated abscisic acid pathway. Overexpression of OsJAZ5 facilitated seed germination. The application of OsJAZ5 might be useful for increasing seed germination for rice direct seeding.

High Concentrations of Se Inhibited the Growth of Rice Seedlings.

Selenium (Se) is crucial for both plants and humans, with plants acting as the main source for human Se intake. In plants, moderate Se enhances growth and increases stress resistance, whereas excessive Se leads to toxicity. The physiological mechanisms by which Se influences rice seedlings' growth are poorly understood and require additional research. In order to study the effects of selenium stress on rice seedlings, plant phenotype analysis, root scanning, metal ion content determination, physiological response index determination, hormone level determination, quantitative PCR (qPCR), and other methods were used. Our findings indicated that sodium selenite had dual effects on rice seedling growth under hydroponic conditions. At low concentrations, Se treatment promotes rice seedling growth by enhancing biomass, root length, and antioxidant capacity. Conversely, high concentrations of sodium selenite impair and damage rice, as evidenced by leaf yellowing, reduced chlorophyll content, decreased biomass, and stunted growth. Elevated Se levels also significantly affect antioxidase activities and the levels of proline, malondialdehyde, metal ions, and various phytohormones and selenium metabolism, ion transport, and antioxidant genes in rice. The adverse effects of high Se concentrations may directly disrupt protein synthesis or indirectly induce oxidative stress by altering the absorption and synthesis of other compounds. This study aims to elucidate the physiological responses of rice to Se toxicity stress and lay the groundwork for the development of Se-enriched rice varieties.

Selection of rice aroma from crossing aromatic rice x T250.7 mutant genotype using DNA marker

Abstract. Ishak I, Fedora F. 2024. Selection of rice aroma from crossing aromatic rice x T250.7 mutant genotype using DNA marker. Biodiversitas 25: 2191-2197. Breeding fragrant rice using conventional approach was considered ineffective due to time consuming, therefore molecular breeding using DNA markers was introduced. This research aimed to utilize marker-assisted selection (MAS) for detection of fragrance gene in rice related to molecular breeding. Crossing between aromatic rice (Sinar 1) and non-aromatic rice (T250.7 mutant genotype) showed the results at F1 plants and found the DNA fragment size of 257 bp for fragrance genes in rice was at TS1, TS2, TS3, TS4, TS5, and TS.7 genotypes, whereas, TS6 did not show a fragment DNA at 257 bp. It was predicted that the TS6 genotype did not cross between variety Sinar 1 with T250.7. The F2 seeds were selected and morphologically, they had slender seeds and continued planting for F2 plants. Based on DNA marker identification, it revealed that 75% of the rice plants carried aroma. Similarly, the selected F3 plants continued to be planted and molecular identification indicated that aroma segregation was the same as that in the previous generation. Plants with the number of accessions of 721, 761, and 7210 were predicted to have homogeneity in aroma after detection using molecular markers. The MAS in breeding programs can shorten the duration of rice selection.

Exogenous proline regulates pectin demethylation by rescuing pectin methylesterase functioning of cell wall from Cr(VI) toxicity in rice plants

BackgroundPlants are equipped with several sophisticated mechanisms to deal with heavy metals (HMs) toxicity. Cell walls, which are rich in pectin, are important in the sequestration and compartmentalization of HMs. Pectin demethylation is carried out by pectin methylesterase (PME), which is a crucial activity in cell walls for the adsorption of HMs. This study focused on the factors that contribute to chromium (Cr) adsorption in rice plants exposed to Cr(VI) treatments without proline (Pro) “Cr(VI)” and with Pro “Pro + Cr(VI)” application.ResultsThe results exhibited that when rice plants were treated with Cr(VI), their PME activity decreased, because Cr(VI) was bound to certain isoforms of PME and prevented the demethylation of pectin. The application of Pro increased PME activity by promoting the transcription of several PME-related genes. These genes were recognized on the basis of their similarity with PME genes in Arabidopsis. Gene expression variation factors (GEVFs) between the “Cr(VI)” and “Pro + Cr(VI)” treatments revealed that OsPME7 and OsPME9 have the highest positive GEVF values than other OsPME genes of rice. In addition, Pro application increased pectin content significantly in rice plants exposed to Cr(VI) stress. Proline application also leads to an increased concentration of Cr in rice roots compared with “Cr(VI)” treatments alone.ConclusionsThese findings suggest that Pro increased Cr(VI) adsorption in cell walls of rice plants by enhancing the PME activity and pectin content when exposed to “Cr(VI)” treatments, mainly regulated by OsPME7 and OsPME9.Graphical

Systems genomics of salinity stress response in rice.

Populations can adapt to stressful environments through changes in gene expression. However, the role of gene regulation in mediating stress response and adaptation remains largely unexplored. Here, we use an integrative field dataset obtained from 780 plants of Oryza sativa ssp. indica (rice) grown in a field experiment under normal or moderate salt stress conditions to examine selection and evolution of gene expression variation under salinity stress conditions. We find that salinity stress induces increased selective pressure on gene expression. Further, we show that trans-eQTLs rather than cis-eQTLs are primarily associated with rice's gene expression under salinity stress, potentially via a few master-regulators. Importantly, and contrary to the expectations, we find that cis-trans reinforcement is more common than cis-trans compensation which may be reflective of rice diversification subsequent to domestication. We further identify genetic fixation as the likely mechanism underlying this compensation/reinforcement. Additionally, we show that cis- and trans-eQTLs are under different selection regimes, giving us insights into the evolutionary dynamics of gene expression variation. By examining genomic, transcriptomic, and phenotypic variation across a rice population, we gain insights into the molecular and genetic landscape underlying adaptive salinity stress responses, which is relevant for other crops and other stresses.

Insights into the virulence profiles and molecular diversity of Xanthomonas oryzae pv. oryzae isolates associated with bacterial blight of rice in major districts of Karnataka, India

Bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating disease affecting rice worldwide. In this study, to characterize Xoo isolates and evaluate the effectiveness of various BLB-resistant genes in rice, a set of 29 rice differentials each carrying, single or combinations of BLB resistant genes were used for characterizing Xoo isolates under glasshouse condition. None of the tested isolates were able to show complete virulence across all Near Isogenic Lines (NILs). All isolates varied with degrees of resistance and susceptibility. Notably, isolate KXo-3 exhibited high virulence. Among single BLB resistant genes, IRBB-8 (xa8) and IRBB-13 (xa13) were found highly promising while among two gene combinations, IRBB-51 (Xa4+xa13), IRBB-52 (Xa4+Xa21) and IRBB-53 (xa5+xa13). Among three and four gene combination of BB resistant genes, IRBB-56 (Xa4+xa5+xa13), IRBB-57 (Xa4+xa5+Xa21), IRBB-58 (Xa4+xa13+Xa21), IRBB-59 (Xa5+xa13+Xa21), IRBB-60 (Xa4+xa5+xa13+Xa21), IRBB-63 (xa5+Xa7+xa13) and IRBB-65 (Xa4+Xa7+xa13+Xa21) were resistant against almost all the Xoo isolates. Based on disease reactions, Xoo isolates were grouped into 6 pathotypes, as IXoPt # 1, 2, 4, 5, 17 and 22. IXoPt-22 was highly virulent and IXoPt-1 was least virulent. IXoPt-2 was widely distributed with the maximum distribution frequency of 23.8. Molecular diversity using IS1112 repetitive elements showed that the Xoo isolates were genetically very diverse with three major clusters, I, II and III. The haplotype diversity resulted in 27 haplotypes generation. The isolates of Belagavi, Haveri and Davanagere were very diverse with distinct haplotypes. These findings enhance our understanding of BLB resistance mechanisms and Xoo population diversity, aiding in the development of effective disease management strategies in rice cultivation.

OsWRKY12 negatively regulates the drought-stress tolerance and secondary cell wall biosynthesis by targeting different downstream transcription factor genes in rice

With the increasing occurrence of global warming, drought is becoming a major constraint for plant growth and crop yield. Plant cell walls experience continuous changes during the growth, development, and in responding to stressful conditions. The plant WRKYs play pivotal roles in regulating the secondary cell wall (SCW) biosynthesis and helping plant defend against abiotic stresses. qRT-PCR evidence showed that OsWRKY12 was affected by drought and ABA treatments. Over-expression of OsWRKY12 decreased the drought tolerance of the rice transgenics at the germination stage and the seedling stage. The transcription levels of drought-stress-associated genes as well as those genes participating in the ABA biosynthesis and signaling were significantly different compared to the wild type (WT). Our results also showed that less lignin and cellulose were deposited in the OsWRKY12-overexpressors, and heterogenous expression of OsWRKY12 in atwrky12 could lower the increased lignin and cellulose contents, as well as the improved PEG-stress tolerance, to a similar level as the WT. qRT-PCR results indicated that the transcription levels of all the genes related to lignin and cellulose biosynthesis were significantly decreased in the rice transgenics than the WT. Further evidence from yeast one-hybrid assay and the dual-luciferase reporter system suggested that OsWRKY12 could bind to promoters of OsABI5 (the critical component of the ABA signaling pathway) and OsSWN3/OsSWN7 (the key positive regulators in the rice SCW thickening), and hence repressing their expression. In conclusion, OsWRKY12 mediates the crosstalk between SCW biosynthesis and plant stress tolerance by binding to the promoters of different downstream genes.

Transcription factor OsWRKY11 induces rice heading at low concentrations but inhibits rice heading at high concentrations.

The heading date of rice is a crucial agronomic characteristic that influences its adaptability to different regions and its productivity potential. Despite the involvement of WRKY transcription factors in various biological processes related to development, the precise mechanisms through which these transcription factors regulate the heading date in rice have not been well elucidated. The present study identified OsWRKY11 as a WRKY transcription factor which exhibits a pivotal function in the regulation of the heading date in rice through a comprehensive screening of a clustered regularly interspaced palindromic repeats (CRISPR) ‒ CRISPR-associated nuclease 9 mutant library that specifically targets the WRKY genes in rice. The heading date of oswrky11 mutant plants and OsWRKY11-overexpressing plants was delayed compared with that of the wild-type plants under short-day and long-day conditions. Mechanistic investigation revealed that OsWRKY11 exerts dual effects on transcriptional promotion and suppression through direct and indirect DNA binding, respectively. Under normal conditions, OsWRKY11 facilitates flowering by directly inducing the expression of OsMADS14 and OsMADS15. The presence of elevated levels of OsWRKY11 protein promote formation of a ternary protein complex involving OsWRKY11, Heading date 1 (Hd1), and Days to heading date 8 (DTH8), and this complex then suppresses the expression of Ehd1, which leads to a delay in the heading date. Subsequent investigation revealed that a mild drought condition resulted in a modest increase in OsWRKY11 expression, promoting heading. Conversely, under severe drought conditions, a significant upregulation of OsWRKY11 led to the suppression of Ehd1 expression, ultimately causing a delay in heading date. Our findings uncover a previously unacknowledged mechanism through which the transcription factor OsWRKY11 exerts a dual impact on the heading date by directly and indirectly binding to the promoters of target genes.

Integrative genomic and transcriptomic analysis of Xanthomonas oryzae pv. oryzae pathotype IV, V, and IX in China reveals rice defense-responsive genes

Bacterial blight of rice is a devastating disease caused by the gram-negative bacteria Xanthomonas oryzae pv. oryzae (Xoo). Chinese Xoo strain pathotypes IV, V, and IX are the major virulent Xoo strain types in South China sequentially from the 1990s to the present. Here, we report the isolation of GD0201 and GD0202, which belong to pathotypes IV and IX, respectively, and the complete genome sequence and transcriptomic analysis of GD0201 (IV), GD1358 (V), and GD0202 (IX). We found that resistance genes xa5, Xa23, and Xa27 confer strong resistance to all three Xoo strains, indicating that they are currently good choices for resistance rice breeding. The genome analysis reveals fewer TAL and non-TAL effector coding genes in GD0202 than in the other two strains, potentially contributing to its strong virulence. Transcriptomic analysis of ZH11 inoculated with the three Xoo strains strongly suggests that three Xoo strains for better infection repress the ethylene response factor (ERF) gene family members. Furthermore, weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) analysis revealed 14 hub genes potentially associated with rice response to the three Xoo strains. The expression of several hub genes was validated to be induced by all three Xoo strains, suggesting its role in bacterial blight disease response to Xoo strains. Genomic analysis of the Xoo strains belonging to pathotypes IV, V, and IX, identification of effectors and genes related to Xoo virulence in rice plants will provide insights into understanding the molecular mechanism underlying rice-Xoo interaction and the gene expression pattern in response to Xoo infection.

Identification and Functional Analysis of KH Family Genes Associated with Salt Stress in Rice.

Salinity stress has a great impact on crop growth and productivity and is one of the major factors responsible for crop yield losses. The K-homologous (KH) family proteins play vital roles in regulating plant development and responding to abiotic stress in plants. However, the systematic characterization of the KH family in rice is still lacking. In this study, we performed genome-wide identification and functional analysis of KH family genes and identified a total of 31 KH genes in rice. According to the homologs of KH genes in Arabidopsis thaliana, we constructed a phylogenetic tree with 61 KH genes containing 31 KH genes in Oryza sativa and 30 KH genes in Arabidopsis thaliana and separated them into three major groups. In silico tissue expression analysis showed that the OsKH genes are constitutively expressed. The qRT-PCR results revealed that eight OsKH genes responded strongly to salt stresses, and OsKH12 exhibited the strongest decrease in expression level, which was selected for further study. We generated the Oskh12-knockout mutant via the CRISPR/Cas9 genome-editing method. Further stress treatment and biochemical assays confirmed that Oskh12 mutant was more salt-sensitive than Nip and the expression of several key salt-tolerant genes in Oskh12 was significantly reduced. Taken together, our results shed light on the understanding of the KH family and provide a theoretical basis for future abiotic stress studies in rice.

Genome-wide characterization and functional analysis of heat shock transcription factors in wild and cultivated rice (Oryza sativa L.)

Due to domestication, the genetic diversity in cultivated rice is lower than that of wild rice species. Recent progress in sequencing the genomes of wild and cultivated rice genus is an important step in identifying their genetic differences associated with abiotic and biotic stresses-resistant ability. Thus, in this study, heat shock transcription factor (Hsf) genes in 6 wild rice accessions, including Oryza barthii, Oryza glumaepatula, Oryza meridionalis, Oryza nivara, Oryza punctate and Oryza rufipogon, and 1 cultivated rice variety Nipponbare were firstly identified, and the evolution of rice Hsf genes were analyzed. Totally, 22, 23, 24, 24, 25, 25 and 25 Hsf genes were identified in the 7 tested rice genomes, Oryza barthii, Oryza glumaepatula, Oryza meridionalis, Oryza nivara, Oryza punctate, Oryza rufipogon and Oryza sativa, respectively. The evolutionary analysis of Hsf genes revealed that Oryza rufipogon was the immediate ancestral progenitor of Oryza sativa. The variation in the number of Hsf genes between wild and cultivated rice genotypes was mainly due to segmental duplication and whole genome duplication (WGD) events, which might contribute to the different stress-tolerant ability between the wild and cultivated rice varieties. Secondly, the function of the Hsf genes in wild and cultivated rice in response to salinity stress was compared. Under salt stress, four Hsf genes, HsfA2b, HsfB4b, HsfB4c and HsfB4d, were exclusively down-regulated in Oryza rufipogon shoots. Whilst, another four Hsf genes, HsfA7a, HsfB2c, HsfC1b and HsfC2b, were up-regulated in the shoots of both the cultivated rice and its ancestral progenitor Oryza rufipogon. Meanwhile, genomic comparison results showed that 1, 1, 5 and 5 nsSNPs were detected in these 4 Hsf genes respectively, which might alter the functions of these genes. RT-qPCR results further confirmed the expression profile of one of these 4 Hsf genes, HsfB2c. The expression pattern of this gene in the cultivated rice variety in response to salt stress was similar with the previously known salt stress-tolerant gene, WRKY28. This indicated that these 4 Hsf genes might be among the conserved genes related to salt stress tolerance in the wild and cultivated rice. In summary, these results provide novel clues to the further functional studies of Hsf genes in rice for breeding programs.

In-locus gene silencing in plants using genome editing.

Gene silencing is crucial in crop breeding for desired trait development. RNA interference (RNAi) has been used widely but is limited by ectopic expression of transgenes and genetic instability. Introducing an upstream start codon (uATG) into the 5'untranslated region (5'UTR) of a target gene may 'silence' the target gene by inhibiting protein translation from the primary start codon (pATG). Here, we report an efficient gene silencing method by introducing a tailor-designed uATG-containing element (ATGE) into the 5'UTR of genes in plants, occupying the original start site to act as a new pATG. Using base editing to introduce new uATGs failed to silence two of the tested three rice genes, indicating complex regulatory mechanisms. Precisely inserting an ATGE adjacent to pATG achieved significant target protein downregulation. Through extensive optimization, we demonstrated this strategy substantially and consistently downregulated target protein expression. By designing a bidirectional multifunctional ATGE4, we enabled tunable knockdown from 19% to 89% and observed expected phenotypes. Introducing ATGE into Waxy, which regulates starch synthesis, generated grains with lower amylose, revealing the value for crop breeding. Together, we have developed a programmable and robust method to knock down gene expression in plants, with potential for biological mechanism exploration and crop enhancement.

The Characteristics and Expression Analysis of the Tomato SlRBOH Gene Family under Exogenous Phytohormone Treatments and Abiotic Stresses.

Respiratory burst oxidase homologs (RBOHs), also known as NADPH oxidases, contribute significantly to the production of ROS in plants, alongside other major sources such as photosynthesis and electron transport in chloroplasts. It has been shown that plant RBOHs play an active role in plant adversity response and electron transport. However, the phylogenetic analysis and characterization of the SlRBOH gene family in tomatoes have not been systematically studied. This study identified 11 SlRBOH genes in the tomato genome using a genome-wide search approach. The physicochemical properties, chromosomal localization, subcellular localization, secondary structure, conserved motifs, gene structure, phylogenetics, collinear relationships, cis-acting elements, evolutionary selection pressures, tissue expressions, and expression patterns under exogenous phytohormones (ABA and MeJA) and different abiotic stresses were also analyzed. We found that the SlRBOHs are distributed across seven chromosomes, collinearity reflecting their evolutionary relationships with corresponding genes in Arabidopsis thaliana and rice. Additionally, all the SlRBOH members have five conserved domains and 10 conserved motifs and have similar gene structures. In addition, the results of an evolutionary selection pressure analysis showed that SlRBOH family members evolved mainly by purifying selection, making them more structurally stable. Cis-acting element analyses showed that SlRBOHs were responsive to light, hormone, and abiotic stresses. Tissue expression analysis showed that SlRBOH family members were expressed in all tissues of tomato to varying degrees, and most of the SlRBOHs with the strongest expression were found in the roots. In addition, the expressions of tomato SlRBOH genes were changed by ABA, MeJA, dark period extension, NaCl, PEG, UV, cold, heat, and H2O2 treatments. Specifically, SlRBOH4 was highly expressed under NaCl, PEG, heat, and UV treatments, while SlRBOH2 was highly expressed under cold stress. These results provide a basis for further studies on the function of SlRBOHs in tomato.