Heavy metal exposure is a serious environmental stress in plants. However, plants have evolved several strategies to improve their heavy metal tolerance. Heavy metal-associated proteins (HMPs) participate in heavy metal detoxification. Here, we identified 46 and 55 HMPs in rice and Arabidopsis, respectively, and named them OsHMP 1–46 and AtHMP 1–55 according to their chromosomal locations. The HMPs from both plants were divided into six clades based on the characteristics of their heavy metal-associated domains (HMA). The HMP gene structures and motifs varied greatly among the different classifications. The HMPs had high collinearity and were segmentally duplicated. A cis-element analysis revealed that the HMPs may be regulated by different transcription factors. An expression profile analysis disclosed that only eight OsHMPs were constitutive in rice tissues. Of these, the expression of OsHMP37 was far higher than that of the other seven genes while OsHMP28 was expressed exclusively in the roots. For Arabidopsis, nine AtHMPs presented with very high transcript levels in all organs. Most of the selected OsHMPs were differentially expressed in various tissues under different heavy metal stresses. Only OsHMP09, OsHMP18, and OsHMP22 showed higher expression levels in all tissues under different heavy metal stresses. In contrast, most of the selected AtHMPs had nearly constant expression levels in different tissues under various heavy metal stresses.The AtHMP20, AtHMP23, AtHMP25, AtHMP31, AtHMP35, AtHMP46 expression levels under different heavy metal stresses were higher in the leaves and roots. The foregoing discoveries elucidated HMP evolution in monocotyledonous and dicotyledonous plants and may helpful functionally characterize HMPs in the future.

Background: Salt stress is one of the main abiotic stresses that limits rice production worldwide. Rice salt tolerance at the bud burst stage directly affects the seedling survival rate and the final yield in the direct seeding cultivation model. However, the reports on quantitative trait locus (QTL) mapping and map-based cloning for salt tolerance at the bud burst stage are limited. Results: Here, an F_2:3 population derived from a cross between IR36 (salt-sensitive) and Weiguo (salt-tolerant) was used to identify salt-tolerant QTL interval at the bud burst stage using a whole-genome sequencing-based QTL-seq containing 40 extreme salt-tolerant and 40 extreme salt-sensitive individuals. A major QTL, qRSL7, related to relative shoot length (RSL) was detected on chromosome 7 using ΔSNP index algorithms and Euclidean Distance (ED) algorithms. According to single nucleotide polymorphisms (SNPs) between the parents, 25 Kompetitive allele-specific PCR (KASP) markers were developed near qRSL7, and regional QTL mapping was performed using 199 individuals from the F_2:3 population. We then confirmed and narrowed down qRSL7 to a 222 kb genome interval. Additionally, RNA sequencing (RNA-seq) was performed for IR36 and Weiguo at 36 h after salt stress and control condition at the bud burst stage, and 5 differentially expressed genes (DEGs) were detected in the candidate region. The qRT-PCR results showed the same expression patterns as the RNA-seq data. Furthermore, sequence analysis revealed a 1 bp Indel difference in Os07g0569700 (OsSAP16) between IR36 and Weiguo. OsSAP16 encodes a stress-associated protein whose expression is increased under drought stress. Conclusion: These results indicate that OsSAP16 was the candidate gene of qRSL7. The results is useful for gene cloning of qRSL7 and for improving the salt tolerance of rice varieties by marker assisted selection (MAS).

Salinity-alkalinity stress is one of the major factors limiting rice production. The damage caused by alkaline salt stress to rice growth is more severe than that caused by neutral salt stress. At present, the genetic resources (quantitative trait loci (QTLs) and genes) that can be used by rice breeders to improve alkalinity tolerance are limited. Here, we assessed the alkalinity tolerance of rice at the seedling stage and performed a genome-wide association study (GWAS) based on genotypic data including 788,396 single-nucleotide polymorphisms (SNPs) developed by re-sequencing 295 japonica rice varieties. We used the score of alkalinity tolerance (SAT), the concentrations of Na+ and K+ in the shoots (SNC and SKC, respectively) and the Na+/K+ ratio of shoots (SNK) as indices to assess alkalinity tolerance at the seedling stage in rice. Based on population structure analysis, the japonica rice panel was divided into three subgroups. Linkage disequilibrium (LD) analysis showed that LD decay occurred at 109.77 kb for the whole genome and varied between 13.79 kb and 415.77 kb across the 12 chromosomes, at which point the pairwise squared correlation coefficient (r2) decreased to half of its maximum value. A total of eight QTLs significantly associated with the SAT, SNC and SNK were identified by genome-wide association mapping. A common QTL associated with the SAT, SNC and SNK on chromosome 3 at the position of 15.0 Mb, which explaining 13.36~13.64% of phenotypic variation, was selected for further analysis. The candidate genes were filtered based on LD decay, Gene Ontology (GO) enrichment, RNA sequencing data, and quantitative real-time PCR (qRT-PCR) analysis. Moreover, sequence analysis revealed one 7-bp insertion/deletion (indel) difference in LOC_Os03g26210 (OsIRO3) between the alkalinity-tolerant and alkalinity-sensitive rice varieties. OsIRO3 encodes a bHLH-type transcription factor and has been shown to be a negative regulator of the Fe-deficiency response in rice. Based on these results, OsIRO3 maybe a novel functional gene associated with alkalinity tolerance in japonica rice. This study provides resources for improving alkalinity tolerance in rice, and the functional molecular marker could be verified to breed new rice varieties with alkalinity tolerance via marker-assisted selection (MAS).

Background: Salinity-alkalinity stress is one of the major factors limiting rice production. Damage caused by alkaline salt stress is more severe than that caused by neutral salt stress. Alkali tolerance at the bud stage in rice directly affects seedling survival and final yield when using the direct seeding cultivation model. However, genetic resources (QTLs and genes) for rice breeders to improve alkali tolerance are limited. In this study, we combined linkage mapping and a genome-wide association study (GWAS) to analyze the genetic structure of this trait in japonica rice at the bud stage. Results: A population of 184 recombinant inbred lines (RILs) was utilized to map quantitative trait loci (QTLs) for the root length under control condition (RL), alkaline stress (ARL) and relative root length (RRL) at the bud stage. A major QTL related to alkali tolerance at the rice bud stage, qAT11, was detected on chromosome 11. Interestingly, a GWAS identified a lead SNP (Chr_21,999,659) in qAT11 that was significantly associated with alkaline tolerance. After filtering by linkage disequilibrium (LD), haplotype analysis, quantitative real-time PCR, we obtained three candidate genes (LOC_Os11g37300, LOC_Os11g37320 and LOC_Os11g37390). In addition, we performed phenotype verification on the CRISPR/Cas9 mutant of LOC_Os11g37390. Conclusion: Based on these results, LOC_Os11g37300, LOC_Os11g37320 and LOC_Os11g37390 were the candidate genes contributing to alkaline tolerance in japonica rice. This study provides resources for breeding aimed at improving rice responses to alkalinity stress.

Trihelix transcription factors play a role in plant growth, development and various stress responses. Here, we identified 41 trihelix family genes in the rice genome. These OsMSLs (Myb/SANT-LIKE) were located on twelve chromosomes. Synteny analysis indicated only six duplicated gene pairs in the rice trihelix family. Phylogenetic analysis of these OsMSLs and the trihelix genes from other species divided them into five clusters. OsMSLs from different groups significantly diverged in terms of gene structure and conserved functional domains. However, all OsMSLs contained the same five cis-elements. Some of these were responsive to light and dehydration stress. All OsMSLs expressed in four tissues and six developmental stages of rice but with different expression patterns. Quantitative real-time PCR analysis revealed that the OsMSLs responded to abiotic stresses including drought and high salt stress and stress signal molecule including ABA (abscisic acid), hydrogen peroxide. OsMSL39 were simultaneously expressed under all treatments, while OsMSL28 showed high expression under hydrogen peroxide, drought, and high salt treatments. Moreover, OsMSL16/27/33 displayed significant expression under ABA and drought treatments. Nevertheless, their responses were regulated by light. The expression levels of the 12 chosen OsMSLs differed between light and dark conditions. In conclusion, our results helped elucidate the biological functions of rice trihelix genes and provided a theoretical basis for further characterizing their biological roles in responding to abiotic stresses.

Caffeic acid O-methyltransferase (COMT) is one of the core enzymes involved in lignin synthesis. However, there is no systematic study on the rice COMT gene family. We identified 33 COMT genes containing the methyltransferase-2 domain in the rice genome using bioinformatic methods and divided them into Group I (a and b) and Group II. Motifs, conserved domains, gene structure and SNPs density are related to the classification of OsCOMTs. The tandem phenomenon plays a key role in the expansion of OsCOMTs. The expression levels of fourteen and thirteen OsCOMTs increased or decreased under salt stress and drought stress, respectively. OsCOMTs showed higher expression levels in the stem. The lignin content of rice was measured in five stages; combined with the expression analysis of OsCOMTs and multiple sequence alignment, we found that OsCOMT8, OsCOMT9 and OsCOMT15 play a key role in the synthesis of lignin. Targeted miRNAs and gene ontology annotation revealed that OsCOMTs were involved in abiotic stress responses. Our study contributes to the analysis of the biological function of OsCOMTs, which may provide information for future rice breeding and editing of the rice genome.