Rice (Oryza sativa) is one of the most important crops grown worldwide, and saline–alkali stress seriously affects the yield and quality of rice. It is imperative to elucidate the molecular mechanisms underlying rice response to saline–alkali stress. In this study, we conducted an integrated analysis of the transcriptome and metabolome to elucidate the effects of long-term saline–alkali stress on rice. High saline–alkali stress (pH > 9.5) induced significant changes in gene expression and metabolites, including 9347 differentially expressed genes (DEGs) and 693 differentially accumulated metabolites (DAMs). Among the DAMs, lipids and amino acids accumulation were greatly enhanced. The pathways of the ABC transporter, amino acid biosynthesis and metabolism, glyoxylate and dicarboxylate metabolism, glutathione metabolism, TCA cycle, and linoleic acid metabolism, etc., were significantly enriched with DEGs and DAMs. These results suggest that the metabolites and pathways play important roles in rice’s response to high saline–alkali stress. Our study deepens the understanding of mechanisms response to saline–alkali stress and provides references for molecular design breeding of saline–alkali resistant rice.

Glutaredoxins (GRX) are key oxidoreductases that modulate protein redox states during plant development and stress responses. Alkaligrass (Puccinellia tenuiflora) is a highly salt-tolerant forage grass, but its GRX gene family (PutGRXs) remains uncharacterized, unlike those in Arabidopsis and other plants. We identified 25 PutGRX genes in the P. tenuiflora genome. Phylogenetic analysis revealed close evolutionary ties to monocotyledonous rice (Oryza sativa). Based on gene structure and conserved domains, PutGRXs were classified into three groups: five CGFS-type, eleven CPYC-type, and nine CC-type GRXs. Promoter analysis identified numerous cis-acting elements linked to abiotic stresses (e.g., light, drought, heat, cold) and hormone responses, suggesting a pivotal role in stress adaptation. Tissue-specific expression profiling showed differential PutGRX expression in roots, leaves, stems, flowers, and sheaths, with most genes responding to NaCl, NaHCO3, and Na2CO3 stresses. Functional characterization of chloroplast-localized PutGrxS12 demonstrated its importance in plant growth and ROS scavenging under salinity stress. This study offers the first comprehensive genomic and functional analysis of the PutGRX family in P. tenuiflora, highlighting its conservation, classification, and stress-responsive roles. Our findings advance understanding of GRX-mediated stress tolerance and provide potential targets for engineering salt-resistant crops.

Reverse transcription quantitative real‐time polymerase chain reaction (RT‐qPCR) is an accurate and convenient method for mRNA quantification. Selection of optimal reference gene(s) is an important step in RT‐qPCR experiments. However, the stability of housekeeping genes in spinach (Spinacia oleracea) under various abiotic stresses is unclear. Evaluating the stability of candidate genes and determining the optimal gene(s) for normalization of gene expression in spinach are necessary to investigate the gene expression patterns during development and stress response. In this study, ten housekeeping genes, 18S ribosomal RNA (18S rRNA), actin, ADP ribosylation factor (ARF), cytochrome c oxidase subunit 5C (COX), cyclophilin (CYP), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (H3), 50S ribosomal protein L2 (RPL2), and tubulin alpha chain (TUBα) from spinach, were selected as candidates in roots, stems, leaves, flowers, and seedlings in response to high temperature, CdCl₂, NaCl, NaHCO₃, and Na₂CO₃ stresses. The expression of these genes was quantified by RT‐qPCR and evaluated by NormFinder, BestKeeper, and geNorm. 18S rRNA, actin, ARF, COX, CYP, EF1α, GAPDH, H3, and RPL2 were detected as optimal reference genes for gene expression analysis of different organs and stress responses. The results were further confirmed by the expression pattern normalized with different reference genes of two heat‐responsive genes. Here, we optimized the detection method of the gene expression pattern in spinach. Our results provide the optimal candidate reference genes which were crucial for RT‐qPCR analysis.

Rubredoxin is one of the simplest iron–sulfur (Fe–S) proteins. It is found primarily in strict anaerobic bacteria and acts as a mediator of electron transfer participation in different biochemical reactions. The PutRUB gene encoding a chloroplast-localized rubredoxin family protein was screened from a yeast full-length cDNA library of Puccinellia tenuiflora under NaCl and NaHCO₃ stress. We found that PutRUB expression was induced by abiotic stresses such as NaCl, NaHCO₃, CuCl₂ and H₂O₂. These findings suggested that PutRUB might be involved in plant responses to adversity. In order to study the function of this gene, we analyzed the phenotypic and physiological characteristics of PutRUB transgenic plants treated with NaCl and NaHCO₃. The results showed that PutRUB overexpression inhibited H₂O₂ accumulation, and enhanced transgenic plant adaptability to NaCl and NaHCO₃ stresses. This indicated PutRUB might be involved in maintaining normal electron transfer to reduce reactive oxygen species (ROS) accumulation.

Rubredoxins are a class of iron-containing proteins that play an important role in the reduction of superoxide in some anaerobic bacteria and also act as electron carriers in many biochemical processes. Unlike the more widely studied about rubredoxin proteins in anaerobic bacteria, very few researches about the function of rubredoxins have been proceeded in plants. Previous studies indicated that rubredoxins inA. thalianamay play a critical role in responding to oxidative stress. In order to identify more rubredoxins in plants that maybe have similar functions as the rubredoxin-like protein ofA. thaliana, we identified and analyzed plant rubredoxin proteins using bioinformatics-based methods. Totally, 66 candidate rubredoxin proteins were identified based on public databases, exhibiting lengths of 187–360 amino acids with molecular weights of 19.856–37.117 kDa. The results of subcellular localization showed that these candidate rubredoxins were localized to the chloroplast, which might be consistent with the fact that rubredoxins were predominantly expressed in leaves. Analyses of conserved motifs indicated that these candidate rubredoxins contained rubredoxin and PDZ domains. The expression patterns of rubredoxins in glycophyte and halophytic plant under salt/drought stress revealed that rubredoxin is one of the important stress response proteins. Finally, the coexpression network of rubredoxin inArabidopsis thalianaunder abiotic was extracted from ATTED-II to explore the function and regulation relationship of rubredoxin inArabidopsis thaliana. Our results showed that putative rubredoxin proteins containing PDZ and rubredoxin domains, localized to the chloroplast, may act with other proteins in chloroplast to responses to abiotic stress in higher plants. These findings might provide value inference to promote the development of plant tolerance to some abiotic stresses and other economically important crops.

Puccinellia tenuiflora is a monocotyledonous halophyte that is able to survive in extreme saline soil environments at an alkaline pH range of 9–10. In this study, we transformed full-length cDNAs of P. tenuiflora into Saccharomyces cerevisiae by using the full-length cDNA over-expressing gene-hunting system to identify novel salt-tolerance genes. In all, 32 yeast clones overexpressing P. tenuiflora cDNA were obtained by screening under NaCl stress conditions; of these, 31 clones showed stronger tolerance to NaCl and were amplified using polymerase chain reaction (PCR) and sequenced. Four novel genes encoding proteins with unknown function were identified; these genes had no homology with genes from higher plants. Of the four isolated genes, two that encoded proteins with two transmembrane domains showed the strongest resistance to 1.3 M NaCl. RT-PCR and northern blot analysis of P. tenuiflora cultured cells confirmed the endogenous NaCl-induced expression of the two proteins. Both of the proteins conferred better tolerance in yeasts to high salt, alkaline and osmotic conditions, some heavy metals and H₂O₂ stress. Thus, we inferred that the two novel proteins might alleviate oxidative and other stresses in P. tenuiflora.