Background: Optimization of a highly efficient transient expression system is critical for the study of gene function, particularly in those plants in which stable transformation methods are not widely available. Agrobacterium tumefaciens‑mediated transient transformation is a simple and low-cost method that has been developed and applied to a wide variety of plant species. However, the transient expression in spinach (Spinacia oleracea L.) is still not reported. Results: We developed a transient expression system in spinach leaves of the Sp75 and Sp73 varieties. Several factors influencing the transformation efficiency were optimized such as Agrobacterium strain, spinach seedling stage, leaf position, and the expression time after injection. Agrobacterium strain GV3101 (pSoup-p19) was more efficient than AGL1 in expressing recombinant protein in spinach leaves. In general, Sp75 leaves were more suitable than Sp73 leaves, regardless of grow stage. At four-leaf stage, higher intensity and efficiency of transient expression were observed in group 1 (G1) of Sp75 at 53 h after injection (HAI) and in G1 of Sp73 at 64 HAI. At six-leaf stage of Sp75, group 3 (G3) at 72 HAI were the most effective condition for transient expression. Using the optimized expression system, we detected the subcellular localization of a transcriptional co-activator SoMBF1c and a NADPH oxidase SoRbohF. We also detected the interaction of the protein kinase SoCRK10 and the NADPH oxidase SoRbohB. Conclusion: This study established a method of highly efficient transient expression mediated by Agrobacterium in spinach leaves. The transient expression system will facilitate the analysis of gene function and lay a solid foundation for molecular design breeding of spinach.

The germin-like protein (GLP) family plays vital roles for plant growth, stress adaptation, and defense; however, its evolutionary dynamics and functional diversity in halophytes remain poorly characterized. Here, we present the genome-wide analysis of the GLP family in the halophytic forage alkaligrass (Puccinellia tenuiflora), which identified 54 PutGLPs with a significant expansion compared to other plant species. Phylogenetic analysis revealed monocot-specific clustering, with 41.5% of PutGLPs densely localized to chromosome 7, suggesting tandem duplication as a key driver of family expansion. Collinearity analysis confirmed evolutionary conservation with monocot GLPs. Integrated gene structure and motif analysis revealed conserved cupin domains (BoxB and BoxC). Promoter cis-acting elements analysis revealed stress-responsive architectures dominated by ABRE, STRE, and G-box motifs. Tissue-/organ-specific expression profiling identified root- and flower-enriched PutGLPs, implying specialized roles in stress adaptation. Dynamic expression patterns under salt-dominated stresses revealed distinct regulatory pathways governing ionic and alkaline stress responses. Functional characterization of PutGLP37 demonstrated its cell wall localization, dual superoxide dismutase (SOD) and oxalate oxidase (OXO) enzymatic activities, and salt stress tolerance in Escherichia coli, yeast (Saccharomyces cerevisiae INVSc1), and transgenic Arabidopsis. This study provides critical insights into the evolutionary innovation and stress adaptive roles of GLPs in halophytes.

CRISPR/Cas9 is a valuable tool and has been extensively employed to perform gene editing in plants. However, CRISPR/Cas9 has not been successfully used in spinach, an important leafy vegetable crop. Here, we established a CRISPR/Cas9-based gene-editing system for spinach hairy roots and edited two cellulose synthase-like D (CSLD) genes (SoCSLD2 and SoCSLD3) that were involved in root-hair formation of spinach hairy roots. Four mutation types (i.e., replacement, insertion, deletion, and combined mutations) were observed, among which the replacement accounted for the vast majority (about 64.1%). Mutation rate differed largely among different targets. Seven homozygous/bi-allelic and eight heterozygous/chimeric mutants of SoCSLD2 were obtained from 15 independent transgenic hairy root lines. All of the seven homozygous/bi-allelic mutant lines displayed bulking and short root hairs, which resembled the characteristics of Arabidopsis atcsld2 mutants. The transcriptomic analysis further revealed that multiple gene expressions for cell-wall modulation and membrane trafficking were disturbed, which might result in the inhibition of root hair growth in socsld2 mutants. This indicates that SoCSLD2 was successfully knocked out in spinach root hairs using the CRISPR/Cas9-based gene editing system.

Spinach (Spinacia oleracea L.), a globally consumed, nutrient-dense vegetable, contains diverse vitamins and minerals. However, elevated temperatures can constrain yield by interrupting leaf development and photosynthetic efficiency. The mitochondrial transcription termination factor (mTERF) family, which regulates organellar gene expression, plays crucial roles in plant growth and photosynthetic regulation. Thus, characterization of the spinach mTERF (SomTERF) family is critical for elucidating thermotolerance mechanisms in this crop. In this study, we systematically identified 31 SomTERF genes from the spinach genome, which are distributed across five chromosomes and nine unassembled genomic scaffolds. Subcellular localization predictions indicated that these proteins predominantly target chloroplasts and mitochondria. Conserved domain analyses confirmed that all SomTERF proteins possess canonical mTERF domains and ten conserved motifs. Phylogenetic clustering segregated these proteins into nine distinct subgroups (I–IX), with significant divergence observed in gene copy numbers among subgroups. Cis-element screening identified an abundance of heat-, cold-, and hormone-responsive motifs within SomTERF promoter regions. Notably, seven members (including SomTERF5) exhibited pronounced enrichment of heat shock elements (HSEs). Organ-specific expression profiling revealed preferential leaf expression of these seven genes. Comparative RT-qPCR in heat-sensitive (Sp73) and heat-tolerant (Sp75) cultivars under thermal stress demonstrated genotype-dependent expression dynamics. Functional validation of SomTERF5 was achieved through cloning, and transgenic Arabidopsis overexpressing SomTERF5 showed significantly enhanced thermotolerance, as evidenced by improved survival rates following heat treatment. Yeast two-hybrid (Y2H) assays further revealed physical interaction between SomTERF5 and SopTAC2. This study provides a comprehensive foundation for understanding mTERF-mediated developmental regulation and advanced molecular breeding strategies for developing heat-resilient spinach varieties.

CrRLK1L receptor-like kinases have attracted significant interest in plant biology owing to their pivotal roles in development, stress response, and signal transduction. However, our understanding of their importance in the extreme saline-alkali tolerant mechanisms of the halophyte alkaligrass ( Puccinellia tenuiflora ) remains limited. In this work, we analyzed the homology and evolutionary relationships of the CrRLK1L gene family across 30 species. Through genome-wide analysis we identified 25 PutCrRLK1L genes, which revealed conserved malectin-like and kinase domain, unique intronless structures, and stress-/hormone-responsive promoter elements. Transcriptomic and RT-qPCR analyses revealed salinity response of nine PutCrRLK1L genes’ expression in roots and leaves. Functional characterization of FER and antioxidant enzyme activities demonstrated that PutFER1 -overexpressing Arabidopsis lines exhibited enhanced root growth under salt stress and reduced ROS accumulation compared to wild-type plants. These findings may open new avenues for research into halophyte-specific salt tolerance mechanisms and provide potential candidate genes for improving crop resilience to saline-alkaline soils.