High temperature induces stomatal opening; however, uncontrolled stomatal opening is dangerous for plants in response to high temperature. We identified a high-temperature sensitive (hts) mutant from the ethyl methane sulfonate (EMS)-induced maize (Zea mays) mutant library that is linked to a single base change in MITOGEN-ACTIVATED PROTEIN KINASE 20 (ZmMPK20). Our data demonstrated that hts mutants exhibit substantially increased stomatal opening and water loss rate, as well as decreased thermotolerance, compared to wild-type plants under high temperature. ZmMPK20-knockout mutants showed similar phenotypes as hts mutants. Overexpression of ZmMPK20 decreased stomatal apertures, water loss rate, and enhanced plant thermotolerance. Additional experiments showed that ZmMPK20 interacts with MAP KINASE KINASE 9 (ZmMKK9) and E3 ubiquitin ligase RPM1 INTERACTING PROTEIN 2 (ZmRIN2), a maize homolog of Arabidopsis (Arabidopsis thaliana) RIN2. ZmMPK20 prevented ZmRIN2 degradation by inhibiting ZmRIN2 self-ubiquitination. ZmMKK9 phosphorylated ZmMPK20 and enhanced the inhibitory effect of ZmMPK20 on ZmRIN2 degradation. Moreover, we employed virus-induced gene silencing (VIGS) to silence ZmMKK9 and ZmRIN2 in maize and heterologously overexpressed ZmMKK9 or ZmRIN2 in Arabidopsis. Our findings demonstrated that ZmMKK9 and ZmRIN2 play negative regulatory roles in high-temperature–induced stomatal opening. Accordingly, we propose that the ZmMKK9–ZmMPK20–ZmRIN2 cascade negatively regulates high-temperature–induced stomatal opening and balances water loss and leaf temperature, thus enhancing plant thermotolerance.

Guar is a commercially important legume crop known for guar gum. Guar is tolerant to various abiotic stresses, but the mechanisms involved in its salinity tolerance are not well established. This study aimed to understand molecular mechanisms of salinity tolerance in guar. RNA sequencing (RNA-Seq) was employed to study the leaf and root transcriptomes of salt-tolerant (Matador) and salt-sensitive (PI 340261) guar genotypes under control and salinity. Our analyses identified a total of 296,114 unigenes assembled from 527 million clean reads. Transcriptome analysis revealed that the gene expression differences were more pronounced between salinity treatments than between genotypes. Differentially expressed genes associated with stress-signaling pathways, transporters, chromatin remodeling, microRNA biogenesis, and translational machinery play critical roles in guar salinity tolerance. Genes associated with several transporter families that were differentially expressed during salinity included ABC, MFS, GPH, and P-ATPase. Furthermore, genes encoding transcription factors/regulators belonging to several families, including SNF2, C₂H₂, bHLH, C3H, and MYB were differentially expressed in response to salinity. This study revealed the importance of various biological pathways during salinity stress and identified several candidate genes that may be used to develop salt-tolerant guar genotypes that might be suitable for cultivation in marginal soils with moderate to high salinity or using degraded water.

Salinity is a major abiotic stress limiting plant productivity. The Salt Overly Sensitive (SOS) pathway, comprising SOS1, SOS2, and SOS3, is central to ion homeostasis under salt stress. While the pathway has been extensively studied in model plants, its components remain uncharacterized in Medicago sativa (alfalfa), a moderately salt-tolerant forage crop of global economic importance. Here, we report the functional characterization of MsSOS2 , a CIPK family gene and putative ortholog of AtSOS2 . Sequence analysis revealed that MsSOS2 possesses conserved domains essential for kinase activity, including a kinase domain, activation loop, the phosphatidic acid (PA)-binding site, and NAF motif. Phylogenetic analysis placed MsSOS2 with AtSOS2 and OsSOS2. Yeast-two-hybrid assays confirmed the interaction between MsSOS2 and MsSOS3, mimicking the AtSOS2 – AtSOS3 complex. Transgenic expression of MsSOS2 in the Arabidopsis sos2 mutant restored salinity tolerance during seed germination, seedling development, and late vegetative growth. Ion profiling showed reduced Na accumulation and enhanced K retention in MsSOS2 -expressing lines under salinity. We propose that MsSOS2 activates AtSOS1 to facilitate Na⁺ efflux and disrupts AtCBL10-mediated inhibition of AKT1 to promote K⁺ uptake. This study provides the first functional validation of an SOS pathway component in alfalfa and highlights MsSOS2 as a promising candidate for improving salinity tolerance in legumes.