Heat stress has become a major threat to crop production due to global warming; however, the mechanisms underlying plant high-temperature sensing are not well known. In plants, the membrane-anchored receptor-like kinases (RLKs) relay environmental signals into the cytoplasm. In a previous study, we isolated a wall-associated RLK-like (WAKL) gene CaWAKL20 from pepper (Capsicum annuum L.). Here, the amino acid sequence of CaWAKL20 was characterized and found to consist of conserved domains of WAK/WAKL family, including an extracellular region containing a GUB-WAK binding domain and a degenerated EGF2-like domain; a transmembrane region; and an intercellular region with an STKc catalytic domain. Moreover, CaWAKL20 transcription was inhibited by heat stress, whereas it was induced by both ABA and H2O2 treatments. Silencing of CaWAKL20 enhanced pepper thermotolerance, while overexpression decreased Arabidopsis thermotolerance. Additionally, Arabidopsis lines overexpressing CaWAKL20 showed less sensitivity to ABA during seed germination and root growth. Finally, the survival rate of Arabidopsis seedlings under heat stress treatment was enhanced by ABA pre-treatment, while it was compromised by the overexpression of CaWAKL20. Furthermore, the heat-induced expression of several ABA-responsive genes and some key regulator genes for thermotolerance was decreased in Arabidopsis CaWAKL20-overexpression lines. These results suggest that CaWAKL20 negatively modulates plant thermotolerance by reducing the expression of ABA-responsive genes, laying a foundation for further investigation into the functional mechanisms of WAKs/WAKLs in plants undergoing environmental stresses.

Adverse environmental conditions have a detrimental impact on crop growth and development, and cause protein denaturation or misfolding. The binding protion (BiP) plays an important protective role by alleviating endoplasmic reticulum (ER) stress induced by misfolded proteins. In this study, we characterized three BiP genes (CaBiP1, CaBiP2 and CaBiP3) in pepper, an economically important vegetable and spice species. The role of CaBiP1 in plant tolerance to ER stress and adverse environmental conditions (including heat, salinity, osmotic and drought stress) were investigated. All the expected functional and signaling domains were detected in three BiP proteins, but the motifs and exon-intron distribution differed slightly in CaBiP3. CaBiP1 and CaBiP2 were constitutively expressed in all the tested tissues under both normal and stressed conditions, whereas CaBiP3 was mainly expressed following stress. Silencing of CaBiP1 reduced pepper tolerance to ER stress and various environment stresses, and was accompanied by increased H2O2 accumulation, MDA content, relative electric leakage, water loss rate, and a reduction in soluble protein content and relative water content in the leaves. Conversely, overexpression of CaBiP1 in Arabidopsis enhanced tolerance to ER stress and multiple environment stresses, as demonstrated by an increase in germination rate, root length, survival rate, soluble protein content, relative water content, the unfolded protein response (UPR) pathway, and a decrease in water loss rate. Our results suggest that CaBiP1 may contribute to plant tolerance to abiotic stresses by reducing ROS accumulation, increasing the water-retention ability, and stimulating UPR pathways and expression of stress-related genes.

In plants,WUSCHEL-related homeobox1(WOX1) homologs promote lamina mediolateral outgrowth. However, the downstream components linking WOX1 and lamina development remain unclear. In this study, we revealed the roles of WOX1 in palmate leaf expansion in cucumber (Cucumis sativus). A cucumbermango fruit(mf) mutant, resulting from truncation of a WOX1-type protein (CsWOX1), displayed abnormal lamina growth and defects in the development of secondary and smaller veins.CsWOX1was expressed in the middle mesophyll and leaf margins and rescued defects of theArabidopsis wox1 prsdouble mutant. Transcriptomic analysis revealed that genes involved in auxin polar transport and auxin response were highly associated with leaf development. Analysis of the cucumbermf rl(round leaf) double mutant revealed that CsWOX1 functioned in vein development via PINOID (CsPID1)-controlled auxin transport. Overexpression ofCsWOX1in cucumber (CsWOX1-OE) affected vein patterning and produced ‘butterfly-shaped’ leaves. CsWOX1 physically interacted with CsTCP4a, which may account for the abnormal lamina development in themfmutant line and the smaller leaves in theCsWOX1-OE plants. Our findings demonstrated that CsWOX1 regulates cucumber leaf vein development by modulating auxin polar transport; moreover, CsWOX1 regulates leaf size by controllingCIN-TCPgenes.

Cucumber (Cucumis sativus L.) has been a model for the study of sex differentiation over the last two decades. Cucumber sex differentiation is mainly under genetic control, but plant growth regulators can also influence or even change it. However, the effect of exogenous auxin application on cucumber sex differentiation is not well understood at the physiological level. In this study, we explored the effects of different exogenous auxin concentrations on cucumber varieties with different mutant sex-controlling genotypes and found that there was a dosage effect of exogenous indole-3-acetic acid (IAA) on the enhancement of cucumber femaleness. Several ACC synthetase (ACS) family members responded directly to exogenous IAA, increasing endogenous ethylene synthesis, and this process appeared to be independent of the previously identified sex-related ACC oxidase CsACO2. We further demonstrated that ENHANCER OF SHOOT REGENERATION 2 (ESR2) responded to exogenous auxin induction by binding to ERE cis-acting element regions in the CsACS2 promoter, directly activating CsACS2 expression and thus increasing endogenous ethylene content, which may induce femaleness. These findings reveal that exogenous auxin increases cucumber femaleness by inducing a sex-controlling gene and promoting ethylene synthesis.

The nuclear factor Y (NF-Y) transcription factor contains three subfamilies: NF-YA, NF-YB, and NF-YC. The NF-Y family have been reported to be key regulators in plant growth and stress responses. However, little attention has been given to these genes in melon (Cucumis melo L.). In this study, twenty-five NF-Ys were identified in the melon genome, including six CmNF-YAs, eleven CmNF-YBs, and eight CmNF-YCs. Their basic information (gene location, protein characteristics, and subcellular localization), conserved domains and motifs, and phylogeny and gene structure were subsequently analyzed. Results showed highly conserved motifs exist in each subfamily, which are distinct between subfamilies. Most CmNF-Ys were expressed in five tissues and exhibited distinct expression patterns. However, CmNF-YA6, CmNF-YB1/B2/B3/B8, and CmNF-YC6 were not expressed and might be pseudogenes. Twelve CmNF-Ys were induced by cold stress, indicating the NF-Y family plays a key role in melon cold tolerance. Taken together, our findings provide a comprehensive understanding of CmNF-Y genes in the development and stress response of melon and provide genetic resources for solving the practical problems of melon production.