Pumpkin CmoDREB2A enhances salt tolerance of grafted cucumber through interaction with CmoNAC1 to regulate H2O2 and ABA signaling and K+/Na+ homeostasis.

Pumpkin CmoNAC1 enhances salt tolerance in grafted cucumbers. However, the potential interactions with other proteins that may co-regulate salt tolerance alongside CmoNAC1 have yet to be explored. In this study, we identified pumpkin CmoDREB2A as a pivotal transcription factor that interacts synergistically with CmoNAC1 in the co-regulation of salt tolerance. Both transcription factors were observed to bind to each other's promoters, forming a positive regulatory loop of their transcription. Knockout of CmoDREB2A in the root resulted in reduced salt tolerance in grafted cucumbers, whereas overexpression demonstrated the opposite effect. Multiple assays in our study provided evidence of the protein interaction between CmoDREB2A and CmoNAC1. Exploiting this interaction, CmoDREB2A facilitated the binding of CmoNAC1 to the promoters of CmoRBOHD1, CmoNCED6, CmoAKT1;2, and CmoHKT1;1, inducing H2O2 and ABA synthesis and increasing the K+/Na+ ratio in grafted cucumbers under salt stress. Additionally, CmoNAC1 also promoted the binding of CmoDREB2A to CmoHAK5;1/CmoHAK5;2 promoters, further contributing to the K+/Na+ homeostasis. In summary, these findings reveal a crucial mechanism of CmoNAC1 and CmoDREB2A forming a complex enhancing salt tolerance in grafted cucumbers.

CmoPIP1-4 confers drought tolerance in pumpkin by altering hydrogen sulfide signaling.

Drought is a major limiting factor for the growth and development of pumpkins. Plasma membrane intrinsic proteins (PIPs) are major water channels that play a crucial role in the regulation of cellular water status and solute trafficking during drought conditions. CmoPIP1-4 is a plasma membrane-localized protein that is significantly upregulated in roots and leaves under drought-stress conditions. In this study, the overexpression of CmoPIP1-4 enhances drought resistance in yeast. In contrast, CRISPR-mediated CmoPIP1-4 knockout in pumpkin roots increased drought sensitivity. This increased drought sensitivity of CmoPIP1-4 knockout plants is associated with a decline in the levels of hydrogen sulfide (H2S) and abscisic acid (ABA), accompanied by an increase in water loss caused by greater levels of transpiration and stomatal conductance. In addition, the sensitivity of CmoPIP1-4 CRISPR plants is further aggravated by reduced antioxidative enzyme activity, decreased proline and sugar contents, and extensive root damage. Furthermore, expression profiles of genes such as CmoHSP70s, CmoNCED3, CmoNCED4, and others involved in metabolic activities were markedly reduced in CmoPIP1-4 CRISPR plants. Moreover, we also discovered an interaction between the drought-responsive gene CmoDCD and CmoPIP1-4, indicating their potential role in activating H2S-mediated signaling in pumpkin, which could confer drought tolerance. The findings of our study collectively demonstrate CmoPIP1-4 plays a crucial role in the regulation of H2S-mediated signaling, influencing stomatal density and aperture in pumpkin plants, and thereby enhancing their drought tolerance.

Multiplex gene editing reveals cucumber MILDEW RESISTANCE LOCUS O family roles in powdery mildew resistance.

Powdery mildew (PM) is one of the most widespread and prevalent diseases that affects a wide range of crops. In cucumber (Cucumis sativus L.), previous forward genetic studies have identified MILDEW RESISTANCE LOCUS O 8 (CsMLO8) as necessary but alone insufficient for cucumber PM resistance, and suggested the involvement of other members of the CsMLO family. However, the function of other CsMLO family members in cucumber remains largely unknown. Here, we developed a highly efficient multiplex gene editing system in cucumber to generate a series of Csmlo mutants from all the 13 family members. Systematic analysis of these mutants revealed growth effects of these CsMLO family members on development and PM resistance. Importantly, we obtained the Csmlo1/8/11 triple mutant with complete resistance to PM. Transcriptome and proteome analysis of PM-resistant Csmlo mutants suggested that the kinesin-like calmodulin-binding protein (KCBP)-interacting Ca2+-binding protein (CsKIC), calmodulin-like protein 28 (CsCML28) and Ca2+-dependent protein kinase 11 (CsCPK11)-mediated calcium signaling pathway is involved in PM resistance. CsMLO8 interacted directly with CsKIC, and the simultaneous silencing of both genes resulted in a phenotype that resembled the silencing of CsKIC alone. Silencing CsCML28 and CsCPK11 increased susceptibility to PM, whereas overexpressing CsCPK11 through genetic transformation enhanced cucumber's PM resistance, demonstrating their positive regulatory roles in PM resistance. Given the importance of PM resistance for cucurbit crops, this research provides unprecedented insights into the function of the proteins encoded by the CsMLO gene family as well as the plant defense response to PM pathogen.

CmoNAC1 in pumpkin rootstocks improves salt tolerance of grafted cucumbers by binding to the promoters of CmoRBOHD1, CmoNCED6, CmoAKT1;2 and CmoHKT1;1 to regulate H2O2, ABA signaling and K+/Na+ homeostasis.

The NAC transcription factor is a type of plant-specific transcription factor that can regulate plant salt tolerance, but the underlying mechanism is unclear in grafted vegetables. H2O2 and ABA in pumpkin rootstocks can be transported to cucumber scion leaves, promoting stomatal closure to improve salt tolerance of grafted cucumbers. Despite these observations, the regulatory mechanism is unknown. Here, our research revealed that CmoNAC1 is a key transcription factor that regulates H2O2 and ABA signaling in pumpkin roots under salt stress. The function of CmoNAC1 was analyzed using root transformation and RNA-seq, and we found that pumpkin CmoNAC1 promoted the production of H2O2 and ABA via CmoRBOHD1 and CmoNCED6, respectively, and regulated K+/Na+ homeostasis via CmoAKT1;2, CmoHKT1;1, and CmoSOS1 to improve salt tolerance of grafted cucumbers. Root knockout of CmoNAC1 resulted in a significant decrease in H2O2 (52.9% and 32.1%) and ABA (21.8% and 42.7%) content and K+/Na+ ratio (81.5% and 56.3%) in leaf and roots of grafted cucumber, respectively, while overexpression showed the opposite effect. The root transformation experiment showed that CmoNCED6 could improve salt tolerance of grafted cucumbers by regulating ABA production and K+/Na+ homeostasis under salt stress. Finally, we found that CmoNAC1 bound to the promoters of CmoRBOHD1, CmoNCED6, CmoAKT1;2, and CmoHKT1;1 using yeast one-hybrid, luciferase, and electrophoretic mobility shift assays. In conclusion, pumpkin CmoNAC1 not only binds to the promoters of CmoRBOHD1 and CmoNCED6 to regulate the production of H2O2 and ABA signals in roots, but also binds to the promoters of CmoAKT1;2 and CmoHKT1;1 to increase the K+/Na+ ratio, thus improving salt tolerance of grafted cucumbers.

The essential role of jasmonate signaling in Solanum habrochaites rootstock-mediated cold tolerance in tomato grafts.

Tomato (Solanum lycopersicum) is among the most important vegetables across the world, but cold stress usually affects its yield and quality. The wild tomato species Solanum habrochaites is commonly utilized as rootstock for enhancing resistance against abiotic stresses in cultivated tomato, especially cold resistance. However, the underlying molecular mechanism remains unclear. In this research, we confirmed that S. habrochaites rootstock can improve the cold tolerance of cultivated tomato scions, as revealed by growth, physiological, and biochemical indicators. Furthermore, transcriptome profiling indicated significant differences in the scion of homo- and heterografted seedlings, including substantial changes in jasmonic acid (JA) biosynthesis and signaling, which were validated by RT-qPCR analysis. S. habrochaites plants had a high basal level of jasmonate, and cold stress caused a greater amount of active JA-isoleucine in S. habrochaites heterografts. Moreover, exogenous JA enhanced while JA inhibitor decreased the cold tolerance of tomato grafts. The JA biosynthesis-defective mutant spr8 also showed increased sensitivity to cold stress. All of these results demonstrated the significance of JA in the cold tolerance of grafted tomato seedlings with S. habrochaites rootstock, suggesting a future direction for the characterization of the natural variation involved in S. habrochaites rootstock-mediated cold tolerance.

An efficient root transformation system for CRISPR/Cas9-based analyses of shoot-root communication in cucurbit crops.

Cucurbit crops are suitable models for studying long-distance signaling in horticultural plants. Although thousands of substances are graft transmissible in cucurbits, functional studies have been hampered by the lack of efficient genetic transformation systems. Here, we report a convenient and efficient root transformation method for several cucurbit crops that will facilitate studies of functional genes and shoot-root crosstalk. We obtained healthy plants with completely transformed roots and non-transgenic shoots within 6 weeks. Furthermore, we combined this root transformation method with grafting, which allowed for gene manipulation in the rootstock. We validated our system by exploring salt tolerance mechanisms using a cucumber (Cucumis sativus)/pumpkin (Cucurbita moschata Duch.) (scion/rootstock) graft in which the sodium transporter gene High-affinity K+ transporter1 (CmoHKT1;1) was edited in the pumpkin rootstock, and by overexpressing the pumpkin tonoplast Na+/H+ antiporter gene Sodium hydrogen exchanger4 (CmoNHX4) in cucumber roots.