Multilayer regulatory landscape and new regulators identification for bud dormancy release and bud break in Populus.

For trees originating from boreal and temperate regions, the dormancy-to-active transition, also known as bud dormancy release and bud break, are crucial processes that allow trees to reactive growth in the spring. The molecular mechanisms underlying these two processes remain poorly understood. Here, through integrative multiomics analysis of the transcriptome, DNA methylome, and proteome, we gained insights into the reprogrammed cellular processes associated with bud dormancy release and bud break. Our findings revealed multilayer regulatory landscapes governing bud dormancy release and bud break regulation, providing a valuable reference framework for future functional studies. Based on the multiomics analysis, we have determined a novel long intergenic noncoding RNA named Phenology Responsive Intergenic lncRNA 1 (PRIR1) plays a role in the activation of bud break. that the molecular mechanism of PRIR1 has been preliminary explored, and it may partially promote bud break by activating its neighbouring gene, EXORDIUM LIKE 5 (PtEXL5), which has also been genetically confirmed as an activator for bud break. This study has revealed a lncRNA-mediated regulatory mechanism for the control of bud break in Populus, operating independently of known regulatory pathways.

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 (PMR) 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 PMR. 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 PMR. 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 PMR, demonstrating their positive regulatory roles in PMR. Given the importance of PMR 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.

Overexpression of the tonoplast sugar transporter CmTST2 in melon fruit increases sugar accumulation.

Fruits are an important part of the human diet and sugar content is a major criterion used to evaluate fruit quality. Melon fruit accumulate high sugar concentrations during their development; however, the mechanism through which these sugars are transported into the vacuoles of fruit cells for storage remains unclear. In this study, three tonoplast sugar transporters (TSTs), CmTST1, CmTST2, and CmTST3, were isolated from melon plants. Analysis of subcellular localization revealed that all these proteins were targeted to the tonoplast, and evaluation of spatial expression and promoter-GUS activity indicated that they had different expression patterns in the plant. RT-PCR and qRT-PCR results indicated that CmTST2 exhibited the highest expression level among the TST isoforms during melon fruit development. Histochemical and immunohistochemistry localization experiments were performed to identify the tissue- and cell-type localization of CmTST2 in the fruit, and the results indicated that both its transcription and translation were in the mesocarp and vascular cells. Overexpressing the CmTST2 gene in strawberry fruit and cucumber plants by transient expression and stable transformation, respectively, both increased sucrose, fructose, and glucose accumulation in the fruit. The results indicate that CmTST2 plays an important role in sugar accumulation in melon fruit.