Evaluation of cucumber UBL5 promoter as a tool for transgene expression and genome editing in plants.

Transgene expression and genome editing can help improve cucumber varieties to better respond to climate change. This study aimed to evaluate the applicability of the CsUBL5 promoter in transgene expression and genome editing in cucumber. The CsUBL5 promoter was cloned and analyzed to identify cis-elements that respond to abiotic signals, hormones, signal molecules, and nutrient treatments. 5' deletion constructs of the promoter were tested for their ability to drive GUS reporter expression in cucumber cotyledons, Arabidopsis seedlings, and tobacco leaves, and their response to various treatments including SA, light, drought, IAA, and GA was determined. The results showed that the CsUBL5 promoter effectively drove transgene expression in these plants, and their expressions under treatments were consistent with the predicted cis-elements, with some exceptions. Furthermore, the pCsUBL5-749 deletion construct can improve genome editing efficiency in cucumber when driving Cas9 expression. The editing efficiency of two sgRNAs targeting the ATG6 gene in cucumber was up to 4.6-fold higher using pCsUBL5-749 compared to a rice UBI promoter, although the effects of changing promoter on the editing efficiency is sgRNA specific. These findings highlight the potential utility of the CsUBL5 promoter for improving cucumber varieties through genetic engineering and genome editing. It also demonstrates the importance of modulating Cas9 expression to increase genome editing efficiency in cucumbers.

In Vivo Proximity Cross-Linking and Immunoprecipitation of Cell Wall Epitopes Identify Proteins Associated with the Biosynthesis of Matrix Polysaccharides.

Identification of proteins involved in cell wall matrix polysaccharide biosynthesis is crucial to understand plant cell wall biology. We utilized in vivo cross-linking and immunoprecipitation with cell wall antibodies that recognized xyloglucan, xylan, mannan, and homogalacturonan to capture proteins associated with matrix polysaccharides in Arabidopsis protoplasts. The use of cross-linkers allowed us to capture proteins actively associated with cell wall polymers, including those directly interacting with glycans via glycan-protein (GP) cross-linkers and those associated with proteins linked to glycans via a protein-protein (PP) cross-linker. Immunoprecipitations led to the identification of 65 Arabidopsis protein IDs localized in the Golgi, ER, plasma membrane, and others without subcellular localization data. Among these, we found several glycosyltransferases directly involved in polysaccharide synthesis, along with proteins related to cell wall modification and vesicle trafficking. Protein interaction networks from DeepAraPPI and AtMAD databases showed interactions between various IDs, including those related to cell-wall-associated proteins and membrane/vesicle trafficking proteins. Gene expression and coexpression analyses supported the presence and relevance of the proteins to the cell wall processes. Reverse genetic studies using T-DNA insertion mutants of selected proteins revealed changes in cell wall composition and saccharification, further supporting their potential roles in cell wall biosynthesis. Overall, our approach represents a novel approach for studying cell wall polysaccharide biosynthesis and associated proteins, providing advantages over traditional immunoprecipitation techniques. This study provides a list of putative proteins associated with different matrix polysaccharides for further investigation and highlights the complexity of cell wall biosynthesis and trafficking within plant cells.