RESUMEN
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.
RESUMEN
Plant cell wall biosynthesis is a complex process that requires proteins and enzymes from glycan synthesis to wall assembly. We show that disruption of At3g50120 (DUF247-1), a member of the DUF247 multigene family containing 28 genes in Arabidopsis, results in alterations to the structure and composition of cell wall polysaccharides and reduced growth and plant size. An ELISA using cell wall antibodies shows that the mutants also exhibit ~50% reductions in xyloglucan (XyG), glucuronoxylan (GX) and heteromannan (HM) epitopes in the NaOH fraction and ~50% increases in homogalacturonan (HG) epitopes in the CDTA fraction. Furthermore, the polymer sizes of XyGs and GXs are reduced with concomitant increases in short-chain polymers, while those of HGs and mHGs are slightly increased. Complementation using 35S:DUF247-1 partially recovers the XyG and HG content, but not those of GX and HM, suggesting that DUF247-1 is more closely associated with XyGs and HGs. DUF247-1 is expressed throughout Arabidopsis, particularly in vascular and developing tissues, and its disruption affects the expression of other gene members, indicating a regulatory control role within the gene family. Our results demonstrate that DUF247-1 is required for normal cell wall composition and structure and Arabidopsis growth.
RESUMEN
Phenotypic analysis of cassava root crowns (CRCs) so far has been limited to visual inspection and very few measurements due to its laborious process in the field. Here, we developed a platform for acquiring 3D CRC models using close-range photogrammetry for phenotypic analysis. The state of the art is a low cost and easy to set up 3D acquisition requiring only a background sheet, a reference object and a camera, compatible with field experiments in remote areas. We tested different software with CRC samples, and Agisoft and Blender were the most suitable software for generating high-quality 3D models and data analysis, respectively. We optimized the workflow by testing different numbers of images for 3D reconstruction and found that a minimum of 25 images per CRC can provide high quality 3D models. Up to ten traits, including 3D crown volumes, 3D crown surface, root density, surface-to-volume ratio, root numbers, root angle, crown diameter, cylinder soil volume, CRC compactness and root length can be extracted providing novel parameters for studying cassava storage roots. We applied this platform to partial-inbred cassava populations and demonstrated that our platform provides reliable 3D CRC modelling for phenotypic analysis, analysis of genetic variances and supporting breeding selection.