Defining the role of cellulose synthase-like M genes in the plant cell wall
The advent of high-throughput sequencing technologies has enabled the rapid accumulation of genetic data in the 21st century, and this, coupled with a low sequencing budget, has led to over 500 sequenced genomes of plants currently. Genomic data and resources are now publicly available in databases and online sources, providing opportunities for using functional genomics to understand the role of genes of interest. The cellulose synthase-like M (CslM) subfamily is the latest identified member of the CesA superfamily. There are now 10 cellulose synthase-like subfamilies in plants with many of them implicated in the biosynthesis of the backbones of non-cellulosic polysaccharides in plant cell walls. However, the function of some subfamilies remains cryptic. Recent research proposes that CslM genes may have a role in the synthesis of type II arabinogalactan linkages of pectin RG-I or AGP (arabinogalactan proteins). However, the glycosylating sugar is unspecified. Previous studies have also shown that a subset of CslM genes, CslM2, adds a glucuronic acid to the C-3 position of triterpenoid aglycones during saponin biosynthesis in plants. However, this has not yet been investigated for tomato (Solanum lycopersicum) saponins, which are steroidal glycoalkaloids (SGA). The work in this thesis has delved more into the role of tomato CslM genes in cell wall polysaccharide biosynthesis and saponin glycosylation. Transcript analysis showed that both CslM1 and CslM2a have different patterns of expression in developmental tissues, however, both genes are active in young fruits and expressed poorly in mature fruits. Overexpression of CslM1 in transgenic tomato lines significantly increased the levels of galacturonic acid, galactose, and glucose in leaf tissues. However, CRISPR/Cas9 edited lines of CslM1 displayed no major phenotypic changes but showed a reduction in cellulose composition, which may be a pleiotropic effect. In contrast, CslM2a edited lines showed a striking male sterile phenotype, named nop (no pollen) accompanied by growth defects and parthenocarpy. Assessment using a suite of cell wall antibodies revealed reduced levels and defects in pectin RG-I and AGP components in the pollen mother cells (PMC) and tapetum cell walls, likely leading to pollen abortion. Moreover, α-tomatine, the major SGA in tomatoes, was completely absent in cslm2a/nop lines, indicating a key role for the CSLM2a protein in tomato SGA glycosylation. Collectively, this study suggests galactose as a likely candidate nucleotide sugar for CslM1 and CslM2a activity in the cell wall, consistent with previous research. The findings also indicate that tomato CslM2a may be a putative galactosyltransferase in SGA biosynthesis, in addition to its established role as a glucuronosyltransferase in the triterpenoid saponin biosynthesis pathway. The work in this thesis confirms the unusual dual-function of members of this cellulose synthase-like family, particularly of CslM2a in tomato. It suggests that CSLM proteins may be promiscuous in plants, fulfilling more than one function. Their roles may be further expanded as we explore this fascinating gene family and perhaps revisit other Csl subfamilies with this in mind.
Cellulose synthase-like, CslM, CRISPR/Cas9, tapetum, pollen mother cell, tomatine
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