Hassan Ali S, O'Donovan Lisa A, Cowley James M, Akomeah Belinda, Phillips Renee J, Pettolino Filomena, Schultz Carolyn J, Burton Rachel A
School of Agriculture, Food and Wine, University of Adelaide, PMB1, Glen Osmond, South Australia 5064, Australia.
CSIRO Agriculture and Food, GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia.
Plant Cell Physiol. 2025 Jan 29;66(1):101-119. doi: 10.1093/pcp/pcae145.
Diversification of the cellulose synthase superfamily of glycosyltransferases has provided plants with the ability to synthesize varied cell wall polysaccharides such as xyloglucan, mannans, and the mixed-linkage glucans of cereals. Surprisingly, some but not all members of the cellulose synthase-like M (CslM) gene family have recently been shown to be involved in the glycosylation of the aglycone core of a range of triterpenoid saponins. However, no cell wall activity has yet been attributed to any of the CslM gene family members. Here, evolution of the CslM gene family in eudicots is explored to better understand the differences between the two metabolically distinct classes of CslMs (CslM1 and CslM2) and the very closely related CslGs. To achieve this, a robust tBLASTn approach was developed to identify CslM1, CslM2, and CslG sequences using diagnostic peptides, suitable for complex genomes using unannotated and short-read datasets. To ascertain whether both CslM1 and CslM2 proteins have cell wall functions, in addition to the 'saponin' role of CslM2, tomato CslM1 and CslM2 genes were ectopically expressed in Arabidopsis thaliana by stable transformation and in the transient Nicotiana benthamiana system. Transformed plants were analysed with immunofluorescence, immunogold transmission electron microscopy, and cell wall polysaccharides were extracted for monosaccharide linkage analysis. Our results support a role for both CslM1 and CslM2 in the biosynthesis of type II arabinogalactan linkages, generating new insight into how the diverse functions of CslMs can coexist and providing clear targets for future research.
糖基转移酶纤维素合酶超家族的多样化使植物能够合成多种细胞壁多糖，如木葡聚糖、甘露聚糖和谷物中的混合连接葡聚糖。令人惊讶的是，最近发现纤维素合酶样M（CslM）基因家族的一些成员（但不是全部）参与了一系列三萜皂苷苷元核心的糖基化。然而，尚未发现CslM基因家族的任何成员具有细胞壁活性。在这里，我们探索了双子叶植物中CslM基因家族的进化，以更好地理解两种代谢上不同的CslM类（CslM1和CslM2）与密切相关的CslG之间的差异。为了实现这一目标，我们开发了一种强大的tBLASTn方法，使用诊断肽来识别CslM1、CslM2和CslG序列，适用于使用未注释和短读数据集的复杂基因组。为了确定CslM1和CslM2蛋白是否都具有细胞壁功能，除了CslM2的“皂苷”作用外，通过稳定转化在拟南芥中异位表达番茄CslM1和CslM2基因，并在瞬时烟草系统中表达。用免疫荧光、免疫金透射电子显微镜对转化植株进行分析，并提取细胞壁多糖进行单糖连接分析。我们的结果支持CslM1和CslM2在II型阿拉伯半乳聚糖连接生物合成中的作用，为CslM的多种功能如何共存提供了新的见解，并为未来的研究提供了明确的目标。
