In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthases (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum using sequence similarity-based approaches, and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1, was closely related to other putative red algal CESA genes. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared with plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal CESAs. The relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are probably functional differences between the algal and land plant CESAs.

中文翻译：

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来自海洋红藻 Calliarthron tuberculosum (Corallinales) 的纤维素合酶 CtCESA1 的功能表征

在陆地植物和藻类中，纤维素对于加强细胞壁和防止由于物理力而破裂很重要。尽管我们对纤维素合酶 (CESA) 生产纤维素的理解已经在几种陆地植物和细菌物种中取得了重大进展，但红藻中缺乏这种基本蛋白质的功能表征。在这里，我们使用基于序列相似性的方法鉴定钙化红藻 Calliarthron tuberculosum 中的 CESA 基因候选者，并阐明它们与来自不同分类群的其他 CESA 的系统发育关系。一个候选基因 CtCESA1 与其他推定的红藻 CESA 基因密切相关。为了测试 CtCESA1 是否编码真正的纤维素合酶，从昆虫和酵母表达系统中表达和纯化 CtCESA1 蛋白。CtCESA1 在葡萄糖示踪测定中显示出葡聚糖合酶活性。与植物和细菌 CESA 活性相比，CtCESA1 活性相对较低。在体外试验中，预测的来自 CtCESA1 的 N 末端淀粉结合结构域结合红藻佛罗里达淀粉提取物，代表了红藻 CESA 中不存在于其他谱系的 CESA 中的独特结构域。当将 CtCESA1 基因导入拟南芥 cesa 突变体时，红藻 CtCESA1 部分挽救了原代细胞壁 cesa6 突变体的生长缺陷，但不能部分挽救 cesa3 或次生细胞壁 cesa7 突变体的生长缺陷。荧光标记的 CtCESA1 定位于拟南芥 cesa6 突变体背景中的质膜。本研究提供了验证红藻 CESA 序列注释的功能证据。CtCESA1的活性相对较低，