Molecular and Cellular Biology Program, Ohio University, Athens, OH, 45701-2979, USA.
Department of Environmental & Plant Biology, Ohio University, Athens, OH, 45701-2979, USA.
BMC Plant Biol. 2020 May 18;20(1):221. doi: 10.1186/s12870-020-02420-5.
Arabinogalactan-proteins (AGPs) are one of the most complex protein families in the plant kingdom and are present in the cell walls of all land plants. AGPs are implicated in diverse biological processes such as plant growth, development, reproduction, and stress responses. AGPs are extensively glycosylated by the addition of type II arabinogalactan (AG) polysaccharides to hydroxyproline residues in their protein cores. Glucuronic acid (GlcA) is the only negatively charged sugar added to AGPs and the functions of GlcA residues on AGPs remain to be elucidated.
Three members of the CAZy GT14 family (GLCAT14A-At5g39990, GLCAT14B-At5g15050, and GLCAT14C-At2g37585), which are responsible for transferring glucuronic acid (GlcA) to AGPs, were functionally characterized using a CRISPR/Cas9 gene editing approach in Arabidopsis. RNA seq and qRT-PCR data showed all three GLCAT genes were broadly expressed in different plant tissues, with GLCAT14A and GLCAT14B showing particularly high expression in the micropylar endosperm. Biochemical analysis of the AGPs from knock-out mutants of various glcat single, double, and triple mutants revealed that double and triple mutants generally had small increases of Ara and Gal and concomitant reductions of GlcA, particularly in the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants. Moreover, AGPs isolated from all the glcat mutants displayed significant reductions in calcium binding compared to WT. Further phenotypic analyses found that the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants exhibited significant delays in seed germination, reductions in root hair length, reductions in trichome branching, and accumulation of defective pollen grains. Additionally, both glcat14b glcat14c and glcat14a glcat14b glcat14c displayed significantly shorter siliques and reduced seed set. Finally, all higher-order mutants exhibited significant reductions in adherent seed coat mucilage.
This research provides genetic evidence that GLCAT14A-C function in the transfer of GlcA to AGPs, which in turn play a role in a variety of biochemical and physiological phenotypes including calcium binding by AGPs, seed germination, root hair growth, trichome branching, pollen development, silique development, seed set, and adherent seed coat mucilage accumulation.
阿拉伯半乳聚糖蛋白（AGPs）是植物王国中最复杂的蛋白质家族之一，存在于所有陆地植物的细胞壁中。AGPs 参与多种生物学过程，如植物生长、发育、繁殖和应激反应。AGPs 通过在其蛋白核心的羟脯氨酸残基上添加 II 型阿拉伯半乳聚糖（AG）多糖而广泛糖基化。只有带负电荷的糖——葡萄糖醛酸（GlcA）被添加到 AGPs 中，而 GlcA 残基在 AGPs 中的功能仍有待阐明。
通过 CRISPR/Cas9 基因编辑方法在拟南芥中对负责将葡萄糖醛酸（GlcA）转移到 AGPs 的 CAZy GT14 家族的三个成员（GLCAT14A-At5g39990、GLCAT14B-At5g15050 和 GLCAT14C-At2g37585）进行了功能表征。RNA seq 和 qRT-PCR 数据显示，所有三个 GLCAT 基因在不同的植物组织中广泛表达，其中 GLCAT14A 和 GLCAT14B 在珠孔端胚乳中表达水平特别高。对各种 glcat 单、双和三突变体的 AGPs 的生化分析表明，双和三突变体通常 Ara 和 Gal 略有增加，而 GlcA 相应减少，特别是在 glcat14a glcat14b 和 glcat14a glcat14b glcat14c 突变体中。此外，与 WT 相比，所有 glcat 突变体分离的 AGPs 的钙结合能力均显著降低。进一步的表型分析发现，glcat14a glcat14b 和 glcat14a glcat14b glcat14c 突变体的种子萌发明显延迟，根毛长度减少，毛状体分支减少，花粉粒缺陷积累。此外，glcat14b glcat14c 和 glcat14a glcat14b glcat14c 突变体的长角果明显变短，种子产量降低。最后，所有更高阶的突变体的附着种皮粘液的积累明显减少。
本研究提供了遗传证据，证明 GLCAT14A-C 负责将 GlcA 转移到 AGPs 上，而 AGPs 又在包括 AGPs 对钙的结合、种子萌发、根毛生长、毛状体分支、花粉发育、长角果发育、种子产量和附着种皮粘液积累在内的各种生化和生理表型中发挥作用。
