Celiz-Balboa Jonathan, Largo-Gosens Asier, Parra-Rojas Juan Pablo, Arenas-Morales Verónica, Sepulveda-Orellana Pablo, Salinas-Grenet Hernán, Saez-Aguayo Susana, Orellana Ariel
Centro de Biotecnología Vegetal, Universidad Andrés Bello, Santiago, Chile.
FONDAP Center for Genome Regulation, Facultad de Ciencias de la Vida, Universidad Andrés Bello, Santiago, Chile.
Front Plant Sci. 2020 Dec 8;11:594544. doi: 10.3389/fpls.2020.594544. eCollection 2020.
Nucleotide sugar transporters (NSTs) are Golgi-localized proteins that play a role in polysaccharide biosynthesis by transporting substrates (nucleotide sugars) from the cytosol into the Golgi apparatus. In Arabidopsis, there is an NST subfamily of six members, called URGTs, which transport UDP-rhamnose and UDP-galactose . URGTs are very similar in protein sequences, and among them, URGT1 and URGT2 are highly conserved in protein sequence and also showed very similar kinetic parameters toward UDP-rhamnose and UDP-galactose . Despite the similarity in sequence and function, mutants in led to a specific reduction in galactose in rosette leaves. In contrast, mutants in showed a decrease in rhamnose content in soluble mucilage from seeds. Given these specific and quite different chemotypes, we wonder whether the differences in gene expression could explain the observed differences between the mutants. Toward that end, we analyzed whether URGT2 could rescue the phenotype and by performing a promoter swapping experiment. We analyzed whether the expression of the coding sequence, controlled by the promoter, could rescue the rosette phenotype. A similar strategy was used to determine whether URGT1 could rescue the mucilage phenotype. Expression analysis of the swapped genes, using qRT-PCR, was similar to the native and genes in wild-type plants. To monitor the protein expression of the swapped genes, both URGTs were tagged with green fluorescent protein (GFP). Confocal microscopy analyses of the swapped lines containing URGT2-GFP showed fluorescence in motile dot-like structures in rosette leaves. Swapped lines containing URGT1-GFP showed fluorescence in dot-like structures in the seed coat. Finally, the expression of in mutants rescued galactose reduction in rosette leaves. In the same manner, the expression of in mutants recovered the content of rhamnose in soluble mucilage. Hence, our results showed that their expression in different organs modulates the role of URGT1 and URGT2. Likely, this is due to their presence in different cellular contexts, where other proteins, acting in partnership, may drive their functions toward different pathways.
核苷酸糖转运蛋白（NSTs）是定位于高尔基体的蛋白质，通过将底物（核苷酸糖）从细胞质转运到高尔基体中来参与多糖的生物合成。在拟南芥中，有一个由六个成员组成的NST亚家族，称为URGTs，它们负责转运UDP-鼠李糖和UDP-半乳糖。URGTs在蛋白质序列上非常相似，其中，URGT1和URGT2在蛋白质序列上高度保守，并且对UDP-鼠李糖和UDP-半乳糖也表现出非常相似的动力学参数。尽管在序列和功能上相似，但 突变体导致莲座叶中半乳糖含量特异性降低。相反， 突变体显示种子可溶性黏液中鼠李糖含量降低。鉴于这些特定且差异较大的化学类型，我们想知道基因表达的差异是否可以解释突变体之间观察到的差异。为此，我们通过进行启动子交换实验分析了URGT2是否可以挽救 表型以及 。我们分析了由 启动子控制的 编码序列的表达是否可以挽救 莲座叶表型。使用了类似的策略来确定URGT1是否可以挽救 黏液表型。使用qRT-PCR对交换基因进行表达分析，结果与野生型植物中的天然 和 基因相似。为了监测交换基因的蛋白质表达，两个URGT都用绿色荧光蛋白（GFP）进行了标记。对含有URGT2-GFP的交换系进行共聚焦显微镜分析，结果显示莲座叶中有运动性点状结构发出荧光。含有URGT1-GFP的交换系在种皮的点状结构中显示荧光。最后， 在 突变体中的表达挽救了莲座叶中半乳糖的减少。同样， 在 突变体中的表达恢复了可溶性黏液中鼠李糖的含量。因此，我们的结果表明它们在不同器官中的表达调节了URGT1和URGT2的作用。可能是由于它们存在于不同的细胞环境中，在那里其他协同作用的蛋白质可能会将它们的功能导向不同的途径。
