Active protein ubiquitination regulates xylem vessel functionality

Xylem vessels function in the long-distance conduction of water in land plants. The NAC transcription factor VASCULAR-RELATED NAC-DOMAIN7 (VND7) is a master regulator of xylem vessel cell differentiation in Arabidopsis (Arabidopsis thaliana). We previously isolated seiv (suppressor of ectopic xylem vessel cell differentiation induced by VND7) mutants, which are suppressor mutants of VND7-inducible xylem vessel cell differentiation. Here, we report that the responsible genes for seiv3, seiv4, seiv6, and seiv9 are protein ubiquitination-related genes encoding PLANT U-BOX46 (PUB46), uncharacterized F-BOX protein, PUB36, and UBIQUITIN-SPECIFIC PROTEASE1, respectively. We also found the decreased expression of genes downstream of VND7 and abnormal xylem transport activity in the seiv mutants. Upon VND7 induction, ubiquitinated levels from 492 and 180 protein groups were up- and down-regulated, respectively. Proteins for cell wall biosynthesis and protein transport were ubiquitinated by the VND7 induction, whereas such active protein ubiquitination was not observed in the seiv mutants. We detected the ubiquitination of three lysine residues in VND7: K94, K105, and K260. Substituting K94 with arginine significantly decreased the transactivation activity of VND7, suggesting that the ubiquitination of K94 is crucial for regulating VND7 activity. Our findings highlight the crucial roles of target protein ubiquitination in regulating xylem vessel activity.木质部导管负责陆生植物体内水分的长距离运输。NAC转录因子维管相关NAC结构域7（VASCULAR-RELATED NAC-DOMAIN7，VND7）是拟南芥（Arabidopsis thaliana）木质部导管细胞分化的核心调控因子。我们此前成功分离得到seiv（VND7诱导异位木质部导管细胞分化抑制因子，suppressor of ectopic xylem vessel cell differentiation induced by VND7）突变体，该类突变体可抑制VND7诱导的木质部导管细胞分化过程。本研究明确了seiv3、seiv4、seiv6与seiv9的突变对应功能基因分别为与蛋白质泛素化相关的基因，它们依次编码植物U-box蛋白46（PLANT U-BOX46，PUB46）、未表征的F-box蛋白、PUB36以及泛素特异性蛋白酶1。我们还发现，seiv突变体中VND7下游基因的表达水平显著下调，且木质部运输活性出现异常。在VND7诱导处理后，共有492个蛋白质组的泛素化水平显著上调，180个蛋白质组的泛素化水平显著下调。VND7诱导可使参与细胞壁生物合成与蛋白质运输的蛋白质发生泛素化修饰，而seiv突变体中未观察到此类活跃的蛋白质泛素化现象。我们检测到VND7蛋白上存在三个赖氨酸残基的泛素化修饰位点：K94、K105与K260。将K94替换为精氨酸后，VND7的转录激活活性显著降低，这表明K94位点的泛素化修饰对调控VND7的活性至关重要。本研究结果凸显了靶蛋白泛素化修饰在调控木质部导管活性过程中的关键作用。