BIG/DARK OVEREXPRESSION OF CAB1/TRANSPORT INHIBITOR RESPONSE3 is a 0.5-MDa protein associated with multiple functions in Arabidopsis (Arabidopsis thaliana) signalling and development. However, the biochemical functions of BIG are unknown. We investigated a role for BIG in the Arg/N-degron pathways, in which substrate protein fate is influenced by the N-terminal (Nt) residue. We crossed a big loss-of-function allele to two N-degron pathway E3 ligase mutants, proteolysis6 (prt6) and prt1, and examined the stability of protein substrates. Stability of model substrates was enhanced in prt6-1 big-2 and prt1-1 big-2 relative to the respective single mutants and the abundance of the PRT6 physiological substrates, HYPOXIA-RESPONSIVE ERF2 (HRE2) and VERNALIZATION2 (VRN2) was similarly increased in prt6 big double mutants. Hypoxia marker expression was enhanced in prt6 big double mutants; this constitutive response required arginyltransferase activity and RAP-type ERFVII transcription factors. Transcriptomic analysis of roots not only demonstrated increased expression of multiple hypoxia-responsive genes in the double mutant relative to prt6, but also revealed other roles for PRT6 and BIG, including regulation of suberin deposition through both ERFVII-dependent and independent mechanisms, respectively. Our results show that BIG acts together with PRT6 to regulate the hypoxia response and broader processes in Arabidopsis.

中文翻译：

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BIG 增强 Arg/N-degron 途径介导的蛋白质降解，调节拟南芥缺氧反应和木栓质沉积

CAB1/转运抑制剂反应3 的大/暗过度表达是一种 0.5-MDa 蛋白，与拟南芥 (Arabidopsis thaliana) 信号传导和发育中的多种功能相关。然而，BIG 的生化功能尚不清楚。我们研究了 BIG 在 Arg/N-degron 途径中的作用，其中底物蛋白的命运受到 N 末端 (Nt) 残基的影响。我们将一个大的功能缺失等位基因与两个 N-降解决定子途径 E3 连接酶突变体 proteothesis6 (prt6) 和 prt1 交叉，并检查了蛋白质底物的稳定性。相对于各自的单突变体，prt6-1 big-2 和 prt1-1 big-2 中模型底物的稳定性得到增强，并且 PRT6 生理底物、缺氧响应 ERF2 (HRE2) 和 VERNALIZATION2 (VRN2) 的丰度同样增加在 prt6 大双突变体中。 prt6 big 双突变体缺氧标记表达增强；这种组成性反应需要精氨酰转移酶活性和 RAP 型 ERFVII 转录因子。根的转录组分析不仅证明了双突变体中多个缺氧反应基因相对于 prt6 的表达增加，而且还揭示了 PRT6 和 BIG 的其他作用，包括分别通过 ERFVII 依赖和独立机制调节木栓质沉积。我们的结果表明，BIG 与 PRT6 一起调节拟南芥的缺氧反应和更广泛的过程。