Department of Chemistry and Biochemistry, University of Regina, Regina, SK, S4S 0A2, Canada.
Planta. 2022 May 19;255(6):129. doi: 10.1007/s00425-022-03909-z.
PpORS-produced 2'-oxo-5-pentacosylresorcinol (2'-oxo-C-RL) restored dehydration tolerance in ors-3, a knockout mutant of PpORS. Feeding experiments with [C]-2'-oxo-C-RL suggested the role of PpORS products in cuticular polymer that confer dehydration resistance. 2'-Oxoalkylresorcinol synthase from the moss Physcomitrium (Physcomitrella) patens (PpORS) is the earliest diverged member of plant type III polyketide synthases, and produces very-long-chain 2'-oxoalkylresorcinols in vitro. Targeted knockouts of PpORS (ors) exhibited an abnormal phenotype (increased susceptibility to dehydration), and a defective cuticle in ors was suggested (Li et al., Planta 247:527-541, 2018). In the present study, we investigated chemical rescue of the ors phenotype and also metabolic fates of the PpORS products in the moss. Using C-CoA as substrate, 2'-oxo-5-pentacosylresorcinol (2'-oxo-C-RL) and two minor pyrones were first enzymatically prepared as total in vitro products. When a knockout mutant (ors-3) and control strains were grown in the presence of the total in vitro products or purified 2'-oxo-C-RL, the ability of ors-3 and the control to survive dehydration stress increased in a dose-dependent manner. Structurally analogous long-chain alkylresorcinols also rescued the ors phenotype, although less efficiently. When the moss was grown in the presence of C-radiolabeled 2'-oxo-C-RL, 96% of the radioactivity was recovered only after acid hydrolysis. These findings led us to propose that 2'-oxoalkylresorcinols are the functional in planta products of PpORS and are incorporated into cuticular biopolymers that confer resistance to dehydration. In addition, the earliest diverging ORS clade in phylogenetic trees of plant type III PKSs exclusively comprises bryophyte enzymes that share similar active site substitutions with PpORS. Further studies on these bryophyte enzymes may shed light on their roles in early plant evolution and offer a novel strategy for improving dehydration tolerance in plants.
PpORS 产生的 2'-氧代-5-二十五烷基间苯二酚（2'-氧代-C-RL）恢复了 PpORS 敲除突变体 ors-3 的脱水耐受性。用 [C]-2'-氧代-C-RL 进行的饲喂实验表明，PpORS 产物在赋予脱水抗性的角质聚合物中发挥作用。来自苔藓Physcomitrium（Physcomitrella）patens（PpORS）的 2'-氧代烷基间苯二酚合酶是植物 III 型聚酮合酶中最早分化的成员，在体外产生非常长链的 2'-氧代烷基间苯二酚。PpORS（ors）的靶向敲除表现出异常表型（对脱水的敏感性增加），并且推测 ors 的角质层有缺陷（Li 等人，Planta 247：527-541，2018）。在本研究中，我们研究了 ors 表型的化学挽救以及苔藓中 PpORS 产物的代谢命运。使用 C-CoA 作为底物，首次在体外酶法制备了 2'-氧代-5-二十五烷基间苯二酚（2'-氧代-C-RL）和两种次要吡喃酮作为总产物。当敲除突变体（ors-3）和对照菌株在总体外产物或纯化的 2'-氧代-C-RL 的存在下生长时，ors-3 和对照菌株在脱水应激下存活的能力以剂量依赖性方式增加。结构类似的长链烷基间苯二酚也能挽救 ors 表型，尽管效率较低。当苔藓在 C 放射性标记的 2'-氧代-C-RL 的存在下生长时，仅在酸水解后 96%的放射性可回收。这些发现使我们提出 2'-氧代烷基间苯二酚是 PpORS 的功能性植物内产物，并被整合到赋予抗脱水性的角质生物聚合物中。此外，系统发育树中最早分化的 ORS 分支仅包含具有与 PpORS 相似活性位点取代的苔藓酶，属于植物 III 型 PKS。对这些苔藓酶的进一步研究可能揭示它们在早期植物进化中的作用，并为提高植物的脱水耐受性提供一种新策略。
