Stress-Induced Legume Root Nodule Senescence. Physiological, Biochemical, and Structural Alterations

Nitrate-fed and dark-stressed bean (Phaseolus vulgaris) and pea (Pisum sativum) plants were used to study nodule senescence. In bean, 1 d of nitrate treatment caused a partially reversible decline in nitrogenase activity and an increase in O(2) diffusion resistance, but minimal changes in carbon metabolites, antioxidants, and other biochemical parameters, indicating that the initial decrease in nitrogenase activity was due to O(2) limitation. In pea, 1 d of dark treatment led to a 96% decline in nitrogenase activity and sucrose, indicating sugar deprivation as the primary cause of activity loss. In later stages of senescence (4 d of nitrate or 2–4 d of dark treatment), nodules showed accumulation of oxidized proteins and general ultrastructural deterioration. The major thiol tripeptides of untreated nodules were homoglutathione (72%) in bean and glutathione (89%) in pea. These predominant thiols declined by approximately 93% after 4 d of nitrate or dark treatment, but the loss of thiol content can be only ascribed in part to limited synthesis by γ-glutamylcysteinyl, homoglutathione, and glutathione synthetases. Ascorbate peroxidase was immunolocalized primarily in the infected and parenchyma (inner cortex) nodule cells, with large decreases in senescent tissue. Ferritin was almost undetectable in untreated bean nodules, but accumulated in the plastids and amyloplasts of uninfected interstitial and parenchyma cells following 2 or 4 d of nitrate treatment, probably as a response to oxidative stress.

本研究以硝酸盐饲喂且经受暗胁迫的菜豆（Phaseolus vulgaris）与豌豆（Pisum sativum）植株为实验材料，探究根瘤衰老（nodule senescence）过程。在菜豆中，1天的硝酸盐处理可导致固氮酶活性（nitrogenase activity）出现部分可逆性下降，并提升氧气扩散阻力（O₂ diffusion resistance），但其对碳代谢产物、抗氧化剂及其他生化参数仅造成轻微变化，这表明固氮酶活性的初始下降源于氧气限制（O₂ limitation）。在豌豆中，1天的暗处理可使固氮酶活性与蔗糖（sucrose）含量分别下降96%，提示糖匮乏（sugar deprivation）是固氮酶活性丧失的主要诱因。在衰老后期（硝酸盐处理4天或暗处理2~4天），根瘤组织出现氧化蛋白积累与普遍的超微结构退化（ultrastructural deterioration）。未处理根瘤中的主要巯基三肽（thiol tripeptides）为菜豆中的同型谷胱甘肽（homoglutathione，占比72%）与豌豆中的谷胱甘肽（glutathione，占比89%）。经4天硝酸盐或暗处理后，这类优势巯基化合物的含量下降约93%，但巯基含量的损失仅可部分归因于γ-谷氨酰半胱氨酸合成酶（γ-glutamylcysteine synthetase）、同型谷胱甘肽合成酶（homoglutathione synthetase）与谷胱甘肽合成酶（glutathione synthetase）的合成能力受限。抗坏血酸过氧化物酶（ascorbate peroxidase）主要免疫定位于受感染的根瘤细胞与内皮层（inner cortex）薄壁组织（parenchyma）细胞中，在衰老组织中其表达量大幅下降。铁蛋白（ferritin）在未处理的菜豆根瘤中几乎无法检测到，但在硝酸盐处理2天或4天后，会在未感染的间质细胞与薄壁组织细胞的质体（plastids）及淀粉体（amyloplasts）中积累，这大概率是对氧化应激（oxidative stress）的响应。