Nitric Oxide Deficiency Accelerates Chlorophyll Breakdown and Stability Loss of Thylakoid Membranes during Dark-Induced Leaf Senescence in Arabidopsis

Nitric oxide (NO) has been known to preserve the level of chlorophyll (Chl) during leaf senescence. However, the mechanism by which NO regulates Chl breakdown remains unknown. Here we report that NO negatively regulates the activities of Chl catabolic enzymes during dark-induced leaf senescence. The transcriptional levels of the major enzyme genes involving Chl breakdown pathway except for RED CHL CATABOLITE REDUCTASE (RCCR) were dramatically up-regulated during dark-induced Chl degradation in the leaves of Arabidopsis NO-deficient mutant nos1/noa1 that exhibited an early-senescence phenotype. The activity of pheide a oxygenase (PAO) was higher in the dark-induced senescent leaves of nos1/noa1 compared with wild type. Furthermore, the knockout of PAO in nos1/noa1 background led to pheide a accumulation in the double mutant pao1 nos1/noa1, which retained the level of Chl during dark-induced leaf senescence. The accumulated pheide a in darkened leaves of pao1 nos1/noa1 was likely to inhibit the senescence-activated transcriptional levels of Chl catabolic genes as a feed-back inhibitory effect. We also found that NO deficiency led to decrease in the stability of photosynthetic complexes in thylakoid membranes. Importantly, the accumulation of pheide a caused by PAO mutations in combination with NO deficiency had a synergistic effect on the stability loss of thylakoid membrane complexes in the double mutant pao1 nos1/noa1 during dark-induced leaf senescence. Taken together, our findings have demonstrated that NO is a novel negative regulator of Chl catabolic pathway and positively functions in maintaining the stability of thylakoid membranes during leaf senescence.

已知一氧化氮（NO）可在叶片衰老过程中维持叶绿素（Chl）水平，但其调控叶绿素分解的分子机制尚未阐明。本研究发现，在黑暗诱导的叶片衰老进程中，NO负向调控叶绿素分解代谢酶的活性。呈现早衰表型的拟南芥NO缺陷型突变体nos1/noa1，其叶片在黑暗诱导的叶绿素降解过程中，除红色叶绿素分解代谢物还原酶（RED CHL CATABOLITE REDUCTASE，RCCR）外，参与叶绿素分解通路的主要酶编码基因的转录水平均显著上调。与野生型相比，nos1/noa1黑暗诱导衰老叶片中的脱镁叶绿酸a加氧酶（pheide a oxygenase，PAO）活性更高。进一步研究显示，在nos1/noa1遗传背景中敲除PAO后，双突变体pao1 nos1/noa1体内出现脱镁叶绿酸积累，该积累可在黑暗诱导的叶片衰老过程中维持叶绿素水平。pao1 nos1/noa1黑暗处理叶片中积累的脱镁叶绿酸，可能通过反馈抑制效应，抑制叶绿素分解代谢基因的衰老激活型转录水平。本研究同时发现，NO缺陷会导致类囊体膜上光合复合物的稳定性降低。尤为关键的是，在黑暗诱导的叶片衰老过程中，PAO突变结合NO缺陷所引发的脱镁叶绿酸积累，对双突变体pao1 nos1/noa1的类囊体膜复合物稳定性丧失具有协同增强效应。综上，本研究证实NO是叶绿素分解代谢通路的新型负调控因子，并在叶片衰老过程中正向维持类囊体膜的稳定性。