Nitric oxide deficiency accelerates chlorophyll breakdown and stability loss of thylakoid membranes during dark-induced leaf senescence in Arabidopsis.

Fang Liu,Fang-Qing Guo

doi:10.1371/journal.pone.0056345

Abstract

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.

Highlights

Chlorophyll (Chl) molecules play a central role in the initial and indispensable processes of photosynthesis, such as harvesting light energy and driving electron transfer
Given that the depletion of endogenous Nitric oxide (NO) leads to an early leaf senescence phenotype in the mutant plants [43,44], we investigated whether NO plays a role in the regulation of Chl breakdown pathway during leaf senescence using the NO-deficient mutant nos1/noa1
In contrast to the four genes mentioned above, the mRNA level of red chl catabolite reductase (RCCR) was downregulated at day one and was kept at similar levels in both the wild type and nos1/noa1 mutant leaves through to day 5 in darkness, which is consistent with the previous results that RCCR expression is constitutively active throughout the process of leaf senescence [60,61]

Summary

Introduction

Chlorophyll (Chl) molecules play a central role in the initial and indispensable processes of photosynthesis, such as harvesting light energy and driving electron transfer. Recent studies questioned the involvement of CLHs in Chl breakdown in vivo during leaf senescence [7,8] even though the two CLHs present in Arabidopsis exhibited chlorophyllase activity in vitro [6]. The resulting pheophorbide (pheide) a is converted into a primary fluorescent chlorophyll catabolite (pFCC), which requires two enzymes including pheide a oxygenase (PAO) and red chl catabolite reductase (RCCR) [10,11,12,13]. PAO catalyzes the cleavage of the porphyrin ring, resulting in the red chlorophyll catabolite, which is further reduced in to pFCC by RCCR. The resulting primary fluorescent catabolite pFCCs are exported from the plastid and after further modification they are imported into their final destination, the vacuole by a primary active transport system [14,15]

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Conclusion