7-Hydroxymethyl chlorophyll a reductase functions in metabolic channeling of chlorophyll breakdown intermediates during leaf senescence

Yasuhito Sakuraba,Ye-Sol Kim,Soo-Cheul Yoo,Stefan Hörtensteiner,Nam-Chon Paek

doi:10.1016/j.bbrc.2012.11.050

Abstract

During natural or dark-induced senescence, chlorophyll degradation causes leaf yellowing. Recent evidence indicates that chlorophyll catabolic enzymes (CCEs) interact with the photosynthetic apparatus; for example, five CCEs (NYC1, NOL, PPH, PAO and RCCR) interact with LHCII. STAY-GREEN (SGR) and CCEs interact with one another in senescing chloroplasts; this interaction may allow metabolic channeling of potentially phototoxic chlorophyll breakdown intermediates. 7-Hydroxymethyl chlorophyll a reductase (HCAR) also acts as a CCE, but HCAR functions during leaf senescence remain unclear. Here we show that in Arabidopsis, HCAR-overexpressing plants exhibited accelerated leaf yellowing and, conversely, hcar mutants stayed green during dark-induced senescence. Moreover, HCAR interacted with LHCII in in vivo pull-down assays, and with SGR, NYC1, NOL and RCCR in yeast two-hybrid assays, indicating that HCAR is a component of the proposed SGR-CCE-LHCII complex, which acts in chlorophyll breakdown. Notably, HCAR and NOL are expressed throughout leaf development and are drastically down-regulated during dark-induced senescence, in contrast with SGR, NYC1, PPH and PAO, which are up-regulated during dark-induced senescence. Moreover, HCAR and NOL are highly up-regulated during greening of etiolated seedlings, strongly suggesting a major role for NOL and HCAR in the chlorophyll cycle during vegetative stages, possibly in chlorophyll turnover.

Highlights

During senescence, loss of green leaf color is caused by chlorophyll (Chl) degradation, a process in which Chl is converted to a primary fluorescent Chl catabolite through irreversible, consecutive reactions in chloroplasts
hydroxymethyl Chl a reductase (HCAR) interacted with light-harvesting complex II (LHCII) in in vivo pull-down assays, and with SGR, NON-YELLOW COLORING 1 (NYC1), NOL and red Chl catabolite (RCC) reductase (RCCR) in yeast two-hybrid assays, indicating that HCAR is a component of the proposed SGR-chlorophyll catabolic enzyme (CCE)-LHCII complex, which acts in chlorophyll breakdown
For example we showed that SGR and five CCEs (NYC1, NOL, PPH, pheophorbide a oxygenase (PAO) and RCCR) interact directly or indirectly with each other in vivo and in vitro [19]

Summary

Introduction

Loss of green leaf color is caused by chlorophyll (Chl) degradation, a process in which Chl is converted to a primary fluorescent Chl catabolite (pFCC) through irreversible, consecutive reactions in chloroplasts. During natural or dark-induced senescence, the rice and Arabidopsis nyc mutants show a stay-green phenotype with dominant retention of Chl b. Arabidopsis hcar mutants exhibit a stay-green phenotype during dark-induced senescence. PAO-deficient pao/acd and RCCR-deficient acd mutants exhibit severe leaf necrosis phenotypes because of excessive accumulation of phototoxic pheophorbide a and RCC, respectively [10,13]. These severe cell-death phenotypes imply that a finely tuned regulation mechanism exists in wild type to avoid the accumulation of phototoxic Chl breakdown intermediates during Chl degradation

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