Analysis of an Arabidopsis heat‐sensitive mutant reveals that chlorophyll synthase is involved in reutilization of chlorophyllide during chlorophyll turnover

Abstract

SUMMARYChlorophylls, the most abundant pigments in the photosynthetic apparatus, are constantly turned over as a result of the degradation and replacement of the damage-prone reaction center D1 protein of photosystem II. Results from isotope labeling experiments suggest that chlorophylls are recycled by reutilization of chlorophyllide and phytol, but the underlying mechanism is unclear. In this study, by characterization of a heat-sensitive Arabidopsis mutant we provide evidence of a salvage pathway for chlorophyllide a. A missense mutation in CHLOROPHYLL SYNTHASE (CHLG) was identified and confirmed to be responsible for a light-dependent, heat-induced cotyledon bleaching phenotype. Following heat treatment, mutant (chlg-1) but not wild-type seedlings accumulated a substantial level of chlorophyllide a, which resulted in a surge of phototoxic singlet oxygen. Immunoblot analysis suggested that the mutation destabilized the chlorophyll synthase proteins and caused a conditional blockage of esterification of chlorophyllide a after heat stress. Accumulation of chlorophyllide a after heat treatment occurred during recovery in the dark in the lightgrown but not the etiolated seedlings, suggesting that the accumulated chlorophyllides were not derived from de novo biosynthesis but from de-esterification of the existing chlorophylls. Further analysis of the triple mutant harboring the CHLG mutant allele and null mutations of CHLOROPHYLLASE1 (CLH1) and CLH2 indicated that the known chlorophyllases are not responsible for the accumulation of chlorophyllide a in chlg-1. Taken together, our results show that chlorophyll synthase acts in a salvage pathway for chlorophyll biosynthesis by re-esterifying the chlorophyllide a produced during chlorophyll turnover.

Identification of a Chlorophyll Dephytylase Involved in Chlorophyll Turnover in Arabidopsis

“…When the normalized chlorophyll dephytylase activities were plotted against the levels of Chlides present in different lines after HS treatment, we observed a good correlation between these two variables (see correlation coefficients in Figure 6D). Although reduced CHLG level can also result in accumulation of Chlide a (Lin et al, 2014), the levels of CHLG in cld1-1 and transgenic lines were comparable to that of the wild type before and after HS treatment ( Figure 3A). Of note, the level of CHLG was reduced ;40% by HS treatment in all lines tested, with that of CLD1 (both the wild type and the G193D mutant) largely unchanged ( Figure 3A).…”

“…In a previous effort to identify new thermotolerance components in Arabidopsis, several dlt mutant alleles were isolated, and the genetic identities of dlt1-1, dlt1-2, and dlt4-1 (or chlg1-1) were revealed (Wu et al, 2013;Lin et al, 2014). In this study, we characterized and cloned the dlt3-1 allele, which we renamed cld1-1 based on its molecular function identified in this study.…”

“…Analysis of the fate of isotope-labeled chlorophylls in the cyanobacterium Synechocystis sp PCC6803 suggests that a substantial portion of the chlorophyll a released during PSII repair is recycled via deand rephytylation steps (Vavilin and Vermaas, 2007). Recently, studies of an Arabidopsis mutant with reduced chlorophyll synthase (CHLG) suggested that CHLG is involved in chlorophyll recycling by catalyzing the rephytylation of chlorophyllide a (Chlide a) derived from chlorophyll dephytylation (Lin et al, 2014). The enzyme responsible for the dephytylation step has not been identified.…”

“…With its enzymatic function, CLH has been proposed to be involved in chlorophyll turnover and breakdown (Jacob-Wilk et al, 1999;Tsuchiya et al, 1999;Morita et al, 2009). However, genetic studies of Arabidopsis CLH mutants do not support this proposition Lin et al, 2014;Zhang et al, 2014). Instead of dephytylation of chlorophyll a, chlorophyll breakdown in leaf senescence is mainly initiated by removing the magnesium ion from chlorophyll a to yield Phein a by Mg-dechelatase (Shimoda et al, 2016).…”

Chlorophyll Synthase under Epigenetic Surveillance Is Critical for Vitamin E Synthesis, and Altered Expression Affects Tocopherol Levels in Arabidopsis

“…Because chlorophyll synthase has dual functions in phytol and chlorophyll synthesis Ischebeck et al, 2006;Lin et al, 2014), the impact of suppressed or enhanced CHLSYN expression on tocopherol concentrations could not be easily predicted. Our findings indicate that suppression of CHLSYN expression increases tocopherol concentrations in leaves, whereas enhanced expression of this gene reduces tocopherol concentrations in leaves (Fig.…”