Identification of a Novel Chloroplast Protein AtNYE1 Regulating Chlorophyll Degradation during Leaf Senescence in Arabidopsis

Abstract

A dramatic increase of chlorophyll (Chl) degradation occurs during senescence of vegetative plant organs and fruit ripening. Although the biochemical pathway of Chl degradation has long been proposed, little is known about its regulatory mechanism. Identification of Chl degradation-disturbed mutants and subsequently isolation of responsible genes would greatly facilitate the elucidation of the regulation of Chl degradation. Here, we describe a nonyellowing mutant of Arabidopsis (Arabidopsis thaliana), nye1-1, in which 50% Chl was retained, compared to less than 10% in the wild type (Columbia-0), at the end of a 6-d dark incubation. Nevertheless, neither photosynthesis-nor senescence-associated process was significantly affected in nye1-1. Characteristically, a significant reduction in pheophorbide a oxygenase activity was detected in nye1-1. However, no detectable accumulation of either chlorophyllide a or pheophorbide a was observed. Reciprocal crossings revealed that the mutant phenotype was caused by a monogenic semidominant nuclear mutation. We have identified AtNYE1 by positional cloning. Dozens of its putative orthologs, predominantly appearing in higher plant species, were identified, some of which have been associated with Chl degradation in a few crop species. Quantitative polymerase chain reaction analysis showed that AtNYE1 was drastically induced by senescence signals. Constitutive overexpression of AtNYE1 could result in either pale-yellow true leaves or even albino seedlings. These results collectively indicate that NYE1 plays an important regulatory role in Chl degradation during senescence by modulating pheophorbide a oxygenase activity.

Stay-green protein, defective in Mendel’s green cotyledon mutant, acts independent and upstream of pheophorbide a oxygenase in the chlorophyll catabolic pathway

“…1A). After 8d dark incubation, 84% of chl was retained in JI2775, thus showing a stronger retention than stay-green Lolium temulentum (Roca et al, 2004) or Arabidopsis nye1 (Ren et al, 2007). Increase in the chl a to b ratio is a characteristic of senescence-related chl breakdown (Pružinská et al, 2005).…”

“…PAO protein extraction and activity measurement PAO was isolated from Pisum chloroplast membranes by Triton X-100 solubilization according to a standard procedure Pružinská et al, 2005;Ren et al, 2007). PAO activity was monitored in vitro essentially as described (Pružinská et al, 2007) but using 0.06 μg μl -1 of His 6 -AtRCCR (Pružinská et al, 2007), and 100 U of catalase (Sigma) to replace red chl catabolite-forming factor (Pružinská et al, 2005).…”

“…These mutants are defective in a gene identified in the Bf993 mutant of Festuca pratensis (Thomas, 1987). The gene was termed SID (senescenceinduced degradation), but orthologous genes from rice, pea and Arabidopsis have now been designated SGR (STAY-GREEN) (Jiang et al, 2007;Park et al, 2007;Sato et al, 2007), SGN (Park et al, 2007) or NYE1 (nonyellowing) (Ren et al, 2007).…”

“…In all cases, introns were present at exactly the same positions within the coding regions indicating that SGR genes had existed prior to monocot and dicot divergence about 200 million years ago and that divergence is characterized by the occurrence of the additional intron. Consequently SGR-like genes are also found in lower plants, such as Gymnosperms (Ren et al, 2007), proteins. An interesting feature of the monocot and dicot sequences in clade I and II, and of two P. patens sequences was the presence of a novel conserved motif near the C-terminal ends (Fig.…”

“…Two functional SGR genes have been identified in Arabidopsis, but so far only for the senescence-regulated AtSGR1 (At4g22920) has an important function in chl breakdown been demonstrated (Armstead et al, 2007;Ren et al, 2007). In order to analyze whether AtSGR2, whose expression is also up-regulated during senescence (Park et al, 2007), is involved in chl catabolism as well, a homozygous T-DNA insertion line (SALK_003830C) was obtained from the SALK resource (Alonso et al, 2003).…”