A Phosphofructokinase B-Type Carbohydrate Kinase Family Protein, NARA5, for Massive Expressions of Plastid-Encoded Photosynthetic Genes in Arabidopsis

Taro Ogawa,Takehiko Aoki,Kenji Nishimura,Akiho Yokota,Ken-Ichi Tomizawa,Hiroki Ashida,Hisabumi Takase

doi:10.1104/pp.109.139683

Abstract

To date, there have been no reports on screening for mutants defective in the massive accumulation of Rubisco in higher plants. Here, we describe a screening method based on the toxic accumulation of ammonia in the presence of methionine sulfoximine, a specific inhibitor of glutamine synthetase, during photorespiration initiated by the oxygenase reaction of Rubisco in Arabidopsis (Arabidopsis thaliana). Five recessive mutants with decreased amounts of Rubisco were identified and designated as nara mutants, as they contained a mutation in genes necessary for the achievement of Rubisco accumulation. The nara5-1 mutant showed markedly lower levels of plastid-encoded photosynthetic proteins, including Rubisco. Map-based cloning revealed that NARA5 encoded a chloroplast phosphofructokinase B-type carbohydrate kinase family protein of unknown function. The NARA5 protein fused to green fluorescent protein localized in chloroplasts. We conducted expression analyses of photosynthetic genes during light-induced greening of etiolated seedlings of nara5-1 and the T-DNA insertion mutant, nara5-2. Our results strongly suggest that NARA5 is indispensable for hyperexpression of photosynthetic genes encoded in the plastid genome, particularly rbcL.

Highlights

To date, there have been no reports on screening for mutants defective in the massive accumulation of Rubisco in higher plants
We identified mutants with low Rubisco activity by selecting plants that were resistant to the inhibitory effects of Met sulfoximine (MSX) on photorespiratory metabolism
Wild-type Arabidopsis plants are killed by MSX as a result of excessive NH3 accumulation from photorespiration, which relies on the Rubisco oxygenase activity

Summary

Introduction

There have been no reports on screening for mutants defective in the massive accumulation of Rubisco in higher plants. Many factors participate in the processes, such as transcription of rbcL and RbcS (Manzara and Gruissem, 1988; Tyagi and Gaur, 2003; Shiina et al, 2005), posttranscriptional regulation and translation of rbcL (Barkan and Goldschmidt-Clermont, 2000; Zerges, 2000; Marin-Navarro et al, 2007), transport of SSU precursors through chloroplast envelopes (Kessler and Schnell, 2006), posttranslational modification of LSU and SSU (Houtz and Portis, 2003; Houtz et al, 2008), and folding and association of these subunits in the stroma (Roy and Andrews, 2000) Most of these factors are not specific to Rubisco synthesis but are selectively or universally involved in the synthesis of other chloroplast proteins. These factors were identified through biochemical and reverse, partly forward, genetic analyses based on visible phenotypes or chlorophyll fluorescence

Methods

Results

Conclusion