A Mutant Impaired in the Production of Plastome-Encoded Proteins Uncovers a Mechanism for the Homeostasis of Isoprenoid Biosynthetic Enzymes inArabidopsisPlastids

Abstract

The plastid-localized methylerythritol phosphate (MEP) pathway synthesizes the isoprenoid precursors for the production of essential photosynthesis-related compounds and hormones. We have identified an Arabidopsis thaliana mutant, rif1, in which posttranscriptional upregulation of MEP pathway enzyme levels is caused by the loss of function of At3g47450, a gene originally reported to encode a mitochondrial protein related to nitric oxide synthesis. However, we show that nitric oxide is not involved in the regulation of the MEP pathway and that the encoded protein is a plastid-targeted homolog of the Bacillus subtilis YqeH protein, a GTPase required for proper ribosome assembly. Consistently, in rif1 seedlings, decreased levels of plastome-encoded proteins were observed, with the exception of ClpP1, a catalytic subunit of the plastidial Clp protease complex. The unexpected accumulation of ClpP1 in plastids with reduced protein synthesis suggested a compensatory mechanism in response to decreased Clp activity levels. In agreement, a negative correlation was found between Clp protease activity and MEP pathway enzyme levels in different experiments, suggesting that Clp-mediated degradation of MEP pathway enzymes might be a mechanism used by individual plastids to finely adjust plastidial isoprenoid biosynthesis to their functional and physiological states.

Hunting for Plant Nitric Oxide Synthase Provides New Evidence of a Central Role for Plastids in Nitric Oxide Metabolism

“…Furthermore, a recombinant version of the bacterial YqeH protein was active in complementing the mutant nos1/rif1 phenotype when fused to the N-terminal targeting peptide of the NOA1/RIF1 protein (Sudhamsu et al, 2008) or when specifically targeted to plastids (Flores-Pé rez et al, 2008) but not when targeted to mitochondria (Figure 1). In agreement with the YqeH-like activity of the NOA1/RIF1 protein being required only in plastids, the ultrastructure of etioplasts and chloroplasts but not mitochondria was affected in rif1 seedlings (Flores-Pé rez et al, 2008). These results, together with the observed decrease in the levels of proteins encoded by the plastid genome (plastome) in rif1 chloroplasts (Flores-Pé rez et al, 2008), suggest that NOA1/RIF1 might bind plastidial ribosomes and be required for their normal function and therefore for proper protein synthesis in plastids.…”

“…A plastidial localization was recently reported for the diatom NOA1 homolog (Vardi et al, 2008). The untagged Arabidopsis protein was imported into isolated wild-type chloroplasts, whereas a GFP fusion that fully rescued the rif1 phenotype was found to be localized in chloroplasts (Flores-Pé rez et al, 2008). Substitution of the putative N-terminal organelle-targeting sequence of the NOA1/RIF1 protein by a previously characterized plastid-targeting sequence resulted in a chimeric protein that was able to restore fosmidomycin sensitivity and normal greening and growth when expressed in rif1 plants (Figure 1).…”

“…As recently suggested (Moreau et al, 2008), the decreased NO production in the noa1/rif1 mutant might result from pleiotropic effects of defective plastids. Impaired production of plastomeencoded proteins in the mutant is likely the cause of the defects observed in plastid development (Flores-Pé rez et al, 2008), which in turn might result in the increased production of reactive oxygen species (ROS) detected in mutant plants Zhao et al, 2007aZhao et al, , 2007b. Interaction of elevated levels of ROS with NO synthesized in plastids would reduce the amount of NO that could react in the available NO detection assays (Moreau et al, 2008), explaining the reduced levels detected in noa1/rif1 plants.…”

“…Moreover, a role for NOS1 unrelated to NO synthesis was suggested by the observation that not all the phenotypes observed in the mutant can be rescued by NO supplementation. For example, the accumulation of plastid-targeted enzymes of the methylerythritol pathway causing fosmidomycin resistance in a recently isolated nos1 allele named resistant to inhibition by fosmidomycin1 (rif1) was unaffected by treatment with an NO donor (Flores-Pé rez et al, 2008). These results and the failure to reproduce the published results on the detection of NOS activity of the recombinant NOS1 protein (Crawford et al, 2006;Zemojtel et al, 2006a;Moreau et al, 2008) and the absence of such activity in bacterial homologs (Sudhamsu et al, 2008) led to the conclusion that NOS1 is not a NOS.…”

Molecular components of stress-responsive plastid retrograde signaling networks and their involvement in ammonium stress

“…Recently, it has been found that enzymes of the MEP pathway require posttranslational modification, such as can be seen in the stromal ClpPR proteolytic complex. 48 Therefore, it is possible that the AMOS1/ EGY1 metalloprotease activates MEP pathway enzymes in such a manner, modulating MEcPP levels, to regulate the expression of ammonium-stress-responsive genes in the nucleus.…”