Clp Protease and OR Directly Control the Proteostasis of Phytoene Synthase, the Crucial Enzyme for Carotenoid Biosynthesis in Arabidopsis

Abstract

Phytoene synthase (PSY) is the crucial plastidial enzyme in the carotenoid biosynthetic pathway. However, its post-translational regulation remains elusive. Likewise, Clp protease constitutes a central part of the plastid protease network, but its substrates for degradation are not well known. In this study, we report that PSY is a substrate of the Clp protease. PSY was uncovered to physically interact with various Clp protease subunits (i.e., ClpS1, ClpC1, and ClpD). High levels of PSY and several other carotenogenic enzyme proteins overaccumulate in the clpc1, clpp4, and clpr1-2 mutants. The overaccumulated PSY was found to be partially enzymatically active. Impairment of Clp activity in clpc1 results in a reduced rate of PSY protein turnover, further supporting the role of Clp protease in degrading PSY protein. On the other hand, the ORANGE (OR) protein, a major post-translational regulator of PSY with holdase chaperone activity, enhances PSY protein stability and increases the enzymatically active proportion of PSY in clpc1, counterbalancing Clp-mediated proteolysis in maintaining PSY protein homeostasis. Collectively, these findings provide novel insights into the quality control of plastid-localized proteins and establish a hitherto unidentified post-translational regulatory mechanism of carotenogenic enzymes in modulating carotenoid biosynthesis in plants.

Ectopic expression of ORANGE promotes carotenoid accumulation and fruit development in tomato

“…The same profile of carotenoid accumulation in green fruit of the PSY1 line suggests an increase in PSY activity in the AtOR His lines. Indeed, OR is known to post‐transcriptionally regulate PSY enzyme activity (Welsch et al ., ; Zhou et al ., ). In comparison with the PSY1 line that causes an up to 1.7‐fold increase in total carotenoids in mature green tomato fruit (Fraser et al ., ), a much high fold enrichment (6.4‐fold) was observed at the MG stage in the AtOR His lines.…”

“…OR encodes a plastidial DnaJ cysteine‐rich domain‐containing protein and is highly conserved among plant species (Lu et al ., ; Tzuri et al ., ). Recent studies reveal that OR has dual roles in post‐transcriptionally regulating PSY and promoting chromoplast biogenesis for carotenoid accumulation in plants (Chayut et al ., ; Park et al ., ; Welsch et al ., ; Yuan et al ., 2015a; Zhou et al ., ). Expression of a wild‐type OR ( OR WT ) results in carotenoid accumulation in multiple plant species (Bai et al ., ; Berman et al ., ; Park et al ., ; Yuan et al ., 2015a), likely due to its role in regulating PSY activity.…”

Extreme variation in rates of evolution in the plastid Clp protease complex

“…It is the most abundant stromal protease in developing chloroplasts and has been shown to degrade various chloroplast proteins, e.g. the cytochrome b 6 f complex, a copper transporter, glutamyl‐tRNA reductase and phytoene synthase (Majeran et al ., ; Nishimura and van Wijk, ; Apitz et al ., ; Nishimura et al ., ; Welsch et al ., ). Consistent with the significance of the Clp complex, plastid‐encoded ClpP1 as well as most nuclear‐encoded subunits are essential for plant growth and viability (Kuroda and Maliga, ; Clarke et al ., ; Zheng et al ., ; Koussevitzky et al ., ; Kim et al ., ; Nishimura and van Wijk, ).…”

The organ-specific differential roles of rice DXS and DXR, the first two enzymes of the MEP pathway, in carotenoid metabolism in Oryza sativa leaves and seeds

“…In Arabidopsis, AtDXS proteins have been reported to be post-translationally regulated by chloroplast biogenesis 6 (CLB6, a hydroxy-2-methyl-butenyl 4-diphosphate reductase), Hsp100 chaperones, and several Clp proteases [37], and the proteostasis of Arabidopsis PSY proteins are controlled directly by the Clp protease and ORANGE protein [38]. In Catharanthus roseus, the stability of DXR proteins is highly dependent on Clp protease-mediated degradation [39], and the enzyme activity of PDS proteins is more stably sustained in the βcarotene-enhanced tomato fruits, even if its transcripts are downregulated [34].…”