A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

Abstract

The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.Chloroplasts not only serve as the site of photosynthesis but also are responsible for the production of many essential metabolites in higher plants. Although a descendant of photosynthetic prokaryotic organisms through endosymbiosis, the chloroplast has evolved many unique features including complex regulatory networks to achieve the coordinated expressions of plastid genome and nuclear genome and the coordination of chloroplast development with leaf development (1, 2). Given the importance of the chloroplast, much research has been directed at dissecting the intricate pathways that regulate chloroplast development. One fruitful way has been the identification of mutants that are defective in nuclear genes for chloroplast proteins. Large scale genetic screens have revealed that mutants of nuclear genes for chloroplast proteins can give rise to myriads of visible leaf color phenotypes ranging from albino, yellow, and pale green to virescence and variegation (3).Among the many types of mutants that are associated with chloroplast defects are the intriguing virescent mutants. These mutants display young and emerging tissues that are deficient of photosynthetic pigments, a sign that is often associated with underdeveloped plastids (2, 4). As leaves expand, however, these tissues gradually become greener along the leaf proximaldistal axis. At the whole plant level, the ce...

The Plastid and Mitochondrial Peptidase Network in Arabidopsis thaliana: A Foundation for Testing Genetic Interactions and Functions in Organellar Proteostasis

“…Module IV contains 10 plastid (putative) proteostasis factors: CAAX3, LON-like 2, EGY2, RBL8, four out the six thylakoid lumenal peptidases in the network (the lumenal DEG5/8 complex, D1 protein C-terminal peptidase CTPA, and its homolog CTPA-like-1), thylakoid AT5G27290, a homolog of thylakoid VIRESCENT3 (VIR3; AT1G56180), and a a/b-hydrolase. We note that it is not clear that AT5G27290 or VIR3 itself are actual metallo-peptidases as suggested by Qi et al (2016), since they lack homology to other peptidases and are not assigned to a clan/family in MEROPS. However, they do have a typical Zn binding domain (as also demonstrated experimentally for VIR3; Qi et al, 2016), but this is not unique to metallo-proteases.…”

“…We note that it is not clear that AT5G27290 or VIR3 itself are actual metallo-peptidases as suggested by Qi et al (2016), since they lack homology to other peptidases and are not assigned to a clan/family in MEROPS. However, they do have a typical Zn binding domain (as also demonstrated experimentally for VIR3; Qi et al, 2016), but this is not unique to metallo-proteases. The other (nonpeptidase) lumenal proteins (mostly isomerases and biogenesis factors) are strongly enriched in this module (31 edges).…”

Essentials of Proteolytic Machineries in Chloroplasts

“…Given the importance of FtsH and Clp in photosynthesis, another chloroplast AAA+ protease, Lon, may well play a crucial role in chloroplast physiology (van Wijk, 2015), but its dual targets make its genetic studies challenging. The conserved VIR3 protein family seems to play a role in early chloroplast biogenesis (Qi et al, 2016). Other proteases such as NANA and SCO4 are plant specific (Paparelli et al, 2012;Albrecht-Borth et al, 2013), which suggests unique proteolytic regulatory pathways in chloroplasts.…”

“…SCO4 appears to participate in the acclimation to high light. VIR3 VIR3 (VIRESCENT3) is a putative zinc metalloprotease associated with the stroma lamella (Qi et al, 2016). It contains two TMDs with the N and C termini protruding into the stromal side where the catalytic HExxH motif of the C-terminal tail seems to be exposed.…”

Heavy-ion beam mutagenesis identified an essential gene for chloroplast development under cold stress conditions during both early growth and tillering stages in rice

“…24) VIR3 encodes a putative chloroplast metalloprotease. 25) In maize, one gene, Etched1, which encodes a protein similar to the eukaryotic transcription factor TFIIS, has been reported as a causative gene of virescence. 26) Although there are many genes related to the virescent phenotype as described above, little is known about the mechanism underlying this phenotype.…”