GUN1 influences the accumulation of NEP-dependent transcripts and chloroplast protein import in Arabidopsis cotyledons upon perturbation of chloroplast protein homeostasis

Abstract

Correct chloroplast development and function require coordinated expression of chloroplast and nuclear genes. This is achieved through chloroplast signals that modulate nuclear gene expression in accordance with the chloroplast's needs. Genetic evidence indicates that GUN1, a chloroplast-localized pentatricopeptide-repeat (PPR) protein with a C-terminal Small MutS-Related (SMR) domain, is involved in integrating multiple developmental and stress-related signals in both young seedlings and adult leaves. Recently, GUN1 was found to interact physically with factors involved in chloroplast protein homeostasis, and with enzymes of tetrapyrrole biosynthesis in adult leaves that function in various retrograde signaling pathways. Here we show that, following perturbation of chloroplast protein homeostasis i) by growth in lincomycin-containing medium, or ii) in mutants defective in either the FtsH protease complex (ftsh), plastid ribosome activity (prps21-1 and prpl11-1) or plastid protein import and folding (cphsp70-1), GUN1 influences NEP-dependent transcript accumulation during cotyledon greening and also intervenes in chloroplast protein import. import Chloroplast biogenesis is achieved through a cascade of events that include cytosolic synthesis of chloroplasttargeted proteins, followed by their import, assembly and folding, while unfolded/misfolded proteins and damaged or malformed chloroplasts are degraded (Sakamoto et al., 2008; Jarvi and López-Jauez, 2013; Izumi and Nakamura, 2018; Otegui, 2018). Most chloroplast-targeted proteins enter the chloroplasts via the protein complexes TOC ('translocon at outer envelope of chloroplast') and TIC ('translocon at inner envelope of chloroplast'). On the cytosolic side, precursor proteins are directed to the Toc34 and Toc159 receptors through the interaction of their chloroplast-targeting peptides with the chaperone heat shock protein 90 (Hsp90) and the guidance complex formed by Hsp70 and a 14-3-3 protein. Active transport through the chloroplast envelope is then mediated by an ATP-dependent import motor, consisting of Hsp70, Hsp90 and the stromal Hsp93 (ClpC2) proteins (Kessler and Schnell, 2009; Sjuts et al., 2017).On the stromal side, transcription of the plastid-encoded genes in higher plants requires two different RNA polymerases: the nuclear-encoded RNA polymerase (NEP), also termed RpoTp (located specifically in the chloroplasts and encoded by the RPOT3 gene) or RpoTmp (located both in mitochondria and plastids and encoded by the RPOT2 gene) and the plastid-encoded RNA polymerase (PEP) (Yu et al., 2014; Börner et al., 2015). NEP is a monomeric T3-T7 bacteriophage-type RNA polymerase that is mainly responsible for transcribing housekeeping genes, whereas PEP is a bacterial-type multisubunit enzyme largely tasked with transcribing photosynthesis-related genes. Chloroplast development is associated with a shift in the primary RNA polymerase from NEP to PEP. Furthermore, compensatory responses can be observed between the two RNA polymerases, as depletion of PEP activ...

Role of plastids and mitochondria in the early development of seedlings in dark growth conditions

“…Among six GUN proteins, five (GUN2-6) belong to the tetrapyrrole biosynthetic pathway (TBP) and control branching pathways downstream of protoporphyrin IX, whereas GUN1 is not an enzyme of TBP (Wu and Bock, 2021). GUN1 is a plastidlocalized, nuclear-encoded protein with pentatricopeptide repeat (PPR) and small MutS-related (SMR) domains involved in RNA binding and DNA repair, respectively (Tadini et al, 2016;Tadini et al, 2020). It has been suggested that GUN1 integrates several signals associated with plastid dysfunction, particularly those generated by inhibitors of plastid differentiation, such as norflurazon or lincomycin, or defective TBP (Sullivan and Gray, 1999;Koussevitzky et al, 2007;Tadini et al, 2016;Tadini et al, 2020).…”

“…GUN1 is a plastidlocalized, nuclear-encoded protein with pentatricopeptide repeat (PPR) and small MutS-related (SMR) domains involved in RNA binding and DNA repair, respectively (Tadini et al, 2016;Tadini et al, 2020). It has been suggested that GUN1 integrates several signals associated with plastid dysfunction, particularly those generated by inhibitors of plastid differentiation, such as norflurazon or lincomycin, or defective TBP (Sullivan and Gray, 1999;Koussevitzky et al, 2007;Tadini et al, 2016;Tadini et al, 2020). GUN1 was shown to interact with nuclear-encoded plastid RNA polymerase (NEP), multiple organellar RNA editing factor 2 (MORF2), a GUN1 interactor (FUG1), and plastid ribosomal protein S1 (PRPS1) (Tadini et al, 2016;Zhao et al, 2019) (Figure 4).…”

“…In response to lincomycin treatment, GUN1 regulates the expression of the genes encoding the chloroplast subunits TIC (TRANSLOCON AT THE INNER ENVELOPE MEMBRANE OF CHLOROPLASTS) and TOC (TRANSLOCON AT THE OUTER ENVELOPE MEMBRANE OF CHLOROPLASTS) to link the malfunctioning chloroplast protein synthesis with the impaired protein import ability (Tadini et al, 2020). In addition, GUN1 interacts with HEAT SHOCK COGNATE PROTEIN 70-1 (HSC70-1), which is associated with the TIC complex and facilitates the import of plastid proteins (Wu et al, 2019;Tadini et al, 2020;Wu and Bock, 2021). When GUN1 is absent, unimported preproteins are prominent, and the degree of protein ubiquitination in the cytosol is much higher (Tadini et al, 2020;Wu and Bock, 2021).…”

“…In addition, GUN1 interacts with HEAT SHOCK COGNATE PROTEIN 70-1 (HSC70-1), which is associated with the TIC complex and facilitates the import of plastid proteins (Wu et al, 2019;Tadini et al, 2020;Wu and Bock, 2021). When GUN1 is absent, unimported preproteins are prominent, and the degree of protein ubiquitination in the cytosol is much higher (Tadini et al, 2020;Wu and Bock, 2021). As a result, the chaperones HEAT SHOCK PROTEIN 90 (HSP90), HEAT SHOCK PROTEIN 70 (HSP70), and HEAT SHOCK COGNATE PROTEIN 70-4 (HSC70-4) are found to accumulate more in the cytosol (Wu et al, 2018).…”

“…As a result, the chaperones HEAT SHOCK PROTEIN 90 (HSP90), HEAT SHOCK PROTEIN 70 (HSP70), and HEAT SHOCK COGNATE PROTEIN 70-4 (HSC70-4) are found to accumulate more in the cytosol (Wu et al, 2018). Their accumulation ultimately results in the induction of PhANGs expression (Wu et al, 2018;Tadini et al, 2020). The authors conclude that the nucleus might sense the accumulation of preproteins as a stress plastid signal that acts by either activating positive or inhibiting negative transcriptional regulators in the nucleus for the expression of PhANGs (Wu et al, 2018).…”