Abiotic stress-induced chloroplast proteome remodelling: a mechanistic overview

Abiotic stress-induced chloroplast proteome remodelling: a mechanistic overview Abstract The chloroplast houses photosynthesis in all green plants, and is therefore of fundamental importance to the viability and productivity of plants, ecosystems, and agriculture. Chloroplasts are, however, extremely vulnerable to environmental stress, on account of the inherent volatility of oxygenic photosynthesis. To counteract this sensitivity, sophisticated systems of chloroplast stress acclimation have evolved, and many of these involve broad proteome changes. Here, we provide an overview of the interlocking and mutually dependent mechanisms of abiotic stress-induced chloroplast proteome remodelling. Topics that are covered in this context include: nucleus to chloroplast signalling mechanisms, with a particular emphasis on the nuclear control of the chloroplast genome; chloroplast to nucleus signalling; and the roles of chloroplast pre-protein import regulation and chloroplast proteases. Abiotic stress, anterograde signalling, chloroplasts, plastids, pre-proteins, proteases, retrograde signalling, ROS Introduction The chloroplast is the defining plant organelle (Jarvis and López-Juez, 2013). Chloroplasts are the site of photosynthesis in all green plants, and also house the biosynthesis of a number of important molecules, including amino acids and fatty acids. As such, the chloroplast is key to plant productivity and survival. It is, however, exquisitely sensitive to environmental stress, due, in large part, to the inherent volatility of oxygenic photosynthesis. Under a range of abiotic stress conditions, the photosynthetic machinery becomes overexcited, producing damaging reactive oxygen species (ROS) that threaten the health and viability of the plant. As a consequence, chloroplasts must respond to abiotic stressors, and this often involves broad proteome changes. The chloroplast proteome is rapidly modified in response to a range of abiotic stressors, and this modification is key to the plant’s stress response (Taylor et al., 2009). The environmental regulation of chloroplast proteomes can also occur over a longer period of time, for example during the acclimation of plants to cold temperatures (Goulas et al., 2006). A large number of chloroplast proteins have been shown to change in abundance in response to one or more environmental stressors. Proteins involved in photosynthesis are particularly responsive to stress (Taylor et al., 2009). Two specific examples are the photosynthetic proteins ribulose-5-phosphate-3-epimerase and PsbO1, which both show large decreases in abundance under cold stress (Goulas et al., 2006). Chloroplast proteome remodelling is a complex and multifaceted process. Part of the reason for this complexity is the fact that the chloroplast is the endosymbiotic descendant of an independent prokaryotic ancestor, and retains a small genome. The chloroplast proteome is thus shaped by two genomes, and dynamically regulated through complex, bidirectional communication with the nucleus. Multiple systems contribute to proteome remodelling, and this review will explore the mechanisms involved in the phenomenon in response to abiotic stress. The review will first discuss the contribution made to proteome remodelling by nucleus to chloroplast signalling, with a particular emphasis on the role of plastome regulation by nuclear factors. Secondly, the role of chloroplast to nucleus communication will be discussed, and an overview of the pathways likely to contribute to proteome remodelling will be provided. Lastly, the involvement of chloroplast pre-protein import regulation and of chloroplast proteases will be explored. The separation of these mechanisms into discrete sections is, of course, artificial, but it serves as a useful conceptual framework. As the topic of this review is broad, it cannot be exhaustive; instead, key examples are introduced to illustrate each section. Nucleus–chloroplast communication As discussed, chloroplasts, along with other plastids, are the well-integrated descendants of once free-living cyanobacterial ancestors, and, as such, retain genomes. However, over the course of evolution, the plastid genome—the plastome—has significantly decreased in size. Many protein-encoding genes have been transferred to the nucleus, and others have been lost entirely. Despite this, the plastome retains a number of important genes, and thus two genomes contribute to chloroplast function. The co-ordination of the two genomes is therefore of paramount importance, and necessitates bidirectional communication between the plastid and nucleus (Woodson and Chory, 2008; Jarvis and López-Juez, 2013). The nucleus houses most of the genes required for chloroplast function, and these control the development, maintenance, and remodelling of the chloroplast proteome; prominent amongst these are those necessary for photosynthesis, and those that regulate the plastome (Terry and Smith, 2013). Nucleus to chloroplast communication is known as ‘anterograde’ signalling (Fig. 1). Interestingly, communication is bidirectional; the chloroplast is able to convey information on its physiological or developmental state to the nucleus, and this is known as ‘retrograde’ signalling. Fig. 1. View largeDownload slide An overview of the various mechanisms that drive stress-induced chloroplast proteome remodelling. The nucleus encodes a large number of chloroplast-destined proteins, and these include proteins that regulate the chloroplast genome (the plastome) under stress conditions. Nucleus-encoded, chloroplast proteins are imported into the organelles via the translocon at the outer chloroplast membrane (TOC) and the translocon at the inner chloroplast membrane (TIC) complexes. Import of proteins through this TOC–TIC system is an important component of nucleus to chloroplast communication, which is known as ‘anterograde signalling’. Some chloroplast-targeted proteins can be degraded in the cytosol via the CHIP/Hsp70 E3 ubiquitin ligase system, which sends them for 26S proteasomal degradation. Under stress conditions, the TOC complex is targeted for degradation by the outer membrane-bound E3 ubiquitin ligase SP1, which suppresses the import of photosynthetic proteins, thus attenuating photosynthetic ROS production and promoting survival of the plant. Chloroplast-resident proteases, particularly filamentation temperature-sensitive H (FtsH) and the caseinolytic protease (Clp), turn over mature proteins, and this can drive stress-induced proteome remodelling. In turn, the chloroplast is able to communicate its status to the nucleus via ‘retrograde signalling’; this regulates nucleus-encoded chloroplast genes, particularly under stress conditions. A number of pathways are known to contribute to this behaviour; shown are the SAL1/PAP, MEcPP, and ROS pathways. ROS signalling is complex, and involves multiple ROS types and downstream signalling components. Fig. 1. View largeDownload slide An overview of the various mechanisms that drive stress-induced chloroplast proteome remodelling. The nucleus encodes a large number of chloroplast-destined proteins, and these include proteins that regulate the chloroplast genome (the plastome) under stress conditions. Nucleus-encoded, chloroplast proteins are imported into the organelles via the translocon at the outer chloroplast membrane (TOC) and the translocon at the inner chloroplast membrane (TIC) complexes. Import of proteins through this TOC–TIC system is an important component of nucleus to chloroplast communication, which is known as ‘anterograde signalling’. Some chloroplast-targeted proteins can be degraded in the cytosol via the CHIP/Hsp70 E3 ubiquitin ligase system, which sends them for 26S proteasomal degradation. Under stress conditions, the TOC complex is targeted for degradation by the outer membrane-bound E3 ubiquitin ligase SP1, which suppresses the import of photosynthetic proteins, thus attenuating photosynthetic ROS production and promoting survival of the plant. Chloroplast-resident proteases, particularly filamentation temperature-sensitive H (FtsH) and the caseinolytic protease (Clp), turn over mature proteins, and this can drive stress-induced proteome remodelling. In turn, the chloroplast is able to communicate its status to the nucleus via ‘retrograde signalling’; this regulates nucleus-encoded chloroplast genes, particularly under stress conditions. A number of pathways are known to contribute to this behaviour; shown are the SAL1/PAP, MEcPP, and ROS pathways. ROS signalling is complex, and involves multiple ROS types and downstream signalling components. Anterograde signalling In response to changing environmental conditions, the nucleus exerts tight regulatory control over the chloroplast proteome, and this regulation occurs at many levels. The nuclear transcriptome is remodelled in response to environmental cues by chloroplast-dependent and chloroplast-independent mechanisms; this ultimately changes the identity and abundance of chloroplast-destined pre-proteins. Additionally, the expression of the plastome is subject to stringent regulation by a battery of nuclear factors, while the mature chloroplast proteome is actively regulated by a suite of nuclear-encoded proteases. This section will explore the anterograde regulation of the chloroplast proteome in response to stress. There will not be a detailed discussion of chloroplast-independent transcriptional reprogramming of nuclear-encoded, chloroplast genes, as this is an extremely broad subject and is beyond the scope of the review. Interested readers are encouraged to consult Saibo et al. (2009). The focus of this section will instead be on (i) the regulation of chloroplast-targeted pre-proteins as they move through the cytosol to the organelle; and (ii) the nuclear regulation of the plastome. Nucleus-encoded proteases will be discussed later. The regulation of chloroplast-targeted pre-proteins within the cytosol Chloroplast-targeted pre-proteins can be marked for ubiquitin–proteasome system (UPS)-mediated degradation as they travel through the cytosol. In addition to preventing the accumulation of toxic aggregates in the cytosol, this can help to shape the chloroplast proteome, and, in some cases, it probably acts in concert with changes in chloroplast pre-protein import behaviour (discussed at length in the ‘Chloroplast pre-protein import’ section). The heat shock cognate 70-4 (Hsc70-4) has a role in this process, as does the E3 ubiquitin ligase C-terminus of Hsc70-interacting protein (CHIP) (Shen et al., 2007a; Lee et al., 2009). The transcripts of both genes are up-regulated in the albino plastid protein import 2 (ppi2) mutant, which is defective in the synthesis of Toc159, a major receptor component of the chloroplast import machinery (see the ‘Chloroplast pre-protein import’ section for a more detailed discussion). The ppi2 mutant cannot efficiently import nuclear-encoded chloroplast pre-proteins, and these consequently accumulate in the cytosol. Hsc70-4 and CHIP physically interact with each other, and function within a cytosolic complex. Hsc70-4 binds the transit peptide sequences of target proteins, and recruits CHIP, which acts as an E3 ligase for the UPS, ubiquitinating the substrate and marking it for 26S proteasomal degradation. Other chloroplast-targeted proteins have since been shown to be recognized by the UPS in the cytosol, and, again, this recognition has been shown to be dependent on the transit peptide of the substrates (Sako et al., 2014). Interestingly, the filamentation temperature-sensitive H (FtsH) protease is a target of the CHIP system, as is ClpP4, a component of the caseinolytic protease (Clp) (Shen et al., 2007a, b). This regulation of chloroplast proteases is likely to have roles in abiotic stress tolerance—under high light, CHIP overexpression suppresses the light-induced increase in ClpP4 and FtsH, suggesting that the complex regulates the levels of both proteases, perhaps under stress conditions. The regulation of plastome gene expression by nuclear proteins The plastome contains a number of essential genes. While the plastome encodes core components of its own transcription and translation machineries, the nucleus exerts control over plastome activity via a broad suite of proteins. Nuclear factors regulate plastome gene expression transcriptionally, post-transcriptionally, translationally, and post-translationally, and this form of anterograde regulation has roles in plant adaptation to abiotic stress. Transcriptional regulation of plastome gene expression Plastome genes are transcribed via two classes of RNA polymerases: the plastid-encoded polymerase (PEP), and the nuclear-encoded polymerases (NEPs) (Kanamaru et al., 1999; Lerbs-Mache, 2011). PEP activity is dependent on PEP-associated proteins (‘PAPs’), as well as bacterial-type σ70 subunits (sigma factors); these are key to the initiation and specificity of plastome transcription. Thus, while the chloroplast contains its own genome, its transcription is under the strict control of the nucleus, which exerts its control via NEPs and, indirectly, PEP. Although plastome transcription is tightly regulated, there is a surprisingly weak correlation between transcript and chloroplast protein abundance, and it has been argued that most regulation of plastid gene expression occurs post-transcriptionally (Woodson and Chory, 2008). Despite this, there are documented cases of stress- and environmentally induced plastome transcriptional changes (Pfannschmidt, 2003). An interesting example is provided by the sigma factor SIG5, which is induced by a variety of abiotic stressors: high light, high salt, osmotic stress, and low temperature (Nagashima et al., 2004). SIG5 guides PEP to the psbD promoter; psbD is an operon encoding three proteins including the D2 PSI reaction centre component and the antenna protein CP43. Thus, under certain abiotic stress conditions, plastome genes encoding components of the photosynthetic machinery are transcriptionally regulated by nuclear-encoded SIG5 and, presumably, by other factors as well. The scale of the contribution that such regulation makes to dynamic chloroplast proteome remodelling is, however, currently unclear. Post-transcriptional regulation As discussed, it is thought that post-transcriptional regulation is the dominant force that shapes plastid gene regulation, and this appears to be particularly the case in mature chloroplasts (Sun and Zerges, 2015). Nuclear-encoded proteins control the transcription, splicing, editing, and stability of plastome-encoded mRNAs (Stern et al., 2010). In some cases, this regulation has been explicitly linked to dynamic chloroplast proteome remodelling, and examples of this behaviour are discussed below. In the green alga Chlamydomonas reinhardtii, the plastome gene encoding cytochrome f, a key subunit of the cytochrome b6f complex, is post-transcriptionally regulated by nuclear-encoded proteins (Raynaud et al., 2007). Two proteins have been implicated: maturation of cytochrome b6f petA mRNA (MCA1) and translation of cytochrome b6f petA mRNA (TCA1). MCA1 belongs to the pentatricopeptide repeat (PPR) family of RNA-binding proteins, and it binds and stabilizes photosynthetic electron transfer A (petA, which encodes cytochrome f) mRNA, allowing it to accumulate. Similarly, TCA1 binds the 5′-untranslated region (UTR) of petA mRNA, promoting its translation. The two proteins thus enable the efficient expression of the petA gene. Interestingly, they appear to contribute to chloroplast proteome remodelling in response to nitrogen limitation. Under nitrogen starvation, the cytochrome b6f complex is rapidly degraded (Wei et al., 2014) (as discussed in more detail in the ‘Chloroplast proteases’ section), and MCA1 and TCA1 also decrease in abundance, preventing further biogenesis of the cytochrome b6f complex. Intriguingly, there is also evidence that anterograde post-transcriptional gene regulation has a role in the adaptation of the C. reinhardtii proteome to iron-limiting conditions (Lefebvre-Legendre et al., 2015). Under such conditions, PSI components are targeted for turnover, perhaps on account of the system's high iron content (Moseley et al., 2002). It appears that a nuclear-encoded protein called translation of psaA1 (TAA1) complements this process by regulating the stability and translation of plastome-encoded psaA mRNA, which codes for a key component of PSI (Lefebvre-Legendre et al., 2015). The stability of psaA mRNA is impaired in taa1 mutants, and TAA1 is indispensable for PsaA translation. Furthermore, the TAA1 protein strongly decreases in abundance in response to iron limitation, implying a role for the protein in the nutrient limitation-induced remodelling of the C. reinhardtii chloroplast proteome. Retrograde signalling During the development and subsequent operation of the chloroplast, the organelle communicates with the nucleus, altering the nuclear transcriptome through a number of complex pathways. This communication—known as ‘retrograde signalling’—is vital for the biogenesis, homeostasis, and stress-induced remodelling of the chloroplast proteome. Retrograde signalling can be divided into two broad categories: biogenic and operational signalling (Pogson et al., 2008; Terry and Smith, 2013). Biogenic signalling drives chloroplast biogenesis, the process through which the chloroplasts develop from their proplastid or etioplast progenitors. Operational signalling occurs after chloroplast maturation, and serves to regulate the nuclear transcriptome under changing environmental conditions. As chloroplast proteome biogenesis is not within the scope of this review, we focus here on operational signalling pathways as they relate to chloroplast proteome remodelling. Thus, the well-studied genomes uncoupled (GUN) mutants will not be discussed, as they have principally been studied in relation to chloroplast biogenesis. A number of operational signalling pathways have been proposed, with varying degrees of experimental support. The relevant pathways are discussed below. Reactive oxygen species (ROS) Oxygenic photosynthesis is a volatile process which is highly sensitive to environmental disruption. Under a range of abiotic stress conditions, the photosynthetic machinery generates ROS, which can severely threaten the health and viability of the plant. ROS do, however, have an important role in plant stress signalling, acting as retrograde signals to modify the nuclear transcriptome under unfavourable environmental conditions (Tripathy and Oelmüller, 2012). Two ROS molecules have been intensively studied in this context: 1O2 (singlet oxygen), which is typically produced at PSII, and H2O2 (hydrogen peroxide), which is generally produced by PSI. Interestingly, different ROS molecules may modify the expression of different sets of nuclear genes, as described below. Singlet oxygen As discussed, different ROS molecules induce distinct transcriptional responses, and teasing these apart has presented major challenges. A breakthrough came with the identification of the flu mutant (Meskauskiene et al., 2001). This Arabidopsis mutant lacks a functional FLU protein (a key negative regulator of chlorophyll biosynthesis), and consequently accumulates the chlorophyll biosynthesis intermediate protochlorophyllide in the dark. Protochlorophyllide is strongly photosensitizing, and dark-treated flu mutants specifically accumulate singlet oxygen when moved into the light (op den Camp et al., 2003). The flu mutant allowed several groups to tease apart the distinctive roles of singlet oxygen signalling. Other mutants, such as chlorina 1, which lacks the light harvesting complex (LHC) of PSII and accumulates singlet oxygen, have also provided major insights (Ramel et al., 2013). Singlet oxygen is produced at PSII through the excitation of ground-state triplet oxygen, and it may be the major ROS produced under high-light conditions (Triantaphylidès and Havaux, 2009; González-Pérez et al., 2011). This ROS acts to change the expression of a set of nuclear genes, termed singlet oxygen-regulated genes (SORGs). Many of the implicated genes have roles in photosynthesis and carbon metabolism, as well as in plastid mRNA processing. Although singlet oxygen signalling has mostly been studied in relation to operational signalling, a role for the ROS within the context of biogenic signalling has recently been proposed (Page et al., 2017). Singlet oxygen signalling triggers programmed cell death and growth suppression under high-light conditions (op den Camp et al., 2003), and this activity is dependent on the action of two chloroplast-localized proteins: Executor (Ex) 1 and 2 (Wagner et al., 2004; Lee et al., 2007; for a review, see Kim and Apel, 2013). However, under moderate light intensity, the two proteins drive abiotic stress acclimation, partly through chloroplast proteome remodelling (Uberegui et al., 2015; Carmody et al., 2016). The flu ex1 ex2 triple mutant demonstrates the importance of the Ex1/Ex2 proteins in this process, as it displays abrogated regulation of SORG expression following a dark to light shift (Lee et al., 2007). Interestingly, it has recently emerged that the plastid protease FtsH2 has a role in Ex1/Ex2 signalling: the protease associates with Ex1, and the var2 flu double mutant (var2 lacks functional FtsH2) shows dampened SORG expression changes in response to dark to light shifts, with no change in the case of 85% of tested SORGs (Wang et al., 2016; Dogra et al., 2017). This demonstrates an intriguing link between retrograde signalling and plastid proteolysis, although the mechanistic basis of the link is currently unclear. Surprisingly, the Ex1/Ex2 pathway may not be the only singlet oxygen signalling pathway. Under very high light conditions, oxidation of PSII-associated carotenoids occurs, and two of the oxidation products—β-cyclocitral and dihydroactinidiolide—seem to change the expression of a subset of nuclear genes (Ramel et al., 2012; Shumbe et al., 2014). β-Cyclocitral is an oxidized derivative of β-carotene, and it acts as a signalling molecule under high-light stress (Ramel et al., 2013). Exogenous β-cyclocitral treatment induces the misregulation of a large number of SORGs in Arabidopsis (Ramel et al., 2012). The downstream pathway components of β-cyclocitral signalling are still poorly understood; however, methylene blue sensitivity 1 (MBS1), a small zinc finger protein, has been implicated in both C. reinhardtii (Shao et al., 2013) and Arabidopsis (Shumbe et al., 2017). Interestingly, the genes regulated by the β-cyclocitral pathway substantially differ from those regulated by the Ex1/Ex2 pathway; one study found that the two categories overlap by just 20 genes (Dogra et al., 2017). This suggests that singlet oxygen may signal through two distinctive pathways which regulate different subsets of SORGs. There is evidence to suggest that the subgranal location of singlet oxygen production may influence the signalling mode (Wang et al., 2016). Hydrogen peroxide As in the case of singlet oxygen, untangling the specific role of hydrogen peroxide within the context of broader ROS signalling has presented technical challenges. Major insights came through the creation of an RNAi line that specifically suppresses the expression of thylakoid membrane-bound ascorbate peroxidase (tAPX), a factor that is key to chloroplast hydrogen peroxide regulation (Maruta et al., 2012). This transgenic line shows altered regulation of a large number of cold stress-related and pathogen resistance genes; these were named Responsive to tAPX Silencing (RTS) genes by the authors. Interestingly, some of the implicated genes encode chloroplast proteins. In another successful approach, a chloroplast transit peptide–glycolate oxidase (GO) fusion gene was overexpressed in Arabidopsis, and this specifically induced hydrogen peroxide accumulation in the chloroplast (Fahnenstich et al., 2008; Balazadeh et al., 2012). GO converts glycolate to glyoxylate, producing hydrogen peroxide in the process. The induction of hydrogen peroxide production in the chloroplast through this method was again shown to change the regulation of a large number of nuclear genes. Hydrogen peroxide is comparatively stable, and may, in some circumstances, act directly as a mobile retrograde signalling molecule. Interestingly, it was recently suggested that a subpopulation of chloroplasts located next to the nucleus transfers hydrogen peroxide directly, by-passing the cytosol (Exposito-Rodriguez et al., 2017). A considerable number of studies have attempted to identify components of the hydrogen peroxide signal transduction network(s), and a number of putative pathway components have been identified. These include a diverse variety of transcription factors, mitogen-activated protein kinases (MAPKs), and miRNAs (reviewed by Petrov and Van Breusegem, 2012). MEcPP Another relevant operational signalling pathway was discovered in a screen for mutant plants with enhanced expression of hydroxyperoxide lyase (HPL)—a nuclear-encoded, stress-inducible gene that encodes a chloroplast enzyme involved in the synthesis of defence-related molecules such as the hormone jasmonic acid (JA). In the screen, the constitutively expressing HPL (ceh1) mutant was discovered (Xiao et al., 2012). This mutant hyperaccumulates methylerythritol cyclodiphosphate (MEcPP), an intermediate of the methylerythritol phosphate (MEP) pathway for plastid isoprenoid biosynthesis, due to a lesion in an enzyme of the pathway. MEcPP accumulates in response to high-light or wounding stress, and strongly and specifically up-regulates the expression of HPL. Interestingly, MEcPP accumulation also triggers salicylic acid (SA) biosynthesis, although genetic experiments have demonstrated that the mutant’s HPL up-regulation is not dependent on this change in hormone abundance—when the SA-deficient eds16-1 mutant is crossed into the ceh1 background, the plants still show strong HPL up-regulation (Xiao et al., 2012). MEcPP is involved in activation of stress responses (Benn et al., 2016), and regulates the expression of a broad range of nuclear genes, partially through hormone signalling. Recent studies have linked MEcPP to the regulation of genes key to the endoplasmic reticulum unfolded protein response (UPR) (Walley et al., 2015), alongside a variety of JA-regulated genes (Lemos et al., 2016). Interestingly, another recent study found that many nuclear genes are misregulated in ceh and ceh eds16-1 (Bjornson et al., 2017): under gene set enrichment analysis, a number of patterns emerged, and it is noteworthy that gene sets involved in photosynthesis and carbon fixation were misregulated. SAL1–PAP In recent years, the phosphonucleotide 3′-phosphoadenosine 5′-phosphate (PAP) has emerged as a key player in a novel, operational, retrograde signalling pathway (Estavillo et al., 2011). PAP is produced as a by-product of sulphur metabolism, and, under favourable environmental conditions, it is degraded by the chloroplast phosphatase SAL1 (Quintero et al., 1996; Estavillo et al., 2011). SAL1 is inactivated in response to oxidative stress, which leads to PAP accumulation in the chloroplast, and from there it moves through the cytosol into the nucleus. Within the nucleus, PAP regulates a number of genes: mutants of SAL1 show misregulation of ~1800 genes, many of which are involved in the stress response, osmoprotection, and starch metabolism (Wilson et al., 2009). The SAL1–PAP pathway is also involved in physiological responses to abiotic stress; recent work has identified a link between the pathway and stomatal closure, a key component of the plant drought response (Pornsiriwong et al., 2017). Interestingly, it has been proposed that SAL1 is a direct sensor of ROS/chloroplast redox poise (Chan et al., 2016). SAL1 activity is suppressed under oxidative stress conditions, and this allows PAP to accumulate. It is argued that plant SAL1 is unusually susceptible to redox inactivation, and that this physical property explains its putative role as an oxidative stress receptor. Chloroplast pre-protein import As discussed, most proteins required for chloroplast function are encoded by genes that reside in the central, nuclear genome. The products of these genes must therefore be imported across the chloroplast double membrane, and this transport occurs post-translationally via a mechanism dependent on the co-ordinate activity of two large, multiprotein complexes. These complexes—the translocon at the outer chloroplast membrane, or TOC, and the translocon at the inner chloroplast membrane, or TIC—reside in the outer and inner envelope membranes, respectively, and co-operatively import cytosolically translated pre-proteins into the organelle (for a review, see Jarvis, 2008). The TOC complex is exposed to the cytosol, and comprises three major components: Toc33 and Toc159, which are both GTPases involved in pre-protein (substrate) recognition; and Toc75, which forms a channel through which the entry of pre-proteins proceeds. The Toc33 and Toc159 receptors are encoded by small gene families, leading to the formation of different receptor isoforms with varying substrate preferences. Interestingly, there is evidence that chloroplast pre-protein import is modified in response to abiotic stress (Fig. 1). For example, under heat stress conditions, nuclear-encoded chloroplast proteins accumulate in the cytosol (Heckathorn et al., 1998). This behaviour is rapidly reversible, which implies that the short-term suppression of chloroplast pre-protein import could have a role in plant abiotic stress tolerance. In keeping with this hypothesis, it has also been shown that the import of pre-Rubisco small subunit (pRSS) into isolated heat- or chill-stressed pea chloroplasts is retarded (Dutta et al., 2009), and that pRSS has reduced ability to bind the outer membranes of isolated chloroplasts under heat shock conditions. Interestingly, the same study found that the Toc159 protein decreases in abundance in response to heat treatment. Although it appears that chloroplast import is a stress-responsive and surprisingly dynamic process, until recently little was known about its regulation. Within the last 5 years, a considerable amount of progress has been made. It was recently shown that TOC complex components, including Toc33, Toc75, and Toc159, are targeted by suppressor of ppi1 locus 1 (SP1), an E3 ubiquitin ligase that localizes to the chloroplast outer membrane (Ling et al., 2012). In response to developmental or environmental cues, SP1 promotes the degradation of TOC complexes, thus suppressing plastid pre-protein import. The SP1 protein has important physiological roles: sp1 Arabidopsis mutants show delayed plastid interconversions, with plants displaying defects in de-etiolation and leaf senescence. This implies that SP1 has a role in plastid proteome remodelling within developmental contexts, which it may achieve by altering the ratios of different TOC component isoforms with different pre-protein preferences (Ling et al., 2012). Interestingly, SP1 also promotes plant survival under various abiotic stress conditions: While sp1 mutants are more sensitive to oxidative, osmotic, and salt stresses, SP1 overexpression enhances resistance to these conditions (Ling and Jarvis, 2015). In addition, sp1 mutants and SP1 overexpressor lines show increased and decreased hydrogen peroxide accumulation, respectively, when osmotically stressed. Furthermore, these phenotypes have been linked to changes in chloroplast pre-protein import efficiency under stress conditions: in sp1 mutants, the import of key photosystem proteins was enhanced, whereas, in the overexpressor lines, the opposite behaviour was observed. Taken together, a picture emerges in which stress induces the rapid, UPS-mediated breakdown of TOC complexes, and this acts to suppress the import of pre-proteins key to photosynthesis. This import suppression probably attenuates photosynthesis, and dampens the accumulation of harmful ROS. From the work described here, it is clear that the regulation of pre-protein import has key roles within the stress-induced remodelling of the chloroplast proteome. Further study is needed to determine how SP1 is regulated, and to probe the conditions under which its activity becomes important. Chloroplast proteases Chloroplasts contain a considerable number of proteases; ~20 have so far been described, and these are composed of >50 components (Nishimura et al., 2016b). Plastid-resident proteases can be divided into three major categories: serine, metallo, and aspartyl proteases. A number of proteases housed within the plastid are of bacterial origin, whereas others are specific to plants and algae. Some of the most intensively studied, and, in this case relevant, plastid proteases are the Clp and FtsH proteases. The Clp proteases are serine proteases that are abundant within the chloroplasts of green algae and higher plants (Olinares et al., 2011). FtsH proteases contain ATPase associated with various cellular activities (AAA) and Zn2+ metalloprotease domains, and are found in both mitochondria and chloroplasts (Wagner et al., 2012). Proteases are important for a large number of plastid processes. For example, some are involved in the processing of newly imported pre-proteins: the metalloprotease stromal processing peptidase (SPP) removes transit peptides from pre-proteins at an early stage of translocation, upon emergence into the stroma, and the transit peptides are then degraded by the action of a number of other plastid-resident proteases such as pre-sequence peptidases 1 and 2 (Prep1/2) and organellar oligopeptidase (OOP) (for a detailed review, see Nishimura et al., 2016a). In addition, a number of other plastid proteases have roles in plastid proteome homeostasis and the removal of damaged proteins, particularly within the context of the photosynthetic machinery. These processes are not covered here, however, as the focus is on the contribution plastid proteases make to abiotic stress-induced remodelling of the plastid proteome (Fig. 1). Almost all of the literature relevant here involves the model green alga C. reinhardtii. A number of studies have shown that the thylakoid proteome of the alga is extensively remodelled in response to nutrient stress. Under nitrogen deprivation, Rubisco, as well as all of the components of the cytochrome b6f complex and its biogenesis factors, are selectively degraded (Wei et al., 2014). The degradation of the cytochrome b6f complex and related factors is dependent on the action of the thylakoid-bound FtsH protease, and, to a lesser extent, Clp, as well as an unknown, thylakoid lumen-localized protease. Interestingly, the degradation of the photosynthetic machinery is accompanied by an increase in the abundance of l-amino acid oxidase 1 (LAO1), a nitrogen-scavenging enzyme, as well as increases in the abundance of various proteins involved in metabolic reprogramming, implying that the proteases act within a broad functional transition. In addition, sulphur deprivation has also been shown to trigger the FtsH-mediated degradation of the cytochrome b6f complex, as well as of PSII (Malnoë et al., 2014). Taken together, these studies clearly implicate plastid proteases in the stress-induced remodelling of the chloroplast proteome, at least in the case of C. reinhardtii. Concluding remarks The chloroplast proteome is dynamically modified in response to abiotic stress. Here, we have provided an overview of the interconnected systems that drive this phenomenon. From the work discussed, it is clear that the process is complex and multifaceted. The nucleus houses most chloroplast genes, including those that regulate the behaviour of the plastome, chloroplast pre-protein import, and chloroplast proteolysis; and it exerts tight regulatory control over the chloroplast proteome under abiotic stress conditions. The chloroplast can, in turn, regulate the nuclear genome to influence its own proteome indirectly. 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Google Scholar CrossRef Search ADS © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

Abiotic stress-induced chloroplast proteome remodelling: a mechanistic overview

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0022-0957
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1460-2431
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10.1093/jxb/ery053
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Abstract

Abstract The chloroplast houses photosynthesis in all green plants, and is therefore of fundamental importance to the viability and productivity of plants, ecosystems, and agriculture. Chloroplasts are, however, extremely vulnerable to environmental stress, on account of the inherent volatility of oxygenic photosynthesis. To counteract this sensitivity, sophisticated systems of chloroplast stress acclimation have evolved, and many of these involve broad proteome changes. Here, we provide an overview of the interlocking and mutually dependent mechanisms of abiotic stress-induced chloroplast proteome remodelling. Topics that are covered in this context include: nucleus to chloroplast signalling mechanisms, with a particular emphasis on the nuclear control of the chloroplast genome; chloroplast to nucleus signalling; and the roles of chloroplast pre-protein import regulation and chloroplast proteases. Abiotic stress, anterograde signalling, chloroplasts, plastids, pre-proteins, proteases, retrograde signalling, ROS Introduction The chloroplast is the defining plant organelle (Jarvis and López-Juez, 2013). Chloroplasts are the site of photosynthesis in all green plants, and also house the biosynthesis of a number of important molecules, including amino acids and fatty acids. As such, the chloroplast is key to plant productivity and survival. It is, however, exquisitely sensitive to environmental stress, due, in large part, to the inherent volatility of oxygenic photosynthesis. Under a range of abiotic stress conditions, the photosynthetic machinery becomes overexcited, producing damaging reactive oxygen species (ROS) that threaten the health and viability of the plant. As a consequence, chloroplasts must respond to abiotic stressors, and this often involves broad proteome changes. The chloroplast proteome is rapidly modified in response to a range of abiotic stressors, and this modification is key to the plant’s stress response (Taylor et al., 2009). The environmental regulation of chloroplast proteomes can also occur over a longer period of time, for example during the acclimation of plants to cold temperatures (Goulas et al., 2006). A large number of chloroplast proteins have been shown to change in abundance in response to one or more environmental stressors. Proteins involved in photosynthesis are particularly responsive to stress (Taylor et al., 2009). Two specific examples are the photosynthetic proteins ribulose-5-phosphate-3-epimerase and PsbO1, which both show large decreases in abundance under cold stress (Goulas et al., 2006). Chloroplast proteome remodelling is a complex and multifaceted process. Part of the reason for this complexity is the fact that the chloroplast is the endosymbiotic descendant of an independent prokaryotic ancestor, and retains a small genome. The chloroplast proteome is thus shaped by two genomes, and dynamically regulated through complex, bidirectional communication with the nucleus. Multiple systems contribute to proteome remodelling, and this review will explore the mechanisms involved in the phenomenon in response to abiotic stress. The review will first discuss the contribution made to proteome remodelling by nucleus to chloroplast signalling, with a particular emphasis on the role of plastome regulation by nuclear factors. Secondly, the role of chloroplast to nucleus communication will be discussed, and an overview of the pathways likely to contribute to proteome remodelling will be provided. Lastly, the involvement of chloroplast pre-protein import regulation and of chloroplast proteases will be explored. The separation of these mechanisms into discrete sections is, of course, artificial, but it serves as a useful conceptual framework. As the topic of this review is broad, it cannot be exhaustive; instead, key examples are introduced to illustrate each section. Nucleus–chloroplast communication As discussed, chloroplasts, along with other plastids, are the well-integrated descendants of once free-living cyanobacterial ancestors, and, as such, retain genomes. However, over the course of evolution, the plastid genome—the plastome—has significantly decreased in size. Many protein-encoding genes have been transferred to the nucleus, and others have been lost entirely. Despite this, the plastome retains a number of important genes, and thus two genomes contribute to chloroplast function. The co-ordination of the two genomes is therefore of paramount importance, and necessitates bidirectional communication between the plastid and nucleus (Woodson and Chory, 2008; Jarvis and López-Juez, 2013). The nucleus houses most of the genes required for chloroplast function, and these control the development, maintenance, and remodelling of the chloroplast proteome; prominent amongst these are those necessary for photosynthesis, and those that regulate the plastome (Terry and Smith, 2013). Nucleus to chloroplast communication is known as ‘anterograde’ signalling (Fig. 1). Interestingly, communication is bidirectional; the chloroplast is able to convey information on its physiological or developmental state to the nucleus, and this is known as ‘retrograde’ signalling. Fig. 1. View largeDownload slide An overview of the various mechanisms that drive stress-induced chloroplast proteome remodelling. The nucleus encodes a large number of chloroplast-destined proteins, and these include proteins that regulate the chloroplast genome (the plastome) under stress conditions. Nucleus-encoded, chloroplast proteins are imported into the organelles via the translocon at the outer chloroplast membrane (TOC) and the translocon at the inner chloroplast membrane (TIC) complexes. Import of proteins through this TOC–TIC system is an important component of nucleus to chloroplast communication, which is known as ‘anterograde signalling’. Some chloroplast-targeted proteins can be degraded in the cytosol via the CHIP/Hsp70 E3 ubiquitin ligase system, which sends them for 26S proteasomal degradation. Under stress conditions, the TOC complex is targeted for degradation by the outer membrane-bound E3 ubiquitin ligase SP1, which suppresses the import of photosynthetic proteins, thus attenuating photosynthetic ROS production and promoting survival of the plant. Chloroplast-resident proteases, particularly filamentation temperature-sensitive H (FtsH) and the caseinolytic protease (Clp), turn over mature proteins, and this can drive stress-induced proteome remodelling. In turn, the chloroplast is able to communicate its status to the nucleus via ‘retrograde signalling’; this regulates nucleus-encoded chloroplast genes, particularly under stress conditions. A number of pathways are known to contribute to this behaviour; shown are the SAL1/PAP, MEcPP, and ROS pathways. ROS signalling is complex, and involves multiple ROS types and downstream signalling components. Fig. 1. View largeDownload slide An overview of the various mechanisms that drive stress-induced chloroplast proteome remodelling. The nucleus encodes a large number of chloroplast-destined proteins, and these include proteins that regulate the chloroplast genome (the plastome) under stress conditions. Nucleus-encoded, chloroplast proteins are imported into the organelles via the translocon at the outer chloroplast membrane (TOC) and the translocon at the inner chloroplast membrane (TIC) complexes. Import of proteins through this TOC–TIC system is an important component of nucleus to chloroplast communication, which is known as ‘anterograde signalling’. Some chloroplast-targeted proteins can be degraded in the cytosol via the CHIP/Hsp70 E3 ubiquitin ligase system, which sends them for 26S proteasomal degradation. Under stress conditions, the TOC complex is targeted for degradation by the outer membrane-bound E3 ubiquitin ligase SP1, which suppresses the import of photosynthetic proteins, thus attenuating photosynthetic ROS production and promoting survival of the plant. Chloroplast-resident proteases, particularly filamentation temperature-sensitive H (FtsH) and the caseinolytic protease (Clp), turn over mature proteins, and this can drive stress-induced proteome remodelling. In turn, the chloroplast is able to communicate its status to the nucleus via ‘retrograde signalling’; this regulates nucleus-encoded chloroplast genes, particularly under stress conditions. A number of pathways are known to contribute to this behaviour; shown are the SAL1/PAP, MEcPP, and ROS pathways. ROS signalling is complex, and involves multiple ROS types and downstream signalling components. Anterograde signalling In response to changing environmental conditions, the nucleus exerts tight regulatory control over the chloroplast proteome, and this regulation occurs at many levels. The nuclear transcriptome is remodelled in response to environmental cues by chloroplast-dependent and chloroplast-independent mechanisms; this ultimately changes the identity and abundance of chloroplast-destined pre-proteins. Additionally, the expression of the plastome is subject to stringent regulation by a battery of nuclear factors, while the mature chloroplast proteome is actively regulated by a suite of nuclear-encoded proteases. This section will explore the anterograde regulation of the chloroplast proteome in response to stress. There will not be a detailed discussion of chloroplast-independent transcriptional reprogramming of nuclear-encoded, chloroplast genes, as this is an extremely broad subject and is beyond the scope of the review. Interested readers are encouraged to consult Saibo et al. (2009). The focus of this section will instead be on (i) the regulation of chloroplast-targeted pre-proteins as they move through the cytosol to the organelle; and (ii) the nuclear regulation of the plastome. Nucleus-encoded proteases will be discussed later. The regulation of chloroplast-targeted pre-proteins within the cytosol Chloroplast-targeted pre-proteins can be marked for ubiquitin–proteasome system (UPS)-mediated degradation as they travel through the cytosol. In addition to preventing the accumulation of toxic aggregates in the cytosol, this can help to shape the chloroplast proteome, and, in some cases, it probably acts in concert with changes in chloroplast pre-protein import behaviour (discussed at length in the ‘Chloroplast pre-protein import’ section). The heat shock cognate 70-4 (Hsc70-4) has a role in this process, as does the E3 ubiquitin ligase C-terminus of Hsc70-interacting protein (CHIP) (Shen et al., 2007a; Lee et al., 2009). The transcripts of both genes are up-regulated in the albino plastid protein import 2 (ppi2) mutant, which is defective in the synthesis of Toc159, a major receptor component of the chloroplast import machinery (see the ‘Chloroplast pre-protein import’ section for a more detailed discussion). The ppi2 mutant cannot efficiently import nuclear-encoded chloroplast pre-proteins, and these consequently accumulate in the cytosol. Hsc70-4 and CHIP physically interact with each other, and function within a cytosolic complex. Hsc70-4 binds the transit peptide sequences of target proteins, and recruits CHIP, which acts as an E3 ligase for the UPS, ubiquitinating the substrate and marking it for 26S proteasomal degradation. Other chloroplast-targeted proteins have since been shown to be recognized by the UPS in the cytosol, and, again, this recognition has been shown to be dependent on the transit peptide of the substrates (Sako et al., 2014). Interestingly, the filamentation temperature-sensitive H (FtsH) protease is a target of the CHIP system, as is ClpP4, a component of the caseinolytic protease (Clp) (Shen et al., 2007a, b). This regulation of chloroplast proteases is likely to have roles in abiotic stress tolerance—under high light, CHIP overexpression suppresses the light-induced increase in ClpP4 and FtsH, suggesting that the complex regulates the levels of both proteases, perhaps under stress conditions. The regulation of plastome gene expression by nuclear proteins The plastome contains a number of essential genes. While the plastome encodes core components of its own transcription and translation machineries, the nucleus exerts control over plastome activity via a broad suite of proteins. Nuclear factors regulate plastome gene expression transcriptionally, post-transcriptionally, translationally, and post-translationally, and this form of anterograde regulation has roles in plant adaptation to abiotic stress. Transcriptional regulation of plastome gene expression Plastome genes are transcribed via two classes of RNA polymerases: the plastid-encoded polymerase (PEP), and the nuclear-encoded polymerases (NEPs) (Kanamaru et al., 1999; Lerbs-Mache, 2011). PEP activity is dependent on PEP-associated proteins (‘PAPs’), as well as bacterial-type σ70 subunits (sigma factors); these are key to the initiation and specificity of plastome transcription. Thus, while the chloroplast contains its own genome, its transcription is under the strict control of the nucleus, which exerts its control via NEPs and, indirectly, PEP. Although plastome transcription is tightly regulated, there is a surprisingly weak correlation between transcript and chloroplast protein abundance, and it has been argued that most regulation of plastid gene expression occurs post-transcriptionally (Woodson and Chory, 2008). Despite this, there are documented cases of stress- and environmentally induced plastome transcriptional changes (Pfannschmidt, 2003). An interesting example is provided by the sigma factor SIG5, which is induced by a variety of abiotic stressors: high light, high salt, osmotic stress, and low temperature (Nagashima et al., 2004). SIG5 guides PEP to the psbD promoter; psbD is an operon encoding three proteins including the D2 PSI reaction centre component and the antenna protein CP43. Thus, under certain abiotic stress conditions, plastome genes encoding components of the photosynthetic machinery are transcriptionally regulated by nuclear-encoded SIG5 and, presumably, by other factors as well. The scale of the contribution that such regulation makes to dynamic chloroplast proteome remodelling is, however, currently unclear. Post-transcriptional regulation As discussed, it is thought that post-transcriptional regulation is the dominant force that shapes plastid gene regulation, and this appears to be particularly the case in mature chloroplasts (Sun and Zerges, 2015). Nuclear-encoded proteins control the transcription, splicing, editing, and stability of plastome-encoded mRNAs (Stern et al., 2010). In some cases, this regulation has been explicitly linked to dynamic chloroplast proteome remodelling, and examples of this behaviour are discussed below. In the green alga Chlamydomonas reinhardtii, the plastome gene encoding cytochrome f, a key subunit of the cytochrome b6f complex, is post-transcriptionally regulated by nuclear-encoded proteins (Raynaud et al., 2007). Two proteins have been implicated: maturation of cytochrome b6f petA mRNA (MCA1) and translation of cytochrome b6f petA mRNA (TCA1). MCA1 belongs to the pentatricopeptide repeat (PPR) family of RNA-binding proteins, and it binds and stabilizes photosynthetic electron transfer A (petA, which encodes cytochrome f) mRNA, allowing it to accumulate. Similarly, TCA1 binds the 5′-untranslated region (UTR) of petA mRNA, promoting its translation. The two proteins thus enable the efficient expression of the petA gene. Interestingly, they appear to contribute to chloroplast proteome remodelling in response to nitrogen limitation. Under nitrogen starvation, the cytochrome b6f complex is rapidly degraded (Wei et al., 2014) (as discussed in more detail in the ‘Chloroplast proteases’ section), and MCA1 and TCA1 also decrease in abundance, preventing further biogenesis of the cytochrome b6f complex. Intriguingly, there is also evidence that anterograde post-transcriptional gene regulation has a role in the adaptation of the C. reinhardtii proteome to iron-limiting conditions (Lefebvre-Legendre et al., 2015). Under such conditions, PSI components are targeted for turnover, perhaps on account of the system's high iron content (Moseley et al., 2002). It appears that a nuclear-encoded protein called translation of psaA1 (TAA1) complements this process by regulating the stability and translation of plastome-encoded psaA mRNA, which codes for a key component of PSI (Lefebvre-Legendre et al., 2015). The stability of psaA mRNA is impaired in taa1 mutants, and TAA1 is indispensable for PsaA translation. Furthermore, the TAA1 protein strongly decreases in abundance in response to iron limitation, implying a role for the protein in the nutrient limitation-induced remodelling of the C. reinhardtii chloroplast proteome. Retrograde signalling During the development and subsequent operation of the chloroplast, the organelle communicates with the nucleus, altering the nuclear transcriptome through a number of complex pathways. This communication—known as ‘retrograde signalling’—is vital for the biogenesis, homeostasis, and stress-induced remodelling of the chloroplast proteome. Retrograde signalling can be divided into two broad categories: biogenic and operational signalling (Pogson et al., 2008; Terry and Smith, 2013). Biogenic signalling drives chloroplast biogenesis, the process through which the chloroplasts develop from their proplastid or etioplast progenitors. Operational signalling occurs after chloroplast maturation, and serves to regulate the nuclear transcriptome under changing environmental conditions. As chloroplast proteome biogenesis is not within the scope of this review, we focus here on operational signalling pathways as they relate to chloroplast proteome remodelling. Thus, the well-studied genomes uncoupled (GUN) mutants will not be discussed, as they have principally been studied in relation to chloroplast biogenesis. A number of operational signalling pathways have been proposed, with varying degrees of experimental support. The relevant pathways are discussed below. Reactive oxygen species (ROS) Oxygenic photosynthesis is a volatile process which is highly sensitive to environmental disruption. Under a range of abiotic stress conditions, the photosynthetic machinery generates ROS, which can severely threaten the health and viability of the plant. ROS do, however, have an important role in plant stress signalling, acting as retrograde signals to modify the nuclear transcriptome under unfavourable environmental conditions (Tripathy and Oelmüller, 2012). Two ROS molecules have been intensively studied in this context: 1O2 (singlet oxygen), which is typically produced at PSII, and H2O2 (hydrogen peroxide), which is generally produced by PSI. Interestingly, different ROS molecules may modify the expression of different sets of nuclear genes, as described below. Singlet oxygen As discussed, different ROS molecules induce distinct transcriptional responses, and teasing these apart has presented major challenges. A breakthrough came with the identification of the flu mutant (Meskauskiene et al., 2001). This Arabidopsis mutant lacks a functional FLU protein (a key negative regulator of chlorophyll biosynthesis), and consequently accumulates the chlorophyll biosynthesis intermediate protochlorophyllide in the dark. Protochlorophyllide is strongly photosensitizing, and dark-treated flu mutants specifically accumulate singlet oxygen when moved into the light (op den Camp et al., 2003). The flu mutant allowed several groups to tease apart the distinctive roles of singlet oxygen signalling. Other mutants, such as chlorina 1, which lacks the light harvesting complex (LHC) of PSII and accumulates singlet oxygen, have also provided major insights (Ramel et al., 2013). Singlet oxygen is produced at PSII through the excitation of ground-state triplet oxygen, and it may be the major ROS produced under high-light conditions (Triantaphylidès and Havaux, 2009; González-Pérez et al., 2011). This ROS acts to change the expression of a set of nuclear genes, termed singlet oxygen-regulated genes (SORGs). Many of the implicated genes have roles in photosynthesis and carbon metabolism, as well as in plastid mRNA processing. Although singlet oxygen signalling has mostly been studied in relation to operational signalling, a role for the ROS within the context of biogenic signalling has recently been proposed (Page et al., 2017). Singlet oxygen signalling triggers programmed cell death and growth suppression under high-light conditions (op den Camp et al., 2003), and this activity is dependent on the action of two chloroplast-localized proteins: Executor (Ex) 1 and 2 (Wagner et al., 2004; Lee et al., 2007; for a review, see Kim and Apel, 2013). However, under moderate light intensity, the two proteins drive abiotic stress acclimation, partly through chloroplast proteome remodelling (Uberegui et al., 2015; Carmody et al., 2016). The flu ex1 ex2 triple mutant demonstrates the importance of the Ex1/Ex2 proteins in this process, as it displays abrogated regulation of SORG expression following a dark to light shift (Lee et al., 2007). Interestingly, it has recently emerged that the plastid protease FtsH2 has a role in Ex1/Ex2 signalling: the protease associates with Ex1, and the var2 flu double mutant (var2 lacks functional FtsH2) shows dampened SORG expression changes in response to dark to light shifts, with no change in the case of 85% of tested SORGs (Wang et al., 2016; Dogra et al., 2017). This demonstrates an intriguing link between retrograde signalling and plastid proteolysis, although the mechanistic basis of the link is currently unclear. Surprisingly, the Ex1/Ex2 pathway may not be the only singlet oxygen signalling pathway. Under very high light conditions, oxidation of PSII-associated carotenoids occurs, and two of the oxidation products—β-cyclocitral and dihydroactinidiolide—seem to change the expression of a subset of nuclear genes (Ramel et al., 2012; Shumbe et al., 2014). β-Cyclocitral is an oxidized derivative of β-carotene, and it acts as a signalling molecule under high-light stress (Ramel et al., 2013). Exogenous β-cyclocitral treatment induces the misregulation of a large number of SORGs in Arabidopsis (Ramel et al., 2012). The downstream pathway components of β-cyclocitral signalling are still poorly understood; however, methylene blue sensitivity 1 (MBS1), a small zinc finger protein, has been implicated in both C. reinhardtii (Shao et al., 2013) and Arabidopsis (Shumbe et al., 2017). Interestingly, the genes regulated by the β-cyclocitral pathway substantially differ from those regulated by the Ex1/Ex2 pathway; one study found that the two categories overlap by just 20 genes (Dogra et al., 2017). This suggests that singlet oxygen may signal through two distinctive pathways which regulate different subsets of SORGs. There is evidence to suggest that the subgranal location of singlet oxygen production may influence the signalling mode (Wang et al., 2016). Hydrogen peroxide As in the case of singlet oxygen, untangling the specific role of hydrogen peroxide within the context of broader ROS signalling has presented technical challenges. Major insights came through the creation of an RNAi line that specifically suppresses the expression of thylakoid membrane-bound ascorbate peroxidase (tAPX), a factor that is key to chloroplast hydrogen peroxide regulation (Maruta et al., 2012). This transgenic line shows altered regulation of a large number of cold stress-related and pathogen resistance genes; these were named Responsive to tAPX Silencing (RTS) genes by the authors. Interestingly, some of the implicated genes encode chloroplast proteins. In another successful approach, a chloroplast transit peptide–glycolate oxidase (GO) fusion gene was overexpressed in Arabidopsis, and this specifically induced hydrogen peroxide accumulation in the chloroplast (Fahnenstich et al., 2008; Balazadeh et al., 2012). GO converts glycolate to glyoxylate, producing hydrogen peroxide in the process. The induction of hydrogen peroxide production in the chloroplast through this method was again shown to change the regulation of a large number of nuclear genes. Hydrogen peroxide is comparatively stable, and may, in some circumstances, act directly as a mobile retrograde signalling molecule. Interestingly, it was recently suggested that a subpopulation of chloroplasts located next to the nucleus transfers hydrogen peroxide directly, by-passing the cytosol (Exposito-Rodriguez et al., 2017). A considerable number of studies have attempted to identify components of the hydrogen peroxide signal transduction network(s), and a number of putative pathway components have been identified. These include a diverse variety of transcription factors, mitogen-activated protein kinases (MAPKs), and miRNAs (reviewed by Petrov and Van Breusegem, 2012). MEcPP Another relevant operational signalling pathway was discovered in a screen for mutant plants with enhanced expression of hydroxyperoxide lyase (HPL)—a nuclear-encoded, stress-inducible gene that encodes a chloroplast enzyme involved in the synthesis of defence-related molecules such as the hormone jasmonic acid (JA). In the screen, the constitutively expressing HPL (ceh1) mutant was discovered (Xiao et al., 2012). This mutant hyperaccumulates methylerythritol cyclodiphosphate (MEcPP), an intermediate of the methylerythritol phosphate (MEP) pathway for plastid isoprenoid biosynthesis, due to a lesion in an enzyme of the pathway. MEcPP accumulates in response to high-light or wounding stress, and strongly and specifically up-regulates the expression of HPL. Interestingly, MEcPP accumulation also triggers salicylic acid (SA) biosynthesis, although genetic experiments have demonstrated that the mutant’s HPL up-regulation is not dependent on this change in hormone abundance—when the SA-deficient eds16-1 mutant is crossed into the ceh1 background, the plants still show strong HPL up-regulation (Xiao et al., 2012). MEcPP is involved in activation of stress responses (Benn et al., 2016), and regulates the expression of a broad range of nuclear genes, partially through hormone signalling. Recent studies have linked MEcPP to the regulation of genes key to the endoplasmic reticulum unfolded protein response (UPR) (Walley et al., 2015), alongside a variety of JA-regulated genes (Lemos et al., 2016). Interestingly, another recent study found that many nuclear genes are misregulated in ceh and ceh eds16-1 (Bjornson et al., 2017): under gene set enrichment analysis, a number of patterns emerged, and it is noteworthy that gene sets involved in photosynthesis and carbon fixation were misregulated. SAL1–PAP In recent years, the phosphonucleotide 3′-phosphoadenosine 5′-phosphate (PAP) has emerged as a key player in a novel, operational, retrograde signalling pathway (Estavillo et al., 2011). PAP is produced as a by-product of sulphur metabolism, and, under favourable environmental conditions, it is degraded by the chloroplast phosphatase SAL1 (Quintero et al., 1996; Estavillo et al., 2011). SAL1 is inactivated in response to oxidative stress, which leads to PAP accumulation in the chloroplast, and from there it moves through the cytosol into the nucleus. Within the nucleus, PAP regulates a number of genes: mutants of SAL1 show misregulation of ~1800 genes, many of which are involved in the stress response, osmoprotection, and starch metabolism (Wilson et al., 2009). The SAL1–PAP pathway is also involved in physiological responses to abiotic stress; recent work has identified a link between the pathway and stomatal closure, a key component of the plant drought response (Pornsiriwong et al., 2017). Interestingly, it has been proposed that SAL1 is a direct sensor of ROS/chloroplast redox poise (Chan et al., 2016). SAL1 activity is suppressed under oxidative stress conditions, and this allows PAP to accumulate. It is argued that plant SAL1 is unusually susceptible to redox inactivation, and that this physical property explains its putative role as an oxidative stress receptor. Chloroplast pre-protein import As discussed, most proteins required for chloroplast function are encoded by genes that reside in the central, nuclear genome. The products of these genes must therefore be imported across the chloroplast double membrane, and this transport occurs post-translationally via a mechanism dependent on the co-ordinate activity of two large, multiprotein complexes. These complexes—the translocon at the outer chloroplast membrane, or TOC, and the translocon at the inner chloroplast membrane, or TIC—reside in the outer and inner envelope membranes, respectively, and co-operatively import cytosolically translated pre-proteins into the organelle (for a review, see Jarvis, 2008). The TOC complex is exposed to the cytosol, and comprises three major components: Toc33 and Toc159, which are both GTPases involved in pre-protein (substrate) recognition; and Toc75, which forms a channel through which the entry of pre-proteins proceeds. The Toc33 and Toc159 receptors are encoded by small gene families, leading to the formation of different receptor isoforms with varying substrate preferences. Interestingly, there is evidence that chloroplast pre-protein import is modified in response to abiotic stress (Fig. 1). For example, under heat stress conditions, nuclear-encoded chloroplast proteins accumulate in the cytosol (Heckathorn et al., 1998). This behaviour is rapidly reversible, which implies that the short-term suppression of chloroplast pre-protein import could have a role in plant abiotic stress tolerance. In keeping with this hypothesis, it has also been shown that the import of pre-Rubisco small subunit (pRSS) into isolated heat- or chill-stressed pea chloroplasts is retarded (Dutta et al., 2009), and that pRSS has reduced ability to bind the outer membranes of isolated chloroplasts under heat shock conditions. Interestingly, the same study found that the Toc159 protein decreases in abundance in response to heat treatment. Although it appears that chloroplast import is a stress-responsive and surprisingly dynamic process, until recently little was known about its regulation. Within the last 5 years, a considerable amount of progress has been made. It was recently shown that TOC complex components, including Toc33, Toc75, and Toc159, are targeted by suppressor of ppi1 locus 1 (SP1), an E3 ubiquitin ligase that localizes to the chloroplast outer membrane (Ling et al., 2012). In response to developmental or environmental cues, SP1 promotes the degradation of TOC complexes, thus suppressing plastid pre-protein import. The SP1 protein has important physiological roles: sp1 Arabidopsis mutants show delayed plastid interconversions, with plants displaying defects in de-etiolation and leaf senescence. This implies that SP1 has a role in plastid proteome remodelling within developmental contexts, which it may achieve by altering the ratios of different TOC component isoforms with different pre-protein preferences (Ling et al., 2012). Interestingly, SP1 also promotes plant survival under various abiotic stress conditions: While sp1 mutants are more sensitive to oxidative, osmotic, and salt stresses, SP1 overexpression enhances resistance to these conditions (Ling and Jarvis, 2015). In addition, sp1 mutants and SP1 overexpressor lines show increased and decreased hydrogen peroxide accumulation, respectively, when osmotically stressed. Furthermore, these phenotypes have been linked to changes in chloroplast pre-protein import efficiency under stress conditions: in sp1 mutants, the import of key photosystem proteins was enhanced, whereas, in the overexpressor lines, the opposite behaviour was observed. Taken together, a picture emerges in which stress induces the rapid, UPS-mediated breakdown of TOC complexes, and this acts to suppress the import of pre-proteins key to photosynthesis. This import suppression probably attenuates photosynthesis, and dampens the accumulation of harmful ROS. From the work described here, it is clear that the regulation of pre-protein import has key roles within the stress-induced remodelling of the chloroplast proteome. Further study is needed to determine how SP1 is regulated, and to probe the conditions under which its activity becomes important. Chloroplast proteases Chloroplasts contain a considerable number of proteases; ~20 have so far been described, and these are composed of >50 components (Nishimura et al., 2016b). Plastid-resident proteases can be divided into three major categories: serine, metallo, and aspartyl proteases. A number of proteases housed within the plastid are of bacterial origin, whereas others are specific to plants and algae. Some of the most intensively studied, and, in this case relevant, plastid proteases are the Clp and FtsH proteases. The Clp proteases are serine proteases that are abundant within the chloroplasts of green algae and higher plants (Olinares et al., 2011). FtsH proteases contain ATPase associated with various cellular activities (AAA) and Zn2+ metalloprotease domains, and are found in both mitochondria and chloroplasts (Wagner et al., 2012). Proteases are important for a large number of plastid processes. For example, some are involved in the processing of newly imported pre-proteins: the metalloprotease stromal processing peptidase (SPP) removes transit peptides from pre-proteins at an early stage of translocation, upon emergence into the stroma, and the transit peptides are then degraded by the action of a number of other plastid-resident proteases such as pre-sequence peptidases 1 and 2 (Prep1/2) and organellar oligopeptidase (OOP) (for a detailed review, see Nishimura et al., 2016a). In addition, a number of other plastid proteases have roles in plastid proteome homeostasis and the removal of damaged proteins, particularly within the context of the photosynthetic machinery. These processes are not covered here, however, as the focus is on the contribution plastid proteases make to abiotic stress-induced remodelling of the plastid proteome (Fig. 1). Almost all of the literature relevant here involves the model green alga C. reinhardtii. A number of studies have shown that the thylakoid proteome of the alga is extensively remodelled in response to nutrient stress. Under nitrogen deprivation, Rubisco, as well as all of the components of the cytochrome b6f complex and its biogenesis factors, are selectively degraded (Wei et al., 2014). The degradation of the cytochrome b6f complex and related factors is dependent on the action of the thylakoid-bound FtsH protease, and, to a lesser extent, Clp, as well as an unknown, thylakoid lumen-localized protease. Interestingly, the degradation of the photosynthetic machinery is accompanied by an increase in the abundance of l-amino acid oxidase 1 (LAO1), a nitrogen-scavenging enzyme, as well as increases in the abundance of various proteins involved in metabolic reprogramming, implying that the proteases act within a broad functional transition. In addition, sulphur deprivation has also been shown to trigger the FtsH-mediated degradation of the cytochrome b6f complex, as well as of PSII (Malnoë et al., 2014). Taken together, these studies clearly implicate plastid proteases in the stress-induced remodelling of the chloroplast proteome, at least in the case of C. reinhardtii. Concluding remarks The chloroplast proteome is dynamically modified in response to abiotic stress. Here, we have provided an overview of the interconnected systems that drive this phenomenon. From the work discussed, it is clear that the process is complex and multifaceted. The nucleus houses most chloroplast genes, including those that regulate the behaviour of the plastome, chloroplast pre-protein import, and chloroplast proteolysis; and it exerts tight regulatory control over the chloroplast proteome under abiotic stress conditions. The chloroplast can, in turn, regulate the nuclear genome to influence its own proteome indirectly. 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Journal

Journal of Experimental BotanyOxford University Press

Published: Mar 14, 2018

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