JXB Advance Access originally published online on January 31, 2006
Journal of Experimental Botany 2006 57(9):1871-1881; doi:10.1093/jxb/erj008
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FOCUS PAPER |
Arabidopsis variegation mutants: new insights into chloroplast biogenesis
Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa 50011, USA
To whom correspondence should be addressed. Fax: +1 515 294 1337. E-mail: rodermel{at}iastate.edu
Received 8 July 2005; Accepted 23 September 2005
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Abstract |
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 Top Abstract Introductionimmutansvar2References |
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Plant variegations are characterized by the presence of white sectors in normally green tissues and organs. Whereas the white sectors contain defective plastids that lack coloured pigments, the green sectors contain morphologically normal chloroplasts. Variegation mutants are defective in chloroplast developmental processes and arise due to mutations in nuclear or organellar genes. Despite their widespread occurrence in nature, only a few variegations have been studied at the molecular level. In this review, recent progress toward understanding two Arabidopsis variegations, immutans (im) and var2 is summarized. Both im and var2 are caused by nuclear recessive mutations and the responsible genes have been cloned and characterized. IMMUTANS functions as a chloroplast terminal oxidase that transfers electrons from the plastoquinol pool to oxygen. It appears to be a versatile electron sink, especially early in chloroplast development, when its function is crucial for carotenoid biosynthesis, and in excess light, when it serves as a ‘safety valve’. IM also probably functions in chlororespiration. VAR2 encodes a chloroplast FtsH metalloprotease (termed AtFtsH2). Along with other AtFtsH proteins (AtFtsH1, 5 and 8), it forms complexes in the thylakoid membrane that are probably involved in the process of PSII repair during photoinhibition. A model has been proposed to explain the mechanism of var2 variegation, which suggests that threshold levels of FtsH complexes are required for green sector formation. It is concluded that studies on im and var2 have provided novel insights into nuclear–chloroplast interactions and, especially, into mechanisms of photoprotection.
Key words: Immutans, metalloprotease, photo-oxidation, photoprotection, terminal oxidase, thylakoid, var2
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Introduction |
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TopAbstractIntroductionimmutansvar2References |
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The endosymbiotic theory of evolution postulates that chloroplasts originated from endosymbiotic cyanobacteria and that, during the process of symbiogenesis, many symbiont genes were lost or transferred to the nucleus. Consistent with this theory, plastid genomes encode
80–100 proteins, while 2500–3500 nucleus-encoded proteins are predicted to be targeted to the chloroplast (Goldschmidt-Clermont, 1998; Abdallah et al., 2000; Peltier et al., 2002). Both anterograde (nucleus-to-chloroplast) and retrograde (plastid-to-nucleus) signals have evolved to facilitate the co-ordinate expression of nuclear and chloroplast genes. Anterograde mechanisms are elicited in response to exogenous or endogenous signals and regulate the expression of nuclear genes for plastid proteins. These include structural proteins in the plastid, as well as regulatory proteins that are necessary for the expression of plastid genes. The translational regulation of rbcL expression (for the Rubisco large subunit, LS) in the plastid by the abundance of nuclear-encoded (rbcS) small subunits (SS) is a classic example of how anterograde traffic co-ordinates the accumulation of gene products from nuclear and chloroplast genomes (Rodermel, 1999). Conversely, early observations that the transcription of nuclear genes for chloroplast proteins, such as Lhcb and rbcS, is down-regulated in tissues treated with the carotenoid biosynthesis inhibitor, norflurazon (a bleaching herbicide), and in mutants with defective chloroplasts gave rise to the notion of retrograde signalling. Retrograde signals are produced in response to the developmental and/or metabolic state of the plastid and regulate the expression of nuclear genes (reviewed in Rodermel, 2001b; Surpin et al., 2002). Thus, the development of a fully-functional chloroplast is dependent on nuclear-organelle interactions. Variegation mutants have played a prominent role in the history of genetics and led to the discovery of non-Mendelian inheritance (Tilney-Bassett, 1975). Variegated plants have green and white (or yellow) sectors in normally green organs of the plant. Whereas the green sectors contain normal-appearing chloroplasts, the white sectors have abnormal plastids that lack well-developed lamellar structures and are deficient in chlorophyll and carotenoid pigments (reviewed in Rodermel, 2001a). Sectoring in these mutants arises as a consequence of mutations in nuclear or plastid genes that disrupt a process that is required for normal chloroplast development; the lack of pigments is often a secondary consequence of the primary lesion. Unlike albino plants, variegation mutants are non-lethal and offer excellent opportunities to study nuclear-organelle interactions. However, only a few variegations have been studied at the molecular level.
This laboratory has long been interested in variegation mutants of Arabidopsis as a means of understanding nuclear-organelle interactions. Relatively few variegations have been reported in Arabidopsis, despite the fact that a large number of colour mutants (usually uniformly pale-green, yellow, or all-white) are found in many types of mutagenesis screens. Initial studies focused on immutans, then branched out to a mutant that was thought might be an allele of immutans. This turned out to be an incorrect assumption and, rather, led to the identification of another variegation mutant, var2. This mutant has been studied further. In this manuscript, an overview is provided of what is known about immutans and var2. Both mutants have yielded novel insights into the mechanisms of nuclear–chloroplast interactions and chloroplast biogenesis.
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immutans |
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TopAbstractIntroductionimmutansvar2References |
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The immutans (im) variegation mutant is one of the oldest Arabidopsis mutants. It was first described and partially characterized nearly 50 years ago by Rédei in the US and Röbbelen in Germany (reviewed in Rodermel, 2001a; Aluru and Rodermel, 2004). They isolated independent alleles of the gene. Green- and white-sectoring in im is caused by a nuclear recessive gene and the gene has a normal Mendelian mode of transmission (Fig. 1). Light intensity and quality modulate white sector formation, with increased light enhancing white sector formation. Progeny of im seeds display the same parental variegation pattern regardless of whether they are derived from branches of green or white sectors. This indicates that both types of sectors have a uniform genetic constitution and that variegation is not due to the action of a transposable element. It also suggests that a bi-directional switch occurs between the green and white phenotype whereby the plastid composition can be reversed, i.e. green plastids can be converted to white plastids and vice versa. Because of this phenotypic reversibility, and because of the mutant's inability to convert permanently from an all-green to an albino phenotype, Rédei called the mutant immutans (for ‘immutable’) (Rédei, 1975).
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immutans is impaired in carotenoid biosynthesis
Whereas im green sectors contain a normal complement of chlorophylls and carotenoids, the white tissues accumulate phytoene, a non-coloured C40 carotenoid intermediate (Wetzel et al., 1994). This finding suggested that im is blocked at the phytoene desaturase (PDS) step of carotenoid biosynthesis. Coloured carotenoids (which are produced downstream of the PDS step) dissipate excess light energy absorbed by the light-harvesting complexes as heat, and a lack of coloured carotenoids results in photo-oxidation of the contents of the plastid under high light conditions (Demmig-Adams and Adams, 1996). Therefore, the accumulation of phytoene in im white tissues suggested that the mutant is incapable of producing enough carotenoids to avoid photo-oxidation; i.e. im is a classical carotenoid mutant. Consistent with this idea, green sector formation in im is enhanced by low-light conditions and high light intensity favours white sector formation.
Map-based cloning and sequencing of IM revealed that the gene product bears similarity to mitochondrial alternative oxidase (AOX) (Carol et al., 1999; Wu et al., 1999). Alternative oxidases are inner membrane proteins that function as terminal oxidases in the alternative pathway of mitochondrial respiration by transferring electrons from ubiquinol to water using molecular oxygen as a terminal acceptor (Vanlerberghe and McIntosh, 1997). Prior to the cloning of IM, it was known that PDS activity requires several redox components, including plastoquinone and molecular oxygen (reviewed by Carol and Kuntz, 2001). Therefore the sequence similarity of IM to AOX suggested that IM is a component of the phytoene desaturation pathway and functions as a terminal oxidase by transferring electrons from the plastoquinol pool to molecular oxygen (Fig. 2). In accordance with this notion, recombinant IM protein has plastoquinol oxidase activity in vitro (Josse et al., 2000), and IM has been implicated in the transfer of electrons from PSII to molecular oxygen via the plastoquinol pool in a PSI-deficient Chlamydomonas mutant (Cournac et al., 2000). The in vitro and in vivo data thus support the idea that IM functions as a terminal oxidase, and further suggest that its role in plastid metabolism is more widespread than carotenoid biosynthesis. This will be more fully discussed later.
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The active site iron binding sites are essential for IM activity
The resemblance of the AOX and IM sequences raises questions about the similarity of the two proteins regarding structure/function relationships. According to a recent structural model of AOX by Andersson and Nordlund (1999), the hydrophobic regions of AOX are not membrane-spanning (as originally thought), but interfacial as in other di-iron carboxylate (RNR R2) proteins. A similar model for IM has been proposed (Fig. 3) (Berthold et al., 2000; Rodermel, 2001a). According to this model, E136, E175, H178, E227, E296, and H299 serve as Fe-binding sites in the reaction centre of IM. To test this hypothesis, site-directed mutagenesis experiments were conducted in which the four E residues were mutated to A, D, or H and the two H residues were mutated to A, E, or N (A Fu and S Rodermel, unpublished data). In addition, H177, E224, and H298 were mutated to A as internal controls. The recombinant proteins were expressed in E. coli and IM activity was assayed according to Josse et al. (2000, 2003). In this assay, O2 consumption is measured in membranes isolated from E. coli that have been transformed with various mutant IM sequences. The addition to the membranes of NADH as an electron donor results in the formation of reduced quinone (by membrane-bound NADH dehydrogenase). Electrons are then transferred to molecular oxygen via IM or the cytochrome pathway. IM activity is inhibited by pyrogallol analogues, such as propyl gallate and octyl gallate, but is insensitive to cyanide. Thus, O2 consumption occurs by the cytochrome pathway in the absence of KCN, but by IM activity in the presence of KCN. KCN and n-propyl gallate (n-PG) together abolish O2 consumption. IM becomes engaged in this system only when the cytochrome pathway is blocked.
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It was found that amino acid changes in any of the six putative Fe-binding sites cause IM to lose its cyanide-resistant, propyl gallate-sensitive oxygen consumption activity (A Fu and S Rodermel, unpublished data). On the other hand, proteins with mutations in H177, E224, and H298, which are adjacent to the putative Fe-ligands, exhibit wild-type activity. These results confirm the importance of the six iron-binding sites for IM activity. These results were confirmed by in planta experiments in which the mutant IM sequences were transformed into a null allele of immutans: the six Fe ligand mutations were not able to revert the variegation phenotype to normal, whereas normal plants were obtained when im was transformed with the H177, E224, and H298 mutations.
In contrast to AOX, IM has an additional 48 bp sequence (16 amino acids) located near its C-terminus. This sequence comprises all of exon 8 and is found in nearly all IM-like homologues. After expression in E. coli, the mutant protein (i.e. lacking exon 8) accumulates normally, but shows no in vitro activity (A Fu and S Rodermel, unpublished data). However, the mutant protein behaves differently in planta. Fortuitously, an allele of im that lacks exon-8 was discovered. This mutant produces normal levels of mRNA, but no IM protein accumulates. This appears to be due to turnover, inasmuch as in vitro-expressed IM that lacks the exon 8 sequence is less stable than wild-type IM following import into isolated chloroplasts. Taken together, these data suggest that exon 8 sequences are important for IM function, folding, and/or stability.
IM plays a global role in plastid metabolism
Previous data from light shift experiments indicated that IM is expressed early in cotyledon development (Röbbelen, 1968; Wetzel et al., 1994). During this ‘light-responsive’ phase, the phenotype of the cotyledons is irreversibly determined depending on the light environment. However, IM expression is not restricted to green tissues, as demonstrated by promoter/GUS fusion, northern, RT-PCR, and western blotting experiments. IM appears to be expressed ubiquitously in Arabidopsis tissues and organs throughout development. Consistent with these findings, the development of multiple plastid-types, including chloroplasts, amyloplasts (in roots), and etioplasts (in dark-grown seedlings) is impaired in im (Aluru et al., 2001). This raises the question of whether IM plays a global role in plastid metabolism, e.g. whether it is active in non-green tissues that do not accumulate appreciable levels of carotenoids.
Carotenogenesis occurs in all plant tissues, and thus this is one possible function of IM in non-green tissues. As a notable example, carotenoids are the precursors of ABA, which is found in all tissues. Another function of IM that might not be restricted to leaves is chlororespiration (oxidation of PQ in the dark), where IM is probably the long sought-after terminal oxidase of this process. In chlororespiration, NADPH is oxidized by plastid membrane-bound NADPH dehydrogenase, generating plastoquinol; the plastoquinol is then oxidized by IM to generate water. Evidence for this role of IM has come from experiments in PSI-deficient Chlamydomonas and in tobacco that overexpress Arabidopsis IM (Cournac et al., 2000; Joet et al., 2002). In addition, IM, like the Ndh complex, is localized in the stromal lamellae where cyclic electron transfer reactions around PSI occur (Joet et al., 2002; Lennon et al., 2003). This has led to the suggestion that IM plays a role in regulating the cyclic flow of electrons around PSI.
Another function of IM is as a ‘safety valve’ in photo-oxidative stress. IM protein levels increase under low light in double antisense tobacco plants lacking two hydrogen-peroxide detoxifying enzymes, catalase and ascorbate peroxidase (Rizhsky et al., 2002). IM is also induced under high light conditions in wild-type Arabidopsis and tobacco. These studies suggest that IM is involved in detoxifying excess electrons (i.e. is a ‘safety valve’). Experiments with ndh mutants have revealed that the Ndh complex plays a role in protection against photo-oxidative stress (Martin et al., 1996). Considered together, the data suggest a working hypothesis in which chlororespiratory processes are involved in photoprotection through the oxidation of stromal reductants.
Altered rates of photosynthesis in im green leaf sectors
Light microscopy has revealed that, except for leaves, the morphology of im organs and tissues is not altered (Aluru et al., 2001). As shown in Fig. 4, the green sectors of im are thicker than normal due to an increase in epidermal and mesophyll cell sizes, and an increase in air space volume. The white sectors have a normal thickness but the palisade cells do not expand normally. Alterations in leaf anatomy are not common in chloroplast development mutants, and such perturbations have been interpreted as due to an impairment in plastid-to-nucleus signalling pathways that impact cell and leaf differentiation. This has been observed, for example, in Arabidopsis cla1, cue1, and pac; in dcl of tomato; and in dag of Antirrhinum majus (Aluru et al., 2001; Rodermel, 2001a).
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In addition to anatomical changes, other physiological and biochemical changes have been observed in im leaves (Aluru et al., 2001). The green leaf sectors have higher than normal chlorophyll a/b ratios and increased light-dependent oxygen evolution rates under CO2-saturating conditions, when compared with the wild type. These changes/adaptations occur even in plants grown under normal light conditions. One hypothesis is that the increases in photosynthesis are a means of compensating for a lack of photosynthesis in the white sectors. For example, the green/white sectoring pattern is established early in leaf development, and it is therefore possible that im plants have a dramatic increase in sink demand since there is decreased total source area.
To test this hypothesis, various aspects of photosynthesis and photosynthate partitioning in the spotty allele of immutans were measured. Figure 5 shows that im green leaf sectors have elevated rates of photosynthesis as monitored by 14CO2 uptake. These results confirm the earlier oxygen evolution data suggesting that the green sectors have elevated rates of photosynthesis (Aluru et al., 2001). The green sectors also have enhanced levels of starch and sucrose (on a chlorophyll basis) when compared with the wild-type, and they partition more newly-fixed carbon into soluble carbohydrate. Global transcription profiling and protein analyses revealed that expression of photosynthetic and carbon assimilation genes is unaltered in the green sectors, which suggests that the enhanced photosynthesis in theses sectors is not due to an up-regulation of gene expression (M Aluru and S Rodermel, unpublished data). Rather, the increases are probably due to enhanced activities and activation states of key regulatory enzymes, such as Rubisco and sucrose phosphate synthase (Fig. 6).
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In contrast to the green sectors, the white sectors of im accumulate low levels of sucrose, indicating there is a sucrose gradient between the green and white sectors (sink demand). Import of sucrose is vital to maintain metabolism in sink tissues. Plant invertases hydrolyse sucrose into glucose and fructose and are considered to contribute to sink strength by maintaining a gradient of sucrose from source to sink tissues (for reviews see Sturm, 1999; Tymowska-Lalanne and Kreis, 1998). In particular, cell wall invertases influence resource allocation between source–sink tissues and thereby control plant growth and development (Heyer et al., 2004). The white sectors of im have higher cell wall invertase activities than the green sectors (Fig. 7), which is consistent with the movement of sucrose from source to sink (M Aluru and S Rodermel, unpublished data). Although flux has not been measured directly from the im green to the im white tissues, these data are consistent with the idea that sink demand plays a role in enhancing sucrose production and partitioning in the green tissues. However, further experiments need to be performed to understand and clarify the exact mechanism(s).
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Mechanism of im variegation
Arabidopsis im is a classic example of a variegation in which the cells of the plant have a uniform genetic constitution (i.e. mutant) but the mutant phenotype is expressed only in the white sectors: despite a lack of IM, im is variegated, not albino. How do the green sectors form?
During the first critical days of seedling germination and photomorphogenesis, proplastids in the leaf meristem are converted into chloroplasts. This involves an elaboration of the thylakoid membrane, the synthesis and assembly of the photosynthetic apparatus, and an increase in chlorophyll and carotenoid biosynthesis (Mullet, 1988). Carotenoids act as photoprotective agents by quenching excess light energy absorbed by the light-harvesting complexes (Demmig-Adams and Adams, 1996). This study's working hypothesis is that IM plays a major role as an electron sink during the early events of thylakoid membrane formation when the membrane might become transiently over-reduced due to uneven production of the components of the electron transport chain. For instance, IM would be present to dissipate excess electrons in case PSII, but not PSI, were functional. Under high light conditions, a lack of IM would thus be expected to generate over-reduced membranes and toxic oxygen radicals. Without carotenoids, the developing plastid would become photo-oxidized.
How can one explain the generation of a normal-appearing chloroplast in im? One possibility is that that there is a compensating IM function, for instance, a redox component downstream from the plastoquinone pool, such as the cyt b6/f complex, PSI, or even other terminal oxidases (Peltier and Cournac, 2002). Consistent with this idea is the high likelihood that there are intrinsic differences in the rates of the various reactions involved in light capture versus use. As Niyogi (1999) points out, photoprotection is a ‘balancing act’ comprised of many different activities. It is therefore speculated that im plastids can tolerate high light below a certain threshold of these activities, but that above this threshold, photodamage occurs. The crucial element of this model is that the threshold varies from plastid-to-plastid. For instance, some plastids have more or less of the compensating activity, or of some other photoprotective activity, with the net result being a heterogeneity of light damage from plastid-to-plastid.
Regardless of precisely how chloroplasts form, it is assumed that variegation per se is a consequence of the sorting-out of white versus green plastids early in leaf development (Tilney-Bassett, 1978). At this stage of development, chloroplast divisions occur concomitantly with cell division and cell elongation (Mullet, 1988). Although the responsible mechanisms are obscure, it is thought that this process also involves the parcelling of different plastid types into different cells. This might be a random or directed process. Nevertheless, the net consequence of this sorting-out process is the formation of sectors, which represent clones of cells containing either all-green or all-white plastids. Once the process of plastid division has ceased (well before the attainment of full leaf expansion), plastid- and cell-type are fixed and cannot be reversed. Only nascent daughter plastids have the potential of changing their state, depending on the existing conditions that either promote or inhibit photodamage.
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var2 |
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TopAbstractIntroductionimmutansvar2References |
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The Arabidopsis yellow variegated mutant was isolated by Rédei in the 1950s (GP Rédei, personal communication). It was initially thought this mutant might be an allele of im, but it was found, instead, that it is allelic to another Arabidopsis variegation mutant, var2 (Martínez-Zapater, 1993).
Like im, var2 is caused by a nuclear recessive gene mutation and has green- and white-sectored leaves (Chen et al., 1999; Takechi et al., 2000). Also like im, cells in the green sectors of var2 have normal-appearing chloroplasts, while plastids in the cells of the white sectors are vacuolated, lack organized membrane structures, and contain a large number of plastoglobuli-like bodies. Yet, some cells in the white sectors of these mutants are heteroplastidic and contain plastids in various developmental stages, including plastids resembling normally developed chloroplasts (Chen et al., 1999). Such ‘plastid autonomous’ behaviour is also found in im (Wetzel et al., 1994). In contrast to im, the cotyledons of var2 are not variegated.
Map-based procedures were used to clone VAR2 and it was found that it encodes a homologue of FtsH, an ATP dependent metalloprotease (Chen et al., 2000). A T-DNA tagged allele of VAR2 has also been reported (Takechi et al., 2000). VAR2 (also designated AtFtsH2) is predicted to contain two transmembrane-spanning domains in its N-terminus and a large C-terminus that contains functional domains for ATP- and metal- binding (described below). Consistent with a membrane localization, chloroplast import assays have demonstrated that VAR2 is targeted to thylakoid membranes with its large C-terminus facing the stroma (Chen et al., 2000).
FtsH gene family
The ftsH (filamentation temperature sensitive) gene was first identified in E. coli (Ogura et al., 1997). It encodes a plasma membrane-bound metalloprotease. FtsH is a member of the large and diverse AAA (ATPase associated with diverse cellular activities) protein family. All members of this family contain either one or two conserved 200–250 amino acid ATP-binding domains (termed the ‘AAA cassette’) that contain several well-conserved regions including Walker A, Walker B, and ‘second region of homology’ (SRH) (Beyer, 1997). FtsH proteins contain a single AAA cassette in their C-terminus. Also in the C-terminus is a conserved zinc-binding domain (HEXXH). It is thought that these functional regions impart chaperone and protease activity to the enzyme. All FtsH proteins appear to be localized to membranes.
FtsH was first identified in higher plants when an E. coli FtsH antibody was used to detect a western blot signal corresponding to a protein of the expected size of E. coli FtsH in spinach chloroplasts. An FtsH-like cDNA was subsequently isolated in Arabidopsis (designated AtFtsH1) (Lindahl et al., 1996). FtsH genes have been found to be present as a multigene family in all prokaryotic and eukaryotic photosynthetic organisms examined. For example, for those species whose genomes have been sequenced, the cyanobacterium Synechocystis sp. PCC 6803 contains four FtsH genes (Kaneko et al., 1996); at least nine FtsH genes are present in the rice genome (Yu et al., 2005); and in Arabidopsis thaliana there are 12 FtsH genes (Sokolenko et al., 2002; Sakamoto et al., 2003; Yu et al., 2004). Of the 12 Arabidopsis genes, eight comprise four highly-conserved gene pairs (AtFtsH1/5, AtFtsH2/8, AtFtsH3/10, AtFtsH7/9) (Sakamoto et al., 2003; Yu et al., 2004), while AtFtsH4 and AtFtsH11 are also related to a much lesser degree. Nine of the 12 FtsH gene products are located in chloroplasts (AtFtsH1/2/6/7/8/9/11/12) and three are targeted to mitochondria (AtFtsH3/4/10) (Chen et al., 2000; Sakamoto et al., 2002, 2003; Yu et al., 2004).
Phylogenetic comparisons of the structures and protein sequences of rice and Arabidopsis FtsH genes revealed that a ‘core’ complement of FtsH genes existed before the monocot/dicot divergence (Yu et al., 2005; Fig. 8). The subsequent evolution of these genes was characterized by extensive gene duplication, especially in Arabidopsis (Vision et al., 2000).
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FtsH function in plants
In E. coli, FtsH has both chaperone and protease activities and is involved in the degradation of a variety of protein substrates (Suzuki et al., 1997). In higher plants, FtsH appears to be involved in the degradation of unassembled cytochrome b6f Rieske FeS proteins in thylakoid membranes (Ostersetzer and Adam, 1997), as well as in the N-gene-mediated hypersensitive reaction against tobacco mosaic virus infection in tobacco (Seo et al., 2000). FtsH proteins might also participate in membrane fusion and/or translocation events since the pepper Pftf (Plastid fusion and/or translocation factor) protein shares high similarity with FtsH (Hugueney et al., 1995). However, the best characterized function of FtsH is its involvement in photosystem II (PSII) photodamage and repair (Nixon et al., 2005). It is well-documented that the D1 reaction centre protein of PSII is the target of reactive oxygen species formed during photosynthesis, and that photodamaged D1 is turned-over and replaced by a newly-synthesized copy. Evidence that FtsH is involved in the D1 turnover process was first published by Lindahl et al. (2000), and it is now thought that turnover is a two-step process in plants: (i) the photodamaged D1 protein (
32 kDa) is cleaved by DegP2, a serine protease, into a 23 kDa fragment and an 9 kDa fragment (Haußühl et al., 2001); and (ii) the 23 kDa fragment is degraded by AtFtsH1. However, it seems that the absence of DegP2 activity in Synechocystis does not lead to the inhibition of D1 turnover, suggesting that FtsH itself might be sufficient for the turnover process in this species (Nixon et al., 2005). In addition to AtFtsH1, AtFtsH2 and AtFtsH5 might be involved in D1 turnover inasmuch as var2 and var1, an Arabidopsis variegation that is due to a mutation in the nuclear gene for another chloroplast FtsH homologue, AtFtsH5 or VAR1 (Sakamoto et al., 2002), are more prone to PSII photoinhibition, and the D1 degradation process is impaired in var2 (Bailey et al., 2002). Interestingly, an insertional mutant of slr0228, one of the four FtsH genes in Synechocystis, exhibits impaired D1 turnover, suggesting that involvement of FtsH in D1 turnover is conserved in both prokaryotic and eukaryotic photosynthetic organisms (Silva et al., 2003). However, the same mutant also shows a significant reduction in the amount of photosystem I, something not seen in the higher plant AtFtsH mutants (Mann et al., 2000). This reduction might be a secondary effect of impaired D1 turnover, or it might suggest that, at least in Synechocystis, FtsH has a more general role in the maintenance of photosynthetic membranes.
FtsH oligomeric complex formation in chloroplast thylakoid membrane
In E. coli, FtsH forms homocomplexes (Akiyama et al., 1995) and heterocomplexes with HflK/C (Kihara et al., 1996). In plants, higher molecular weight complexes containing chloroplast FtsH have also been observed when thylakoid membranes are fractionated by gel filtration, or by sucrose gradient centrifugation and two-dimensional green gels (Sakamoto et al., 2003; Yu et al., 2004, 2005). Sakamoto et al. (2003) have also shown by co-IP that AtFtsH2 and 5 might directly interact with each other and that AtFtsH2 might form homocomplexes. However, the results of these studies are difficult to interpret because polyclonal antibodies were used that detect multiple isoforms.
The stability of the AtFtsH2/8 pair and the AtFtsH5/1 pair are mutually-dependent because the levels of AtFtsH2 and AtFtsH5 are co-ordinately reduced in amount in var1 and var2 mutants (Yu et al., 2004). Using two-dimensional green gel analysis, it was possible to detect two AtFtsH-containing bands that migrate close to one another on second dimension gels (SDS-PAGE) containing Arabidopsis thylakoid membrane proteins. Four AtFtsH proteins could be identified in the two protein bands by mass spectrometry: an upper band that contains AtFtsH1 and 5 and a lower band that contains AtFtsH2 and 8. These two bands are co-ordinately decreased in amount in var2 and var1. Because these reductions occur post-translationally, the data are consistent with the idea that proteins in each band interact with one another and that excess subunits are turned over (Yu et al., 2004). Using an AtFtsH1-specific antibody and an AtFtsH2 polyclonal antibody (which detects AtFtsH2 and AtFtsH8), it was found by co-IP that AtFtsH1 interacts with AtFtsH2/8 (Yu et al., 2005). This suggests that interactions occur between the proteins in each of the two bands. A recent report using isoelectric focusing in the first dimension and SDS–PAGE in the second dimension identified the same four AtFtsH proteins from Arabidopsis thylakoid membranes as were identified on the two-imensional green gels (Sinvany-Villalobo et al., 2004). GFP-tagging has also shown that these four proteins are targeted to thylakoids (Sakamoto et al., 2003). Taken together, these data support the notion that there are at least four FtsH proteins located in chloroplast thylakoid membranes.
Mechanism of var2 and var1 variegation
One of the most intriguing features of var2 is its variegation phenotype. As with im, how can one explain the formation of green sectors in a uniform genetic background (i.e. var2/var2)? Similar to im, early explanations for this centred on the notion of compensating activities (Chen et al., 2000; Rodermel, 2001). These have now been refined and a model has been presented that is based on the finding that AtFtsH is a multigene family (Yu et al., 2004, 2005). This model is described below. A similar mechanism cannot be formulated for IM, which is a single gene in Arabidopsis.
Model
The closest homologue of AtFtsH2 is AtFtsH8 (90% amino acid identity) (Yu et al., 2004; Sinvany-Villalobo et al., 2004). To test whether AtFtsH8 is able to compensate for AtFtsH2, AtFtsH8 cDNA was overexpressed in var2 (Yu et al., 2004). It was found that the var2 variegation phenotype was abolished in the transgenic plants, and that normal-appearing plants were generated. This suggests that AtFtsH8 is able to replace the activity of AtFtsH2 in chloroplasts. As mentioned above, two-dimensional green gel analyses revealed that ‘upper’ and ‘lower’ AtFtsH-containing bands are co-ordinately reduced in amount in var2. Similar analyses of the overexpression plants showed that levels of both bands were restored back to wild-type levels. This suggests that AtFtsH8 is able to replace AtFtsH2 in thylakoid membrane AtFtsH complexes.
Based on these observations, a model was proposed to explain the mechanism of var2 variegation (Yu et al., 2004; Fig. 9). In this model, two pairs of FtsH proteins, AtFtsH1 and 5 and AtFtsH2 and 8, form oligomeric complexes in the thylakoid membrane and a threshold level of complexes is required for normal chloroplast function and green sector formation. When complex levels fall below the threshold, chloroplast function will be impaired and white sectors form. It was also proposed that proteins within each pair are interchangeable and that the abundance of proteins in each pair is matched with that of the other pair, with excess subunits being turned over post-translationally. Thus, overexpression of AtFtsH8 in var2 will result in a ‘lower band’ containing normal AtFtsH protein levels, and this will serve to stabilize AtFtsH1 and 5 in the ‘upper band’.
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In further support of the model, the level of AtFtsH1 was manipulated in var1, which lacks AtFtsH5 (Yu et al., 2005). As with var2, the upper and lower AtFtsH-containing bands were co-ordinately reduced in amount in var1. In agreement with the model, the var1 variegation was rescued by AtFtsH1 overexpression, and AtFtsH2 and 8 protein levels were restored to wild-type levels.
Recent overexpression and antisense experiments have revealed that AtFtsH1 and AtFtsH5 share redundant functions, like AtFtsH2 and AtFtsH8 (Yu et al., 2005). All four genes also have similar expression patterns as revealed by RT-PCR and promoter–GUS fusion gene studies: all four are predominantly expressed in green photosynthetic tissues (Yu et al., 2004, 2005). However, overexpression of AtFtsH2 failed to rescue the var1 variegation. This suggests that the two pairs of FtsH proteins might play distinct structural or functional roles.
It has been reported that AtFtsH1, 2, 5, and 8 are expressed at different levels with AtFtsH2 being the most abundantly expressed, followed by AtFtsH5, AtFtsH1, and AtFtsH8 at both the transcript and protein levels (Sinvany-Villalobo et al., 2004). These quantitative differences in gene expression correlate well with the mutant phenotypes: var2 has the highest degree of variegation; var1 is only slightly variegated; and mutants of AtFtsH1 and AtFtsH8 resemble the wild type and do not display any variegation (Sakamoto et al., 2003; F Yu and S Rodermel, unpublished data). The rationale underlying these differences in expression is not clear. However, one possibility is that AtFtsH gene expression is optimized to achieve high protein concentrations in the chloroplast, similar to the proposed rationale for the proliferation of the rbcS (Rubisco small subunit) gene family (Rodermel, 1999). In the case of AtFtsH, this would allow complex formation to occur that is above the threshold required for normal chloroplast function.
var2 suppressor screening
To gain a better understanding of FtsH function and the mechanism of var2 variegation, a second-site suppressor screen was carried out to isolate mutants that modify the var2 variegation phenotype. One normal-appearing, non-variegated suppressor was identified and cloned by map-based methods (Park and Rodermel, 2004). The responsible gene was found to be ClpC2, which encodes a class 1 Hsp100 chaperone, containing two conserved ATP-binding domains. ClpC2 is located in the chloroplast stroma. Suppression of variegation is expressed in nuclear recessive plants (i.e. clpC2/clpC2). The single mutant plants do not have a visible phenotype, but they have vastly reduced levels of ClpC2 due to a splice site mutation in ClpC2. clpC2 and var2 act antagonistically, and thus it was suggested that whereas VAR2 promotes thylakoid membrane biogenesis, ClpC2 normally serves to suppress this process, perhaps by enhancing photo-oxidative stress while the photosynthetic apparatus is being assembled. Further characterization of this mutant and other suppressor lines will help us to understand the mechanisms of variegation and the regulation of chloroplast development.
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* Both investigators contributed equally to this paper and should be considered as first authors.
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