Nuclear—organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis

The immutans (im) variegation mutant of Arabidopsis thaliana contains green- and white-sectored leaves due to the action of a nuclear recessive gene. The mutation is somatically unstable, and the degree of sectoring is influenced by light and temperature. Whereas the cells in the green sectors contain normal chloroplasts, the cells in the white sectors are heteroplastidic and contain non-pigmented plastids that lack organized lamellar structures, as well as small pigmented plastids and/or rare normal chloroplasts. This indicates that the plastids in im white cells are not affected equally by the nuclear mutation and that the expression of immutans is ‘plastid autonomous’. In contrast to other variegation mutants with heteroplastidic cells, the defect in im is not maternally inherited. immutans thus represents a novel type of nuclear gene-induced variegation mutant. It has also been found that the white tissues of immutans accumulate phytoene, a non-colored C₄₀ carotenoid intermediate. This suggests that immutans controls, either directly or indirectly, the activity of phytoene desaturase (PDS), the enzyme that converts phytoene to zeta-carotene in higher plants. However, im is not the structural gene for PDS. A secondary effect of carotenoid deficiency, both in immutans and in wild-type plants treated with a herbicide that blocks carotenoid synthesis, is an increase in acid ribonuclease activity in white tissue. It is concluded that the novel variegation generated by the immutans mutation should offer great insight into the complex circuitry that regulates nuclear—organelle interactions.     

Variegation mutants and mechanisms of chloroplast biogenesis

Variegated plants typically have green‐ and white‐sectored leaves. Cells in the green sectors contain normal‐appearing chloroplasts, whereas cells in the white sectors lack pigments and appear to be blocked at various stages of chloroplast biogenesis. Variegations can be caused by mutations in nuclear, chloroplast or mitochondrial genes. In some plants, the green and white sectors have different genotypes, but in others they have the same (mutant) genotype. One advantage of variegations is that they provide a means of studying genes for proteins that are important for chloroplast development, but for which mutant analysis is difficult, either because mutations in a gene of interest are lethal or because they do not show a readily distinguishable phenotype. This paper focuses on Arabidopsis variegations, for which the most information is available at the molecular level. Perhaps the most interesting of these are variegations caused by defective nuclear gene products in which the cells of the mutant have a uniform genotype. Two questions are of paramount interest: (1) What is the gene product and how does it function in chloroplast biogenesis? (2) What is the mechanism of variegation and why do green sectors arise in plants with a uniform (mutant) genotype? Two paradigms of variegation mechanism are described: immutans (im) and variegated2 (var2). Both mechanisms emphasize compensating activities and the notion of plastid autonomy, but redundant gene products are proposed to play a role in var2, but not in im. It is hypothesized that threshold levels of certain activities are necessary for normal chloroplast development.     

Impaired Chloroplast Biogenesis in Immutans,an Arabidopsis Variegation Mutant,Modifies Developmental Programming,Cell Wall Composition and Resistance to Pseudomonas syringae

The immutans (im) variegation mutation of Arabidopsis has green- and white- sectored leaves due to action of a nuclear recessive gene. IM codes for PTOX, a plastoquinol oxidase in plastid membranes. Previous studies have revealed that the green and white sectors develop into sources (green tissues) and sinks (white tissues) early in leaf development. In this report we focus on white sectors, and show that their transformation into effective sinks involves a sharp reduction in plastid number and size. Despite these reductions, cells in the white sectors have near-normal amounts of plastid RNA and protein, and surprisingly, a marked amplification of chloroplast DNA. The maintenance of protein synthesis capacity in the white sectors might poise plastids for their development into other plastid types. The green and white im sectors have different cell wall compositions: whereas cell walls in the green sectors resemble those in wild type, cell walls in the white sectors have reduced lignin and cellulose microfibrils, as well as alterations in galactomannans and the decoration of xyloglucan. These changes promote susceptibility to the pathogen Pseudomonas syringae. Enhanced susceptibility can also be explained by repressed expression of some, but not all, defense genes. We suggest that differences in morphology, physiology and biochemistry between the green and white sectors is caused by a reprogramming of leaf development that is coordinated, in part, by mechanisms of retrograde (plastid-to-nucleus) signaling, perhaps mediated by ROS. We conclude that variegation mutants offer a novel system to study leaf developmental programming, cell wall metabolism and host-pathogen interactions.     

Arabidopsis Chloroplast FtsH,vat2 and Suppressors of var2 Leaf Variegation: a Review

Variegation mutants are ideal model systems to study chloroplast biogenesis.We are interested in variegations whose green and whitesectored leaves arise as a consequence of the action of nuclear recessive genes.In this review,we focus on the Arabidopsis var2 variegation mutant,and discuss recent progress toward understanding the function of VAR2 and the mechanism of var2-mediated variegation.VAR2 is a subunit of the chloroplast FtsH complex,which is involved in turnover of the Photosystem Ⅱ reaction center D1 protein,as well as in other processes required for the development and maintenance of the photosynthetic apparatus.The cells in green sectors of var2have normal-appearing chloroplasts whereas cells in the white sectors have abnormal plastids that lack pigments and organized lameliae.To explain the mechanism of var2 variegation,we have proposed a threshold model in which the formation of chloroplasts is due to the presence of activities/processes that are able to compensate for a lack of VAR2.To gain insight into these activities,second-site suppressor screens have been carried out to obtain mutants with nonvariegation phenotypes.Cloning and characterization of several var2 suppressor lines have uncovered several mechanisms of variegation suppression,including an unexpected link between var2 variegation and chloroplast translation.     

Arabidopsis Chloroplast FtsH,var2 and Suppressors of var2 Leaf Variegation:a Review

Variegation mutants are ideal model systems to study chloroplast biogenesis. We are interested in variegations whose green and whitesectored leaves arise as a consequence of the action of nuclear recessive genes. In this review, we focus on the Arabidopsis var2 variegation mutant, and discuss recent progress toward understanding the function of VAR2 and the mechanism of var2-mediated variegation. VAR2 is a subunit of the chloroplast FtsH complex, which is involved in turnover of the Photosystem II reaction center D1 protein, as well as in other processes required for the development and maintenance of the photosynthetic apparatus. The cells in green sectors of var2 have normal-appearing chloroplasts whereas cells in the white sectors have abnormal plastids that lack pigments and organized lamellae. To explain the mechanism of var2 variegation, we have proposed a threshold model in which the formation of chloroplasts is due to the presence of activities/processes that are able to compensate for a lack of VAR2. To gain insight into these activities, second-site suppressor screens have been carried out to obtain mutants with nonvariegation phenotypes. Cloning and characterization of several var2 suppressor lines have uncovered several mechanisms of variegation suppression, including an unexpected link between var2 variegation and chloroplast translation.     

Alterations in photosynthesis in <Emphasis Type="Italic">Arabidopsis</Emphasis> lacking IMMUTANS,a chloroplast terminal oxidase

Green and white variegation in the Arabidopsis immutans (im) mutant is caused by a nuclear recessive gene. The green sectors contain cells with normal-appearing chloroplasts, while cells in the white sectors have photooxidized plastids lacking organized lamellae. In the present experiments, we found that the green im sectors have enhanced rates of carbon assimilation (monitored by ¹⁴CO₂ uptake) and that there are corresponding increases in the activities of Rubisco and SPS, elevated starch and sucrose pool sizes, and an altered pattern of carbohydrate partitioning that favors sucrose over starch. We hypothesize that these increases are due, at least in part, to interactions with white sectors, perhaps to compensate for reductions in total source tissue. Consistent with this idea, the im white sectors accumulate low levels of sucrose and acid invertase activities are markedly increased in the white versus green cells. This suggests that there is a sucrose gradient between the green and white sectors, and that sucrose is transported from the green to white cells in response to sink demand. The expression of photosynthetic genes is not appreciably altered in the green im sectors versus wild type, but rather there is an up-regulation of genes involved in defense against oxidative stress and down-regulation of genes involved in cell wall biosynthesis. We postulate that changes in photosynthesis in the im green cells are driven by a need for photoprotection (especially early in chloroplast biogenesis) and due to source-sink interactions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Control of chloroplast redox by the IMMUTANS terminal oxidase

Variegation mutants offer excellent opportunities to study interactions between the nucleus-cytoplasm, the chloroplast, and the mitochondrion. Variegation in the immutans ( im ) mutant of Arabidopsis is induced by a nuclear recessive gene and the extent of variegation can be modulated by light and temperature. Whereas the green sectors have morphologically normal chloroplasts, the white sectors are devoid of pigments and accumulate a colourless carotenoid, phytoene. The green sectors are hypothesized to arise from cells that have avoided irreversible photooxidative damage whereas the white sectors originate from cells that are photooxidized. Cloning of the IMMUTANS ( IM ) gene has revealed that IMMUTANS (IM) is a plastid homologue of the mitochondrial alternative oxidase. This finding suggested a model in which IM functions as a redox component of the phytoene desaturation pathway, which requires phytoene desaturase activity. Consistent with this idea, IM has quinol oxidase activity in vitro. Recent studies have revealed that IM plays a more global role in plastid metabolism. For example, it appears to be the elusive terminal oxidase of chlororespiration and also functions as a light stress protein.     

NUCLEAR GENE-INDUCED PLASTOME MUTATIONS IN OENOTHERA HOOKERI. II. PHENOTYPIC DESCRIPTION WITH ELECTRON MICROSCOPY

The nuclear gene pm of Oenothera hookeri T. & G. strain Johansen increases the frequency of plastome mutations. These plastome mutations are visible because they form variegation sectors. A distinctive feature of the pm system is the large number of different phenotypes induced by the plastome mutator. The fine structure of four plastome mutants has been studied using electron microscopy. Each of the four mutants had a unique phenotype which was characterized by the amount of thylakoid development, the number of plastoglobuli, and the dilation of thylakoids with age. The developmental sequence of the phenotype of one of the mutants was studied by following plastid development in progressively larger leaves. The phenotypes of the pm-induced mutants are generally similar to those of spontaneous plastome mutations described in the literature.     

Histone Gene Multiplicity and Position Effect Variegation in DROSOPHILA MELANOGASTER

The histone genes of wild-type Drosophila melanogaster are reiterated 100–150 times per haploid genome and are located in the segment of chromosome 2 that corresponds to polytene bands 39D2-3 to E1-2. The influence of altered histone gene multiplicity on chromatin structure has been assayed by measuring modification of the gene inactivation associated with position effect variegation in genotypes bearing deletions of the 39D-E segment. The proportion of cells in which a variegating gene is active is increased in genotypes that are heterozygous for a deficiency that removes the histone gene complex. Deletions that remove segments adjacent to the histone gene complex have no effect on the expression of variegating genes. Suppression of position effect variegation associated with reduction of histone gene multiplicity applies to both X-linked and autosomal variegating genes. Position effects exerted by both autosomal and sex-chromosome heterochromatin were suppressible by deletions of the histone gene complex. The suppression was independent of the presence of the Y chromosome. A deficiency that deletes only the distal portion of the histone gene complex also has the ability to suppress position effect variegation. Duplication of the histone gene complex did not enhance position effect variegation. Deletion or duplication of the histone gene complex in the maternal genome had no effect on the extent of variegation in progeny whose histone gene multiplicity was normal. These results are discussed with respect to current knowledge of the organization of the histone gene complex and control of its expression.     

Natural foliar variegation without costs? The case of Begonia

Background and Aims

Foliar variegation is recognized as arising from two major mechanisms: leaf structure and pigment-related variegation. Begonia has species with a variety of natural foliar variegation patterns, providing diverse examples of this phenomenon. The aims of this work are to elucidate the mechanisms underlying different foliar variegation patterns in Begonia and to determine their physiological consequences.

Methods

Six species and one cultivar of Begonia were investigated. Light and electron microscopy revealed the leaf structure and ultrastructure of chloroplasts in green and light areas of variegated leaves. Maximum quantum yields of photosystem II were measured by chlorophyll fluorescence. Comparison with a cultivar of Ficus revealed key features distinguishing variegation mechanisms.

Key Results

Intercellular space above the chlorenchyma is the mechanism of variegation in these Begonia. This intercellular space can be located (a) below the adaxial epidermis or (b) below the adaxial water storage tissue (the first report for any taxa), creating light areas on a leaf. In addition, chlorenchyma cell shape and chloroplast distribution within chlorenchyma cells differ between light and green areas. Chloroplasts from both areas showed dense stacking of grana and stroma thylakoid membranes. The maximum quantum yield did not differ significantly between these areas, suggesting minimal loss of function with variegation. However, the absence of chloroplasts in light areas of leaves in the Ficus cultivar led to an extremely low quantum yield.

Conclusions

Variegation in these Begonia is structural, where light areas are created by internal reflection between air spaces and cells in a leaf. Two forms of air space structural variegation occur, distinguished by the location of the air spaces. Both forms may have a common origin in development where dermal tissue becomes loosely connected to mesophyll. Photosynthetic functioning is retained in light areas, and these areas do not include primary veins, potentially limiting the costs of variegation.     

Tetrapyrrole involvement in expression regulation of a nuclear gene of low-molecular-weight plastid protein ELIP

An inhibitor analysis was used for studying the tetrapyrrole role in the regulation of the expression of the nuclear gene encoding a low-molecular-weight protein, a stress plastid light-inducible protein ELIP. 2,2′-Dipyridyl and norflurazon were used as inhibitors. Experiments with dipyridyl demonstrated that tetrapyrroles were involved in the regulation of Elip gene expression, inhibiting it by ～50%. Similar results were obtained when there was photodestruction of the chloroplasts, caused by a plant treatment with norflurazon. The results confirm the involvement of the chloroplasts in the regulation of the nuclear gene expression coding for plastid proteins. Tetrapyrroles are important contributors to this process.     

Variegated tobacco leaves generated by chloroplast FtsH suppression: implication of FtsH function in the maintenance of thylakoid membranes

Mutants lacking a thylakoid membrane-bound metalloprotease, FtsH, are known to cause leaf variegation in Arabidopsis. However, the effect of reduced FtsH levels on leaf variegation has scarcely been examined in other plants. In this study, we performed RNA interference (RNAi) by which FtsH expression was suppressed in tobacco. The resulting FtsH knock-down tobacco plants showed variegation in their leaves, and a negative correlation between the degree of variegation and the level of FtsH, which supported earlier observations in Arabidopsis. A decrease of NtFtsH2 as well as NtFtsH1 suggested that these are the two major isoforms comprising the FtsH complex in tobacco chloroplasts. The RNAi tobacco lines also showed photoinhibition-vulnerable phenotypes, as evidenced by high-light-sensitive PSII activity and retarded degradation of D1 protein. Interestingly, the formation of variegated sectors during leaf development appeared to differ between Arabidopsis and tobacco. In contrast to the formation of variegation in Arabidopsis, the yellow sectors in FtsH RNAi tobacco emerged from green leaves at a late stage of leaf development. A series of cytological observations implied that thylakoid membranes were dismantled after development had already occurred. Late formation of variegation in FtsH RNAi tobacco suggested that the heteromeric FtsH complex is important for maintaining thylakoid membranes.     

Mutations in the Arabidopsis VAR2 locus cause leaf variegation due to the loss of a chloroplast FtsH protease

Variegated plants have green- and white-sectored leaves. Cells in the green sectors contain morphologically normal chloroplasts, whereas cells in the white sectors contain non-pigmented plastids that lack organized lamellar structures. Many variegations are caused by mutations in nuclear genes that affect plastid function, yet in only a few cases have the responsible genes been cloned. We show that mutations in the nuclear VAR2 locus of Arabidopsis cause variegation due to loss of a chloroplast thylakoid membrane protein that bears similarity to the FtsH family of AAA proteins (ATPases associated with diverse cellular activities). Escherichia coli FtsH is a chaperone metalloprotease that functions in a number of diverse membrane-associated events. Although FtsH homologs have been identified in multicellular organisms, their functions and activities are largely unknown; we provide genetic in vivo evidence that VAR2 functions in thylakoid membrane biogenesis. We have isolated four var2 alleles and they have allowed us to define domains of the protein that are required for activity. These include two putative ATP-binding sites. VAR2 protein amounts generally correlate with the severity of the var2 mutant phenotype. One allele lacks detectable VAR2 protein, suggesting that the mechanism of var2 variegation involves the action of a redundant activity in the green sectors. We conclude that redundant activities may be a general mechanism to explain nuclear gene-induced plant variegations.