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.     

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.     

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.     

Cell Lineage of Vein Formation in Variegated Leaves of the C4Grass Stenotaphrum secundatum

Clonal analysis of variegated leaves of the C₄grass, Stenotaphrumsecundatum, indicates that invasions among meristematic layersoccur during the organogenetic stage of leaf development, resultingin long, broad white and green stripes. These layer invasionscease prior to the second phase of leaf development when delimitationof leaf regions occurs. Vein precursors mostly arise duringthe second phase, so that procambial strand formation is superimposedon the lineage makeup of earlier-formed tissue. Anatomical evidenceindicates that procambium arises through formative divisionswithin ground tissue of leaf primordia and that each strandis derived from a variable number (one–four) of groundmeristem precursors. If a developing vein straddles the boundarybetween previously-formed green and white sectors, then themature vein is half green and half white, reflecting its mixedcell lineage. In Stenotaphrum, 24.8% of the sectors observedwere bounded by such ‘half veins’. The temporalrelationship of layer invasion and tissue system delimitationin this species supports the view that positional signals aremore important than lineage history in the determination oftissue type. However, analysis of planes of cell division indeveloping veins indicates, that, once formed, procambial strandsare discrete lineage units that extend longitudinally by proliferativedivisions. Thus, lineage restrictions may play an importantrole in the third stage of leaf development, differentiationof tissues and cells, which also includes the maintenance ofcell identity.Copyright 2000 Annals of Botany Company C₄photosynthesis, cell lineage, clonal analysis, leaf development, St. Augustine’s grass,Stenotaphrum secundatum , variegation, vein formation     

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.     

Tobacco plastid ribosomal protein S18 is essential for cell survival

Plastid genomes contain a conserved set of genes most of which are involved in either photosynthesis or gene expression. Among the ribosomal protein genes present in higher plant plastid genomes, rps18 is special in that it is absent from the plastid genomes of several non-green unicellular organisms, including Euglena longa and Toxoplasma gondii. Here we have tested whether the ribosomal protein S18 is required for translation by deleting the rps18 gene from the tobacco plastid genome. We report that, while deletion of the rps18 gene was readily obtained, no homoplasmic Δrps18 plants or leaf sectors could be isolated. Instead, segregation into homoplasmy led to severe defects in leaf development suggesting that the knockout of rps18 is lethal and the S18 protein is required for cell survival. Our data demonstrate that S18 is indispensable for plastid ribosome function in tobacco and support an essential role for plastid translation in plant development. Moreover, we demonstrate the occurrence of flip-flop recombination on short inverted repeat sequences which generates different isoforms of the transformed plastid genome that differ in the orientation a 70 kb segment in the large single-copy region. However, infrequent occurrence of flip-flop recombination and random segregation of plastid genomes result in the predominant presence of only one of the isoforms in many tissue samples. Implications for the interpretation of chloroplast transformation experiments and vector design are discussed.     

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.     

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.     

The plastome mutator of Oenothera continues to function as originally described

Summary Recently, Lindenhahn et al. (1985) hypothesized that the plastome mutator (pm) system in Oenothera originated through contaiminating cross-pollination and that the variegation was an example of hybrid plastome-genome incompatibility. Their evidence was based on restriction pattern analyses of white sectors which showed wild-type plastome III patterns rather than the wild-type plastome I patterns of the green portions of their plants. Their hypothesis does not adequately account for the results which our laboratories have obtained independently; the pm-system of Oenothera continues to generate many new and different plastome mutations following the genetic parameters as published originally (Epp 1973). Our studies support mutator gene function. The restriction pattern of the chloroplast DNA of five newly isolated pm-induced variegation sectors are reported here to show a restriction pattern identical to the green wild-type plastids. The restriction pattern reported by Lindenhahn et al. (1985) for their white sector plastids is different than we would expect from a pm-induced plastome mutation. Their overall analysis did not utilize many of the salient features of the genetics of Oenothera and of the pm-system. The white sectors they observed are probably due to an accidental contamination by plastome III plastids. Suggestions are made for delineating experimentally plastome mutations and hybrid incompatibility. For future analyses, a comparative study of numerous pm-induced sectors is recommended, since the pm-system readily generates many different plastome mutations with independent origins. This comparison would greatly assist in the interpretation of restriction patterns.     

Two immunological approaches to the detection of ribulose-1,5-bisphosphate carboxylase in guard cell chloroplasts

Two immunological approaches were used to determine if ribulose bisphosphate carboxylase oxygenase (RuBisCo) is present in guard cell chloroplasts. Immunocytochemistry on thin plastic sections using tissue samples that were processed using traditional glutaraldehyde/osmium fixation and then restored to antigenicity with metaperiodate treatment, resulted in labeling over wild-type mesophyll and guard cell plastids of several green and white variegated Pelargonium chimeras. The density of immunogold labeling in guard cell chloroplasts was only about one-seventh of that noted in mesophyll chloroplasts on a square micron basis. Because guard cell chloroplasts are much smaller than mesophyll chloroplasts, and occur at lower quantities/cell, the relative differences in RuBisCo concentration between the cell types indicate that guard cells have only 0.48% of the RuBisCo of mesophyll cells. No reaction was noted over 70S ribosomeless plastids of these chimeras even though adjacent green chloroplasts were heavily stained, indicating the high specificity of the reaction for RuBisCo. Spurr's resin gave the most successful colloidal gold labeling in terms of low background staining and structural detail but L. R. White's resin appeared to be superior for antigen retention. In the white leaf edges of the white and green Pelargonium chimeras, the only green, functional chloroplasts are in the guard cells. When either whole tissue or plastid enriched extracts from this white tissue were electrophoresed, blotted, and probed with anti-RuBisCo a large subunit band was detected, identical to that in the green tissue. These data indicate that a low, but detectable, level of RuBisCo is present in guard cell chloroplasts.     

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.