PLANT PHYSIOLOGY BIOENERGETICS AND PHOTOSYNTHESIS

[BIOENERGETICS AND PHOTOSYNTHESIS] The Pentratricopeptide Repeat Protein DELAYED GREENING1 Is Involved in the Regulation of Early Chloroplast Development and Chloroplast Gene Expression in Arabidopsis

Chi, W., Ma, J., Zhang, D., Guo, J., Chen, F., Lu, C., Zhang, L. — 2008-06-04

An Arabidopsis (Arabidopsis thaliana) mutant that exhibited a delayed greening phenotype (dg1) was isolated from a population of activation-tagged Arabidopsis lines. Young, inner leaves of dg1 mutants were initially very pale, but gradually greened and mature outer leaves, more than 3 weeks old, appeared similar to those of wild-type plants. Sequence and transcription analyses showed that DG1 encodes a chloroplast protein consisting of eight pentratricopeptide repeat domains and that its expression depends on both light and developmental status. In addition, analysis of the transcript profiles of chloroplast genes revealed that plastid-encoded polymerase-dependent transcript levels were markedly reduced, while nucleus-encoded polymerase-dependent transcript levels were increased, in dg1 mutants. Thus, DG1 is probably involved in the regulation of plastid-encoded polymerase-dependent chloroplast gene expression during early stages of chloroplast development.

[BIOENERGETICS AND PHOTOSYNTHESIS] Reduction of Plastid-Localized Carbonic Anhydrase Activity Results in Reduced Arabidopsis Seedling Survivorship

Ferreira, F. J., Guo, C., Coleman, J. R. — 2008-06-04

Carbonic anhydrase (CA; EC 4.2.1.1) catalyzes the interconversion of CO₂ and HCO₃^– and is a major protein constituent of the C₃ higher plant chloroplast where it is presumed to play a role in photosynthetic carbon assimilation. In this study, we have used both RNA antisense and gene knockout lines to specifically reduce the activity of the chloroplast βCA1 polypeptide (At3g01500) in the model plant Arabidopsis (Arabidopsis thaliana). Although able to germinate, seedling establishment of transgenic plants is significantly reduced relative to wild-type plants when grown at ambient levels of CO₂. Growth at elevated (1,500 µL L^–1) CO₂ or on plates supplemented with sucrose restores seedling establishment rates to wild-type levels. Seed from wild-type and transgenic plants exhibited no significant differences in seed protein, lipid content, or reserve mobilization during seedling growth. βCA1-deficient seedlings do, however, exhibit reduced capacity for light-dependent ¹⁴CO₂ assimilation prior to the development of true leaves. The small number of surviving seedlings able to grow and develop are phenotypically similar to wild-type plants, even when subsequently grown at subambient levels of CO₂. Microarray analysis of mature leaves of βCA1-deficient plants shows some differences in transcript abundance, particularly with genes involved in ethylene signaling and response. The data suggest that reduced levels of seedling establishment by βCA1-deficient plants could be the result of poor cotyledon photosynthetic performance at the onset of phototrophic growth and prior to the development of true leaves.

[BIOENERGETICS AND PHOTOSYNTHESIS] The Absence of ALTERNATIVE OXIDASE1a in Arabidopsis Results in Acute Sensitivity to Combined Light and Drought Stress

2008-06-04

Treatment of Arabidopsis (Arabidopsis thaliana) alternative oxidase1a (aox1a) mutant plants with moderate light under drought conditions resulted in a phenotypic difference compared with ecotype Columbia (Col-0), as evidenced by a 10-fold increase in the accumulation of anthocyanins in leaves, alterations in photosynthetic efficiency, and increased superoxide radical and reduced root growth at the early stages of seedling growth. Analysis of metabolite profiles revealed significant changes upon treatment in aox1a plants typical of combined stress treatments, and these were less pronounced or absent in Col-0 plants. These changes were accompanied by alteration in the abundance of a variety of transcripts during the stress treatment, providing a molecular fingerprint for the stress-induced phenotype of aox1a plants. Transcripts encoding proteins involved in the synthesis of anthocyanins, transcription factors, chloroplastic and mitochondrial components, cell wall synthesis, and sucrose and starch metabolism changed, indicating that effects were not confined to mitochondria, where the AOX1a protein is located. Microarray and quantitative reverse transcription-polymerase chain reaction analysis revealed that transcripts typically induced upon stress treatment or involved in antioxidant defense systems, especially chloroplast-located antioxidant defense components, had altered basal levels in untreated aox1a plants, suggesting a significant change in the basal equilibrium of signaling pathways that regulate these components. Taken together, these results indicate that aox1a plants have a greatly altered stress response even when mitochondria or the mitochondrial electron transport chain are not the primary target of the stress and that AOX1a plays a broad role in determining the normal redox balance in the cell.

[BIOENERGETICS AND PHOTOSYNTHESIS] Photosynthetic Utilization of Bicarbonate in Zostera marina Is Reduced by Inhibitors of Mitochondrial ATPase and Electron Transport

Carr, H., Axelsson, L. — 2008-06-04

When Zostera marina was irradiated after a period of darkness, initiation of photosynthetic O₂ evolution occurred in two phases. During a lag phase, lasting 4 to 5 min, photosynthesis was supported by a diffusive entry of CO₂. Photosynthesis then rapidly increased to its full rate. Tris buffer, at a concentration of 50 mm, completely inhibited this increase without affecting CO₂-supported photosynthesis during the lag phase. These results verify that the increase in photosynthesis after the lag phase depended on an activation of bicarbonate (HCO₃^–) utilization through acid zones generated by proton pumps located to the outer cell membrane. In similar experiments, 6.25 µm of the mitochondrial ATPase blocker oligomycin inhibited photosynthetic HCO₃^– utilization by more than 60%. Antimycin A, a selective blocker of mitochondrial electron transport, caused a similar inhibition of HCO₃^– utilization. Measurements at elevated CO₂ concentrations verified that neither oligomycin nor antimycin interfered with linear photosynthetic electron transport or with CO₂ fixation. Thus, a major part of the ATP used for the generation of acid zones involved in HCO₃^– utilization in Z. marina was derived from mitochondrial respiration.

[BIOENERGETICS AND PHOTOSYNTHESIS] Decrease in Manganese Superoxide Dismutase Leads to Reduced Root Growth and Affects Tricarboxylic Acid Cycle Flux and Mitochondrial Redox Homeostasis

2008-04-28

Superoxide dismutases (SODs) are key components of the plant antioxidant defense system. While plastidic and cytosolic isoforms have been extensively studied, the importance of mitochondrial SOD at a cellular and whole-plant level has not been established. To address this, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated in which expression of AtMSD1, encoding the mitochondrial manganese (Mn)SOD, was suppressed by antisense. The strongest antisense line showed retarded root growth even under control growth conditions. There was evidence for a specific disturbance of mitochondrial redox homeostasis in seedlings grown in liquid culture: a mitochondrially targeted redox-sensitive green fluorescent protein was significantly more oxidized in the MnSOD-antisense background. In contrast, there was no substantial change in oxidation of cytosolically targeted redox-sensitive green fluorescent protein, nor changes in antioxidant defense components. The consequences of altered mitochondrial redox status of seedlings were subtle with no widespread increase of mitochondrial protein carbonyls or inhibition of mitochondrial respiratory complexes. However, there were specific inhibitions of tricarboxylic acid (TCA) cycle enzymes (aconitase and isocitrate dehydrogenase) and an inhibition of TCA cycle flux in isolated mitochondria. Nevertheless, total respiratory CO₂ output of seedlings was not decreased, suggesting that the inhibited TCA cycle enzymes can be bypassed. In older, soil-grown plants, redox perturbation was more pronounced with changes in the amount and/or redox poise of ascorbate and glutathione. Overall, the results demonstrate that reduced MnSOD affects mitochondrial redox balance and plant growth. The data also highlight the flexibility of plant metabolism with TCA cycle inhibition having little effect on overall respiratory rates.

[BIOENERGETICS AND PHOTOSYNTHESIS] Mild Reductions in Mitochondrial Citrate Synthase Activity Result in a Compromised Nitrate Assimilation and Reduced Leaf Pigmentation But Have No Effect on Photosynthetic Performance or Growth

2008-04-28

Transgenic tomato (Solanum lycopersicum) plants, expressing a fragment of the mitochondrial citrate synthase gene in the antisense orientation and exhibiting mild reductions in the total cellular activity of this enzyme, displayed essentially no visible phenotypic alteration from the wild type. A more detailed physiological characterization, however, revealed that although these plants were characterized by relatively few changes in photosynthetic parameters they displayed a decreased relative flux through the tricarboxylic acid cycle and an increased rate of respiration. Furthermore, biochemical analyses revealed that the transformants exhibited considerably altered metabolism, being characterized by slight decreases in the levels of organic acids of the tricarboxylic acid cycle, photosynthetic pigments, and in a single line in protein content but increases in the levels of nitrate, several amino acids, and starch. We additionally determined the maximal catalytic activities of a wide range of enzymes of primary metabolism, performed targeted quantitative PCR analysis on all three isoforms of citrate synthase, and conducted a broader transcript profiling using the TOM1 microarray. Results from these studies confirmed that if the lines were somewhat impaired in nitrate assimilation, they were not severely affected by this, suggesting the presence of strategies by which metabolism is reprogrammed to compensate for this deficiency. The results are discussed in the context of carbon-nitrogen interaction and interorganellar coordination of metabolism.

[BIOENERGETICS AND PHOTOSYNTHESIS] Expression Analysis of Genes Associated with the Induction of the Carbon-Concentrating Mechanism in Chlamydomonas reinhardtii

Yamano, T., Miura, K., Fukuzawa, H. — 2008-04-28

Acclimation to varying CO₂ concentrations and light intensities is associated with the monitoring of environmental changes by controlling genetic and physiological responses through CO₂ and light signal transduction. While CO₂ and light signals are indispensable for photosynthesis, and these environmental factors have been proposed as strongly associated with each other, studies linking these components are largely limited to work on higher plants. In this study, we examined the physiological characteristics of a green alga, Chlamydomonas reinhardtii, exposed to various light intensities or CO₂ concentrations. Acclimation to CO₂-limiting conditions by Chlamydomonas requires the induction of a carbon-concentrating mechanism (CCM) to allow the uptake of inorganic carbon (Ci) and increase the affinity for Ci. We revealed that the induction of the CCM is not solely dependent on absolute environmental Ci concentrations but is also affected by light intensity. Using a cDNA array containing 10,368 expressed sequence tags, we also obtained global expression profiles related to the physiological responses. The induction of several CCM-associated genes was strongly affected by high light as well as CO₂ concentrations. We identified novel candidates for Ci transporters and CO₂-responsive regulatory factors whose expression levels were significantly increased during the induction of the CCM.

[BIOENERGETICS AND PHOTOSYNTHESIS] Protein Diffusion and Macromolecular Crowding in Thylakoid Membranes

Kirchhoff, H., Haferkamp, S., Allen, J. F., Epstein, D. B.A., Mullineaux, C. W. — 2008-04-03

The photosynthetic light reactions of green plants are mediated by chlorophyll-binding protein complexes located in the thylakoid membranes within the chloroplasts. Thylakoid membranes have a complex structure, with lateral segregation of protein complexes into distinct membrane regions known as the grana and the stroma lamellae. It has long been clear that some protein complexes can diffuse between the grana and the stroma lamellae, and that this movement is important for processes including membrane biogenesis, regulation of light harvesting, and turnover and repair of the photosynthetic complexes. In the grana membranes, diffusion may be problematic because the protein complexes are very densely packed (approximately 75% area occupation) and semicrystalline protein arrays are often observed. To date, direct measurements of protein diffusion in green plant thylakoids have been lacking. We have developed a form of fluorescence recovery after photobleaching that allows direct measurement of the diffusion of chlorophyll-protein complexes in isolated grana membranes from Spinacia oleracea. We show that about 75% of fluorophores are immobile within our measuring period of a few minutes. We suggest that this immobility is due to a protein network covering a whole grana disc. However, the remaining fraction is surprisingly mobile (diffusion coefficient 4.6 ± 0.4 x 10^–11 cm² s^–1), which suggests that it is associated with mobile proteins that exchange between the grana and stroma lamellae within a few seconds. Manipulation of the protein-lipid ratio and the ionic strength of the buffer reveals the roles of macromolecular crowding and protein-protein interactions in restricting the mobility of grana proteins.

[BIOENERGETICS AND PHOTOSYNTHESIS] Metabolic and Developmental Adaptations of Growing Potato Tubers in Response to Specific Manipulations of the Adenylate Energy Status

Riewe, D., Grosman, L., Zauber, H., Wucke, C., Fernie, A. R., Geigenberger, P. — 2008-04-03

Heterotrophic carbon metabolism has been demonstrated to be limited by oxygen availability in a variety of plant tissues, which in turn inevitably affects the adenylate status. To study the effect of altering adenylate energy metabolism, without changing the oxygen supply, we expressed a plastidially targeted ATP/ADP hydrolyzing phosphatase (apyrase) in tubers of growing potato (Solanum tuberosum) plants under the control of either inducible or constitutive promoters. Inducible apyrase expression in potato tubers, for a period of 24 h, resulted in a decrease in the ATP-content and the ATP-ADP ratio in the tubers. As revealed by metabolic profiling, this was accompanied by a decrease in the intermediates of sucrose to starch conversion and several plastidially synthesized amino acids, indicating a general depression of tuber metabolism. Constitutive tuber-specific apyrase expression did not lead to a reduction of ATP, but rather a decrease in ADP and an increase in AMP levels. Starch accumulation was strongly inhibited and shifted to the production of amylopectin instead of amylose in these tubers. Furthermore, the levels of almost all amino acids were decreased, although soluble sugars and hexose-Ps were highly abundant. Respiration was elevated in the constitutively expressing lines indicating a compensation for the dramatic increase in ATP hydrolysis. The increase in respiration did not affect the internal oxygen tensions in the tubers. However, the tubers developed a ginger-like phenotype having an elevated surface-volume ratio and a reduced mass per tuber. Decreased posttranslational redox activation of ADP-glucose pyrophosphorylase and a shift in the ratio of soluble starch synthase activity to granule-bound starch synthase activity were found to be partially responsible for the alterations in starch structure and abundance. The activity of alcohol dehydrogenase was decreased and pyruvate decarboxylase was induced, but this was neither reflected by an increase in fermentation products nor in the cellular redox state, indicating that fermentation was not yet induced in the transgenic lines. When taken together the combined results of these studies allow the identification of both short- and long-term adaptation of plant metabolism and development to direct changes in the adenylate status.

[BIOENERGETICS AND PHOTOSYNTHESIS] In Vivo Target Sites of Nitric Oxide in Photosynthetic Electron Transport as Studied by Chlorophyll Fluorescence in Pea Leaves

Wodala, B., Deak, Z., Vass, I., Erdei, L., Altorjay, I., Horvath, F. — 2008-04-03

The role of nitric oxide (NO) in photosynthesis is poorly understood as indicated by a number of studies in this field with often conflicting results. As various NO donors may be the primary source of discrepancies, the aim of this study was to apply a set of NO donors and its scavengers, and examine the effect of exogenous NO on photosynthetic electron transport in vivo as determined by chlorophyll fluorescence of pea (Pisum sativum) leaves. Sodium nitroprusside-induced changes were shown to be mediated partly by cyanide, and S-nitroso-N-acetylpenicillinamine provided low yields of NO. However, the effects of S-nitrosoglutathione are inferred exclusively by NO, which made it an ideal choice for this study. Q_A^– reoxidation kinetics show that NO slows down electron transfer between Q_A and Q_B, and inhibits charge recombination reactions of Q_A^– with the S₂ state of the water-oxidizing complex in photosystem II. Consistent with these results, chlorophyll fluorescence induction suggests that NO also inhibits steady-state photochemical and nonphotochemical quenching processes. NO also appears to modulate reaction-center-associated nonphotochemical quenching.