Szilvia Z. Tóth

The physiological role of ascorbate as photosystem II electron donor: protection against photoinactivation in heat-stressed leaves

Previously, we showed that ascorbate (Asc), by donating electrons to photosystem II (PSII), supports a sustained electron transport activity in leaves in which the oxygen-evolving complexes were inactivated with a heat pulse (49°C, 40 s). Here, by using wild-type, Asc-overproducing, and -deficient Arabidopsis (Arabidopsis thaliana) mutants (miox4 and vtc2-3, respectively), we investigated the physiological role of Asc as PSII electron donor in heat-stressed leaves (40°C, 15 min), lacking active oxygen-evolving complexes. Chlorophyll-a fluorescence transients show that in leaves excited with trains of saturating single-turnover flashes spaced 200 ms apart, allowing continual electron donation from Asc to PSII, the reaction centers remained functional even after thousands of turnovers. Higher flash frequencies or continuous illumination (300 μmol photons m−2 s−1) gradually inactivated them, a process that appeared to be initiated by a dramatic deceleration of the electron transfer from TyrZ to P680+, followed by the complete loss of charge separation activity. These processes occurred with half-times of 1.2 and 10 min, 2.8 and 23 min, and 4.1 and 51 min in vtc2-3, the wild type, and miox4, respectively, indicating that the rate of inactivation strongly depended on the Asc content of the leaves. The recovery of PSII activity, following the degradation of PSII proteins (D1, CP43, and PsbO), in moderate light (100 μmol photons m−2 s−1, comparable to growth light), was also retarded in the Asc-deficient mutant. These data show that high Asc content of leaves contributes significantly to the ability of plants to withstand heat-stress conditions.

Experimental evidence for ascorbate-dependent electron transport in leaves with inactive oxygen-evolving complexes.

Previously, we showed that in barley (Hordeum vulgare) leaves with heat-inactivated oxygen-evolving complexes, photosystem II (PSII) has access to a large pool of alternative electron donors. Based on in vitro data, we proposed that this donor was ascorbate, yet this hypothesis has not been substantiated in vivo. In this paper, with the aid of chlorophyll a fluorescence induced by short (5-ms) light pulses and 820-nm absorbance transient measurements on wild-type and ascorbate-deficient (vtc2-1) mutant leaves of Arabidopsis (Arabidopsis thaliana), we show that in heat-treated leaves the rate of electron donation to PSII as well as the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive electron transport toward photosystem I depend on the ascorbate content of the leaves: upon ascorbate treatment, the donation half-time in the wild type and the mutant decreased from 25 to 22 ms and from 55 to 32 ms, respectively. Thermoluminescence measurements show that TyrZ+ is involved in the electron transfer from ascorbate to PSII. These data and the similar ascorbate dependencies of the heat-treated and the tris(hydroxymethyl)aminomethane-treated thylakoid membranes, with maximal donation half-times of about 16 ms, show that ascorbate is capable of supporting a sustained electron transport activity in leaves containing inactivated oxygen-evolving complexes. This alternative electron transport appears to be ubiquitous in the plant kingdom and is present in the green alga Chlamydomonas reinhardtii, and its rate depends on the physiological state of the plants and on environmental conditions. Our data suggest that ascorbate, as an alternative PSII electron donor, plays a physiological role in heat-stressed plants.

Chlorophyll a fluorescence: beyond the limits of the Q A model

Chlorophyll a fluorescence is a non-invasive tool widely used in photosynthesis research. According to the dominant interpretation, based on the model proposed by Duysens and Sweers (1963, Special Issue of Plant and Cell Physiology, pp 353–372), the fluorescence changes reflect primarily changes in the redox state of QA, the primary quinone electron acceptor of photosystem II (PSII). While it is clearly successful in monitoring the photochemical activity of PSII, a number of important observations cannot be explained within the framework of this simple model. Alternative interpretations have been proposed but were not supported satisfactorily by experimental data. In this review we concentrate on the processes determining the fluorescence rise on a dark-to-light transition and critically analyze the experimental data and the existing models. Recent experiments have provided additional evidence for the involvement of a second process influencing the fluorescence rise once QA is reduced. These observations are best explained by a light-induced conformational change, the focal point of our review. We also want to emphasize that—based on the presently available experimental findings—conclusions on α/ß-centers, PSII connectivity, and the assignment of FV/FM to the maximum PSII quantum yield may require critical re-evaluations. At the same time, it has to be emphasized that for a deeper understanding of the underlying physical mechanism(s) systematic studies on light-induced changes in the structure and reaction kinetics of the PSII reaction center are required.

The chl a fluorescence intensity is remarkably insensitive to changes in the chlorophyll content of the leaf as long as the chl a/b ratio remains unaffected.

The effects of changes in the chlorophyll (chl) content on the kinetics of the OJIP fluorescence transient were studied using two different approaches. An extensive chl loss (up to 5-fold decrease) occurs in leaves suffering from either an Mg(2+) or SO(4)(2-) deficiency. The effects of these treatments on the chl a/b ratio, which is related to antenna size, were very limited. This observation was confirmed by the identical light intensity dependencies of the K, J and I-steps of the fluorescence rise for three of the four treatments and by the absence of changes in the F(685 nm)/F(695 nm)-ratio of fluorescence emission spectra measured at 77K. Under these conditions, the F(0) and F(M)-values were essentially insensitive to the chl content. A second experimental approach consisted of the treatment of wheat leaves with specifically designed antisense oligodeoxynucleotides that interfered with the translation of mRNA of the genes coding for chl a/b binding proteins. This way, leaves with a wide range of chl a/b ratios were created. Under these conditions, an inverse proportional relationship between the F(M) values and the chl a/b ratio was observed. A strong effect of the chl a/b ratio on the fluorescence intensity was also observed for barley Chlorina f2 plants that lack chl b. The data suggest that the chl a/b ratio (antenna size) is a more important determinant of the maximum fluorescence intensity than the chl content of the leaf.

The physiological roles and metabolism of ascorbate in chloroplasts.

Ascorbate is a multifunctional metabolite in plants. It is essential for growth control, involving cell division and cell wall synthesis and also involved in redox signaling, in the modulation of gene expression and regulation of enzymatic activities. Ascorbate also fulfills crucial roles in scavenging reactive oxygen species, both enzymatically and nonenzymatically, a well-established phenomenon in the chloroplasts stroma. We give an overview on these important physiological functions and would like to give emphasis to less well-known roles of ascorbate, in the thylakoid lumen, where it also plays multiple roles. It is essential for photoprotection as a cofactor for violaxanthin de-epoxidase, a key enzyme in the formation of nonphotochemical quenching. Lumenal ascorbate has recently also been shown to act as an alternative electron donor of photosystem II once the oxygen-evolving complex is inactivated and to protect the photosynthetic machinery by slowing down donor-side induced photoinactivation; it is yet to be established if ascorbate has a similar role in the case of other stress effects, such as high light and UV-B stress. In bundle sheath cells, deficient in oxygen evolution, ascorbate provides electrons to photosystem II, thereby poising cyclic electron transport around photosystem I. It has also been shown that, by supporting linear electron transport through photosystem II in sulfur-deprived Chlamydomonas reinhardtii cells, in which oxygen evolution is largely inhibited, externally added ascorbate enhances hydrogen production. For fulfilling its multiple roles, Asc has to be transported into the thylakoid lumen and efficiently regenerated; however, very little is known yet about these processes.

Ascorbate accumulation during sulphur deprivation and its effects on photosystem II activity and H2 production of the green alga Chlamydomonas reinhardtii

In nature, H2 production in Chlamydomonas reinhardtii serves as a safety valve during the induction of photosynthesis in anoxia, and it prevents the over-reduction of the photosynthetic electron transport chain. Sulphur deprivation of C. reinhardtii also triggers a complex metabolic response resulting in the induction of various stress-related genes, down-regulation of photosynthesis, the establishment of anaerobiosis and expression of active hydrogenase. Photosystem II (PSII) plays dual role in H2 production because it supplies electrons but the evolved O2 inhibits the hydrogenase. Here, we show that upon sulphur deprivation, the ascorbate content in C. reinhardtii increases about 50-fold, reaching the mM range; at this concentration, ascorbate inactivates the Mn-cluster of PSII, and afterwards, it can donate electrons to tyrozin Z(+) at a slow rate. This stage is followed by donor-side-induced photoinhibition, leading to the loss of charge separation activity in PSII and reaction centre degradation. The time point at which maximum ascorbate concentration is reached in the cell is critical for the establishment of anaerobiosis and initiation of H2 production. We also show that ascorbate influenced H2 evolution via altering the photosynthetic electron transport rather than hydrogenase activity and starch degradation.

Synthetic Antisense Oligodeoxynucleotides to Transiently Suppress Different Nucleus- and Chloroplast-Encoded Proteins of Higher Plant Chloroplasts

Selective inhibition of gene expression by antisense oligodeoxynucleotides (ODNs) is widely applied in gene function analyses; however, experiments with ODNs in plants are scarce. In this work, we extend the use of ODNs in different plant species, optimizing the uptake, stability, and efficiency of ODNs with a combination of molecular biological and biophysical techniques to transiently inhibit the gene expression of different chloroplast proteins. We targeted the nucleus-encoded phytoene desaturase (pds) gene, encoding a key enzyme in carotenoid biosynthesis, the chlorophyll a/b-binding (cab) protein genes, and the chloroplast-encoded psbA gene, encoding the D1 protein. For pds and psbA, the in vivo stability of ODNs was increased by phosphorothioate modifications. After infiltration of ODNs into juvenile tobacco (Nicotiana benthamiana) leaves, we detected a 25% to 35% reduction in mRNA level and an approximately 5% decrease in both carotenoid content and the variable fluorescence of photosystem II. In detached etiolated wheat (Triticum aestivum) leaves, after 8 h of greening, the mRNA level, carotenoid content, and variable fluorescence were inhibited up to 75%, 25%, and 20%, respectively. Regarding cab, ODN treatments of etiolated wheat leaves resulted in an up to 59% decrease in the amount of chlorophyll b, a 41% decrease of the maximum chlorophyll fluorescence intensity, the cab mRNA level was reduced to 66%, and the protein level was suppressed up to 85% compared with the control. The psbA mRNA and protein levels in Arabidopsis (Arabidopsis thaliana) leaves were inhibited by up to 85% and 72%, respectively. To exploit the potential of ODNs for photosynthetic genes, we propose molecular design combined with fast, noninvasive techniques to test their functional effects.

Regulation of ascorbate biosynthesis in green algae has evolved to enable rapid stress-induced response via the VTC2 gene encoding GDP-l-galactose phosphorylase

Ascorbate (vitamin C) plays essential roles in stress resistance, development, signaling, hormone biosynthesis and regulation of gene expression; however, little is known about its biosynthesis in algae. In order to provide experimental proof for the operation of the Smirnoff-Wheeler pathway described for higher plants and to gain more information on the regulation of ascorbate biosynthesis in Chlamydomonas reinhardtii, we targeted the VTC2 gene encoding GDP-l-galactose phosphorylase using artificial microRNAs. Ascorbate concentrations in VTC2 amiRNA lines were reduced to 10% showing that GDP-l-galactose phosphorylase plays a pivotal role in ascorbate biosynthesis. The VTC2 amiRNA lines also grow more slowly, have lower chlorophyll content, and are more susceptible to stress than the control strains. We also demonstrate that: expression of the VTC2 gene is rapidly induced by H2 O2 and 1 O2 resulting in a manifold increase in ascorbate content; in contrast to plants, there is no circadian regulation of ascorbate biosynthesis; photosynthesis is not required per se for ascorbate biosynthesis; and Chlamydomonas VTC2 lacks negative feedback regulation by ascorbate in the physiological concentration range. Our work demonstrates that ascorbate biosynthesis is also highly regulated in Chlamydomonas albeit via mechanisms distinct from those previously described in land plants.

Restriction Enzyme Body Doubles and PCR Cloning: On the General Use of Type IIS Restriction Enzymes for Cloning

The procedure described here allows the cloning of PCR fragments containing a recognition site of the restriction endonuclease (Type IIP) used for cloning in the sequence of the insert. A Type IIS endonuclease - a Body Double of the Type IIP enzyme - is used to generate the same protruding palindrome. Thus, the insert can be cloned to the Type IIP site of the vector without digesting the PCR product with the same Type IIP enzyme. We achieve this by incorporating the recognition site of a Type IIS restriction enzyme that cleaves the DNA outside of its recognition site in the PCR primer in such a way that the cutting positions straddle the desired overhang sequence. Digestion of the PCR product by the Body Double generates the required overhang. Hitherto the use of Type IIS restriction enzymes in cloning reactions has only been used for special applications, the approach presented here makes Type IIS enzymes as useful as Type IIP enzymes for general cloning purposes. To assist in finding Body Double enzymes, we summarised the available Type IIS enzymes which are potentially useful for Body Double cloning and created an online program (http://group.szbk.u-szeged.hu/welkergr/body_double/index.html) for the selection of suitable Body Double enzymes and the design of the appropriate primers.

Water-splitting-based, sustainable and efficient H2 production in green algae as achieved by substrate limitation of the Calvin-Benson-Bassham cycle

BackgroundPhotobiological H2 production has the potential of becoming a carbon-free renewable energy source, because upon the combustion of H2, only water is produced. The [Fe–Fe]-type hydrogenases of green algae are highly active, although extremely O2-sensitive. Sulphur deprivation is a common way to induce H2 production, which, however, relies substantially on organic substrates and imposes a severe stress effect resulting in the degradation of the photosynthetic apparatus.ResultsWe report on the establishment of an alternative H2 production method by green algae that is based on a short anaerobic induction, keeping the Calvin–Benson–Bassham cycle inactive by substrate limitation and preserving hydrogenase activity by applying a simple catalyst to remove the evolved O2. Cultures remain photosynthetically active for several days, with the electrons feeding the hydrogenases mostly derived from water. The amount of H2 produced is higher as compared to the sulphur-deprivation procedure and the process is photoautotrophic.ConclusionOur protocol demonstrates that it is possible to sustainably use algal cells as whole-cell catalysts for H2 production, which enables industrial application of algal biohydrogen production.