Physiologia Plantarum

Carpentier, Sebastien Christian; Coemans, Bert; Podevin, Nancy; Laukens, Kris; Witters, Erwin; Matsumura, Hideo; Terauchi, Ryohei; Swennen, Rony; Panis, Bart

doi: 10.1111/j.1399-3054.2008.01069.xpmid: 18312499

There is no question that protein‐ and RNA‐based measurements are complementary, but which approach has the highest return in the case of a non‐model crop and what is the correlation between mRNA and proteins? We describe and evaluate in detail the advantages and pitfalls of both a proteomics and a transcriptomics approach. The information on the abundance of transcripts was obtained by serial analysis of gene expression (SAGE), while information on the abundance of proteins was obtained via two‐dimensional gel electrophoresis.

Roach, Thomas; Ivanova, Mariyana; Beckett, Richard P.; Minibayeva, Farida V.; Green, Ian; Pritchard, Hugh W.; Kranner, Ilse

doi: 10.1111/j.1399-3054.2007.00986.xpmid: 18452494

Recalcitrant seeds are intolerant of desiccation and cannot be stored in conventional seed banks. Cryopreservation allows storage of the germplasm of some recalcitrant seeded species, but application to a wide range of plant diversity is still limited. The present work aimed at understanding the stresses that accompany the first steps in cryopreservation protocols, wounding and desiccation, both of which are likely to lead to the formation of reactive oxygen species (ROS). Extracellular ROS production was studied in isolated embryonic axes of sweet chestnut (Castanea sativa). Axis excision was accompanied by a burst of superoxide (O2·−), demonstrated by a colorimetric assay using epinephrine, electron spin resonance and staining with nitroblue tetrazolium. Superoxide was immediately produced on the cut surface after isolation of the axis from the seed, with an initial ‘burst’ in the first 5 min. Isolated axes subjected to variable levels of desiccation stress showed a decrease in viability and vigour and increased electrolyte leakage, indicative of impaired membrane integrity. The pattern of O2·− production showed a typical Gaussian pattern in response to increasing desiccation stress. The results indicate a complex interaction between excision and subsequent drying and are discussed with a view of manipulating ROS production for optimisation of cryopreservation protocols.

Moschou, Panagiotis N.; Delis, Ioannis D.; Paschalidis, Konstantinos A.; Roubelakis‐Angelakis, Kalliopi A.

doi: 10.1111/j.1399-3054.2008.01049.xpmid: 18282192

The molecular and biochemical mechanism(s) of polyamine (PA) action remain largely unknown. Transgenic tobacco plants overexpressing polyamine oxidase (PAO) from Zea mays exhibited dramatically increased expression levels of Mpao and high 1,3‐diaminopropane (Dap) content. All fractions of spermidine and spermine decreased significantly in the transgenic lines. Although Dap was concomitantly generated with H2O2 by PAO, the latter was below the detection limits. To show the mode(s) of H2O2 scavenging, the antioxidant machinery of the transgenics was examined. Specific isoforms of peroxidase, superoxide dismutase and catalase were induced in the transgenics but not in the wild‐type (WT), along with increase in activities of additional enzymes contributing to redox homeostasis. One would expect that because the antioxidant machinery was activated, the transgenics would be able to cope with increased H2O2 generated by abiotic stimuli. However, despite the enhanced antioxidant machinery, further increase in the intracellular reactive oxygen species (ROS) by exogenous H2O2, or addition of methylviologen or menadione to transgenic leaf discs, resulted in oxidative stress as evidenced by the lower quantum yield of PSII, the higher ion leakage, lipid peroxidation and induction of programmed cell death (PCD). These detrimental effects of oxidative burst were as a result of the inability of transgenic cells to further respond as did the WT in which induction of antioxidant enzymes was evident soon following the treatments. Thus, although the higher levels of H2O2 generated by overexpression of Mpao in the transgenics, with altered PA homeostasis, were successfully controlled by the concomitant activation of the antioxidant machinery, further increase in ROS was detrimental to cellular functions and induced the PCD syndrome.

Villasuso, Ana Laura; Racagni, Graciela E.; Machado, Estela E.

doi: 10.1111/j.1399-3054.2008.01050.xpmid: 18282190

Phosphorylated derivatives of phosphatidylinositol, in association with phosphatidylinositol 3‐kinase (PI3 kinase, EC 2.7.1.137) and phosphatidylinositol 4‐kinase (PI4 kinase, EC 2.7.1.67), play a key role in regulation of fundamental cell processes. We present evidence for a relationship between α‐amylase (EC 3.2.1.1) secretion regulated by GA and levels of phosphatidylinositol 3‐phosphate and phosphatidylinositol 4‐phosphate (PtdIns(4)P) in barley (Hordeum vulgare). Microsomal membranes were incubated in the presence of [γ‐32P]ATP, and radiolabeled membrane lipids were extracted and separated by TLC using a boric acid system. Treatment of aleurone layers with GA for short or long periods of time increased PI4 kinase activity. To evaluate the effect of PtdIns(4)P levels on GA signaling, we used phenylarsine oxide (PAO), an inhibitor of PI4 kinase activity. PAO reversibly reduced the α‐amylase secretion and protoplast cell vacuolation in a dose‐dependent manner. Wortmannin showed a similar inhibitory effect on α‐amylase secretion and PI4 kinase activity. GA evoked only a long‐term increase in PI3 kinase activity, which was also affected by PAO. The effect of PAO was suppressed by the reducing agent 2,3‐dimercapto‐1‐propanol (BAL), leading to restoration of secretion, vacuolation and PI4 kinase activity. In contrast, the effect of PAO on PI3 kinase activity was not abolished by BAL, suggesting that PI3 kinase is not involved in the secretion process. Likewise, the compound LY294002 inhibited PI3 kinase but had no effect on the secretion process. These findings indicate that PI4 kinase acts as a positive regulator of early GA signaling in aleurone.

Kudo, Toru; Kawai, Akiko; Yamaya, Tomoyuki; Hayakawa, Toshihiko

doi: 10.1111/j.1399-3054.2008.01051.xpmid: 18282189

Regulatory ACT domains serve as amino acid‐binding sites in certain amino acid metabolic enzymes and transcriptional regulators in bacteria. The ACT domain repeat protein (ACR) family in plants is primarily composed of four copies of the domain homologous to those of the bacteria Gln sensor GLND. In the current study, to evaluate the possible involvement of the protein OsACR9 in the Gln‐sensing system related to nitrogen (N) metabolism in rice (Oryza sativa L.), subcellular localization of OsACR9 and its accumulation and cellular distribution in various rice organs were examined by transient expression analysis and immunological methods using a monospecific antibody, respectively. Transient expression analysis of OsACR9 fused with a synthetic green fluorescent protein in cultured rice cells suggested nuclear localization of OsACR9. In rice roots, OsACR9 protein was distributed in epidermis, exodermis, sclerenchyma and vascular parenchyma cells, and its accumulation markedly increased after supply of NH+4. In rice leaf samples, OsACR9 protein was abundant in the vascular parenchyma and mestome‐sheath cells of young leaf blades at the early stage of development and in the vascular parenchyma and phloem‐companion cells of mature leaf sheaths. OsACR9 protein also showed a high level of accumulation in vascular parenchyma cells of dorsal vascular bundles and aleurone cells in young rice grains at the early stage of ripening. The possibility of the nuclear protein OsACR9 acting as a Gln sensor in rice is subsequently discussed through comparison of its spatiotemporal expression with that of Gln‐responsive N‐assimilatory genes.

Tian, Hua; Jiang, Linrong; Liu, Ee; Zhang, Jianjun; Liu, Fang; Peng, Xinxiang

doi: 10.1111/j.1399-3054.2008.01052.xpmid: 18282188

Oxalate, a common constituent in many plants, is known to play important functional roles in plants. However, excess levels of oxalate in edible parts of plants adversely affect their quality as food. Understanding the regulatory mechanism in plants, particularly in food crops, is of both scientific and practical significance. While a number of studies have shown that nitrate can efficiently induce oxalate accumulation in plants, how it elicits such an effect is not well understood. This study aimed to gain a further insight into the mechanism underlying the nitrate‐induced oxalate accumulation. Nitrate‐N efficiently caused oxalate accumulation in rice leaves, depending on the nitrate concentrations and treatment time. In contrast, same nitrogen molar levels of the other N forms such as nitrite, ammonium, glutamate and urea either had no effect on the accumulation or even reduced the oxalate level. When glutamate, glutamine, asparate and asparagine were added into the nutrient solution that already contained saturating concentration of nitrate, both oxalate levels and NR activity were correspondingly decreased. In all of these modes of treatment, the change in NR activity was positively paralleled to that in oxalate levels. For a further confirmation, we generated the transgenic rice plants with a NR interference gene introduced. The result further demonstrated that in the transgenic plants, unlike in wild‐type plants, oxalate was no longer able to accumulate in response to the nitrate treatment even though the endogenous nitrate levels were substantially elevated. Taken together, our results suggest that the nitrate‐induced oxalate accumulation in rice leaves is dependent on the NR‐catalyzed nitrate reduction, rather than on nitrate itself or nitrite reduction or its downstream metabolites.

Lopez, Alex B.; Yang, Yong; Thannhauser, Theodore W.; Li, Li

doi: 10.1111/j.1399-3054.2008.01058.xpmid: 18298413

Phytoene desaturase (PDS; EC 1.14.99.‐) represents one of the key enzymes in the carotenoid biosynthetic pathway and is present in nearly all types of plastids in plants. To further characterize PDS, we isolated the PDS cDNA from cauliflower (BoPDS) and confirmed its function by heterologous expression in a strain of Escherichia coli containing a carotenoid‐producing plasmid. The BoPDS cDNA encodes a predicted mature protein of approximately 55 kDa. In comparison with PDS from a few other plant species, BoPDS exhibited a high enzyme activity in E. coli, and its expression in plastids was independent of carotenoid levels. Plastids were purified from tissues of different plant species including cauliflower curds, tomato fruits, carrot roots and Arabidopsis leaves. By employing both Blue Native PAGE and SDS‐PAGE approaches in conjunction with Western blot analysis, it was found that PDS in these plants existed in two forms. The plastid membrane form was present in a large protein complex of approximately 350 kDa, whereas the stroma version was in an approximately 660 kDa complex.

Albert, Kristian R.; Mikkelsen, Teis N.; Ro‐Poulsen, Helge

doi: 10.1111/j.1399-3054.2008.01065.xpmid: 18312501

An UV‐B‐exclusion experiment was established in high arctic Zackenberg, Northeast Greenland, to investigate the possible effects of ambient UV‐B on plant performance. During almost a whole growing season, canopy gas exchange and Chl fluorescence were measured on Vaccinium uliginosum (bog blueberry). Leaf area, biomass, carbon, nitrogen and UV‐B‐absorbing compounds were determined from a late season harvest. Compared with the reduced UV‐B treatment, the plants in ambient UV‐B were found to have a higher content of UV‐B‐absorbing compounds, and canopy net photosynthesis was as an average 23% lower during the season. By means of the JIP‐test, it was found that the potential of processing light energy through the photosynthetic machinery was slightly reduced in ambient UV‐B. This indicates that not only the UV‐B effects on PSII may be responsible for some of the observed reduction of photosynthesis but also the effects on other parts of the photosynthetic machinery, e.g. the Calvin cycle, might be important. The 60% reduction of the UV‐B irradiance used in this study implies a higher relative change in the UV‐B load than many of the supplemental experiments do, but the substantial effect on photosynthesis clearly indicates that V. uliginosum is negatively affected by the current level of UV‐B.

Holtgrefe, Simone; Gohlke, Jochen; Starmann, Julia; Druce, Samantha; Klocke, Susanne; Altmann, Bianca; Wojtera, Joanna; Lindermayr, Christian; Scheibe, Renate

doi: 10.1111/j.1399-3054.2008.01066.xpmid: 18298409

Cytosolic NAD‐dependent glyceraldehyde 3‐P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S‐nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin‐switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys‐155 and Cys‐159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP‐dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC‐green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate‐valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.

Morandi, Brunella; Corelli Grappadelli, Luca; Rieger, Mark; Lo Bianco, Riccardo

doi: 10.1111/j.1399-3054.2008.01068.xpmid: 18298408

Along with sucrose, sorbitol represents the main photosynthetic product and form of translocated carbon in peach. This study aimed at determining whether peach fruit carbohydrate metabolism is affected by changes in source–sink balance, and specifically whether sorbitol or sucrose availability regulates fruit enzyme activities and growth. In various trials, different levels of assimilate availability to growing fruits were induced in vivo by varying crop load of entire trees, leaf : fruit ratio (L:F) of fruiting shoots, or by interrupting the phloem stream (girdling) to individual fruits. In vitro, fruit tissue was incubated in presence/absence of sorbitol and sucrose. Relative growth rate (RGR), enzyme activities and carbohydrates were measured at different fruit growth stages of various peach cultivars in different years. At stage III, high crop load induced higher acid invertase (AI, EC 3.2.1.26) activities and hexose : sucrose ratios. Both sorbitol and sucrose contents were proportional to L:F, while sorbitol dehydrogenase (SDH, EC 1.1.1.14) activity was the only enzyme activity directly related to L:F in both fruit growth stages. Girdling reduced fruit RGR and all major carbohydrates after 4 days and SDH activity already after 48 h, but it did not affect sucrose synthase (SS, EC 2.4.1.13), AI and neutral invertase (NI, EC 3.2.1.27). Fruit incubation in sorbitol for 24 h induced higher SDH activities than in buffer alone. In general, assimilate availability affected both sorbitol and sucrose metabolism in peach fruit, and sorbitol may function as a signal for modulating SDH activity. Under highly competitive conditions, AI activity may be enhanced by assimilate depletion, providing a mechanism to increase fruit sink strength by increasing hexose concentrations.