Reduced vacuolar ß-1,3-glucan synthesis affects carbohydrate metabolism as well as plastid homeostasis and structure in Phaeodactylum tricornutum.

The ß-1,3-glucan chrysolaminarin is the main storage polysaccharide of diatoms. In contrast to plants and green algae, diatoms and most other algal groups do not accumulate storage polysaccharides in their plastids. The diatom Phaeodactylum tricornutum possesses only a single gene encoding a putative ß-1,3-glucan synthase (PtBGS). Here, we characterize this enzyme by expressing GFP fusion proteins in P. tricornutum and by creating and investigating corresponding gene silencing mutants. We demonstrate that PtBGS is a vacuolar protein located in the tonoplast. Metabolite analyses of two mutant strains with reduced amounts of PtBGS reveal a reduction in their chrysolaminarin content and an increase of soluble sugars and lipids. This indicates that carbohydrates are shunted into alternative pathways when chrysolaminarin production is impaired. The mutant strains show reduced growth and lower photosynthetic capacities, while possessing higher photoprotective abilities than WT cells. Interestingly, a strong reduction in PtBGS expression also results in aberrations of the usually very regular thylakoid membrane patterns, including increased thylakoid thickness, reduced numbers of thylakoids per plastid, and increased numbers of lamellae per thylakoid stack. Our data demonstrate the complex intertwinement of carbohydrate storage in the vacuoles with carbohydrate metabolism, photosynthetic homeostasis, and plastid morphology.

Profiling carbohydrate composition, biohydrogen capacity, and disease resistance in potato

Background: Potato (Solanum tuberosum) is one of the most important sources of carbohydrates in human diet. Because of its high carbohydrate levels it recently has also received attention in biohydrogen production. To exploit the natural variation of potato with respect to resistance to major diseases, carbohydrate levels and composition, and capacity for biohydrogen production we analyzed tubers of native, improved, and genetically modified potatoes, and two other tuberous species for their glucose, fructose, sucrose, and starch content. Results: High-starch potato varieties were evaluated for their potential for Caldicellulosiruptor saccharolyticus-mediated biohydrogen production with Desirée and Rosita varieties delivering the highest biohydrogen amounts. Native line Vega1 and improved line Yagana were both immune to two isolates (A291, A287) of Phytophthora infestans. Conclusions: Our data demonstrate that native potato varieties might have great potential for further improving the multifaceted use of potato in worldwide food and biohydrogen production.

Uridine-ribohydrolase is a key regulator in the uridine degradation pathway of Arabidopsis.

Nucleoside degradation and salvage are important metabolic pathways but hardly understood in plants. Recent work on human pathogenic protozoans like Leishmania and Trypanosoma substantiates an essential function of nucleosidase activity. Plant nucleosidases are related to those from protozoans and connect the pathways of nucleoside degradation and salvage. Here, we describe the cloning of such an enzyme from Arabidopsis thaliana, Uridine-Ribohydrolase 1 (URH1) and the characterization by complementation of a yeast mutant. Furthermore, URH1 was synthesized as a recombinant protein in Escherichia coli. The pure recombinant protein exhibited highest hydrolase activity for uridine, followed by inosine and adenosine, the corresponding K(m) values were 0.8, 1.4, and 0.7 mM, respectively. In addition, URH1 was able to cleave the cytokinin derivative isopentenyladenine-riboside. Promoter beta-glucuronidase fusion studies revealed that URH1 is mainly transcribed in the vascular cells of roots and in root tips, guard cells, and pollen. Mutants expressing the Arabidopsis enzyme or the homolog from rice (Oryza sativa) exhibit resistance toward toxic fluorouridine, fluorouracil, and fluoroorotic acid, providing clear evidence for a pivotal function of URH1 as regulative in pyrimidine degradation. Moreover, mutants with increased and decreased nucleosidase activity are delayed in germination, indicating that this enzyme activity must be well balanced in the early phase of plant development.

Limitation of nocturnal import of ATP into Arabidopsis chloroplasts leads to photooxidative damage.

When grown in short day conditions and at low light, leaves of Arabidopsis plants with mutations in the genes encoding two plastidial ATP/ADP transporters (so-called null mutants) spontaneously develop necrotic lesions. Under these conditions, the mutants also display light-induced accumulation of H(2)O(2) and constitutive expression of genes for copper/zinc superoxide dismutase 2 and ascorbate peroxidase 1. In the light phase, null mutants accumulate high levels of phototoxic protoporphyrin IX but have only slightly reduced levels of Mg protoporphyrin IX. The physiological changes are associated with reduced magnesium-chelatase activity. Since the expression of genes encoding any of the three subunits of magnesium-chelatase is similar in wild type and null mutants, decreased enzyme activity is probably due to post-translational modification which might be due to limited availability of ATP in plastids during the night. Surprisingly, the formation of necrotic lesions was absent when null mutants were grown either in long days and low light intensity or in short days and high light intensity. We ascribe the lack of lesion phenotype to increased nocturnal ATP supply due to glycolytic degradation of starch which may lead to additional substrate-level phosphorylation in the stroma. Thus, nocturnal import of ATP into chloroplasts represents a crucial, previously unknown process that is required for controlled chlorophyll biosynthesis and for preventing photooxidative damage.

Molecular and biochemical analysis of periplastidial starch metabolism in the cryptophyte Guillardia theta.

Starch in synchronously grown Guillardia theta cells accumulates throughout the light phase, followed by a linear degradation during the night. In contrast to the case for other unicellular algae such as Chlamydomonas reinhardtii, no starch turnover occurred in this organism under continuous light. The gene encoding granule-bound starch synthase (GBSS1), the enzyme responsible for amylose synthesis, displays a diurnal expression cycle. The pattern consisted of a maximal transcript abundance around the middle of the light phase and a very low level during the night. This diurnal regulation of GBSS1 transcript abundance was demonstrated to be independent of the circadian clock but tightly light regulated. A similar yet opposite type of regulation pattern was found for two alpha-amylase isoforms and for one of the two plastidic triose phosphate transporter genes investigated. In these cases, however, the transcript abundance peaked in the night phase. The second plastidic triose phosphate transporter gene had the GBSS1 mRNA abundance pattern. Quantification of the GBSS1 activity revealed that not only gene expression but also total enzyme activity exhibited a maximum in the middle of the light phase. To gain a first insight into the transport processes involved in starch biosynthesis in cryptophytes, we demonstrated the presence of both plastidic triose phosphate transporter and plastidic ATP/ADP transporter activities in proteoliposomes harboring either total membranes or plastid envelope membranes from G. theta. These molecular and biochemical data are discussed with respect to the environmental conditions experienced by G. theta and with respect to the unique subcellular location of starch in cryptophytes.

Molecular physiological analysis of the two plastidic ATP/ADP transporters from Arabidopsis.

Arabidopsis (Arabidopsis thaliana) possesses two isoforms of plastidic ATP/ADP transporters (AtNTT1 and AtNTT2) exhibiting similar biochemical properties. To analyze the function of both isoforms on the molecular level, we examined the expression pattern of both genes by northern-blot analysis and promoter-beta-glucuronidase fusions. AtNTT1 represents a sugar-induced gene mainly expressed in stem and roots, whereas AtNTT2 is expressed in several Arabidopsis tissues with highest accumulation in developing roots and young cotyledons. Developing lipid-storing seeds hardly contained AtNTT1 or -2 transcripts. The absence of a functional AtNTT1 gene affected plant development only slightly, whereas AtNTT2T-DNA, AtNTT1-2T-DNA, and RNA interference (RNAi) plants showed retarded plant development, mainly characterized by a reduced ability to generate primary roots and a delayed chlorophyll accumulation in seedlings. Electron microscopic examination of chloroplast substructure also revealed an impaired formation of thylakoids in RNAi seedlings. Moreover, RNAi- and AtNTT1-2T-DNA plants showed reduced accumulation of the nuclear-encoded protein CP24 during deetiolation. Under short-day conditions reduced plastidic ATP import capacity correlates with a substantially reduced plant growth rate. This effect is absent under long-day conditions, strikingly indicating that nocturnal ATP import into chloroplasts is important. Plastidic ATP/ADP transport activity exerts significant control on lipid synthesis in developing Arabidopsis seeds. In total we made the surprising observation that plastidic ATP/ADP transport activity is not required to pass through the complete plant life cycle. However, plastidic ATP/ADP-transporter activity is required for both an undisturbed development of young tissues and a controlled cellular metabolism in mature leaves.

Enhanced resistance to Phytophthora infestans and Alternaria solani in leaves and tubers, respectively, of potato plants with decreased activity of the plastidic ATP/ADP transporter.

Recently, it has been reported that tubers of transgenic potato ( Solanum tuberosum L.) plants with decreased activity of the plastidic ATP/ADP transporter (AATP1) contain less starch, despite having an increased glucose level [P. Geigenberger et al. (2001) Plant Physiol 125:1667-1678]. The metabolic alterations correlated with enhanced resistance to the bacterium Erwinia carotovora. Here it is shown that transgenic potato tubers, possessing less starch yet increased glucose levels due to the expression of a cytoplasm-localized yeast invertase, exhibit drastic susceptibility to E. carotovora. In addition, it is demonstrated that AATP1 anti-sense tubers show an increased capacity to ward off the pathogenic fungus Alternaria solani. In contrast to AATP1 anti-sense tubers, the corresponding leaf tissue does not show changes in carbohydrate accumulation. However, upon elicitor treatment, AATP1 anti-sense leaves possess an increased capacity to release H(2)O(2) and activate various defence-related genes, reactions that are associated with substantially delayed appearance of disease symptoms caused by Phytophthora infestans. Grafting experiments between AATP1 anti-sense plants and wild-type plants indicate the presence of a signal that is generated in AATP1 rootstocks and primes wild-type scions for potentiated activation of cellular defence responses in leaves. Together, the results suggest that (i) the enhanced pathogen tolerance of AATP1 anti-sense tubers is not due to "high sugar resistance", (ii) the increased disease resistance of AATP1 anti-sense tubers is effective against different types of pathogen and (iii) a systemic signal induced by antisensing the plastidic ATP/ADP transporter in potato tubers confers increased resistance to pathogens.

Inhibition of the plastidic ATP/ADP transporter protein primes potato tubers for augmented elicitation of defense responses and enhances their resistance against Erwinia carotovora.

Tubers of transgenic potato (Solanum tuberosum) plants with decreased activity of the plastidic ATP/ADP transporter AATP1 display reduced levels of starch, modified tuber morphology, and altered concentrations of primary metabolites. Here, we demonstrate that the spontaneous production of hydrogen peroxide, the endogenous content of salicylic acid, and the levels of mRNAs of various defense-related genes are similar in tuber discs of wild-type and AATP1(St) antisense plants. However, upon challenging the tissue with fungal elicitors or culture supernatants of the soft rot-causing pathogen Erwinia carotovora subsp. atroseptica, the AATP1(St) antisense tubers exhibit highly potentiated activation of defense responses when compared with wild-type tissue. The augmented defense responses comprise enhanced accumulation of transcripts of five defense-related genes (beta-1,3-GLUCANASE B2 and A1, CHITINASE B3 and A2, and Phe AMMONIA-LYASE) and enhanced elicitation (up to 21-fold) of the early hydrogen peroxide burst. The potentiated activation of cellular defense responses in AATP1(St) antisense tubers is not accompanied by a precedent increase in endogenous salicylic acid levels, but is associated with a strongly enhanced resistance of the tissue to E. carotovora. From these results, we conclude that inhibition of primary metabolic reactions induces a primed state that sensitizes the potato tubers for improved elicitation of various cellular defense responses, which likely contribute to enhanced E. carotovora resistance.