David Riewe

The Potato-Specific Apyrase Is Apoplastically Localized and Has Influence on Gene Expression, Growth, and Development

Apyrases hydrolyze nucleoside triphosphates and diphosphates and are found in all eukaryotes and a few prokaryotes. Although their enzymatic properties have been well characterized, relatively little is known regarding their subcellular localization and physiological function in plants. In this study, we used reverse genetic and biochemical approaches to investigate the role of potato (Solanum tuberosum)-specific apyrase. Silencing of the apyrase gene family with RNA interference constructs under the control of the constitutive 35S promoter led to a strong decrease in apyrase activity to below 10% of the wild-type level. This decreased activity led to phenotypic changes in the transgenic lines, including a general retardation in growth, an increase in tuber number per plant, and differences in tuber morphology. Silencing of apyrase under the control of a tuber-specific promoter led to similar changes in tuber morphology; however, there were no direct effects of apyrase inhibition on tuber metabolism. DNA microarrays revealed that decreased expression of apyrase leads to increased levels of transcripts coding for cell wall proteins involved in growth and genes involved in energy transfer and starch synthesis. To place these results in context, we determined the subcellular localization of the potato-specific apyrase. Using a combination of approaches, we were able to demonstrate that this enzyme is localized to the apoplast. We describe the evidence that underlies both this fact and that potato-specific apyrase has a crucial role in regulating growth and development.

A tyrosine aminotransferase involved in tocopherol synthesis in Arabidopsis

The metabolic function of the predicted Arabidopsis tyrosine aminotransferase (TAT) encoded by the At5g53970 gene was studied using two independent knock-out mutants. Gas chromatography-mass spectrometry based metabolic profiling revealed a specific increase in tyrosine levels, supporting the proposed function of At5g53970 as a tyrosine-specific aminotransferase not involved in tyrosine biosynthesis, but rather in utilization of tyrosine for other metabolic pathways. The TAT activity of the At5g53970-encoded protein was verified by complementation of the Escherichia coli tyrosine auxotrophic mutant DL39, and in vitro activity of recombinantly expressed and purified At5g53970 was found to be specific for tyrosine. To investigate the physiological role of At5g53970, the consequences of reduction in tyrosine utilization on metabolic pathways having tyrosine as a substrate were analysed. We found that tocopherols were substantially reduced in the mutants and we conclude that At5g53970 encodes a TAT important for the synthesis of tocopherols in Arabidopsis.

The central regulation of plant physiology by adenylates.

There have been many recent developments concerning the metabolic, transport and signalling functions of adenylates in plants, suggesting new roles for these compounds as central regulators of plant physiology. For example, altering the expression levels of enzymes involved in the equilibration, salvaging, synthesis and transport of adenylates leads to perturbations in storage, growth and stress responses, implying a role for adenylates as important signals. Furthermore, sensing of the internal energy status involves SNF1-related kinases, which control the expression and phosphorylation of key metabolic enzymes. ATP also acts as an apoplastic signalling molecule to control cell growth and pathogen responses. These new results could shed light on the emerging question of whether energy homeostasis in plant cells differs from mechanisms found in microbes and mammals.

Phenotypic and metabolic responses to drought and salinity of four contrasting lentil accessions

Highlight Automated imaging-based plant phenotyping combined with GC-MS-based metabolite profiling of four lentil accessions differing in their drought and salt tolerance shows common and specific responses and yields characteristic stress markers

Differentiation of endosperm transfer cells of barley: a comprehensive analysis at the micro-scale.

Barley endosperm cells differentiate into transfer cells (ETCs) opposite the nucellar projection. To comprehensively analyse ETC differentiation, laser microdissection-based transcript and metabolite profiles were obtained from laser microdissected tissues and cell morphology was analysed. Flange-like secondary-wall ingrowths appeared between 5 and 7 days after pollination within the three outermost cell layers. Gene expression analysis indicated that ethylene-signalling pathways initiate ETC morphology. This is accompanied by gene activity related to cell shape control and vesicle transport, with abundant mitochondria and endomembrane structures. Gene expression analyses indicate predominant formation of hemicelluloses, glucuronoxylans and arabinoxylans, and transient formation of callose, together with proline and 4-hydroxyproline biosynthesis. Activation of the methylation cycle is probably required for biosynthesis of phospholipids, pectins and ethylene. Membrane microdomains involving sterols/sphingolipids and remorins are potentially involved in ETC development. The transcriptional activity of assimilate and micronutrient transporters suggests ETCs as the main uptake organs of solutes into the endosperm. Accordingly, the endosperm grows maximally after ETCs are fully developed. Up-regulated gene expression related to amino acid catabolism, C:N balances, carbohydrate oxidation, mitochondrial activity and starch degradation meets high demands for respiratory energy and carbohydrates, required for cell proliferation and wall synthesis. At 10 days after pollination, ETCs undergo further differentiation, potentially initiated by abscisic acid, and metabolism is reprogrammed as shown by activated storage and stress-related processes. Overall, the data provide a comprehensive view of barley ETC differentiation and development, and identify candidate genes and associated pathways.

Identification of enzymatic and regulatory genes of plant metabolism through QTL analysis in Arabidopsis.

The biochemical diversity in the plant kingdom is estimated to well exceed 100,000 distinct compounds (Weckwerth, 2003) and 4000 to 20,000 metabolites per species seem likely (Fernie et al., 2004). In recent years extensive progress has been made towards the identification of enzymes and regulatory genes working in a complex network to generate this large arsenal of metabolites. Genetic loci influencing quantitative traits, e.g. metabolites or biomass, may be mapped to associated molecular markers, a method called quantitative trait locus mapping (QTL mapping), which may facilitate the identification of novel genes in biochemical pathways. Arabidopsis thaliana, as a model organism for seed plants, is a suitable target for metabolic QTL (mQTL) studies due to the availability of highly developed molecular and genetic tools, and the extensive knowledge accumulated on the metabolite profile. While intensely studied, in particular since the availability of its complete sequence, the genome of Arabidopsis still comprises a large proportion of genes with only tentative function based on sequence homology. From a total number of 33,518 genes currently listed (TAIR 9, http://www.arabidopsis.org), only about 25% have direct experimental evidence for their molecular function and biological process, while for more than 30% no biological data are available. Modern metabolomics approaches together with continually extended genomic resources will facilitate the task of assigning functions to those genes. In our previous study we reported on the identification of mQTL (Lisec et al., 2008). In this paper, we summarize the current status of mQTL analyses and causal gene identification in Arabidopsis and present evidence that a candidate gene located within the confidence interval of a fumarate mQTL (AT5G50950) encoding a putative fumarase is likely to be the causal gene of this QTL. The total number of genes molecularly identified based on mQTL studies is still limited, but the advent of multi-parallel analysis techniques for measurement of gene expression, as well as protein and metabolite abundances and for rapid gene identification will assist in the important task of assigning enzymes and regulatory genes to the growing network of known metabolic reactions.

Metabolic and Developmental Adaptations of Growing Potato Tubers in Response to Specific Manipulations of the Adenylate Energy Status

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.

Metabolic and transcriptional transitions in barley glumes reveal a role as transitory resource buffers during endosperm filling

Highlight The development and metabolism of barley glumes is tightly associated with grain filling and filial sink strength, which coordinate developmental phase changes in the glumes via metabolic, hormonal, and transcriptional control.

The production of male-sterile wheat plants through split barnase expression is promoted by the insertion of introns and flexible peptide linkers

The successful use of transgenic plants depends on the strong and stable expression of the heterologous genes. In this study, three introns (PSK7-i1 and PSK7-i3 from Petunia and UBQ10-i1 from Arabidopsis) were tested for their ability to enhance the tapetum-specific expression of a split barnase transgene. We also analyzed the effects of introducing multiple copies of flexible peptide linkers that bridged the fusion domains of the assembled protein. The barnase fragments were assembled into a functional cytotoxin via intein-mediated trans-splicing, thus leading to male sterility through pollen ablation. A total of 14 constructs carrying different combinations of introns and peptide linkers were transformed into wheat plants. The resulting populations (between 41 and 301 independent plants for each construct) were assayed for trait formation. Depending on which construct was used, there was an increase of up to fivefold in the proportion of plants exhibiting male sterility compared to the populations harboring unmodified constructs. Furthermore, the average barnase copy number in the plants displaying male sterility could be reduced. The metabolic profiles of male-sterile transgenic plants and non-transgenic plants were compared using gas chromatography–mass spectrometry. The profiles generated from leaf tissues displayed no differences, thus corroborating the anther specificity of barnase expression. The technical advances achieved in this study may be a valuable contribution for future improvement of transgenic crop systems.

Structure annotation and quantification of wheat seed oxidized lipids by high resolution LC-MS/MS

Acyl composition annotation and quantification of hundreds of oxidized and nonoxidized lipids in naturally aged wheat seeds by high-resolution LC-MS/MS reveals enhanced lipid oxidation at ambient versus cold conditions. Lipid oxidation is a process ubiquitous in life, but the direct and comprehensive analysis of oxidized lipids has been limited by available analytical methods. We applied high-resolution liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) to quantify oxidized lipids (glycerides, fatty acids, phospholipids, lysophospholipids, and galactolipids) and implemented a platform-independent high-throughput-amenable analysis pipeline for the high-confidence annotation and acyl composition analysis of oxidized lipids. Lipid contents of 90 different naturally aged wheat (Triticum aestivum) seed stocks were quantified in an untargeted high-resolution LC-MS experiment, resulting in 18,556 quantitative mass-to-charge ratio features. In a posthoc liquid chromatography-tandem mass spectrometry experiment, high-resolution MS/MS spectra (5 mD accuracy) were recorded for 8,957 out of 12,080 putatively monoisotopic features of the LC-MS data set. A total of 353 nonoxidized and 559 oxidized lipids with up to four additional oxygen atoms were annotated based on the accurate mass recordings (1.5 ppm tolerance) of the LC-MS data set and filtering procedures. MS/MS spectra available for 828 of these annotations were analyzed by translating experimentally known fragmentation rules of lipids into the fragmentation of oxidized lipids. This led to the identification of 259 nonoxidized and 365 oxidized lipids by both accurate mass and MS/MS spectra and to the determination of acyl compositions for 221 nonoxidized and 295 oxidized lipids. Analysis of 15-year aged wheat seeds revealed increased lipid oxidation and hydrolysis in seeds stored in ambient versus cold conditions.