Petronia Carillo

A Robot-Based Platform to Measure Multiple Enzyme Activities in Arabidopsis Using a Set of Cycling Assays: Comparison of Changes of Enzyme Activities and Transcript Levels during Diurnal Cycles and in Prolonged Darkness

A platform has been developed to measure the activity of 23 enzymes that are involved in central carbon and nitrogen metabolism in Arabidopsis thaliana. Activities are assayed in optimized stopped assays and the product then determined using a suite of enzyme cycling assays. The platform requires inexpensive equipment, is organized in a modular manner to optimize logistics, calculates results automatically, combines high sensitivity with throughput, can be robotized, and has a throughput of three to four activities in 100 samples per person/day. Several of the assays, including those for sucrose phosphate synthase, ADP glucose pyrophosphorylase (AGPase), ferredoxin-dependent glutamate synthase, glycerokinase, and shikimate dehydrogenase, provide large advantages over previous approaches. This platform was used to analyze the diurnal changes of enzyme activities in wild-type Columbia-0 (Col-0) and the starchless plastid phosphoglucomutase (pgm) mutant, and in Col-0 during a prolongation of the night. The changes of enzyme activities were compared with the changes of transcript levels determined with the Affymetrix ATH1 array. Changes of transcript levels typically led to strongly damped changes of enzyme activity. There was no relation between the amplitudes of the diurnal changes of transcript and enzyme activity. The largest diurnal changes in activity were found for AGPase and nitrate reductase. Examination of the data and comparison with the literature indicated that these are mainly because of posttranslational regulation. The changes of enzyme activity are also strongly delayed, with the delay varying from enzyme to enzyme. It is proposed that enzyme activities provide a quasi-stable integration of regulation at several levels and provide useful data for the characterization and diagnosis of different physiological states. As an illustration, a decision tree constructed using data from Col-0 during diurnal changes and a prolonged dark treatment was used to show that, irrespective of the time of harvest during the diurnal cycle, the pgm mutant resembles a wild-type plant that has been exposed to a 3 d prolongation of the night.

Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana

Tre6P (trehalose 6-phosphate) is implicated in sugar-signalling pathways in plants, but its exact functions in vivo are uncertain. One of the main obstacles to discovering these functions is the difficulty of measuring the amount of Tre6P in plant tissues. We have developed a highly specific assay, using liquid chromatography coupled to MS-Q3 (triple quadrupole MS), to measure Tre6P in the femto-picomole range. The Tre6P content of sucrose-starved Arabidopsis thaliana seedlings in axenic culture increased from 18 to 482 pmol x g(-1) FW (fresh weight) after adding sucrose. Leaves from soil-grown plants contained 67 pmol x g(-1) FW at the end of the night, which rose to 108 pmol x g(-1)FW after 4 h of illumination. Even greater changes in Tre6P content were seen after a 6 h extension of the dark period, and in the starchless mutant, pgm. The intracellular concentration of Tre6P in wild-type leaves was estimated to range from 1 to 15 microM. It has recently been reported that the addition of Tre6P to isolated chloroplasts leads to redox activation of AGPase (ADPglucose pyrophosphorylase) [Kolbe, Tiessen, Schluepmann, Paul, Ulrich and Geigenberger (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 11118-11123]. Using the new assay for Tre6P, we found that rising sugar levels in plants are accompanied by increases in the level of Tre6P, redox activation of AGPase and the stimulation of starch synthesis in vivo. These results indicate that Tre6P acts as a signalling metabolite of sugar status in plants, and support the proposal that Tre6P mediates sucrose-induced changes in the rate of starch synthesis.

Adjustment of growth and central metabolism to a mild but sustained nitrogen-limitation in Arabidopsis

We have established a simple soil-based experimental system that allows a small and sustained restriction of growth of Arabidopsis by low nitrogen (N). Plants were grown in a large volume of a peat-vermiculite mix that contained very low levels of inorganic N. As a control, inorganic N was added in solid form to the peat-vermiculite mix, or plants were grown in conventional nutrient-rich solids. The low N growth regime led to a sustained 20% decrease of the relative growth rate over a period of 2 weeks, resulting in a two- to threefold decrease in biomass in 35- to 40-day-old plants. Plants in the low N regime contained lower levels of nitrate, lower nitrate reductase activity, lower levels of malate, fumarate and other organic acids and slightly higher levels of starch, as expected from published studies of N-limited plants. However, their rosette protein content was unaltered, and total and many individual amino acid levels increased compared with N-replete plants. This metabolic phenotype reveals that Arabidopsis responds adaptively to low N by decreasing the rate of growth, while maintaining the overall protein content, and maintaining or even increasing the levels of many amino acids.

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

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.

Salinity Stress and Salt Tolerance

Salinity is one of the most serious factors limiting the productivity of agricultural crops, with adverse effects on germination, plant vigour and crop yield (R Munns & Tester, 2008). Salinization affects many irrigated areas mainly due to the use of brackish water. Worldwide, more than 45 million hectares of irrigated land have been damaged by salt, and 1.5 million hectares are taken out of production each year as a result of high salinity levels in the soil (R Munns & Tester, 2008). High salinity affects plants in several ways: water stress, ion toxicity, nutritional disorders, oxidative stress, alteration of metabolic processes, membrane disorganization, reduction of cell division and expansion, genotoxicity (Hasegawa, Bressan, Zhu, & Bohnert, 2000; R. Munns, 2002; Zhu, 2007). Together, these effects reduce plant growth, development and survival. During the onset and development of salt stress within a plant, all the major processes such as photosynthesis, protein synthesis and energy and lipid metabolism are affected (Parida & Das, 2005). During initial exposure to salinity, plants experience water stress, which in turn reduces leaf expansion. The osmotic effects of salinity stress can be observed immediately after salt application and are believed to continue for the duration of exposure, resulting in inhibited cell expansion and cell division, as well as stomatal closure (T. J. Flowers, 2004; R. Munns, 2002). During long-term exposure to salinity, plants experience ionic stress, which can lead to premature senescence of adult leaves, and thus a reduction in the photosynthetic area available to support continued growth (Cramer & Nowak, 1992). In fact, excess sodium and more importantly chloride has the potential to affect plant enzymes and cause cell swelling, resulting in reduced energy production and other physiological changes (Larcher 1980). Ionic stress results in premature senescence of older leaves and in toxicity symptoms (chlorosis, necrosis) in mature leaves due to high Na+ which affects plants by disrupting protein synthesis and interfering with enzyme activity (Hasegawa, Bressan, Zhu, & Bohnert, 2000; R. Munns, 2002; R Munns & Termaat, 1986). Many plants have evolved several mechanisms either to exclude salt from their cells or to tolerate its presence within the cells. In this chapter, we mainly discuss about soil salinity, its effects on plants and tolerance mechanisms which permit the plants to withstand stress, with particular emphasis on ion homeostasis, Na+ exclusion and tissue tolerance. Moreover we give a synthetic overview of the two major approaches that have been used to improve stress tolerance: exploitation of natural genetic variations and generation of transgenic plants with novel genes or altered expression levels of the existing genes. A fundamental biological understanding and knowledge of the effects of salt stress on plants is necessary to provide additional

A fluorometric assay for trehalose in the picomole range

BackgroundTrehalose is a non-reducing disaccharide that is used as an osmolyte, transport sugar, carbon reserve and stress protectant in a wide range of organisms. In plants, trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is thought to be a signal of sucrose status. Trehalose itself may play a role in pathogenic and symbiotic plant-microbe interactions, in responses to abiotic stress and in developmental signalling, but its precise functions are unknown. A major obstacle to investigating its function is the technical difficulty of measuring the very low levels of trehalose usually found in plant tissues, as most of the established trehalose assays lack sufficient specificity and/or sensitivity.ResultsA kinetic assay for trehalose was established using recombinant Escherichia coli cytoplasmic trehalase (treF), which was shown to be highly specific for trehalose. Hydrolysis of trehalose to glucose is monitored fluorometrically and the trehalose content of the tissue extract is determined from an internal calibration curve. The assay is linear for 0.2-40 pmol trehalose, and recoveries of trehalose were ≥88%. A. thaliana Col-0 rosettes contain about 20–30 nmol g-1FW of trehalose, increasing to about 50–60 nmol g-1FW in plants grown at 8°C. Trehalose is not correlated with sucrose content, whereas a strong correlation between Tre6P and sucrose was confirmed. The trehalose contents of ear inflorescence primordia from the maize ramosa3 mutant and wild type plants were 6.6±2.6 nmol g-1FW and 19.0±12.7 nmol g-1FW, respectively. The trehalose:Tre6P ratios in the ramosa3 and wild-type primordia were 2.43±0.85 and 6.16±3.45, respectively.ConclusionThe fluorometric assay is highly specific for trehalose and sensitive enough to measure the trehalose content of very small amounts of plant tissue. Chilling induced a 2-fold accumulation of trehalose in A. thaliana rosettes, but the levels were too low to make a substantial quantitative contribution to osmoregulation. Trehalose is unlikely to function as a signal of sucrose status. The abnormal inflorescence branching phenotype of the maize ramosa3 mutant might be linked to a decrease in trehalose levels in the inflorescence primordia or a downward shift in the trehalose:Tre6P ratio.

Durum wheat seedling responses to simultaneous high light and salinity involve a fine reconfiguration of amino acids and carbohydrate metabolism

Durum wheat plants are extremely sensitive to drought and salinity during seedling and early development stages. Their responses to stresses have been extensively studied to provide new metabolic targets and improving the tolerance to adverse environments. Most of these studies have been performed in growth chambers under low light [300-350 µmol m-2 s-1 photosynthetically active radiation (PAR), LL]. However, in nature plants have to face frequent fluctuations of light intensities that often exceed their photosynthetic capacity (900-2000 µmol m-2 s-1 ). In this study we investigated the physiological and metabolic changes potentially involved in osmotic adjustment and antioxidant defense in durum wheat seedlings under high light (HL) and salinity. The combined application of the two stresses decreased the water potential and stomatal conductance without reducing the photosynthetic efficiency of the plants. Glycine betaine (GB) synthesis was inhibited, proline and glutamate content decreased, while γ-aminobutyric acid (GABA), amides and minor amino acids increased. The expression level and enzymatic activities of Δ1-pyrroline-5-carboxylate synthetase, asparagine synthetase and glutamate decarboxylase, as well as other enzymatic activities of nitrogen and carbon metabolism, were analyzed. Antioxidant enzymes and metabolites were also considered. The results showed that the complex interplay seen in durum wheat plants under salinity at LL was simplified: GB and antioxidants did not play a main role. On the contrary, the fine tuning of few specific primary metabolites (GABA, amides, minor amino acids and hexoses) remodeled metabolism and defense processes, playing a key role in the response to simultaneous stresses.

Polymorphism of a new Ty1-copia retrotransposon in durum wheat under salt and light stresses

Long terminal repeat retrotransposons are the most abundant mobile elements in the plant genome and play an important role in the genome reorganization induced by environmental challenges. Their success depends on the ability of their promoters to respond to different signaling pathways that regulate plant adaptation to biotic and abiotic stresses. We have isolated a new Ty1-copia-like retrotransposon, named Ttd1a from the Triticum durum L. genome. To get insight into stress activation pathways in Ttd1a, we investigated the effect of salt and light stresses by RT-PCR and S-SAP profiling. We screened for Ttd1a insertion polymorphisms in plants grown to stress and showed that one new insertion was located near the resistance gene. Our analysis showed that the activation and mobilization of Ttd1a was controlled by salt and light stresses, which strengthened the hypothesis that stress mobilization of this element might play a role in the defense response to environmental stresses.

Durum Wheat Roots Adapt to Salinity Remodeling the Cellular Content of Nitrogen Metabolites and Sucrose

Plants are currently experiencing increasing salinity problems due to irrigation with brackish water. Moreover, in fields, roots can grow in soils which show spatial variation in water content and salt concentration, also because of the type of irrigation. Salinity impairs crop growth and productivity by inhibiting many physiological and metabolic processes, in particular nitrate uptake, translocation, and assimilation. Salinity determines an increase of sap osmolality from about 305 mOsmol kg−1 in control roots to about 530 mOsmol kg−1 in roots under salinity. Root cells adapt to salinity by sequestering sodium in the vacuole, as a cheap osmoticum, and showing a rearrangement of few nitrogen-containing metabolites and sucrose in the cytosol, both for osmotic adjustment and oxidative stress protection, thus providing plant viability even at low nitrate levels. Mainly glycine betaine and sucrose at low nitrate concentration, and glycine betaine, asparagine and proline at high nitrate levels can be assumed responsible for the osmotic adjustment of the cytosol, the assimilation of the excess of ammonium and the scavenging of ROS under salinity. High nitrate plants with half of the root system under salinity accumulate proline and glutamine in both control and salt stressed split roots, revealing that osmotic adjustment is not a regional effect in plants. The expression level and enzymatic activities of asparagine synthetase and Δ1-pyrroline-5-carboxylate synthetase, as well as other enzymatic activities of nitrogen and carbon metabolism, are analyzed.

Ttd1a promoter is involved in DNA–protein binding by salt and light stresses

Stress modulation of retrotransposons may play a role in generating host genetic plasticity in response to environmental stress. Transposable elements have been suggested to contribute to the evolution of genes, by providing cis-regulatory elements leading to changes in expression patterns. Indeed, their promoter elements are similar to those of plant defence genes and may bind similar defence-induced transcription factors. We previously isolated a new Ty1-copia retrontrasposon named Ttd1a and showed its activation and mobilization in salt and light stresses. Here, using a retard mobility assay in Triticum durum L. crude extracts, we showed that the CAAT motif present in the Ttd1a retrotransposon promoter, is involved in DNA–protein binding under salt and light stresses and therefore in the regulation of Ttd1a activity. Data presented in this paper suggest that nuclear proteins can interact with the CAAT motif either directly or indirectly and enhance Ttd1a by a specific ligand-dependent activation under stress.