Rosa Morcuende

Genome-Wide Reprogramming of Primary and Secondary Metabolism, Protein Synthesis, Cellular Growth Processes, and the Regulatory Infrastructure of Arabidopsis in Response to Nitrogen

Transcriptome analysis, using Affymetrix ATH1 arrays and a real-time reverse transcription-PCR platform for >1,400 transcription factors, was performed to identify processes affected by long-term nitrogen-deprivation or short-term nitrate nutrition in Arabidopsis. Two days of nitrogen deprivation led to coordinate repression of the majority of the genes assigned to photosynthesis, chlorophyll synthesis, plastid protein synthesis, induction of many genes for secondary metabolism, and reprogramming of mitochondrial electron transport. Nitrate readdition led to rapid, widespread, and coordinated changes. Multiple genes for the uptake and reduction of nitrate, the generation of reducing equivalents, and organic acid skeletons were induced within 30 min, before primary metabolites changed significantly. By 3 h, most genes assigned to amino acid and nucleotide biosynthesis and scavenging were induced, while most genes assigned to amino acid and nucleotide breakdown were repressed. There was coordinate induction of many genes assigned to RNA synthesis and processing and most of the genes assigned to amino acid activation and protein synthesis. Although amino acids involved in central metabolism increased, minor amino acids decreased, providing independent evidence for the activation of protein synthesis. Specific genes encoding expansin and tonoplast intrinsic proteins were induced, indicating activation of cell expansion and growth in response to nitrate nutrition. There were rapid responses in the expression of many genes potentially involved in regulation, including genes for trehalose metabolism and hormone metabolism, protein kinases and phosphatases, receptor kinases, and transcription factors.

Sugars and circadian regulation make major contributions to the global regulation of diurnal gene expression in Arabidopsis.

The diurnal cycle strongly influences many plant metabolic and physiological processes. Arabidopsis thaliana rosettes were harvested six times during 12-h-light/12-h-dark treatments to investigate changes in gene expression using ATH1 arrays. Diagnostic gene sets were identified from published or in-house expression profiles of the response to light, sugar, nitrogen, and water deficit in seedlings and 4 h of darkness or illumination at ambient or compensation point [CO2]. Many sugar-responsive genes showed large diurnal expression changes, whose timing matched that of the diurnal changes of sugars. A set of circadian-regulated genes also showed large diurnal changes in expression. Comparison of published results from a free-running cycle with the diurnal changes in Columbia-0 (Col-0) and the starchless phosphoglucomutase (pgm) mutant indicated that sugars modify the expression of up to half of the clock-regulated genes. Principle component analysis identified genes that make large contributions to diurnal changes and confirmed that sugar and circadian regulation are the major inputs in Col-0 but that sugars dominate the response in pgm. Most of the changes in pgm are triggered by low sugar levels during the night rather than high levels in the light, highlighting the importance of responses to low sugar in diurnal gene regulation. We identified a set of candidate regulatory genes that show robust responses to alterations in sugar levels and change markedly during the diurnal cycle.

Extension of the visualization tool mapman to allow statistical analysis of arrays, display of coresponding genes, and comparison with known responses

MapMan is a user-driven tool that displays large genomics datasets onto diagrams of metabolic pathways or other processes. Here, we present new developments, including improvements of the gene assignments and the user interface, a strategy to visualize multilayered datasets, the incorporation of statistics packages, and extensions of the software to incorporate more biological information including visualization of coresponding genes and horizontal searches for similar global responses across large numbers of arrays.

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.

Sucrose-feeding leads to increased rates of nitrate assimilation, increased rates of alpha-oxoglutarate synthesis, and increased synthesis of a wide spectrum of amino acids in tobacco leaves

Abstract. To investigate the importance of the sugar supply for the regulation of nitrogen and organic acid metabolism, various sugars and nitrogenous compounds were supplied for 8 h to detached tobacco leaves in low light. (i) In control leaves supplied with water, there was a large decrease of the Nia transcript level, a 50% decline of nitrate reductase (NR) activity, starch increased and sugars remained low, nitrate decreased by 50%, and amino acids increased only slightly during the 8 h incubation. About half of the nitrogen accumulating in amino acids was present in glutamine (Gln). (ii) When 25 mM sucrose was supplied, the in-vivo rate of nitrate assimilation (estimated from the accumulation of ammonium and amino acids) increased 2-fold. The Nia transcript level still decreased, but the decline of NR activity was less pronounced and NR activation was increased. The in-vivo net rate of ammonium assimilation (estimated from the accumulation of amino acids) also doubled after feeding sucrose. Ammonium and glutamate (Glu) decreased and Gln rose markedly, showing that in-vivo activity of glutamine synthetase had been stimulated. Glutamine still accounted for about half of the nitrogen, indicating that sucrose does not selectively stimulate glutamine synthase. Glutamate and aspartate decreased and all the minor amino acids increased, showing that the amino acid biosynthesis pathways are activated by sucrose. There was a decrease of 3-phosphoglycerate (3PGA) and phosphoenolpyruvate (PEP) and a large increase of α-oxoglutarate, showing that the flow of carbon from glycolysis into organic acids has been stimulated by sucrose. (iii) The changes of 3PGA, PEP, α-oxoglutarate, Glu, aspartate and the minor amino acids were smaller when 50 mM glucose was supplied, even though the internal levels of sugars at the end of the incubation resembled those found after feeding 25 mM sucrose. This indicates that the signals that regulate nitrogen and respiratory metabolism are derived from the uptake or metabolism of sucrose, rather than glucose. (iv) A different spectrum of changes was found when 20 mM nitrate was supplied. The estimated rate of nitrate assimilation increased 2-fold, and this was accompanied by an increase of NR activity but not of NR activation. Nitrate-feeding did not lead to a decrease of Glu, and the increase of minor amino acids was slightly smaller than with sucrose. There was a decrease of sugars, starch, and hexose phosphates, but 3PGA and PEP were not significantly decreased and isocitrate increased instead of α-oxoglutarate. (v) A different spectrum of changes was also found when 10 mM Gln was supplied. The estimated rate of nitrate assimilation decreased, and this was accompanied by a decrease of NR activity and NR activation. Glutamate did not decrease, and the increase of minor amino acids was smaller than with sucrose. Starch and sugars remained high and, although hexose phosphates decreased, 3PGA and PEP were not significantly decreased. Isocitrate remained unaltered and the increase of α-oxoglutarate was smaller than after supplying sucrose. (vi) When 25 mM sucrose was added together with 20 mM nitrate or 10 mM Gln, the effect on NR activity, NR activation and the estimated rate of nitrate assimilation was additive to the effect of nitrate, and antagonistic to the effect of Gln. Sucrose still led to a decrease of Glu, an increase of the minor amino acids, a decrease of 3PGA and PEP, and an increase of α-oxoglutarate when it was supplied together with nitrate or Gln. (vii) It is concluded that sucrose initiates a co-ordinate activation of nitrate assimilation, ammonium assimilation, amino acid biosynthesis, and α-oxoglutarate synthesis. Sucrose acts in concert with nitrate and antagonistically to Gln to increase NR activity and nitrate assimilation, and complements the action of nitrate and Gln to increase the flow of nitrogen from ammonium into amino acids, and to increase α-oxoglutarate formation.

Does ear C sink strength contribute to overcoming photosynthetic acclimation of wheat plants exposed to elevated CO2

Wheat plants (Triticum durum Desf., cv. Regallo) were grown in the field to study the effects of contrasting [CO2] conditions (700 versus 370 μmol mol−1) on growth, photosynthetic performance, and C management during the post-anthesis period. The aim was to test whether a restricted capacity of sink organs to utilize photosynthates drives a loss of photosynthetic capacity in elevated CO2. The ambient 13C/12C isotopic composition (δ13C) of air CO2 was changed from –10.2‰ in ambient [CO2] to –23.6‰ under elevated [CO2] between the 7th and the 14th days after anthesis in order to study C assimilation and partitioning between leaves and ears. Elevated [CO2] had no significant effect on biomass production and grain filling, and caused an accumulation of C compounds in leaves. This was accompanied by up-regulation of phosphoglycerate mutase and ATP synthase protein content, together with down-regulation of adenosine diphosphate glucose pyrophosphatase protein. Growth in elevated [CO2] negatively affected Rubisco and Rubisco activase protein content and induced photosynthetic down-regulation. CO2 enrichment caused a specific decrease in Rubisco content, together with decreases in the amino acid and total N content of leaves. The C labelling revealed that in flag leaves, part of the C fixed during grain filling was stored as starch and structural C compounds whereas the rest of the labelled C (mainly in the form of soluble sugars) was completely respired 48 h after the end of labelling. Although labelled C was not detected in the δ13C of ear total organic matter and respired CO2, soluble sugar δ13C revealed that a small amount of labelled C reached the ear. The 12CO2 labelling suggests that during the beginning of post-anthesis the ear did not contribute towards overcoming flag leaf carbohydrate accumulation, and this had a consequent effect on protein expression and photosynthetic acclimation.

Future CO2 concentrations, though not warmer temperatures, enhance wheat photosynthesis temperature responses.

The temperature dependence of C3 photosynthesis is known to vary according to the growth environment. Atmospheric CO2 concentration and temperature are predicted to increase with climate change. To test whether long-term growth in elevated CO2 and temperature modifies photosynthesis temperature response, wheat (Triticum aestivum L.) was grown in ambient CO2 (370 micromol mol(-1)) and elevated CO2 (700 micromol mol(-1)) combined with ambient temperatures and 4 degrees C warmer ones, using temperature gradient chambers in the field. Flag leaf photosynthesis was measured at temperatures ranging from 20 to 35 degrees C and varying CO2 concentrations between ear emergence and anthesis. The maximum rate of carboxylation was determined in vitro in the first year of the experiment and from the photosynthesis-intercellular CO2 response in the second year. With measurement CO2 concentrations of 330 micromol mol(-1) or lower, growth temperature had no effect on flag leaf photosynthesis in plants grown in ambient CO2, while it increased photosynthesis in elevated growth CO2. However, warmer growth temperatures did not modify the response of photosynthesis to measurement temperatures from 20 to 35 degrees C. A central finding of this study was that the increase with temperature in photosynthesis and the photosynthesis temperature optimum were significantly higher in plants grown in elevated rather than ambient CO2. In association with this, growth in elevated CO2 increased the temperature response (activation energy) of the maximum rate of carboxylation. The results provide field evidence that growth under CO2 enrichment enhances the response of Rubisco activity to temperature in wheat.

Transcriptome and metabolome analysis of plant sulfate starvation and resupply provides novel information on transcriptional regulation of metabolism associated with sulfur, nitrogen and phosphorus nutritional responses in Arabidopsis

Sulfur is an essential macronutrient for plant growth and development. Reaching a thorough understanding of the molecular basis for changes in plant metabolism depending on the sulfur-nutritional status at the systems level will advance our basic knowledge and help target future crop improvement. Although the transcriptional responses induced by sulfate starvation have been studied in the past, knowledge of the regulation of sulfur metabolism is still fragmentary. This work focuses on the discovery of candidates for regulatory genes such as transcription factors (TFs) using ‘omics technologies. For this purpose a short term sulfate-starvation/re-supply approach was used. ATH1 microarray studies and metabolite determinations yielded 21 TFs which responded more than 2-fold at the transcriptional level to sulfate starvation. Categorization by response behaviors under sulfate-starvation/re-supply and other nutrient starvations such as nitrate and phosphate allowed determination of whether the TF genes are specific for or common between distinct mineral nutrient depletions. Extending this co-behavior analysis to the whole transcriptome data set enabled prediction of putative downstream genes. Additionally, combinations of transcriptome and metabolome data allowed identification of relationships between TFs and downstream responses, namely, expression changes in biosynthetic genes and subsequent metabolic responses. Effect chains on glucosinolate and polyamine biosynthesis are discussed in detail. The knowledge gained from this study provides a blueprint for an integrated analysis of transcriptomics and metabolomics and application for the identification of uncharacterized genes.

Elevated CO2 and temperature differentially affect photosynthesis and resource allocation in flag and penultimate leaves of wheat

Differences in acclimation to elevated growth CO2 (700 µmol mol−1, EC) and elevated temperature (ambient +4 °C, ET) in successive leaves of wheat were investigated in field chambers. At a common measurement CO2, EC increased photosynthesis and the quantum yield of electron transport (Φ) early on in the growth of penultimate leaves, and later decreased them. In contrast, EC did not change photosynthesis, and increased Φ at later growth stages in the flag leaf. Contents of chlorophyll (Chl), ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO), and total soluble protein were initially higher and subsequently lower in penultimate than flag leaves. EC decreased RuBPCO protein content relative to soluble protein and Chl contents throughout the development of penultimate leaves. On the other hand, EC initially increased the RuBPCO:Chl and Chl a/b ratios, but later decreased them in flag leaves. In the flag leaves but not in the penultimate leaves, ET initially decreased initial and specific RuBPCO activities at ambient CO2 (AC) and increased them at EC. Late in leaf growth, ET decreased Chl contents under AC in both kinds of leaves, and had no effect or a positive one under EC. Thus the differences between the two kinds of leaves were due to resource availability, and to EC-increased allocation of resources to photon harvesting in the penultimate leaves, but to increased allocation to carboxylation early on in growth, and to light harvesting subsequently, in the flag leaves.

Short-term feedback inhibition of photosynthesis in wheat leaves supplied with sucrose and glycerol at two temperatures

AbstractThe inhibition of photosynthesis by reduced sink demand or low rates of end product synthesis was investigated by supplying detached wheat (Triticum aestivum L. cv. Tauro) leaves with 50 mM sucrose, 50 mM glycerol or water through the transpiration stream for 2 h, either at 23 or 12 °C. Lowering the temperature and sucrose and glycerol feeding decreased photosynthetic oxygen evolution at high irradiance and saturating CO2. The decrease in temperature reduced the pools of sucrose and starch, and the ratio glucose 6-phosphate (G6P)/fructose 6-phosphate (F6P), while it increased the concentrations of G6P and F6P (hexose phosphates). Sucrose feeding, in contrast to glycerol feeding, increased sucrose, glucose and fructose contents and the G6P/F6P ratio. Sucrose and glycerol incubations at 23 °C, as well as decreasing the temperature in leaves incubated in water, increased the concentration of triose-phosphates (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, TP) and decreased the glycerate 3-phosphate (PGA) content, thus increasing the TP/PGA ratio; they also tended to increase the ribulose 1,5-bisphosphate (RuBP) content and the RuBP/PGA ratio. Sucrose and glycerol feeding at 12 °C and the decrease in temperature of leaves incubated in these solutions decreased TP and RuBP contents and the TP/PGA and RuBP/PGA ratios. The results suggest that the phosphate limitation caused by accumulation of end products, restriction of their synthesis and sequestration of cytosolic phosphate can inhibit photosynthesis through decreased carboxylation of RuBP or, with increased phosphate limitation, through lowered supply of ATP.