Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with g-32 P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated 32 P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granuleassociated phosphoproteins after incubation of intact amyloplasts with g-32 P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein-protein interactions in the control of starch anabolism and catabolism.

Poaceae represent the most important group of crops susceptible to abiotic stress. This large family of monocotyledonous plants, commonly known as grasses, counts several important cultivated species, namely wheat (Triticum aestivum), rice (Oryza sativa), maize (Zea mays), and barley (Hordeum vulgare). These crops, notably, show different behaviors under abiotic stress conditions: wheat and rice are considered sensitive, showing serious yield reduction upon water scarcity and soil salinity, while barley presents a natural drought and salt tolerance. During the green revolution (1940–1960), cereal breeding was very successful in developing high-yield crops varieties; however, these cultivars were maximized for highest yield under optimal conditions, and did not present suitable traits for tolerance under unfavorable conditions. The improvement of crop abiotic stress tolerance requires a deep knowledge of the phenomena underlying tolerance, to devise novel approaches and decipher the key components of agricultural production systems. Approaches to improve food production combining both enhanced water use efficiency (WUE) and acceptable yields are critical to create a sustainable agriculture in the future. This paper analyzes the latest results on abiotic stress tolerance in Poaceae. In particular, the focus will be directed toward various aspects of water deprivation and salinity response efficiency in Poaceae. Aspects related to cell wall metabolism will be covered, given the importance of the plant cell wall in sensing environmental constraints and in mediating a response; the role of silicon (Si), an important element for monocots' normal growth and development, will also be discussed, since it activates a broad-spectrum response to different exogenous stresses. Perspectives valorizing studies on landraces conclude the survey, as they help identify key traits for breeding purposes.

Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP+/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, Km for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg2+ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots.

In barley (Hordeum vulgare L. var. Nure), glutamate synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) are strictly correlated biochemical processes. NADH-GOGAT was the major root isoform, whose activity increased on a medium supplied with NH4+ or NO3-; by contrast, no noticeable variations could be observed in the leaves of plants supplied with nitrogen. In the leaves, the major isoform is Fd-GOGAT, whose activity increased under nitrogen feeding. G6PDH activity increased in the roots supplied with nitrogen; no variations were observed in the leaves. Moreover, an increase of the P2 isoform in the roots was measured, giving 13.6% G6PDH activity localized in the plastids under ammonium, and 25.2% under nitrate feeding conditions. Western blots confirmed that P2-G6PDH protein was induced in the roots by nitrogen. P1-G6PDH protein was absent in the roots and increased in the leaves by nitrogen supply to the plants. The changes measured in cytosolic G6PDH seem correlated to more general cell growth processes, and do not appear to be directly involved in glutamate synthesis. The effects of light on Fd-GOGAT is discussed, together with the possibility for P2-G6PDH to sustain nitrogen assimilation upon illumination.

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Plants respond to changes of nutrient availability in the soil by modulating their root system developmental plan. This response is mediated by systemic changes of the nutritional status and/or by local perception of specific signals. The effect of nitrate on Arabidopsis (Arabidopsis thaliana) root development represents a paradigm of these responses, and nitrate transporters are involved both in local and systemic control. Ammonium (NH 4 + ) represents an important nitrogen (N) source for plants, although toxicity symptoms are often associated with high NH 4 + concentration when this is present as the only N source. The reason for these effects is still controversial, and mechanisms associating ammonium supply and plant developmental programs are completely unknown. We determined in Lotus japonicus the range of ammonium concentration that significantly inhibits the elongation of primary and lateral roots without affecting the biomass of the shoot. The comparison of the growth phenotypes in different N conditions indicated the specificity of the ammonium effect, suggesting that this was not mediated by assimilatory negative feedback mechanisms. In the range of inhibitory NH 4 + conditions, only the LjAMT1;3 gene, among the members of the LjAMT1 family, showed a strong increased transcription that was reflected by an enlarged topology of expression. Remarkably, the short-root phenotype was phenocopied in transgenic lines by LjAMT1;3 overexpression independently of ammonium supply, and the same phenotype was not induced by another AMT1 member. These data describe a new plant mechanism to cope with environmental changes, giving preliminary information on putative actors involved in this specific ammonium-induced response.

Most of the carbon used for starch biosynthesis in cereal endosperms is derived from ADP-glucose (ADP-Glc) synthesized by extra-plastidial AGPase activity, and imported directly across the amyloplast envelope. The properties of the wheat endosperm amyloplast ADP-Glc transporter were analysed with respect to substrate kinetics and specificities using reconstituted amyloplast envelope proteins in a proteoliposome-based assay system, as well as with isolated intact organelles. Experiments with liposomes showed that ADP-Glc transport was dependent on counter-exchange with other adenylates. Rates of ADP-Glc transport were highest with ADP and AMP as counter-exchange substrates, and kinetic analysis revealed that the transport system has a similar affinity for ADP and AMP. Measurement of ADP and AMP efflux from intact amyloplasts showed that, under conditions of ADP-Glc-dependent starch biosynthesis, ADP is exported from the plastid at a rate equal to that of ADP-Glc utilization by starch synthases. Photo-affinity labelling of amyloplast membranes with the substrate analogue 8-azido-[alpha-32P]ADP-Glc showed that the polypeptide involved in substrate binding is an integral membrane protein of 38 kDa. This study shows that the ADP-Glc transporter in cereal endosperm amyloplasts imports ADP-Glc in exchange for ADP which is produced as a by-product of the starch synthase reaction inside the plastid.

Summary• Temperature effects on growth, photosynthesis, respiration and nitrate reductase (NR) were studied in the cryophilic algae Koliella antarctica and ' Chlorella ' saccharophila , and in the mesophilic Chlorella sorokiniana .• Growth rate was measured as increase in optical density. Photosynthesis at saturating light and respiration in darkness were measured as O 2 exchange. NADH : NR was assayed in crude extracts.• The two cryophilic algae grew below 15 ° C, and C. sorokiniana above 20 ° C. Photosynthetic and respiration rates of K. antarctica and ' C .' saccharophila were elevated at 5 ° C, and peaked at 30 ° C. Arrhenius plots from 5 to 25 ° C were linear in K. antarctica , whereas in ' C .' saccharophila and C. sorokiniana they exhibited breaks at 15 and 20 ° C, respectively. Values for activation energy ( E a ) and the factor by which the rate increases with raising the temperature 10 ° C ( Q 10 ) differed. Nitrate reductase had its optimum at 25 ° C in cryophilic algae and at 35 ° C in C. sorokiniana .• We conclude that growth of cryophilic algae at low temperature is favoured by elevated photosynthesis and respiration rates, but that it could be limited by a high respiration : photosynthesis ratio.