ABA and Not Chilling Reduces Heat Requirement to Force Cherry Blossom after Endodormancy Release.

Models used to predict the onset of fruit tree blossom under changed climate conditions should be physiologically based as much as possible. Pure optimized phenology models carry the risk of unrealistic predictions due to a misinterpretation of metabolic processes. This was the motivation determining the relevant phases for chill and heat accumulation, which induces cherry blossom (cv. Summit). Investigations are based on 8 years of observational and analytical data, as well as on controlled experiments. For 'Summit' buds, to be released from endodormancy, 43 chill portions from 1 September are necessary. After endodormancy release (t1), on average on 30 November, no further chilling is required, because no correlation between chill accumulation during ecodormancy and the subsequent heat accumulation until 'Summit' blossom exist. The declining amount of heat, which induces cherry blossom after t1-shown in several forcing experiments-seems to be the result of the declining bud's abscisic acid (ABA) content, up to ~50% until the beginning of ontogenetic development. Shortly after t1, when the bud's ABA content is high, a huge amount of heat is necessary to induce cherry blossom under controlled conditions. Heat requirement reduces during ecodormancy along with the reduction in the ABA content. According to these findings, plant development during ecodormancy is suppressed by low temperatures in the orchard and a slowly declining bud's ABA content. These results should lead to a better consideration of the ecodormancy phase in phenology models.

Selected Plant Metabolites Involved in Oxidation-Reduction Processes during Bud Dormancy and Ontogenetic Development in Sweet Cherry Buds (Prunus avium L.).

Many biochemical processes are involved in regulating the consecutive transition of different phases of dormancy in sweet cherry buds. An evaluation based on a metabolic approach has, as yet, only been partly addressed. The aim of this work, therefore, was to determine which plant metabolites could serve as biomarkers for the different transitions in sweet cherry buds. The focus here was on those metabolites involved in oxidation-reduction processes during bud dormancy, as determined by targeted and untargeted mass spectrometry-based methods. The metabolites addressed included phenolic compounds, ascorbate/dehydroascorbate, reducing sugars, carotenoids and chlorophylls. The results demonstrate that the content of phenolic compounds decrease until the end of endodormancy. After a long period of constancy until the end of ecodormancy, a final phase of further decrease followed up to the phenophase open cluster. The main phenolic compounds were caffeoylquinic acids, coumaroylquinic acids and catechins, as well as quercetin and kaempferol derivatives. The data also support the protective role of ascorbate and glutathione in the para- and endodormancy phases. Consistent trends in the content of reducing sugars can be elucidated for the different phenophases of dormancy, too. The untargeted approach with principle component analysis (PCA) clearly differentiates the different timings of dormancy giving further valuable information.

15N allocation into wheat grains (Triticum aestivum L.) influenced by sowing rate and water supply at flowering under a Mediterranean climate.

This study examined the effects of a reduced wheat sowing rate (250 vs. 500 grains m-2) on grain yield, uptake of 15N into grains, and the incorporation into gluten and non-gluten proteins of wheat under field conditions in the Aegean region. A single 15N application was applied at stem elongation, at flowering, or at both developmental stages. Each 15N treatment included either additional water supply, or no additional water supply at flowering. Sowing rate (either 250 or 500 grains m-2) had no impact on grain yield. Grain yield increased with additional water supply, but at the expense of protein quality, because of a decrease in the protein content of gluten. The 15N content of the gluten and non-gluten proteins at grain maturity was not different among cultivars. 15N applied at both stem elongation and flowering was found in comparable amounts in grains and protein fractions, irrespective of sowing rate.

Barley grains, deficient in cytosolic small subunit of ADP-glucose pyrophosphorylase, reveal coordinate adjustment of C:N metabolism mediated by an overlapping metabolic-hormonal control.

The barley Risø16 mutation leads to inactivation of cytosolic ADP-Glc pyrophosphorylase, and results in decreased ADP-Glc and endospermal starch levels. Here we show that this mutation is accompanied by a decrease in storage protein accumulation and seed size, which indicates that alteration of a single enzymatic step can change the network of storage metabolism as a whole. We used comprehensive transcript, metabolite and hormonal profiling to compare grain metabolism and development of Risø16 and wild-type endosperm. Despite increased sugar availability in mutant endosperm, glycolytic intermediates downstream of hexose phosphates remained unchanged or decreased, while several glycolytic enzymes were downregulated at the transcriptional level. Metabolite and transcript profiling also indicated an inhibition of the tricarboxylic acid cycle at the level of mitochondrial nicotinamide adenine dinucleotide (NAD)-isocitrate dehydrogenase and an attendant decrease in alpha-ketoglutarate and amino acids levels in Risø16, compared with wild type. Decreased levels of cytokinins in Risø16 endosperm suggested co-regulation between starch synthesis, abscisic acid (ABA) deficiency and cytokinin biosynthesis. Comparative cis-element analysis in promoters of jointly downregulated genes in Risø16 revealed an overlap between metabolic and hormonal regulation, which leds to a coordinated downregulation of endosperm-specific and ABA-inducible gene expression (storage proteins) together with repression by sugars (isocitrate dehydrogenase, amylases). Such co-regulation ensured that decreased carbon fluxes into starch lead to a coordinated inhibition of glycolysis, amino acid and storage proteins biosynthesis, which is useful in the prevention of osmotic imbalances and oxidative stress due to increased accumulation of sugars.

Effects of N-application on utilization of 15N and 13C and quality in two wheat cultivars.

We studied N uptake and distribution in wheat, and the incorporation of nitrogen and carbon into gluten and non-gluten proteins using a double-labelling approach with 15N and 13C. Doses of N-fertilizer were split and applied at emergence, onset of stem elongation, and heading at rates of 280/140/140 mg N pot(-1), respectively simulating 90/45/45 kg N ha(-1). Five different combinations of N-fertilizations containing no or 10 % 15N were performed. The recovery of 15N added at the stages emergence, stem elongation or heading were 42, 60, and 64 %. Application of 15N at all three stages yielded in 51 % recovery. Remobilisation of straw N was greater for Golia. The 15N concentration in gluten proteins of Golia show higher values than Gonen. The ratio of 15N gluten/15N non-gluten proteins of Golia were higher, which implies a lower non-gluten protein activity during grain filling. The 13C concentration in gluten and non-gluten proteins did not differ between both cultivars.

Uptake and allocation of carbon and nitrogen in Vicia narbonensis plants with increased seed sink strength achieved by seed-specific expression of an amino acid permease.

Over-expressing an amino acid permease in Vicia narbonensis seeds increases sink strength for N that is evident from the higher seed protein content and seed weight. Here, the effect of increased seed sink strength of line AAP-12 on growth, development, and on whole plant carbon and nitrogen uptake and partitioning is analysed. AAP-12 plants have a prolonged growth period. Accumulation and partitioning of dry matter and N in leaves, stems, and pods are higher whereas remobilization to the seeds is delayed, indicating that the switch from growth to reserve allocation and remobilization is delayed. Measuring uptake and allocation of (15)N-ammonia applied via the roots revealed a higher and longer label uptake period during maturation. Measuring whole plant carbon fixation and allocation after (13)C labelling shows higher levels at maturation, particularly in seeds, indicating higher seed sink strength for C and increased allocation into maturing seeds. Levels of cytokinins were dramatically increased in AAP-12 seeds indicating its role in nitrogen-mediated growth stimulation. AAP-12 seeds have higher natural abundances for (13)C indicating increased C fixation via PEP carboxylase in order to meet the higher demand of carbon acceptors for amino acid synthesis. In summary, increased seed sink strength for N in AAP-12 stimulates seed growth, but also that of vegetative organs, which finally leads to a higher ratio of vegetative to seed biomass at maturity and thus a lower harvest index. Therefore, the increased N uptake due to higher seed demand of AAP-12 is partly compensated by growth stimulation of vegetative organs.

Ectopic expression of phosphoenolpyruvate carboxylase in Vicia narbonensis seeds: effects of improved nutrient status on seed maturation and transcriptional regulatory networks.

Seed maturation responds to endogenous and exogenous signals like nutrient status, energy and hormones. We recently showed that phosphoenolpyruvate carboxylase (PEPC) overexpression in Vicia narbonensis seeds alters seed metabolism and channels carbon into organic acids, resulting in greater seed storage capacity and increased protein content. Thus, these lines represent models with altered sink strength and improved nutrient status. Here we analyse seed developmental and metabolic parameters, and C/N partitioning in these seeds. Transgenic embryos take up more carbon and nitrogen. Changes in dry to FW ratio, seed fill duration and major seed components indicate altered seed development. Array-based gene expression analysis of embryos reveals upregulation of seed metabolism, especially during the transition phase and at late maturation, in terms of protein storage and processing, amino acid metabolism, primary metabolism and transport, energy and mitochondrial activity, transcriptional and translational activity, stress tolerance, photosynthesis, cell proliferation and elongation, signalling and hormone action and regulated protein degradation. Stimulated cell elongation is in accordance with upregulated signalling pathways related to gibberellic acid/brassinosteroids. We discuss that activated organic and amino acid production leads to a wide-range activation of nitrogen metabolism, including the machinery of storage protein synthesis, amino acid synthesis, protein processing and deposition, translational activity and the methylation cycle. We suggest that alpha-ketoglutarate (alpha-KG) and/or oxalacetate provide signals for coordinate upregulation of amino acid biosynthesis. Activation of stress tolerance genes indicates partial overlap between nutrient, stress and abscisic acid (ABA) signals, indicating a common interacting or regulatory mechanism between nutrients, stress and ABA. In conclusion, analysis of PEPC overexpressing seeds identified pathways responsive to metabolic and nutrient control on the transcriptional level and its underlying signalling mechanisms.

Ectopic expression of an amino acid transporter (VfAAP1) in seeds of Vicia narbonensis and pea increases storage proteins.

Storage protein synthesis is dependent on available nitrogen in the seed, which may be controlled by amino acid import via specific transporters. To analyze their rate-limiting role for seed protein synthesis, a Vicia faba amino acid permease, VfAAP1, has been ectopically expressed in pea (Pisum sativum) and Vicia narbonensis seeds under the control of the legumin B4 promoter. In mature seeds, starch is unchanged but total nitrogen is 10% to 25% higher, which affects mainly globulin, vicilin, and legumin, rather than albumin synthesis. Transgenic seeds in vitro take up more [14C]-glutamine, indicating increased sink strength for amino acids. In addition, more [14C] is partitioned into proteins. Levels of total free amino acids in growing seeds are unchanged but with a shift toward higher relative abundance of asparagine, aspartate, glutamine, and glutamate. Hexoses are decreased, whereas metabolites of glycolysis and the tricarboxylic acid cycle are unchanged or slightly lower. Phosphoenolpyruvate carboxylase activity and the phosphoenolpyruvate carboxylase-to-pyruvate kinase ratios are higher in seeds of one and three lines, indicating increased anaplerotic fluxes. Increases of individual seed size by 20% to 30% and of vegetative biomass indicate growth responses probably due to improved nitrogen status. However, seed yield per plant was not altered. Root application of [15N] ammonia results in significantly higher label in transgenic seeds, as well as in stems and pods, and indicates stimulation of nitrogen root uptake. In summary, VfAAP1 expression increases seed sink strength for nitrogen, improves plant nitrogen status, and leads to higher seed protein. We conclude that seed protein synthesis is nitrogen limited and that seed uptake activity for nitrogen is rate limiting for storage protein synthesis.