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This (two-season) study was undertaken to assess the involvement of gibberellins (GAs), cytokinins (CKs), and auxins (AX) in dormancy of the sweet cherry buds ‘Summit’. Our hypothesis consisted in the assumption that representatives of these hormone groups are able to mark the transition between different dormancy phases. Changes in the transition between endo- and ecodormancy and the stages of ontogenetic development were not recognizable by bioactive GA1, GA5, GA7. The transient increase of GA3 during ecodormancy might be interpreted as an indication of the preservation of ecodormancy. The content of the biological active bases tZ, cZ, and DHZ was equal between endo- and ecodormancy. However, the content increased significantly in the first phase of ontogenetic development. The summation of the representatives of the various CKs (total iP-type, total tZ-type, total CK bases, total CK ribosides, total CK nucleotides, total O-glucosides, total N-glucosides, and total CKs) showed no differences regarding their levels during endo- and ecodormancy. These values increased markedly in the subsequent phase. AX increased after ecodormancy. By contrast, from side green until open cluster no differences occurred. As shown for AX, the content of oxIAA increased after ecodormancy. The content of IAAsp was low during endodormancy and increased transiently during ecodormancy and early ontogenetic development. This study revealed that no changes in the content of different bioactive GAs (exception GA3), CKs, and AX occurred during winter rest, and more precisely, during endo- and ecodormancy. These metabolites, therefore, are not suitable to differentiate between these dormancy phases. The ontogenesis is accompanied by specific changes in the content of bioactive molecules, precursors, and conjugation products.

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.

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.

Six phenological models, two simple forcing (F)-models and one sequential chilling/forcing (CF)-model, each with and without day length (DL)-term in the forcing approach were optimised (2001–2010) and validated (2011–2015) on very accurate blossoming data of an experimental sweet cherry orchard at Berlin-Dahlem (cultivar ‘Summit’). In parallel, in 3 seasons (2011/2012–2013/2014) climate chamber experiments were performed in order to determine the end of dormancy for ‘Summit’, which is usually an unknown or uncertain parameter in phenological modelling. Additionally, in the season 2013/2014 an in situ climate change experiment on three trees in the sweet cherry orchard were arranged, which was used to validate the phenological models for distinctly warmer climate conditions at the experimental site. On the basis of our climate chamber experiments we quantified the chilling requirement of ‘Summit’ trees. Thus, we were able to identify a CF-model for the beginning of sweet cherry blossom which is mostly physiologically based and works well for current and for future climate conditions at the experimental site. This paper also shows how phenological models can fail under warmer climates, if either the model is too simple or the model parameters are wrong. Additional, we confirmed that phenological models with DL-term in the forcing approach clearly surpassed the conventional phenological models without this parameter. The reason for this behaviour is extensively discussed.

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.

The effects of biochar (maize biochar – MBC, wood biochar – WBC) and unfermented or fermented hydrochar (HTC) on the euedaphic Collembola Protaphorura fimata and on spring wheat were investigated in greenhouse experiments. The impact of char type, amount of fermented HTC, and MBC-Collembola interactions were assessed. Generally, shoot and root biomass as well as abundance of P. fimata were not affected by the different chars. However, with increasing amounts of fermented HTC the abundance of P. fimata declined, whereas shoot biomass of wheat increased. Moreover, MBC altered root morphology and resulted in thicker roots with higher volume. The latter was not apparent when Collembola were present.

Despite suggested benefits of hydrochars and biochars, little is known about their effects on root development and root-char interactions. To compare effects of two types of biochar (Pyro and Pyreg) and one hydrochar (hydrothermal carbonization (HTC)) on root growth of spring wheat, two rhizobox experiments were set up where physical contact of roots with chars was prevented using nylon gauze. Rhizoboxes were filled with unamended soil as a control or with three different soil-char mixtures (Pyro, Pyreg and HTC). Shoots and roots were harvested before flowering and at tillering in the first and second experiment, respectively. Chemical soil properties (Nt, K, Ct, pH value) were affected differently by the different chars. Both shoot and root dry matter were influenced by chars. Pyro-char had positive effects on root dry matter in both experiments. At tillering, HTC-char affected root length, root surface and number of root tips negatively. Our findings suggest that nutrients released from chars may affect...

Some important stages of plant development, such as dormancy release and beginning of ontogenetic development, cannot be easily observed so that semi-mechanistic phenological models of spring phases mostly does not accurately reflect the control of these physiological stages. For this reason, over 6 years we studied weekly changes of Fresh Weight (FW), Dry Weight (DW), Water (WC), Nitrogen (NC) and Carbon Content (CC) in sweet cherry buds (Prunus avium L., cultivar ‘Summit’), from October to mid of April (stage ‘open cluster’, BBCH 56). Together with an experimental study to determine the date of endodormancy release, we were able to define the annual variability and average duration in the timing of para- endo-, and ecodormancy phase for sweet cherries, cultivar ‘Summit’. The secession of growth in autumn starts when all leafs has fallen down (BBCH 97) and marks the transition from para-to endodormancy. During endodormancy all investigated parameters reached a constant level which also did not change during ecodormancy. Our results showed that ecodormancy can be a relatively long phase in temperate climates, which must be adequately handled in phenological models. Beginning of ontogenetic development was clearly related to steadily rising water contents in cherry buds, which stayed stable during endo- and ecodormancy. Thus, this study highlights the importance of bud’s water content to define dormancy phases as well as beginning of ontogenetic development. The latter one was induced by continuously rising air temperatures above the freezing point. Physiologically, beginning of ontogenetic development is a flexible date which occurred on average 26 days before bud swelling was observed.

In order to develop reliable and physiologically sound models for the plant development in spring, the date of endodormancy release is always a crucial and mostly unknown model parameter. Until present, classical approaches - such as climate chamber experiments - are used to derive this unknown parameter. In these experiments, progressive plant development or significant changes in bud’s fresh weight or water content are measurable markers for dormancy release. This study presents an alternative approach, which is based on four well-known metabolites. For 5 seasons (2011/12-2015/16), the content of abscisic acid (ABA) and sugars such as fructose, sucrose and glucose in sweet cherry flower buds (cultivar ‘Summit’) were weekly analysed between beginning of October and April. These data allow comparing the annual course of these metabolites with the date of endodormancy release, derived from a classical climate chamber experiment, published in a previous study. Results showed that ABA and sucrose are two important metabolites which can help to identify the date of endodormancy release of sweet cherries. On average, ABA content reached a plateau of 5.65 μg g-1 DW-1 during endodormancy, which was maintained for 3-6 weeks. The significant reduction of the ABA content after this period to 4.41 μg g-1 DW-1 on average during ecodormancy was nearly in agreement with the date of endodormancy release of ‘Summit’ on 28 November (332 DOY). The annual cycle of sucrose, which has a cryoprotective effect during winter, is well comprehensible and showed a close relationship to the annual course of minimum air temperature after leaf fall (r=-0.90). The nearly constant level of sucrose during ecodormancy (21.0 mg g-1 DW-1, 5 yr. mean) did not only allow deriving the date of endodormancy release but can also be helpful to define the beginning of ontogenetic development.

The barley Riso16 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 Riso16 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 Riso16, compared with wild type. Decreased levels of cytokinins in Riso16 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 Riso16 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.

Summary 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 α-ketoglutarate (α-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.

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.

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.

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.

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.

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.

Investigations were carried out on Vigna unguiculata L. Walp. to estimate the distribution and utilization of 15N in different organs after stem injection during vegetative, flowering and pod filling stage. During flowering effects of water deficit were also examined. In well watered plants, within 4 days after injection, 65% of 15N accumulated in leaves. This was drastically reduced to 42% by water deficit. 15N accumulation in stems increased under water deficit. The translocation of 15N from the stem base to roots were not altered by water deficit. During pod filling 62% of recovered 15N in the plants had accumulated in seeds, 24% in leaves and 11% in stems within 4 days, whereas the uptake of nitrogen in pod walls and roots remained low (2%). These results demonstrate that the method of injecting very small quantities (1 mg/plant) of 15N into the stem base allows an exact and detailed quantitative assessment of N translocation/distribution with regard to different organs, different growth stages and different treatments.