Klaus-Peter Götz

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.

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.

Distribution and Utilization of 15N in Cowpeas Injected into the Stem Under Influence of Water Deficit

Abstract 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.

Impact of hydro-/biochars on root morphology of spring wheat

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 root morphology of spring wheat. The comparison of different types of chars showed different effects on root growth, shoot growth and soil changes depending on feedstock, production process and the varied amount of chars.

Identification and Timing of Dormant and Ontogenetic Phase for Sweet Cherries in Northeast Germany for Modelling Purposes

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.

Identification of Endodormancy Release for Cherries (Prunus Avium L.) by Abscisic Acid and Sugars

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.

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 Gönen. 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.

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.

Abscisic Acid Related Metabolites in Sweet Cherry Buds (Prunus avium L.)

As our climate changes, plant mechanisms involved for dormancy release become increasingly important for commercial orchards. It is generally believed that abscisic acid (ABA) is a key hormone that responds to various environmental stresses which affects bud dormancy. For this reason, a multi-year study was initiated to obtain data on plant metabolites during winter rest and ontogenetic development in sweet cherry buds (Prunus avium L.). In this paper, we report on metabolites involved in ABA synthesis and catabolism and its effect on bud dormancy in the years 2014/15-2016/17. In previous work, the timings of the different phases of para-, endo-, ecodormancy and ontogenetic development for cherry flower buds of the cultivar ‘Summit’ were determined, based on classical climate chamber experiments and changes in the bud’s water content. Based on these time phases, we focused now on the different aspects of the ABA-metabolism. The results show that there is a continual synthesis of ABA about 5 weeks before leaf fall, and a degradation of ABA during ecodormancy and bud development until the phenological stage ‘open cluster’. This is confirmed by relating the ABA content to that of the total precursor carotenoids, neoxanthin and violaxanthin. The tentative monitoring of individual intermediate metabolites revealed that dihydroxyphaseic acid is the most abundant catabolite of ABA and ABA glucosyl ester is in terms of mass intensity, the most abundant ABA metabolite observed in this study. The results suggest that the direct route for ABA biosynthesis from farnesyl pyrophosphate may also be relevant in cherry flower buds.

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

ABSTRACT 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.