Hardy Rolletschek

ABA biosynthesis and degradation contributing to ABA homeostasis during barley seed development under control and terminal drought-stress conditions

Drought is one of the most severe environmental stress factors limiting crop yield especially when occurring during anthesis and seed filling. This terminal drought is characterized by an excess production of the phytohormone abscisic acid (ABA) which plays an important role during seed development and dormancy. All the genes putatively involved in ABA biosynthesis and inactivation in barley were identified and their expression studied during plant ontogeny under standard and drought-stress conditions to learn more about ABA homeostasis and the possible mode of cross-talk between source and sink tissues. Out of 41 genes related to ABA biosynthesis and inactivation 19 were found to be differentially regulated under drought stress in both flag leaves and developing seed during seed filling. Transcripts of plastid-located enzymes are regulated similarly in flag leaf and seed under terminal drought whereas transcripts of cytosolic enzymes are differentially regulated in the two tissues. Detailed information on the expression of defined gene family members is supplemented by measurements of ABA and its degradation and conjugation products, respectively. Under drought stress, flag leaves in particular contain high concentrations of both ABA and the ABA degradation products phaseic acid (PA) and diphaseic acid (DPA); whereas, in seeds, besides ABA, DPA was mainly found. The measurements also revealed a positive correlation between ABA level and starch content in developing seeds for the following reasons: (i) genes of the ABA controlled SnRK2.6 and RCAR/PP2C-mediated signal transduction pathway to the ABF transcription factor HvABI5 are activated in the developing grain under drought, (ii) novel ABA- and dehydration-responsive cis-elements have been found in the promoters of key genes of starch biosynthesis (HvSUS1, HvAGP-L1) and degradation (HvBAM1) and these transcripts/activity are prominently induced in developing seeds during 12 and 16 DAF, (iii) spraying of fluridone (an ABA biosynthesis inhibitor) to drought-stressed plants results in severely impaired starch content and thousand grain weight of mature seeds.

The oxygen status of the developing seed

Recent applications of oxygen-sensitive microsensors have demonstrated steep oxygen gradients in developing seeds of various crops. Here, we present an overview on oxygen distribution, major determinants of the oxygen status in the developing seed and implications for seed physiology. The steady-state oxygen concentration in different seed tissues depends on developmental parameters, and is determined to a large extent by environmental factors. Photosynthetic activity of the seed significantly diminishes hypoxic constraints, and can even cause transient, local hyperoxia. Changes in oxygen availability cause rapid adjustments in mitochondrial respiration and global metabolism. We argue that nitric oxide (NO) is a key player in the oxygen balancing process in seeds, avoiding fermentation and anoxia in vivo. Molecular approaches aiming to increase oxygen availability within the seed are discussed.

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.

Spatiotemporal Profiling of Starch Biosynthesis and Degradation in the Developing Barley Grain

Barley (Hordeum vulgare) grains synthesize starch as the main storage compound. However, some starch is degraded already during caryopsis development. We studied temporal and spatial expression patterns of genes coding for enzymes of starch synthesis and degradation. These profiles coupled with measurements of selected enzyme activities and metabolites have allowed us to propose a role for starch degradation in maternal and filial tissues of developing grains. Early maternal pericarp functions as a major short-term starch storage tissue, possibly ensuring sink strength of the young caryopsis. Gene expression patterns and enzyme activities suggest two different pathways for starch degradation in maternal tissues. One pathway possibly occurs via α-amylases 1 and 4 and β-amylase 1 in pericarp, nucellus, and nucellar projection, tissues that undergo programmed cell death. Another pathway is deducted for living pericarp and chlorenchyma cells, where transient starch breakdown correlates with expression of chloroplast-localized β-amylases 5, 6, and 7, glucan, water dikinase 1, phosphoglucan, water dikinase, isoamylase 3, and disproportionating enzyme. The suite of genes involved in starch synthesis in filial starchy endosperm is much more complex than in pericarp and involves several endosperm-specific genes. Transient starch turnover occurs in transfer cells, ensuring the maintenance of sink strength in filial tissues and the reallocation of sugars into more proximal regions of the starchy endosperm. Starch is temporally accumulated also in aleurone cells, where it is degraded during the seed filling period, to be replaced by storage proteins and lipids.

Molecular physiology of adventitious root formation in Petunia hybrida cuttings: involvement of wound response and primary metabolism.

Adventitious root formation (ARF) in the model plant Petunia hybrida cv. Mitchell has been analysed in terms of anatomy, gene expression, enzymatic activities and levels of metabolites. This study focuses on the involvement of wound response and primary metabolism. Microscopic techniques were complemented with targeted transcript, enzyme and metabolite profiling using real time polymerase chain reaction (PCR), Northern blot, enzymatic assays, chromatography and mass spectrometry. Three days after severance from the stock plants, first meristematic cells appeared which further developed into root primordia and finally adventitious roots. Excision of cuttings led to a fast and transient increase in the wound-hormone jasmonic acid, followed by the expression of jasmonate-regulated genes such as cell wall invertase. Analysis of soluble and insoluble carbohydrates showed a continuous accumulation during ARF. A broad metabolite profiling revealed a strong increase in organic acids and resynthesis of essential amino acids. Substantial changes in enzyme activities and metabolite levels indicate that specific enzymes and metabolites might play a crucial role during ARF. Three metabolic phases could be defined: (i) sink establishment phase characterized by apoplastic unloading of sucrose and being probably mediated by jasmonates; (ii) recovery phase; and (iii) maintenance phase, in which a symplastic unloading occurs.

Energy status and its control on embryogenesis of legumes. Embryo photosynthesis contributes to oxygen supply and is coupled to biosynthetic fluxes

Legume seeds are heterotrophic and dependent on mitochondrial respiration. Due to the limited diffusional gas exchange, embryos grow in an environment of low oxygen. O2 levels within embryo tissues were measured using microsensors and are lowest in early stages and during night, up to 0.4% of atmospheric O2 concentration (1.1 μm). Embryo respiration was more strongly inhibited by low O2 during earlier than later stages. ATP content and adenylate energy charge were lowest in young embryos, whereas ethanol emission and alcohol dehydrogenase activity were high, indicating restricted ATP synthesis and fermentative metabolism. In vitro and in vivo experiments further revealed that embryo metabolism is O2 limited. During maturation, ATP levels increased and fermentative metabolism disappeared. This indicates that embryos become adapted to the low O2 and can adjust its energy state on a higher level. Embryos become green and photosynthetically active during differentiation. Photosynthetic O2 production elevated the internal level up to approximately 50% of atmospheric O2 concentration (135 μm). Upon light conditions, embryos partitioned approximately 3-fold more [14C]sucrose into starch. The light-dependent increase of starch synthesis was developmentally regulated. However, steady-state levels of nucleotides, free amino acids, sugars, and glycolytic intermediates did not change upon light or dark conditions. Maturing embryos responded to low O2 supply by adjusting metabolic fluxes rather than the steady-state levels of metabolites. We conclude that embryogenic photosynthesis increases biosynthetic fluxes probably by providing O2 and energy that is readily used for biosynthesis and respiration.

Nitrite–nitric oxide control of mitochondrial respiration at the frontier of anoxia

Actively respiring animal and plant tissues experience hypoxia because of mitochondrial O(2) consumption. Controlling oxygen balance is a critical issue that involves in mammals hypoxia-inducible factor (HIF) mediated transcriptional regulation, cytochrome oxidase (COX) subunit adjustment and nitric oxide (NO) as a mediator in vasodilatation and oxygen homeostasis. In plants, NO, mainly derived from nitrite, is also an important signalling molecule. We describe here a mechanism by which mitochondrial respiration is adjusted to prevent a tissue to reach anoxia. During pea seed germination, the internal atmosphere was strongly hypoxic due to very active mitochondrial respiration. There was no sign of fermentation, suggesting a down-regulation of O(2) consumption near anoxia. Mitochondria were found to finely regulate their surrounding O(2) level through a nitrite-dependent NO production, which was ascertained using electron paramagnetic resonance (EPR) spin trapping of NO within membranes. At low O(2), nitrite is reduced into NO, likely at complex III, and in turn reversibly inhibits COX, provoking a rise to a higher steady state level of oxygen. Since NO can be re-oxidized into nitrite chemically or by COX, a nitrite-NO pool is maintained, preventing mitochondrial anoxia. Such an evolutionarily conserved mechanism should have an important role for oxygen homeostasis in tissues undergoing hypoxia.

Surveying the plant’s world by magnetic resonance imaging

Understanding the way in which plants develop, grow and interact with their environment requires tools capable of a high degree of both spatial and temporal resolution. Magnetic resonance imaging (MRI), a technique which is able to visualize internal structures and metabolites, has the great virtue that it is non-invasive and therefore has the potential to monitor physiological processes occurring in vivo. The major aim of this review is to attract plant biologists to MRI by explaining its advantages and wide range of possible applications for solving outstanding issues in plant science. We discuss the challenges and opportunities of MRI in the study of plant physiology and development, plant-environment interactions, biodiversity, gene functions and metabolism. Overall, it is our view that the potential benefit of harnessing MRI for plant research purposes is hard to overrate.

Effects of NaCl-salinity on amino acid and carbohydrate contents of Phragmites australis

Abstract The effects of NaCl-salinity on growth, free amino acid and sugar content and composition were assayed in roots, rhizomes and leaves of Phragmites australis (Cav.) Trin. ex Steud. Juvenile plants produced from freshwater clones, were cultured under greenhouse hydroponic conditions for 21 days. Relative growth rates were highest at a salinity level of 0 and 1.5‰, respectively, but decreased significantly at 10‰. All plants cultured at 35‰ salinity died. The osmolality in rhizomes and leaves increased with salinity. The total contents of free amino acids were highest in rhizomes>leaves>roots. In rhizomes, the amino acid content increased significantly up to four-fold from 0 to 10‰ salinity. This increase was caused by up to 200-fold increase of proline and 11-fold increase of glutamine at 10‰, whilst the share of asparagine and glutamate decreased. Leaves showed a similar response to salinity with increasing amino acid contents, and shares of proline and glutamine whereas roots did not react significantly. The contents of sucrose, glucose and fructose were highest in leaves>rhizomes>roots. In rhizomes of all three clones, the sugar contents increased up to 3.5-fold from 1.5 to 10‰ salinity level, but were lower at 1.5‰ versus the control (0‰). Sugar contents were lowest (roots) and highest (leaves) at 1.5‰ salinity. The sugar composition did not vary significantly except for leaves where the fraction of sucrose decreased with increasing salinity level at all three clones from 89.1 to 61.7% of total dissolved sugar (pooled data). The importance of free amino acids and sugars as osmolytes was similar in rhizomes and leaves (13–15% of total osmolality at 10‰). In rhizomes, free amino acids were more important as osmolyte than sugars, while the opposite was true for leaves. Proline contributed up to 2.7% to total osmolality. It is hypothesised that a strong proline accumulation indicates the exceeding of a critical salinity level.

Antisense-inhibition of ADP-glucose pyrophosphorylase in Vicia narbonensis seeds increases soluble sugars and leads to higher water and nitrogen uptake

Abstract. We previously reported on Vicia narbonensis seeds with largely decreased α-D-glucose-1-phosphate adenyltransferase (AGP; EC 2.7.7.27) due to antisense inhibition [H. Weber et al. (2000) Plant J 24:33–43]. In an extended biochemical analysis we show here that in transgenic seeds both AGP activity and ADP-glucose levels were strongly decreased but starch was only moderately reduced and contained less amylose. The flux control coefficient of AGP to starch accumulation was as low as 0.08, i.e. AGP exerts low control on starch biosynthesis in Vicia seeds. Mature cotyledons of antisense seeds had increased contents of lipids, nitrogen and sulfur. The protein content was higher due, in particular, to increased sulfur-rich albumins. Globulin fractions of storage proteins had a lower ratio of legumin to vicilin. Isolated cotyledons partitioned less [14C]sucrose into starch and more into soluble sugars with no change in the protein fraction. Respiration of isolated cotyledons and activities of the major glycolytic and carbohydrate-metabolizing enzymes were not affected. Sucrose and the hexose-phosphate pool were increased but UDP-glucose, 3-phosphoglyceric acid, phosphoenolpyruvate, pyruvate, ATP and ADP were unchanged or even lower, indicating that carbon partitioning changed from starch to sucrose without affecting the glycolytic and respiratory pathways. Soluble compounds were increased but osmolality remained unchanged, indicating compensatory water influx resulting in higher water contents. Developmental patterns of water and nitrogen accumulation suggest a coupled uptake of amino acids and water into cotyledons. We conclude that, due to higher water uptake, transgenic cotyledons take up more amino acids, which become available for protein biosynthesis leading to a higher protein content. Obviously, a substantial part of amino acid uptake into Vicia seeds occurs passively and is osmotically controlled and driven by water influx.