Drought priming mechanisms in wheat elucidated by in-situ determination of dynamic stomatal behavior.

Stomata play a critical role in balancing photosynthesis and transpiration, which are essential processes for plant growth, especially in response to abiotic stress. Drought priming has been shown to improve drought tolerance. Lots of studies have been done with the response of stomatal behavior to drought stress. However, how the stomatal dynamic movement in intact wheat plants response to drought priming process is not known. Here, a portable microscope was used to take microphotographs in order to in-stiu determination of stomatal behavior. Non-invasive micro-test technology was used for measurements of guard cell K+, H+ and Ca2+ fluxes. Surprisingly, the results found that primed plants close stomatal much faster under drought stress, and reopening the stomatal much quicker under recovery, in relation to non-primed plants. Compared with non-primed plants, primed plants showed higher accumulation of ABA and Ca2+ influx rate in guard cells under drought stress. Furthermore, genes encoding anion channels were higher expressed and K+ outward channels activated, leading to enhanced K+ efflux, resulting in faster stomatal closure in primed plants than non-primed plants. During recovery, both guard cell ABA and Ca2+ influx of primed plants were found to be significantly reducing K+ efflux and accelerating stomatal reopening. Collectively, a portable non-invasive stomatal observation of wheat found that priming promoted faster stomatal closure under drought stress and faster reopening during post-drought recovery in relation to non-primed plants, thereby enhancing overall drought tolerance.

Elevated CO2 Improves the Physiology but Not the Final Yield in Spring Wheat Genotypes Subjected to Heat and Drought Stress During Anthesis.

Heat and drought events often occur concurrently as a consequence of climate change and have a severe impact on crop growth and yield. Besides, the accumulative increase in the atmospheric CO2 level is expected to be doubled by the end of this century. It is essential to understand the consequences of climate change combined with the CO2 levels on relevant crops such as wheat. This study evaluated the physiology and metabolite changes and grain yield in heat-sensitive (SF29) and heat-tolerant (LM20) wheat genotypes under individual heat stress or combined with drought applied during anthesis at ambient (aCO2) and elevated CO2 (eCO2) levels. Both genotypes enhanced similarly the WUE under combined stresses at eCO2. However, this increase was due to different stress responses, whereas eCO2 improved the tolerance in heat-sensitive SF29 by enhancing the gas exchange parameters, and the accumulation of compatible solutes included glucose, fructose, ß-alanine, and GABA to keep water balance; the heat-tolerant LM20 improved the accumulation of phosphate and sulfate and reduced the lysine metabolism and other metabolites including N-acetylornithine. These changes did not help the plants to improve the final yield under combined stresses at eCO2. Under non-stress conditions, eCO2 improved the yield of both genotypes. However, the response differed among genotypes, most probably as a consequence of the eCO2-induced changes in glucose and fructose at anthesis. Whereas the less-productive genotype LM20 reduced the glucose and fructose and increased the grain dimension as the effect of the eCO2 application, the most productive genotype SF29 increased the two carbohydrate contents and ended with higher weight in the spikes. Altogether, these findings showed that the eCO2 improves the tolerance to combined heat and drought stress but not the yield in spring wheat under stress conditions through different mechanisms. However, under non-stress conditions, it could improve mainly the yield to the less-productive genotypes. Altogether, the results demonstrated that more studies focused on the combination of abiotic stress are needed to understand better the spring wheat responses that help the identification of genotypes more resilient and productive under these conditions for future climate conditions.

The effect of individual and combined drought and heat stress under elevated CO2 on physiological responses in spring wheat genotypes.

Abiotic stress due to climate change with continuous rise of atmospheric CO2 concentration is predicted to cause severe changes to crop productivity. Thus, research into wheat cultivars, capable of maintaining yield under limiting conditions is necessary. The aim of this study was to investigate the physiological responses of spring wheat to individual and combined drought- and heat events and their interaction with CO2 concentration. Two heat sensitive (LM19, KU10) and two heat tolerant (LM62, GN5) genotypes were selected and grown under ambient (400 ppm, aCO2) and elevated (800 ppm, eCO2) CO2 concentrations. At the tillering stage, the wheat plants were subjected to different treatments: control, progressive drought, heat and combined drought and heat stress. Our results showed that eCO2 mitigated the negative impact of the moderate stress in all genotypes. However, no distinctive responses were observed in some of the measured parameters between heat sensitive and tolerant genotypes. All genotypes grown at eCO2 had significantly higher net photosynthetic rates and maintained maximum quantum efficiency of PSII photochemistry under heat and combined stress compared to aCO2. Under heat and combined stress, the chlorophyll a:b ratios decreased only in heat tolerant genotypes at eCO2 compared to the control. Furthermore, the heat tolerant genotypes grown at eCO2 showed an increased glucose and fructose contents and a decreased sucrose content under combined stress compared to aCO2. These findings provide new insights into the underlying mechanisms of different genotypic responses to combined abiotic stresses at eCO2 that differ from the response to individual stresses.

Activity in the Arbuscular Mycorrhizal Hyphosphere Warning Neighbouring Plants.

Pathogen infections of the phyllosphere have been investigated in detail, however, the changes induced by these infections on the arbuscular mycorrhizal hyphosphere, and the consequent signalling to the neighbouring plants have been scarcely investigated. Here, our objectives were to document that B.fabae infection of connected Vicia faba plants resulted in changes in the metabolism and microbial community of the hyphosphere, confirming the induction of plant defence in connected plants through gene-expression evaluations. Infected plants were challenged with B. fabae for 72 h. Changes in gene-expression of pathogenesis-related proteins 1,2, and 5 (PR1, PR2, PR5) of both infected- and non-infected plants were analysed, to confirm signalling through the hyphosphere. The primary metabolic profiles and changes in the level of microbiota in the hyphosphere were assessed. Changes in expression of PR1, PR2, and PR5 genes occurred in the neighbouring plants 24 hours after infection. Mannitol levels decreased in presence of AMF. A decrease in the level of actinobacteria in the hyphosphere of infected plants was detected. We conclude that B.fabae infection induced a signalling event through the AM hyphosphere, confirmed by changes in defence gene-expression in non-infected neighbouring plants, influenced primary metabolic activity of-, and affected the microbial composition within-, the AM hyphosphere.

Aphid infestation in the phyllosphere affects primary metabolic profiles in the arbuscular mycorrhizal hyphosphere.

While effects of (a)biotic stress events in the phyllosphere have been studied intensively, possible influences of stress on the arbuscular mycorrhizal hyphosphere has scarcely been investigated. We hypothesised that stress challenge in the phyllosphere could alter primary metabolite profiles of the hyphosphere - the mycelial network connecting plants. Donor plants, connected to receiver plants by mycelial networks, were aphid-challenged during 84 h. Primary metabolite profiles in the hyphosphere were investigated. Gene-expression of plant defence gene PR1 was measured in one of the receiver plants during the challenge. Hexose levels in the hyphosphere increased when donor plants were aphid-challenged. This change in metabolic profile was influenced by leaf sampling from receiver plant. PR1 expression increased in donor plants 48 h after challenge, and consequently 60 h after, in receiver plants. We conclude that aphid infestation of donor plants modified primary carbon metabolism in the hyphosphere. Plant defence response in receiver plants, occurred 12 h after detection of response in the aphid-challenged donor plants. While this work is the first to reveal primary metabolic profiles of the AM hyphosphere, more work is needed to elucidate the possible role of transient changes of hexose metabolism in stress response and signalling processes in the hyphosphere of connected plants.

Physiological and proteome studies of responses to heat stress during grain filling in contrasting wheat cultivars.

Experiments to explore physiological and biochemical differences of the effects of heat stress in ten wheat (Triticum aestivum L.) cultivars have been performed. Based on the response of photosynthesis rates, cell membrane lipid peroxide concentrations and grain yield to heat, six cultivars were clustered as heat-tolerant (cv. '579', cv. '810', cv. '1110', cv. Terice, cv. Taifun and cv. Vinjett) and four as heat-sensitive (cv. '490', cv. '633', cv. '1039' and cv. '1159'). Higher rates of photosynthetic carbon- and light-use were accompanied by lower damage to cell membranes in leaves of tolerant compared to sensitive cultivars under heat stress. The tolerant cv. '810' and the sensitive cv. '1039' were selected for further proteome analysis of leaves. Proteins related to photosynthesis, glycolysis, stress defence, heat shock and ATP production were differently expressed in leaves of the tolerant and sensitive cultivar under heat stress in relation to the corresponding control. The abundance of proteins related to signal transduction, heat shock, photosynthesis, and antioxidants increased, while the abundance of proteins related to nitrogen metabolism decreased in the tolerant cv. '810' under heat stress as compared to the control. Collectively, the results indicate that primarily changes in both the amount and activities of enzymes involved in photosynthesis and antioxidant activities in leaves contributed to higher heat tolerance in the cv. '810' compared to the heat sensitive cv. '1039'.

Improved tolerance to drought stress after anthesis due to priming before anthesis in wheat (Triticum aestivum L.) var. Vinjett.

Drought stress occurring during the reproductive growth stage leads to considerable reductions in crop production and has become an important limiting factor for food security globally. In order to explore the possible role of drought priming (pre-exposure of the plants to mild drought stress) on the alleviation of a severe drought stress event later in development, wheat plants were subjected to single or double mild drought episodes (soil relative water content around 35-40%) before anthesis and/or to a severe drought stress event (soil relative water content around 20-25%) 15 d after anthesis. Here, single or double drought priming before anthesis resulted in higher grain yield than in non-primed plants under drought stress during grain filling. The photosynthesis rate and ascorbate peroxidase activity were higher while malondialdehyde content was lower in primed plants than in the non-primed plants under drought stress during grain filling. Proteins in flag leaves differently expressed by the priming and drought stress were mainly related to photosynthesis, stress defence, metabolism, molecular chaperone, and cell structure. Furthermore, the protein abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit, Rubisco activase and ascorbate peroxidase were upregulated in primed plants compared with non-primed plants under drought stress during grain filling. In conclusion, the altered protein expression and upregulated activities of photosynthesis and ascorbate peroxidase in primed plants may indicate their potential roles in alleviating a later-occurring drought stress episode, thereby contributing to higher wheat grain yield under drought stress during grain filling.

Multiple heat priming enhances thermo-tolerance to a later high temperature stress via improving subcellular antioxidant activities in wheat seedlings.

Seedlings of winter wheat (Triticum aestivum L.) were firstly twice heat-primed at 32/24 °C, and subsequently subjected to a more severe high temperature stress at 35/27 °C. The later high temperature stress significantly decreased plant biomass and leaf total soluble sugars concentration. However, plants experienced priming (PH) up-regulated the Rubisco activase B encoding gene RcaB, which was in accordance with the higher photosynthesis rate in relation to the non-primed plants (NH) under the later high temperature stress. In relation to NH, the major chlorophyll a/b-binding protein gene Cab was down-regulated in PH plants, implying a reduction of the light absorption to protect the photosystem II from excitation energy under high temperature stress. At the same time, under the later high temperature stress PH plants showed significantly higher actual photochemical efficiency, indicating an improvement of light use efficiency due to the priming pre-treatment. Under the later high temperature stress, PH could be maintained a better redox homeostasis than NH, as exemplified by the higher activities of superoxide dismutase (SOD) in chloroplasts and glutathione reductase (GR), and of peroxidase (POD) in mitochondria, which contributed to the lower superoxide radical production rate and malondialdehyde concentration in both chloroplasts and mitochondria. The improved antioxidant capacity in chloroplasts and mitochondria was related to the up-regulated expressions of Cu/Zn-SOD, Mn-SOD and GR in PH. Collectively, heat priming effectively improved thermo-tolerance of wheat seedlings subjected to a later high temperature stress, which could be largely ascribed to the enhanced anti-oxidation at the subcellular level.

PollenCALC: software for estimation of pollen compatibility of self-incompatible allo- and autotetraploid species.

BACKGROUND: Self-incompatibility (SI) is a biological mechanism to avoid inbreeding in allogamous plants. In grasses, this mechanism is controlled by a two-locus system (S-Z). Calculation of male and female gamete frequencies is complex for tetraploid species. We are not aware of any software available for predicting pollen haplotype frequencies and pollen compatibility in tetraploid species. RESULTS: PollenCALC is a software tool written in C++ programming language that can predict pollen compatibility percentages for polyploid species with a two-locus (S, Z) self-incompatibility system. The program predicts pollen genotypes and frequencies based on defined meiotic parameters for allo- or autotetraploid species with a gametophytic S-Z SI system. These predictions can be used to obtain expected values for for diploid and for (allo- or autotetraploidy SI grasses. CONCLUSION: The information provided by this calculator can be used to predict compatibility of pair-crosses in plant breeding applications, to analyze segregation distortion for S and Z genes, as well as linked markers in mapping populations, hypothesis testing of the number of S and Z alleles in a pair cross, and the underlying genetic model.

Distribution of N-glycosylation sequons in proteins: how apart are they?

N-glycosylation is a common protein modification process, which affects a number of properties of proteins. Little is known about the distribution of N-glycosylation sequons, for example, the distance between glycosylated sites and their position in the protein primary sequence. Using a large set of experimentally confirmed eukaryotic N-glycoproteins we analyzed the relative position and distribution of sequons. N-Glycosylation probability was found to be lower in the termini of protein sequences compared to the mid region. N-glycosylated sequons were found much farther from C terminus compared to the N-terminus of the protein sequence and this effect was more pronounced for NXS sequons. The distribution of sequons, modeled based on balls-in-boxes classical occupancy, showed a near-maximum probability. Considerable proportion of sequons was found within a distance of ten amino acids, indicating that the steric hindrance was not a key factor in protein N-glycosylation. Interestingly, the distribution of all sequons present in N-glycoproteins showed a pattern very similar to that of glycosylated sequons. The results indicate that protein N-glycosylation chiefly follows a random design.

Implications of high-temperature events and water deficits on protein profiles in wheat (Triticum aestivum L. cv. Vinjett) grain.

Increased climatic variability is resulting in an increase of both the frequency and the magnitude of extreme climate events. Therefore, cereals may be exposed to more than one stress event in the growing season, which may ultimately affect crop yield and quality. Here, effects are reported of interaction of water deficits and/or a high-temperature event (32°C) during vegetative growth (terminal spikelet) with either of these stress events applied during generative growth (anthesis) in wheat. Influence of combinations of stress on protein fractions (albumins, globulins, gliadins and glutenins) in grains and stress-induced changes on the albumin and gliadin proteomes were investigated by 2-DE and MS. The synthesis of individual protein fractions was shown to be affected by both the type and time of the applied stresses. Identified drought or high-temperature-responsive proteins included proteins involved in primary metabolism, storage and stress response such as late embryogenesis abundant proteins, peroxiredoxins and α-amylase/trypsin inhibitors. Several proteins, e.g. heat shock protein and 14-3-3 protein changed in abundance only under multiple high temperatures.

Waterlogging pretreatment during vegetative growth improves tolerance to waterlogging after anthesis in wheat.

The interaction of multiple waterlogging events during vegetative growth (at the seven- and nine-leaf stage, and at heading) to a waterlogging event during the generative growth stage was studied in wheat (Triticum aestivum L. cv. Yangmai 9). Waterlogging before anthesis was found to effectively enhance tolerance to a waterlogging event after anthesis, as indicated by: (1) increasing net photosynthetsis (P(N)), stomatal conductance (g(s)) and transpiration (Tr) and maintaining high SPAD (soil plant analysis development) values; (2) enhancing use-efficiency of absorbed light energy in the stressed plants due to high maximum and actual quantum yield (F(v)/F(m), Φ(PSII)); (3) increasing activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT); (4) enhancing dry matter accumulation after anthesis and its contribution to grain mass, which further resulting in significantly improved grain yields. The results indicate that hardening by waterlogging applied before anthesis can effectively improve the tolerance of wheat to waterlogging events occurring during the generative growth stage.

Subtle evolutionary changes in the distribution of N-glycosylation sequons in the HIV-1 envelope glycoprotein 120.

Many viruses are known to undergo rapid evolutionary changes under selective pressures. The HIV-1 envelope glycoprotein 120 (gp120) shows extreme selection for NXS/T sequons, the potential sites of N-glycosylation. Although the average number of sequons in gp120 appears to be relatively stable in the recent past, even slight changes in the distribution of sequons may potentially play crucial roles in protein interaction and viral infection. This study tracked the prevalence and distribution of NXS/T sequons in gp120 over a period of 29 years (from 1981 to 2009). The gp120 showed location specific distribution of sequons with higher density in the outer domain of the molecule. The NXT sequon density decreased in the outer domain (despite the increase in the sequon specific amino acid threonine), but increased in the inner domain. By contrast, the NXS sequon density increased specifically in the outer domain. Related changes were also seen in the distribution probabilities of sequons in two domains. The results indicate that the gp120, chiefly in subtype B, is redistributing NXS/T sequons within the molecule with specific selection for NXS sequons. The subtle evolution of sequons in gp120 may have implications in viral resistance and infection.

Evolutionary Pattern of N-Glycosylation Sequon Numbers in Eukaryotic ABC Protein Superfamilies.

Many proteins contain a large number of NXS/T sequences (where X is any amino acid except proline) which are the potential sites of asparagine (N) linked glycosylation. However, the patterns of occurrence of these N-glycosylation sequons in related proteins or groups of proteins and their underlying causes have largely been unexplored. We computed the actual and probabilistic occurrence of NXS/T sequons in ABC protein superfamilies from eight diverse eukaryotic organisms. The ABC proteins contained significantly higher NXS/T sequon numbers compared to respective genome-wide average, but the sequon density was significantly lower owing to the increase in protein size and decrease in sequon specific amino acids. However, mammalian ABC proteins have significantly higher sequon density, and both serine and threonine containing sequons (NXS and NXT) have been positively selected-against the recent findings of only threonine specific Darwinian selection of sequons in proteins. The occurrence of sequons was positively correlated with the frequency of sequon specific amino acids and negatively correlated with proline and the NPS/T sequences. Further, the NPS/T sequences were significantly higher than expected in plant ABC proteins which have the lowest number of NXS/T sequons. Accordingly, compared to overall proteins, N-glycosylation sequons in ABC protein superfamilies have a distinct pattern of occurrence, and the results are discussed in an evolutionary perspective.

"PolyMin": software for identification of the minimum number of polymorphisms required for haplotype and genotype differentiation.

BACKGROUND: Analysis of allelic variation for relevant genes and monitoring chromosome segment transmission during selection are important approaches in plant breeding and ecology. To minimize the number of required molecular markers for this purpose is crucial due to cost and time constraints. To date, software for identification of the minimum number of required markers has been optimized for human genetics and is only partly matching the needs of plant scientists and breeders. In addition, different software packages with insufficient interoperability need to be combined to extract this information from available allele sequence data, resulting in an error-prone multi-step process of data handling. RESULTS: PolyMin, a computer program combining the detection of a minimum set of single nucleotide polymorphisms (SNPs) and/or insertions/deletions (INDELs) necessary for allele differentiation with the subsequent genotype differentiation in plant populations has been developed. Its efficiency in finding minimum sets of polymorphisms is comparable to other available program packages. CONCLUSION: A computer program detecting the minimum number of SNPs for haplotype discrimination and subsequent genotype differentiation has been developed, and its performance compared to other relevant software. The main advantages of PolyMin, especially for plant scientists, is the integration of procedures from sequence analysis to polymorphism selection within a single program, including both haplotype and genotype differentiation.

Water deficits and heat shock effects on photosynthesis of a transgenic Arabidopsis thaliana constitutively expressing ABP9, a bZIP transcription factor.

The effects of water deficits (WD), heat shock (HS), and both (HSWD) on photosynthetic carbon- and light-use efficiencies together with leaf ABA content, pigment composition and expressions of stress- and light harvesting-responsive genes were investigated in ABP9 [ABA-responsive-element (ABRE) binding protein 9] transgenic Arabidopsis (5P2). WD, HS, and HSWD significantly decreased photosynthetic rate (A) and stomatal conductance (g(s)) in wild-type plants (WT). A and g(s) of 5P2 transgenic plants were slightly reduced by a single stress and were hardly modified by HSWD. Although A and electron transport rate (ETR) in 5P2 plants were depressed under optimal growth conditions (control) in relation to WT, they were enhanced under HS and HSWD. These results indicate that ABP9 transgenic plants are less susceptible to stress than the WT. In addition, the increased ABA contents in both WT and 5P2 plants in response to WD and/or HS stresses suggest that declines in A and g(s) might have been due to ABA-induced stomatal closure. Moreover, compared with WT, 5P2 plants exhibited higher ABA content, instantaneous water use efficiency (IWUE), Chl a/b, NPQ, and lower Chl/carotenoid ratios. Finally, altered expression of stress-regulated or light harvesting-responsive genes was observed. Collectively, our results indicate that constitutive expression of ABP9 improves the photosynthetic capacity of plants under stress by adjusting photosynthetic pigment composition, dissipating excess light energy, and elevating carbon-use efficiency as well as increasing ABA content, IWUE, and expression of stress-defensive genes, suggesting an important role of ABP9 in the regulation of plant photosynthesis under stress.

Water mobility in the endosperm of high beta-glucan barley mutants as studied by nuclear magnetic resonance imaging.

(1)H NMR imaging (MRI) was used as a noninvasive technique to study water distribution and mobility in hydrated barley (Hordeum vulgare L.) seeds of accessions with varying content of beta glucan (BG), a highly hygroscopic cell wall component. High contents of BG in barley are unfavorable in malting where it leads to clotting of filters and hazing of beer as well as in animal feed where it hinders the rapid uptake of energy. However, a high content of BG has a positive nutritional effect, as it lowers the cholesterol and the glycaemic index. It was studied whether water distribution and mobility were related to content and location of BG. Water mobility was investigated by following the rate and mode of desiccation in hydrated single seeds. In order to determine the different water components, a multispin echo experiment was set up to reveal the T(2) transverse relaxation rates of water within the seeds. A principal component analysis (PCA) discriminated control seeds from the high-BG mutant seeds. MRI proved efficient in tracing the differences in water-holding capacity of contrasting barley seeds. All accessions showed nonuniform distribution of water at full hydration as well as during desiccation. The embryo retained water even after 36 h of drying, whereas the endosperm showed low and heterogeneous mobility of the water after drying. The relaxation time constants indicated that the BG mutants had regions of much higher water mobility around the ventral crease compared to the control. It is concluded that MRI can be applied to investigate temporal and spatial differences in the location of specific chemical compounds in single seeds.