Nitrogen nutrition effects on δ13C of plant respired CO2 are mostly caused by concurrent changes in organic acid utilisation and remobilisation.

Nitrogen (N) nutrition impacts on primary carbon metabolism and can lead to changes in δ13C of respired CO2. However, uncertainty remains as to whether (1) the effect of N nutrition is observed in all species, (2) N source also impacts on respired CO2 in roots and (3) a metabolic model can be constructed to predict δ13C of respired CO2 under different N sources. Here, we carried out isotopic measurements of respired CO2 and various metabolites using two species (spinach, French bean) grown under different NH4 +:NO3 - ratios. Both species showed a similar pattern, with a progressive 13C-depletion in leaf-respired CO2 as the ammonium proportion increased, while δ13C in root-respired CO2 showed little change. Supervised multivariate analysis showed that δ13C of respired CO2 was mostly determined by organic acid (malate, citrate) metabolism, in both leaves and roots. We then took advantage of nonstationary, two-pool modelling that explained 73% of variance in δ13C in respired CO2. It demonstrates the critical role of the balance between the utilisation of respiratory intermediates and the remobilisation of stored organic acids, regardless of anaplerotic bicarbonate fixation by phosphoenolpyruvate carboxylase and the organ considered.

Changes and their possible causes in δ13C of dark-respired CO2 and its putative bulk and soluble sources during maize ontogeny.

The issues of whether, where, and to what extent carbon isotopic fractionations occur during respiration affect interpretations of plant functions that are important to many disciplines across the natural sciences. Studies of carbon isotopic fractionation during dark respiration in C3 plants have repeatedly shown respired CO2 to be (13)C enriched relative to its bulk leaf sources and (13)C depleted relative to its bulk root sources. Furthermore, two studies showed respired CO2 to become progressively (13)C enriched during leaf ontogeny and (13)C depleted during root ontogeny in C3 legumes. As such data on C4 plants are scarce and contradictory, we investigated apparent respiratory fractionations of carbon and their possible causes in different organs of maize plants during early ontogeny. As in the C3 plants, leaf-respired CO2 was (13)C enriched whereas root-respired CO2 was (13)C depleted relative to their putative sources. In contrast to the findings for C3 plants, however, not only root- but also leaf-respired CO2 became more (13)C depleted during ontogeny. Leaf-respired CO2 was highly (13)C enriched just after light-dark transition but the enrichment rapidly decreased over time in darkness. We conclude that (i) although carbon isotopic fractionations in C4 maize and leguminous C3 crop roots are similar, increasing phosphoenolpyruvate-carboxylase activity during maize ontogeny could have produced the contrast between the progressive (13)C depletion of maize leaf-respired CO2 and (13)C enrichment of C3 leaf-respired CO2 over time, and (ii) in both maize and C3 leaves, highly (13)C enriched leaf-respired CO2 at light-to-dark transition and its rapid decrease during darkness, together with the observed decrease in leaf malate content, may be the result of a transient effect of light-enhanced dark respiration.

Population structure and genome-wide association analysis for frost tolerance in oat using continuous SNP array signal intensity ratios.

KEY MESSAGE: Infinium SNP data analysed as continuous intensity ratios enabled associating genotypic and phenotypic data from heterogeneous oat samples, showing that association mapping for frost tolerance is a feasible option. Oat is sensitive to freezing temperatures, which restricts the cultivation of fall-sown or winter oats to regions with milder winters. Fall-sown oats have a longer growth cycle, mature earlier, and have a higher productivity than spring-sown oats, therefore improving frost tolerance is an important goal in oat breeding. Our aim was to test the effectiveness of a Genome-Wide Association Study (GWAS) for mapping QTLs related to frost tolerance, using an approach that tolerates continuously distributed signals from SNPs in bulked samples from heterogeneous accessions. A collection of 138 European oat accessions, including landraces, old and modern varieties from 27 countries was genotyped using the Infinium 6K SNP array. The SNP data were analyzed as continuous intensity ratios, rather than converting them into discrete values by genotype calling. PCA and Ward's clustering of genetic similarities revealed the presence of two main groups of accessions, which roughly corresponded to Continental Europe and Mediterranean/Atlantic Europe, although a total of eight subgroups can be distinguished. The accessions were phenotyped for frost tolerance under controlled conditions by measuring fluorescence quantum yield of photosystem II after a freezing stress. GWAS were performed by a linear mixed model approach, comparing different corrections for population structure. All models detected three robust QTLs, two of which co-mapped with QTLs identified earlier in bi-parental mapping populations. The approach used in the present work shows that SNP array data of heterogeneous hexaploid oat samples can be successfully used to determine genetic similarities and to map associations to quantitative phenotypic traits.

Opposite carbon isotope discrimination during dark respiration in leaves versus roots - a review.

In general, leaves are (13) C-depleted compared with all other organs (e.g. roots, stem/trunk and fruits). Different hypotheses are formulated in the literature to explain this difference. One of these states that CO2 respired by leaves in the dark is (13) C-enriched compared with leaf organic matter, while it is (13) C-depleted in the case of root respiration. The opposite respiratory fractionation between leaves and roots was invoked as an explanation for the widespread between-organ isotopic differences. After summarizing the basics of photosynthetic and post-photosynthetic discrimination, we mainly review the recent findings on the isotopic composition of CO2 respired by leaves (autotrophic organs) and roots (heterotrophic organs) compared with respective plant material (i.e. apparent respiratory fractionation) as well as its metabolic origin. The potential impact of such fractionation on the isotopic signal of organic matter (OM) is discussed. Some perspectives for future studies are also proposed .

Harden the chloroplast to protect the plant.

The chloroplast is the central switch of the plant's response to cold and light stress. The ability of many plant species to develop a cold tolerant phenotype is dependent on the presence of light and photosynthetic activity during low-temperature growth. Light exposure at low temperature stimulates an over-reduction of the plastoquinone pool as well as the accumulation of reactive oxygen species, and both metabolic conditions generate a retrograde signal controlling nuclear gene expression. At the same time the chloroplast is the target of many cold acclimation processes which are the results of the chloroplast-nucleus cross-talk. Often, the extent of cold acclimation of the chloroplast is tightly correlated with the overall plant tolerance to chilling and freezing temperatures, a finding suggesting that the chloroplast cold acclimation could be the rate limiting factor in the adaptation to low temperature.

The plant phenological online database (PPODB): an online database for long-term phenological data.

We present an online database that provides unrestricted and free access to over 16 million plant phenological observations from over 8,000 stations in Central Europe between the years 1880 and 2009. Unique features are (1) a flexible and unrestricted access to a full-fledged database, allowing for a wide range of individual queries and data retrieval, (2) historical data for Germany before 1951 ranging back to 1880, and (3) more than 480 curated long-term time series covering more than 100 years for individual phenological phases and plants combined over Natural Regions in Germany. Time series for single stations or Natural Regions can be accessed through a user-friendly graphical geo-referenced interface. The joint databases made available with the plant phenological database PPODB render accessible an important data source for further analyses of long-term changes in phenology. The database can be accessed via www.ppodb.de .

Long-Term In Situ Conservation Drove Microevolution of Solina d'Abruzzo Wheat on Adaptive, Agronomic and Qualitative Traits.

Solina is an example of a bread wheat landrace that has been conserved in situ for centuries in Central Italy. A core collection of Solina lines sampled in areas at different altitudes and climatic conditions was obtained and genotyped. A clustering analysis based on a wide SNP dataset generated from DArTseq analysis outlined the existence of two main groups, which, after Fst analysis, showed polymorphism in genes associated with vernalization and photoperiod response. Starting from the hypothesis that the different pedoclimatic environments in which Solina lines were conserved may have shaped the population, some phenotypic characteristics were studied in the Solina core collection. Growth habit, low-temperature resistance, allelic variations at major loci involved in vernalization response, and sensitivity to photoperiod were evaluated, together with seed morphologies, grain colour, and hardness. The two Solina groups showed different responses to low temperatures and to photoperiod-specific allelic variations as well as the different morphology and technological characteristics of the grain. In conclusion, the long-term in situ conservation of Solina in environments sited at different altitudes has had an impact on the evolution of this landrace which, despite its high genetic diversity, remains clearly identifiable and distinct so as to be included in conservation varieties.

Nitrate and ammonium differ in their impact on δ13C of plant metabolites and respired CO2 from tobacco leaves.

The carbon isotopic composition (δ13C) of foliage is often used as proxy for plant performance. However, the effect of N O 3 - vs. N H 4 + supply on δ13C of leaf metabolites and respired CO2 is largely unknown. We supplied tobacco plants with a gradient of N O 3 - to N H 4 + concentration ratios and determined gas exchange variables, concentrations and δ13C of tricarboxylic acid (TCA) cycle intermediates, δ13C of dark-respired CO2, and activities of key enzymes nitrate reductase, malic enzyme and phosphoenolpyruvate carboxylase. Net assimilation rate, dry biomass and concentrations of organic acids and starch decreased along the gradient. In contrast, respiration rates, concentrations of intercellular CO2, soluble sugars and amino acids increased. As N O 3 - decreased, activities of all measured enzymes decreased. δ13C of CO2 and organic acids closely co-varied and were more positive under N O 3 - supply, suggesting organic acids as potential substrates for respiration. Together with estimates of intra-molecular 13C enrichment in malate, we conclude that a change in the anaplerotic reaction of the TCA cycle possibly contributes to 13C enrichment in organic acids and respired CO2 under N O 3 - supply. Thus, the effect of N O 3 - vs. N H 4 + on δ13C is highly relevant, particularly if δ13C of leaf metabolites or respiration is used as proxy for plant performance.

On the 13C/12C isotopic signal of day and night respiration at the mesocosm level.

While there is currently intense effort to examine the (13)C signal of CO(2) evolved in the dark, less is known on the isotope composition of day-respired CO(2). This lack of knowledge stems from technical difficulties to measure the pure respiratory isotopic signal: day respiration is mixed up with photorespiration, and there is no obvious way to separate photosynthetic fractionation (pure c(i)/c(a) effect) from respiratory effect (production of CO(2) with a different delta(13)C value from that of net-fixed CO(2)) at the ecosystem level. Here, we took advantage of new simple equations, and applied them to sunflower canopies grown under low and high [CO(2)]. We show that whole mesocosm-respired CO(2) is slightly (13)C depleted in the light at the mesocosm level (by 0.2-0.8 per thousand), while it is slightly (13)C enriched in darkness (by 1.5-3.2 per thousand). The turnover of the respiratory carbon pool after labelling appears similar in the light and in the dark, and accordingly, a hierarchical clustering analysis shows a close correlation between the (13)C abundance in day- and night-evolved CO(2). We conclude that the carbon source for respiration is similar in the dark and in the light, but the metabolic pathways associated with CO(2) production may change, thereby explaining the different (12)C/(13)C respiratory fractionations in the light and in the dark.

Elevated CO2 Impact on Common Wheat (Triticum aestivum L.) Yield, Wholemeal Quality, and Sanitary Risk.

The rising atmospheric CO2, concentration is expected to exert a strong impact on crop production, enhancing crop growth but threatening food security and safety. An improver wheat, a hybrid, and its parents were grown at elevated CO2, e[CO2] in open field, and their yield and rheological, nutritional, and sanitary quality were assessed. For all cultivars, grain yield increased (+16%) and protein content decreased (-7%), accompanied by a reduction in dough strength. Grain nitrogen yield increased (+24%) only in ordinary bread making cultivars. e[CO2] did not result in significant changes in phenolic acid content and composition, whereas it produced a significant increase in the deoxynivalenol content. Different responses to e[CO2] between cultivars were found for yield parameters, while the effect on qualitative traits was quite similar. In the upcoming wheat cropping systems, agronomic practices and cultivar selection suited to guarantee higher nitrogen responsiveness and minimization of sanitary risk are required.

Consistent patterns in leaf lamina and leaf vein carbon isotope composition across ten herbs and tree species.

Wide-spread post-photosynthetic fractionation processes deplete metabolites and plant compartments in (13)C relative to assimilates to varying degrees. Fragmentation fractionation and exchange of metabolites with distinct isotopic signatures across organ boundaries further modify the patterns of plant isotopic composition. Heterotrophic organs tend to become isotopically heavier than the putative source material as a result of respiratory metabolism. In addition fractionation may occur during metabolite transport across organ and tissue boundaries. Leaf laminae, veins and petioles are leaf compartments that are arranged along a gradient of increasing weight of heterotrophic processes and along a transport chain. Thus, we expect to find consistent patterns of isotopic signatures associated with this gradient. Earlier studies on leaves of Fagus sylvatica, Glycine max, and Saccharum officinarum showed that the organic mass and cellulose of major veins or petioles were consistently more positive than the respective fraction in leaf laminae. The objective of the current study was to assess whether this pattern can be detected in a greater set of plant species. Leaves from ten species were collected in the summer of 2006 outdoors and in glasshouses. Leaf laminae including small veins were separated from the major veins and the isotopic signatures of the organic mass, and the soluble and non-soluble fractions were measured for laminae and veins separately. The organic mass, and the soluble and non-soluble fractions of leaf laminae, were depleted in (13)C relative to the veins in all cases. A general trend for the signature of organic mass being more depleted in (13)C than the soluble fraction is in accordance with well-known patterns of fractionation between metabolites.

Metabolomic responses triggered by arbuscular mycorrhiza enhance tolerance to water stress in wheat cultivars.

Under global climate change forecasts, the pressure of environmental stressors (and in particular drought) on crop productivity is expected to rise and challenge further global food security. The application of beneficial microorganisms may represent an environment friendly tool to secure improved crop performance and yield stability. Accordingly, this current study aimed at elucidating the metabolomic responses triggered by mycorrhizal (Funneliformis mosseae) inoculation of durum (Triticum durum Desf.; cv. 'Mongibello') and bread wheat cultivars (Triticum aestivum L.; cv. 'Chinese Spring') under full irrigation and water deficit regimes. Metabolomics indicated a similar regulation of secondary metabolism in both bread and durum wheat cultivars following water limiting conditions. Nonetheless, a mycorrhizal fungi (AMF) x cultivar interaction could be observed, with the bread wheat cultivar being more affected by arbuscular colonization under water limiting conditions. Discriminant compounds could be mostly related to sugars and lipids, both being positively modulated by AMF colonization under water stress. Moreover, a regulation of metabolites related to oxidative stress and a tuning of crosstalk between phytohormones were also evidenced. Among the latter, the stimulation of the brassinosteroids biosynthetic pathway was particularly evident in inoculated wheat roots, supporting the hypothesis of their involvement in enhancing plant response to water stress and modulation of oxidative stress conditions. This study proposes new insights on the modulation of the tripartite interaction plant-AMF-environmental stress.

Proteomic insight into the mitigation of wheat root drought stress by arbuscular mycorrhizae.

Arbuscular mycorrhizal fungi (AMF) are plant growth promoters that ameliorate plant-water relations and the nutrient uptake of wheat. In this work, two cultivars of Triticum spp., a bread and a durum wheat, grown under drought stress and inoculated or not by AMF, are evaluated through a shotgun proteomic approach. The AMF association had beneficial effects as compared to non-mycorrhizal roots, in both bread and durum wheat. The beneficial symbiosis was confirmed by measuring morphological and physiological traits. In our work, we identified 50 statistically differential proteins in the bread wheat cultivar and 66 differential proteins in the durum wheat cultivar. The findings highlighted a modulation of proteins related to sugar metabolism, cell wall rearrangement, cytoskeletal organization and sulphur-containing proteins, as well as proteins related to plant stress responses. Among differentially expressed proteins both cultivars evidenced a decrease in sucrose:fructan 6-fructosyltransferas. In durum wheat oxylipin signalling pathway was involved with two proteins: increased 12-oxo-phytodienoic acid reductase and decreased jasmonate-induced protein, both related to the biosynthesis of jasmonic acid. Interactome analysis highlighted the possible involvement of ubiquitin although not evidenced among differentially expressed proteins. The AMF association helps wheat roots reducing the osmotic stress and maintaining cellular integrity. BIOLOGICAL SIGNIFICANCE: Drought is one of the major constraints that plants must face in some areas of the world, associated to climate change, negatively affecting the worldwide plant productivity. The adoption of innovative agronomic protocols may represent a winning strategy in facing this challenge. The arbuscular mycorrhizal fungi (AMF) inoculation may represent a natural and sustainable way to mitigate the negative effects due to drought in several crop, ameliorating plant growth and development. Studies on the proteomic responses specific to AMF in drought-stressed plants will help clarify how mycorrhization elicits plant growth, nutrient uptake, and stress-tolerance responses. Such studies also offer the potential to find biological markers and genetic targets to be used during breeding for new drought-resistant varieties.

Occurrence of Fusarium langsethiae and T-2 and HT-2 Toxins in Italian Malting Barley.

T-2 and HT-2 toxins are two of the most toxic members of type-A trichothecenes, produced by a number of Fusarium species. The occurrence of these mycotoxins was studied in barley samples during a survey carried out in the 2011-2014 growing seasons in climatically different regions in Italy. The percentage of samples found positive ranges from 22% to 53%, with values included between 26 and 787 µg/kg. The percentage of samples with a T-2 and HT-2 content above the EU indicative levels for barley of 200 µg/kg ranges from 2% to 19.6% in the 2011-2014 period. The fungal species responsible for the production of these toxins in 100% of positive samples has been identified as Fusarium langsethiae, a well-known producer of T-2 and HT-2 toxins. A positive correlation between the amount of F. langsethiae DNA and of the sum of T-2 and HT-2 toxins was found. This is the first report on the occurrence of F. langsethiae-and of its toxic metabolites T-2 and HT-2-in malting barley grown in Italy.

Intraspecific variability of carbon isotope discrimination and its correlation with grain yield in safflower: prospects for selection in a Mediterranean climate.

The goals of the present study were to obtain a first estimate of intraspecific variability of carbon isotope discrimination (Δ) in safflower, a thistle-like herbaceous plant, and to determine the statistical relationship between Δ and grain yield as well as its components in a collection of 45 accessions of different origins. Grain yield and aboveground biomass, harvest index, average grain weight, and Δ (measured on the bulk leaf organic matter) were investigated in experimental field conditions. A large variability was noted for all traits but a principal component analysis (PCA) allowed to identify several homogeneous groups of accessions. Average grain yield per plant varied between 1 and 39 g. Δ varied between 21.3 and 25.2 ‰, i.e. a large variation of 3.9 ‰. In our experiment, the variation of Δ was not significantly related to that of grain yield in the whole accession sample. However, we found contrasting trends for this relation within accession groups. These initial results motivate further experiments to assess more in depth correlation between Δ and yield in safflower and are encouraging regarding the possibility of using Δ as an effective selection index in safflower to obtain genotypes that efficiently consume water. This study also highlighted one accession that combines the two characters required in the Mediterranean regions, i.e. high yield performance and high water-use efficiency.

Changes in δ(13)C of dark respired CO2 and organic matter of different organs during early ontogeny in peanut plants.

Carbon isotope composition in respired CO2 and organic matter of individual organs were measured on peanut seedlings during early ontogeny in order to compare fractionation during heterotrophic growth and transition to autotrophy in a species with lipid seed reserves with earlier results obtained on beans. Despite a high lipid content in peanut seeds (48%) compared with bean seeds (1.5%), the isotope composition of leaf- and root-respired CO2 as well as its changes during ontogeny were similar to already published data on bean seedlings: leaf-respired CO2 became (13)C-enriched reaching -21.5‰, while root-respired CO2 became (13)C-depleted reaching around -31‰ at the four-leaf stage. The opposite respiratory fractionation in leaves vs. roots already reported for C3 herbs was thus confirmed for peanuts. However, contrarily to beans, the peanut cotyledon-respired CO2 was markedly (13)C-enriched, and its (13)C-depletion was noted from the two-leaf stage onwards only. Carbohydrate amounts being very low in peanut seeds, this cannot be attributed solely to their use as respiratory substrate. The potential role of isotope fractionation during glyoxylate cycle and/or gluconeogenesis on the (13)C-enriched cotyledon-respired CO2 is discussed.

Evaluation of methods for the combination of phenological time series and outlier detection.

There are several applications of combined phenological time series; e.g., trend analysis with long continuous time series, obtaining a compound and representative time series around weather stations for model fitting, data gap filling and outlier detection. Various methods to combine phenological time series have been proposed. We show that all of these methods can be analyzed within the theory of linear models. This has the advantage that the underlying assumptions for each model become transparent providing a theoretical basis for selecting a model for a particular situation. Moreover, the common theoretical background provides a means of comparing methods by Monte-Carlo simulation and with real data. Additionally, we explored the influences of two outlier detection methods. We show that the error called the month-mistake, whose origin is known and which is one of the few mistakes that can be detected in phenological data because of its large deviation, is best detected by the distribution-free 30-day residual rule in combination with a robust estimation procedure based on the minimization of the sum of absolute residuals (L1-norm).

Divergence in delta(13)C of dark respired CO(2) and bulk organic matter occurs during the transition between heterotrophy and autotrophy in Phaseolus vulgaris plants.

Substantial evidence has been published in recent years demonstrating that postphotosynthetic fractionations occur in plants, leading to (13)C-enrichment in heterotrophic (as compared with autotrophic) organs. However, less is known about the mechanism responsible for changes in these responses during plant development. The isotopic signature of both organic matter and respired CO(2) for different organs of French bean (Phaseolus vulgaris) was investigated during early ontogeny, in order to identify the developmental stage at which isotopic changes occur. Isotopic analyses of metabolites and mass balance calculations helped to constrain the metabolic processes involved. At the plant scale, apparent respiratory fractionation was constantly positive in the heterotrophic phase (c. 1 per thousand) and turned negative with autotrophy acquisition (down to -3.08 per thousand). Initially very close to that of the dry seed (-26.83 +/- 0.69 per thousand), isotopic signatures of organic matter and respired CO(2) diverged (in opposite directions) in leaves and roots after onset of photosynthesis. Respired CO(2) reached values up to -20 per thousand in leaves and became (13)C-depleted down to -29 per thousand in roots. It was concluded that isotopic differences between organs occurred subsequent to metabolic changes in the seedling during the transition from heterotrophy to autotrophy. They were especially related to respiration and respiratory fractionation.

Relationships between leaf conductance to CO2 diffusion and photosynthesis in micropropagated grapevine plants, before and after ex vitro acclimatization.

In vitro-cultured plants typically show a low photosynthetic activity, which is considered detrimental to subsequent ex vitro acclimatization. Studies conducted so far have approached this problem by analysing the biochemical and photochemical aspects of photosynthesis, while very little attention has been paid to the role of leaf conductance to CO(2) diffusion, which often represents an important constraint to CO(2) assimilation in naturally grown plants. Mesophyll conductance, in particular, has never been determined in in vitro plants, and no information exists as to whether it represents a limitation to carbon assimilation during in vitro growth and subsequent ex vitro acclimatization. In this study, by means of simultaneous gas exchange and chlorophyll fluorescence measurements, the stomatal and mesophyll conductance to CO(2) diffusion were assessed in in vitro-cultured plants of the grapevine rootstock '41B' (Vitis vinifera 'Chasselas'xVitis berlandieri), prior to and after ex vitro acclimatization. Their impact on electron transport rate partitioning and on limitation of potential net assimilation rate was analysed. In vitro plants had a high stomatal conductance, 155 versus 50 mmol m(-2) s(-1) in acclimatized plants, which ensured a higher CO(2) concentration in the chloroplasts, and a 7% higher electron flow to the carbon reduction pathway. The high stomatal conductance was counterbalanced by a low mesophyll conductance, 43 versus 285 mmol m(-2) s(-1), which accounted for a 14.5% estimated relative limitation to photosynthesis against 2.1% estimated in acclimatized plants. It was concluded that mesophyll conductance represents an important limitation for in vitro plant photosynthesis, and that in acclimatization studies the correct comparison of photosynthetic activity between in vitro and acclimatized plants must take into account the contribution of both stomatal and mesophyll conductance.