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Parthenogenesis is the main technique to produce doubled haploid lines in melon. Although parthenogenesis is a genotype-dependent process and melon has a huge genotypic diversity, we developed a successful protocol for haploid embryo production via pollination with irradiated pollen and a protocol for chromosome doubling of haploid plants of 'Piel de Sapo' genotypes. 'Piel de sapo' genotype has lower efficiencies during the process in comparison with other genotypes, for instance, of the agrestis subspecies. Nevertheless, the doubled haploid lines produced have a great potential as pure parentals for hybrid seed production.

Anther culture is the most used technique to produce doubled haploid lines in rice. This technique is well developed in a wide range of indica rice genotypes. However, in japonica type, and more specifically, the Mediterranean japonica, the protocols are yet to be optimized. Japonica and indica have different androgenic response, as well as different induction and regeneration rates, albinism ratios and chromosome doubling competence. The step-by-step anther culture protocol presented in this chapter allows to regenerate doubled haploid rice plantlets from anther microspores in 8 months. We also include an in vitro chromosome doubling protocol to induce doubled haploids from haploid plantlets by immersion in a colchicine solution. This chromosome doubling protocol complements the anther culture by taking advantage of the regenerated haploid plantlets.

○ The effects are reported here of inoculation with the soil-borne pathogen Fusarium oxysporum f. sp. lycopersici race 1 (FOL-1) on the photosynthetic capacity of tomato plants (Lycopersicon esculentum cv. Roma). ○ Plants were grown in cork compost with a pathogen concentration of C. 1 x 10 5 cfu ml -1 , or without the fungus, for 31 d in controlled environmental chambers. Noninoculated plants were also subjected to progressive drought by withholding water. ○ During the first stage of disease development, FOL-1 wilt decreased the light-saturated rate of CO 2 assimilation. This was accompanied by a decreases in the maximum carboxylation velocity and the capacity for ribulose-1,5-bisphophate regeneration, in the absence of any significant photodamage to PSII, and increased stomatal limitation of the CO 2 supply. As the disease progressed, the maximum quantum efficiency of PSII photochemistry decreased in the leaves. FOL-1 wilt also decreased leaf area, therefore a reduction in the ability of the tomato plants to capture photosynthetically active radiation is also a major factor in determining the depression in the photosynthetic productivity of these infected plants. ○ The effect of the infection by FOL-1 on leaf photosynthesis was mimicked by water stress.

Parthenogenesis is the main technique to produce doubled haploid lines in melon. Although parthenogenesis is a genotype-dependent process and melon has a huge genotypic diversity, we developed a successful protocol for haploid embryo production via pollination with irradiated pollen and a protocol for chromosome doubling of haploid plants of 'Piel de Sapo' genotypes. 'Piel de sapo' genotype has lower efficiencies during the process in comparison with other genotypes, for instance, of the agrestis subspecies. Nevertheless, the doubled haploid lines produced have a great potential as pure parentals for hybrid seed production.

Arundo donax L. (giant reed) is a perennial rhizomatous grass and has been identified as an important non-food biomass crop with capacity for cultivation in marginal and degraded lands where water scarcity conditions frequently occur due to climate change. This review analyzes the effect of water stress on photosynthetic capacity and biomass production in multiple giant reed ecotypes grown in different regions around the world. Furthermore, this review will attempt to explain the reason for the high photosynthetic capacity of giant reed even under changing environmental conditions as well as indicate other morphological reasons that could contribute to maintaining this high photosynthetic rate. Finally, future research in favor of selecting ecotypes with drought tolerance is proposed.

Rice, a major staple, is the most salt-sensitive cereal. High salinity triggers several adaptive responses in rice to cope with osmotic and ionic stress at the physiological, cellular, and molecular levels. A major QTL for salinity tolerance, named Saltol, is present on chromosome 1 of Indian landraces such as Pokkali and Nona Bokra. The early proteomic and physiological responses to salinity in roots and shoots of FL478, an inbred rice line harboring the Saltol QTL, were characterized. Plantlets were cultured in hydroponic cultures with 100 mmol L−1 NaCl and evaluated at 6, 24, and 48 h. At the physiological level, root length significantly increased at 48 h, whereas shoot length was reduced. The Na+/K+ ratio was maintained at lower levels in shoots than in roots, suggesting that roots play a protective role. More than 2000 proteins were detected in both tissues. Roots showed a faster and more coordinated proteomic response than shoots, evident after only 6 h of treatment. These responses showed clear correspondence with those of proteins involved in transcription and translation. Maintenance of mitochondrial activity and amino acid metabolism in roots, and activation of stress-responsive proteins such as dehydrins and PLAT in shoots, may play a key role during the response of the plant to salinity stress. Proteomic and physiological responses showed that roots respond in a more highly adaptive manner than shoots to salinity stress, suggesting that this tissue is critical to the tolerance observed in cultivars harboring Saltol., This project has received funding from the European Union’s Horizon 2020 Research and Innovation Program under grant agreement No[678168]-NEURICE. The authors would also like to acknowledge the PFCHA Program of CONICYT for granting C.L. a BecasChile scholarship [72140224].

Anther culture is the most used technique to produce doubled haploid lines in rice. This technique is well developed in a wide range of indica rice genotypes. However, in japonica type, and more specifically, the Mediterranean japonica, the protocols are yet to be optimized. Japonica and indica have different androgenic response, as well as different induction and regeneration rates, albinism ratios and chromosome doubling competence. The step-by-step anther culture protocol presented in this chapter allows to regenerate doubled haploid rice plantlets from anther microspores in 8 months. We also include an in vitro chromosome doubling protocol to induce doubled haploids from haploid plantlets by immersion in a colchicine solution. This chromosome doubling protocol complements the anther culture by taking advantage of the regenerated haploid plantlets.

Drought is one of the most limiting factors on crop productivity under Mediterranean conditions, where the leguminous species alfalfa (Medicago sativa L.) is extensively cultivated. Whereas the effect of drought on plant performance has been widely described at leaf and nodule levels, less attention has been given to plant-nodule interactions and their implication on metabolites exchange during a regrowth period, when water is limiting. For this purpose, physiological characterization and metabolite profiles in different plant organs and nodules were undertaken under water deficit, including regrowth after removal of aerial parts. In order to study in more detail how nitrogen (N) metabolism was affected by water stress, plants were labelled with N-enriched isotopic air (15N2) using especially designed chambers. Water stress affected negatively water status and photosynthetic machinery. Metabolite profile and isotopic composition analyses revealed that, water deficit induced major changes in the accumulation of amino acids (proline, asparagine, histidine, lysine and cysteine), carbohydrates (sucrose, xylose and pinitol) and organic acids (fumarate, succinate and maleic acid) in the nodules in comparison with other organs. The lower 15N-labeling observed in serine, compared with other amino acids, was related with its high turnover rate, which in turn, indicates its potential implication in photorespiration. Isotopic analysis of amino acids also revealed that proline synthesis in the nodule was a local response to water stress and not associated with a feedback inhibition from the leaves. Water deficit induced extensive reprogramming of whole-plant C and N metabolism, including when the aerial part was removedto trigger regrowth., This study was supported in part by the European research project PERMED (INCO- CT-2004-509140), the Spanish Ministry of Science and Technology (CGL2009-13079-C02) and i-COOP-suelos y legumbres (2016SU0016) funded by the National Research Council. GM was the recipient of a doctoral fellowship (Programa Nacional de Formación de Personal Universitario, AP2005–2916) sponsored by the Spanish Ministry of Education and Science.

Doubled haploids in cucurbit species are produced through in situ parthenogenesis via pollination with irradiated pollen for further use as parental lines for hybrid F1 production. In this study, seven genotypes of melon “Piel de Sapo” were appraised for agronomic traits and pathogen resistances to evaluate its commercial value and used as donor plant material for the parthenogenetic process. Then, in situ parthenogenetic capacity of melon “Piel de Sapo” germplasm was evaluated and optimized. Several steps of the parthenogenetic process were assessed in this study such as melon fruit set after pollination with irradiated pollen, haploid embryo obtention, in vitro germination and growth of parthenogenetic embryos and plantlets, in vitro and in vivo chromosome doubling with colchicine or oryzalin and fruit set of doubled haploid lines. Parthenogenetic efficiencies of “Piel de Sapo” genotypes showed a high genotypic dependency during the whole process. Three different methods were assayed for parthenogenetic embryo detection: one-by-one, X-ray and liquid medium. X-ray radiography of seeds was four times faster than one-by-one method and jeopardized eight times less parthenogenetic embryo obtention than liquid medium. One third of melon fruits set after pollination with irradiated pollen contained at least one parthenogenetic embryo. The 50.94% of the embryos rescued did not develop into plantlets because failed to germinate or plantlet died at the first stages of development because of deleterious gene combination in haploid homozygosity. The distribution of the ploidy-level of the 26 parthenogenetic plantlets obtained was: 73.08% haploid, 23.08% spontaneous doubled haploid and 3.84% mixoploid. Two in vitro chromosome doubling methods, with colchicine or oryzalin, were compared with a third in vivo colchicine method. In vivo immersion of apical meristems in a colchicine solution for 2 h showed the highest results of plant survival, 57.33%, and chromosome doubling, 9.30% mixoploids and 20.93% doubled haploids. Fruit set and seed recovery of doubled haploids lines was achieved. In this study, doubled haploid lines were produced from seven donor genotypes of melon “Piel de Sapo,” however, further improvements are need in order to increase the parthenogenetic efficiency.

Double haploid technique aims to generate pure inbred lines for basic research and as commercial cultivars. This technique consists in a first obtention of haploid plants and a subsequent chromosome doubling, which can be separated in time or overlapped according to the procedure of each species. Since ever, much effort has been focused on haploid production via androgenesis, gynogenesis or parthenogenesis. Haploid plant obtention is the first step and requires more effort to optimize the haploid plant regeneration, and because of that chromosome doubling had usually been lagged behind. Nevertheless, chromosome doubling has recently been of renewed interest in double haploid research to increase double haploid plant obtention by optimizing and analysing different procedures. New antimitotic compounds, procedures and application methods are being published to ensure the success once haploid material has been regenerated. Moreover, haploid inducer-mediated genome-editing CRISPR/Cas9 system is a new breakthrough method to produce haploid plant material and requires chromosome doubling to succeed. This system could be of great importance for those species with non-successful haploid regeneration methods, or for recalcitrant species. In all cases, the new deployment of this system will demand a suitable chromosome doubling protocol. In this review we run through the existing double haploid and chromosome doubling methods to identify potential opportunities to enhance the breeding process in major crops.

Rice is one of the greatest calorie supply for the world population, especially since its production is almost entirely destined to direct human consumption and its demand will increase along with the world population. There are efforts worldwide to increase rice yields by obtaining new improved and stabilized rice lines. The rice anther culture, a fast and cheap technique, allows to obtain double haploid lines in less than one year. We report its application with an improved protocol in four Mediterranean japonica rice genotypes at F2 generation. We performed a screening test for cold-pretreatment at 5.0±0.1°C and concluded that the optimum duration was 9 days as it produced the higher rate of anther-derived callus induction. This revised protocol was successfully applied to the four genotypes, obtaining good results in all the procedure's steps. At the end, more than 100 of double haploid green plants were generated. Moreover, 9 lines obtained from the anther culture procedure showed good qualities for the Spanish market at the growing, farming and grain production level during the field assays. Therefore, we report an improved anther culture procedure for obtaining double haploid lines from temperate japonica rice genotypes showing high commercialization expectance.

Carbon (C) allocation is a key determinant of plant functioning and ecosystem processes. Its dynamic responses to environmental conditions and global change are still poorly understood. This holds particularly true for dwarf shrubs, which are an important component of high mountain vegetation. We studied the short-term C allocation dynamics in two dominant dwarf shrub species, Vaccinium myrtillus and Vaccinium uliginosum, at two subalpine sites, in the Pyrenees and in the Alps, and additionally performed an experimental rain exclusion to simulate summer drought at the site in the Alps. We compared net assimilation rates ( A max ), stomatal conductance ( g s ) and shoot water potentials ( Ψ S ) between the two species and carried out a 13 C pulse labelling experiment to trace their C allocation dynamics, both under natural conditions and in the drought experiment. Under ambient conditions, V. uliginosum showed higher A max and g s and used recent assimilates as a respiratory substrate more slowly than V. myrtillus . C allocation dynamics in current-year shoots differed significantly between the species, showing a progressive decrease after the pulse labelling in V. uliginosum shoots but a progressive increase in V. myrtillus shoots. In the drought experiment, a significant reduction of soil moisture caused predawn Ψ S below −1.2 MPa in both species, which is considered close to the turgor loss point. However, the stomatal conductance and the speed of transfer of newly assimilated C to belowground organs decreased in V. uliginosum only. We found a strong coupling in C allocation dynamics between xylem and phloem tissues of the rhizome, which was maintained under drought. Although both species were largely tolerant to the drought imposed, our results indicate a higher drought sensitivity of V. uliginosum compared to V. myrtillus , with potential implications for community composition and ecosystem C cycling in a future climate.

Doubled haploids have played a major role in cucurbit breeding for the past four decades. In situ parthenogenesis via irradiated pollen is the preferred technique to obtain haploid plantlets whose chromosomes are then doubled in Cucurbitaceae, such as melon, cucumber, pumpkin, squash and winter squash. In contrast to doubled haploid procedures in other species, in situ parthenogenesis in cucurbits presents many limiting factors which impede efficient production of haploids. In addition, it is very time-consuming and labor-intensive. However, the haploid inducer-mediated genome-editing system is a breakthrough technology for producing doubled haploids. Several reports have described using the CRISPR/Cas9 system in cucurbit species, and although its application has many bottlenecks, the targeted knock-out of the CENH3 gene will allow breeders to obtain haploid inducer lines that can be used to obtain parthenogenetic embryos. In this review, we discuss the progress made towards the development of doubled haploids and haploid inducer genotypes using CRISPR/Cas9 technologies in cucurbit species. The present review provides insights for the application of haploid inducer-mediated genome-editing system in cucurbit species

Rice is one of the greatest calorie supply for the world population, especially since its production is almost entirely destined to direct human consumption and its demand will increase along with the world population. There are efforts worldwide to increase rice yields by obtaining new improved and stabilized rice lines. The rice anther culture, a fast and cheap technique, allows to obtain double haploid lines in less than one year. We report its application with an improved protocol in four Mediterranean japonica rice genotypes at F(2) generation. We performed a screening test for cold-pretreatment at 5.0±0.1°C and concluded that the optimum duration was 9 days as it produced the higher rate of anther-derived callus induction. This revised protocol was successfully applied to the four genotypes, obtaining good results in all the procedure’s steps. At the end, more than 100 of double haploid green plants were generated. Moreover, 9 lines obtained from the anther culture procedure showed good qualities for the Spanish market at the growing, farming and grain production level during the field assays. Therefore, we report an improved anther culture procedure for obtaining double haploid lines from temperate japonica rice genotypes showing high commercialization expectance.

Herbivory is one of the most globally distributed disturbances affecting carbon (C)-cycling in trees, yet our understanding of how it alters tree C-allocation to different functions such as storage, growth or rhizodeposition is still limited. Prioritized C-allocation to storage replenishment vs growth could explain the fast recovery of C-storage pools frequently observed in growth-reduced defoliated trees. We performed continuous 13C-labeling coupled to clipping to quantify the effects of simulated browsing on the growth, leaf morphology and relative allocation of stored vs recently assimilated C to the growth (bulk biomass) and non-structural carbohydrate (NSC) stores (soluble sugars and starch) of the different organs of two tree species: diffuse-porous (Betula pubescens Ehrh.) and ring-porous (Quercus petraea [Matt.] Liebl.). Carbon-transfers from plants to bulk and rhizosphere soil were also evaluated. Clipped birch and oak trees shifted their C-allocation patterns above-ground as a means to recover from defoliation. However, such increased allocation to current-year stems and leaves did not entail reductions in the allocation to the rhizosphere, which remained unchanged between clipped and control trees of both species. Betula pubescens and Q. petraea showed differences in their vulnerability and recovery strategies to clipping, the ring-porous species being less affected in terms of growth and architecture by clipping than the diffuse-porous. These contrasting patterns could be partly explained by differences in their C cycling after clipping. Defoliated oaks showed a faster recovery of their canopy biomass, which was supported by increased allocation of new C, but associated with large decreases in their fine root biomass. Following clipping, both species recovered NSC pools to a larger extent than growth, but the allocation of 13C-labeled photo-assimilates into storage compounds was not increased as compared with controls. Despite their different response to clipping, our results indicate no preventative allocation into storage occurred during the first year after clipping in either of the species.

CRISPR/Cas9 system has been widely applied in many plant species to induce mutations in the genome for studying gene function and improving crops. However, to our knowledge, there is no report of CRISPR/Cas9-mediated genome editing in melon (Cucumis melo). In our study, phytoene desaturase gene of melon (CmPDS) was selected as target for the CRISPR/Cas9 system with two designed gRNAs, targeting exons 1 and 2. A construct (pHSE-CmPDS) carrying both gRNAs and the Cas9 protein was delivered by PEG-mediated transformation in protoplasts. Mutations were detected in protoplasts for both gRNAs. Subsequently, Agrobacterium-mediated transformation of cotyledonary explants was carried out, and fully albino and chimeric albino plants were successfully regenerated. A regeneration efficiency of 71% of transformed plants was achieved from cotyledonary explants, a 39% of genetic transformed plants were successful gene edited, and finally, a 42–45% of mutation rate was detected by Sanger analysis. In melon protoplasts and plants most mutations were substitutions (91%), followed by insertions (7%) and deletions (2%). We set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and feasible in melon, generating multi-allelic mutations in both genomic target sites of the CmPDS gene showing an albino phenotype easily detectable after only few weeks after Agrobacterium-mediated transformation.

Flow cytometry trituration methods and the efficiency of lysis buffer solutions efficiency are compared in this study for extraction of nuclei from fresh leaves of rice (Oryza sativa). The rRazor blade sample trituration (chopping) procedure has been widely used to release nuclei from tissues in many plant species, together and combined with different lysis buffers in many plant species for low throughput analysis. AlternativelyIn contrast, the bead beating trituration method has been barely rarely been used for DNA ploidy determination, despite it being proposed as a less tedious alternative procedure to prepare nuclear suspensions when combined with Hanson's lysis buffer. In this study, bead beating was assessed in with rice young rice leaves and compared with the traditional chopping procedure. Each trituration method was combined with one of three nuclear isolation buffers (i.e. Hanson's buffer, Otto's buffers and LB01). Bead beating was applied for the first time using any all three of the assessed Hanson, LB01 and Otto's buffers, resulting in a rapid and suitable effective procedure for ploidy determination in rice fresh rice leaves ploidy determination. In addition, bead beating saved time and money, while reducing the exposure of the user to harmful substances. TIt is concluded that he best results were obtained when Hanson's nuclear isolation buffer was combined with the bead beating trituration method.

Background and aims: The tracing of C assimilation and the subsequent partitioning among plant organs has been a central focus of studies utilising Free Air CO Enrichment (FACE) facilities. The approach makes use of the fossil origin of this carbon, which is depleted in C. However, there is little data for desert environments. The Nevada Desert FACE Facility (NDFF), located in the Mojave Desert, has been one of the main facilities for the study of C dynamics in arid ecosystems and how they respond to rising atmospheric CO concentrations. In this experiment, we studied the incorporation of fixed CO during the previous two years (detectable by its lower δC) in the soil fraction surrounding roots. Methods: The soil was collected monthly in direct vicinity to the roots during a complete growth season, at two depths (5 and 15 cm). Soil samples were dried and fractionated by size (> 50 μm and < 50 μm) by wet sieving, and both size fractions were then analysed for the δC of their organic matter and their carbonates. Results: In the coarse fraction (> 50 μm), δC values ranged between −1 and − 2‰ for carbonates and between −23 and − 25‰ for soil organic matter. These values did not significantly change throughout the experiment and were not affected by depth (5 or 15 cm). In contrast, δC values for both organic and inorganic carbon in the fine fraction (< 50 μm) were much more variable than in the coarse fraction (> 50 μm). The δC values for organic C ranged mostly between −20‰ and − 27‰, and were roughly maintained throughout the sampling period. For inorganic C, the δC values were mostly between 0‰ and − 15‰, and tended to become less negative during the course of the sampling period. Overall the effect of [CO] on δC values of either organic or inorganic carbon was not significant for any experimental condition (plant species, soil depth, soil fraction). Conclusion: Little or no signs of recently fixed CO (C-depleted) were detected in the soils close to the roots, in the coarse fraction (> 50 μm), the fine fraction (< 50 μm), the organic matter, or in carbonates. This indicates a slow C turnover in the studied soils, which can result from a highly conservative use of photoassimilates by plants, including a very low release of organic matter into the soil in the form of dead roots or root exudates, and from a conservative use of available C reserves., The current study was partially funded by the OPTIMA project. RSN also acknowledges research support from the Nevada Agricultural Experiment Station and support for the Nevada Desert FACE Facility from U.S. Department of Energy’s Terrestrial Carbon Processes Program (DE-FG02-03ER63650, DE-FG02-03ER63651) and US DOE National Nuclear Security Administration. CTFC is a member of the CERCA net of research centres of the Catalonia autonomous government.

Anther culture is a fast tool to obtain double haploid plant lines for breeding purposes. In rice, this procedure is commonly performed in two steps: i) induction of calli from anthers and ii) regeneration of plantlets from calli. It has been stated that genotype highly influences the anther culture efficiency, so the media used in each step should be optimized for each variety. In this study, we tested different media modifications of an efficient protocol optimized for a medium sized grain temperate japonica NRVC980385, used as a control, in a long grain temperate japonica rice variety (NRVC20120346), and two long grain tropical japonica varieties (303012 and 303013). We found that the addition of 150 mg l(−1) colchicine to the induction medium worked best for all genotypes except for NRVC20120346, whose best induction was obtained with the colchicine-free medium. Referring to regeneration, increased gelling agent in the medium provided the best rates in NRVC980385, improving our former NRVC980385-optimized anther culture protocol. Sorbitol fortified regeneration medium worked the best in the case of the long grain varieties. The presence of colchicine in the induction medium was also related to a higher obtention of double haploid plantlets. This study highlights that genotype is a key factor in the performance of rice anther culture. It has set a first anther culture study on long grain japonica varieties and optimizes the anther culture protocol for temperate japonica medium grain NRVC980385 with the use of colchicine and other additives that increase osmotic stress.

Switchgrass (Panicum virgatum L.) is a warm perennial grass with valuable characteristics as a biofuel crop. To avoid competition with food crops, biofuel crops will be likely relegated to less productive soils such as marginal lands. Consequently, the salinity and water scarcity problems that commonly affect marginal lands compromise biofuel crop germination, emergence, and seedling establishment. The aims of this study were to study the germination and seedling growth of switchgrass under salinity and water stress and to describe the morpho-anatomical responses of the roots and leaves in the seedlings to these stress conditions. The effect of salt and water stress was assessed using sodium chloride (NaCl) and polyethylene glycol (PEG) 8000 at the same water potentials of − 0.8, − 1.0, and − 1.2 MPa. Seeds were moist prechilled for 7 days at 5 °C and germinated at 30/15 °C (8 h light/16 h dark). NaCl treatments (− 0.8 and − 1.0 MPa) delayed germination rates but did not reduce the final germination percentage, whereas at a lower potential (− 1.2 MPa), the final germination percentage was diminished. The effects of PEG (− 1.0 and − 1.2 MPa) on the germination rate and final percentage were more detrimental than those induced by isosmotic concentrations of NaCl. PEG and NaCl reduced significantly the vigor index of − 0.8 to − 1.2 MPa. The morpho-anatomical changes such as the reduction in the root cross-sectional area and the thickening of the endodermis walls for both stress conditions and aerenchyma formation in the cortex under salinity could significantly contribute in the survival and tolerance during the early seedling stages.

Giant reed (Arundo donax L.) is a C3 perennial rhizomatous grass belonging to the Gramineae family, originating in Asia and later spreading to different subtropical wetlands and warm-temperature regions of Europe, Africa, North America, and Oceania. A wide range of yields is reported in the literature depending on the site, climate, soil type and fertility, inputs, cultivation and harvest practices, and age of plantation. Although it produces flowers, seeds are not fertile. Consequently, its propagation is carried out mainly by rhizomes or stem cuttings. Giant reed has an uncommonly high photosynthetic capacity as compared to other C3 species, and is very similar to those of C4 species. It is able to achieve high photosynthetic rates, up to ∼38 μmol CO2 m−2 s−1 in well-watered treatments, but with substantial transpiration, leading to low or at least lower water use efficiency than many C4 crops (1.19–2.47 g kg−1), but is still more efficient than most C3 species. Radiation use efficiency increases proportionally as nitrogen and available water are increased, and can range from 1.26 to 2.02 g MJ−1, although higher values of 5.74 g MJ−1-intercepted photosynthetically active radiation are also reported. These are much lower than miscanthus, a typical C4 species. Nitrogen use efficiency (NUE) can range from 168.4 to 467 g g−1; single and late harvests, young plantations, and low nitrogen rates lead to increased NUE. Giant reed is also considered as a moderately saline-sensitive plant as it was able to maintain >50% of its relative growth when salinity was

Wheat is one of the most important cereal food crops in the world today. The productivity and quality of this crop is greatly affected by environmental conditions during grain filling. In this study, we have analyzed two genotypes of durum wheat, Blanqueta and Sula (traditional and a modern wheat respectively) in pre-industrial, current and future [CO2]. Plant growth and physiological parameters were analyzed during anthesis and grain filling in order to study the capacity of these plants to create new sinks and their role during the process of the acclimation of photosynthesis. It was observed that plants underwent photosynthetic acclimation at pre-industrial and future [CO2] (up and down-regulation respectively). However, the modern genotype averts the process of down-regulation by creating a new carbon sink (i.e. the spike). Here, we have shown the essential role that the spike plays as a new sink in order to avert the down-regulation of photosynthesis at future [CO2]. Moreover, we have demonstrated that at future [CO2] the growth response will depend on the ability of plants to develop new sinks or expand existing ones This study was supported by Ministerio de Educación y Ciencia de Espana˜ research project CGL2009-13079-C02-02. SA thanks the financial support of Ministerio de Educación y Ciencia de Espana˜ (BES-2010-032433)

The origin of the carbon atoms in CO2 respired by leaves in the dark of several plant species has been studied using 13C/12C stable isotopes. This study was conducted using an open gas exchange system for isotope labeling that was coupled to an elemental analyzer and further linked to an isotope ratio mass spectrometer (EA-IRMS) or coupled to a gas chromatography-combustion-isotope ratio mass spectrometer (GC-C-IRMS). We demonstrate here that the carbon, which is recently assimilated during photosynthesis, accounts for nearly ca. 50% of the carbon in the CO2 lost through dark respiration (Rd) after illumination in fast-growing and cultivated plants and trees and, accounts for only ca. 10% in slow-growing plants. Moreover, our study shows that fast-growing plants, which had the largest percentages of newly fixed carbon of leaf-respired CO2, were also those with the largest shoot/root ratios, whereas slow-growing plants showed the lowest shoot/root values., This study was supported in part by Ministerio de Educación y Ciencia research project AGL2009-13539-C02-01 and the EC research project OPTIMA.

The contribution of carbon and nitrogen reserves to regrowth following shoot removal has been studied in the past. However, important gaps remain in understanding the effect of shoot cutting on nodule performance and its relevance during regrowth. In this study, isotopic labelling was conducted at root and canopy levels with both 15N2 and 13C-depleted CO2 on exclusively nitrogen-fixing alfalfa plants. As expected, our results indicate that the roots were the main sink organs before shoots were removed. Seven days after regrowth the carbon and nitrogen stored in the roots was invested in shoot biomass formation and partitioned to the nodules. The large depletion in nodule carbohydrate availability suggests that root-derived carbon compounds were delivered towards nodules in order to sustain respiratory activity. In addition to the limited carbohydrate availability, the upregulation of nodule peroxidases showed that oxidative stress was also involved during poor nodule performance. Fourteen days after cutting, and as a consequence of the stimulated photosynthetic and N2-fixing machinery, availability of Cnew and Nnew strongly diminished in the plants due to their replacement by C and N assimilated during the post-labelling period. In summary, our study indicated that during the first week of regrowth, root-derived C and N remobilization did not overcome C- and N-limitation in nodules and leaves. However, 14 days after cutting, leaf and nodule performance were re-established., This work has been funded in partby the European research projects, PERMED and OPTIMA, and the Spanish National Research and DevelopmentProgramme-European Regional Development Fund ERDF(AGL2011-30386-C02-01). IA was the recipient of a Ramon y Cajal research grant (Ministerio de Economia y Competitividad).

Plant–plant interactions are key drivers of treeline dynamics. At the Pyrenean treeline, the dwarf shrub Vaccinium myrtillus grows in pure stands or in mixed stands with Vaccinium uliginosum or Rhododendron ferrugineum. They form sparse shrub patches that colonize subalpine grasslands, having dramatic impacts on their structure and functioning. We investigated the role of the two co-occurring shrubs as possible modulators of the structure and performance of V. myrtillus in the Central Pyrenees. We analysed biomass, growth, functional parameters, age distribution, N and C concentrations and isotope compositions (δ15N and δ 13C) of V. myrtillus ramets in pure stands, and stands mixed with V. uliginosum or R. ferrugineum. Volume, above-ground biomass and age of the ramets did not differ between stand types. We found lower δ13C values, indicative of lower water-use efficiency (WUE), in leaves and shoots of V. myrtillus in stands with R. ferrugineum than in pure stands. The N content and δ15N of V. myrtillus leaves and shoots in pure stands were higher than in mixed stands, pointing to a competition for N in mixed stands. Our results indicate that V. myrtillus competes for nutrients with its neighbours, but neither this competition nor the lower WUE affect its above-ground performance. Therefore, the interaction with co-occurring shrubs does not have a major effect on V. myrtillus structure and functioning at treeline and, consequently, should not be considered as a key driver on the dynamics of this species in the encroachment of subalpine grasslands.

We thank Clara Borrull, Noelia Seguer, Estela Illa, Victoria Lafuente, Elena Lahoz and Santiago Pérez for their field and laboratory assistance. We are grateful to CCiT of the University of Barcelona for the use of their facilities and their technical assistance. This work was supported by Conselh Generau d'Aran and the project ARBALMONT/786-2012 (Organismo Autónomo Parques Nacionales, Ministerio de Agricultura, Alimentación y Medio Ambiente, Spain). AAR was funded by an FPU grant (Ministerio de Educación, Cultura y Deporte, Spain) and SP was funded by a Ramón y Cajal fellowship (RYC-2013-14164, Ministerio de Economía y Competitividad, Spain). Climate warming can lead to changes in alpine plant species interactions through modifications in environmental conditions, which may ultimately cause drastic changes in plant communities. We explored the effects of 4 years of experimental warming with open-top chambers (OTC) on Vaccinium myrtillus performance and its interaction with neighbouring shrubs at the Pyrenean treeline ecotone. We examined the effects of warming on height, above-ground (AG) and below-ground (BG) biomass and the C and N concentration and isotope composition of V. myrtillus growing in pure stands or in stands mixed with Vaccinium uliginosum or Rhododendron ferrugineum. We also analysed variations in soil N concentrations, rhizosphere C/N ratios and the functional diversity of the microbial community, and evaluated whether warming altered the biomass, C and N concentration and isotope composition of V. uliginosum in mixed plots. Our results showed that warming induced positive changes in the AG growth of V. myrtillus but not BG, while V. uliginosum did not respond to warming. Vaccinium myrtillus performance did not differ between stand types under increased temperatures, suggesting that warming did not induce shifts in the interaction between V. myrtillus and its neighbouring species. These findings contrast with previous studies in which species interactions changed when temperature was modified. Our results show that species interactions can be less responsive to warming in natural plant communities than in removal experiments, highlighting the need for studies involving the natural assembly of plant species and communities when exploring the effect of environmental changes on plant-plant interactions.

Cessation of traditional management threatens semi-natural grassland diversity through the colonisation or increase of competitive species adapted to nutrient-poor conditions. Regular mowing is one practice that controls their abundance. This study evaluated the ecophysiological mechanisms limiting short- and long-term recovery after mowing for Festuca paniculata, a competitive grass that takes over subalpine grasslands in the Alps following cessation of mowing. We quantified temporal variations in carbon (C) and nitrogen (N) content, starch, fructan and total soluble sugars in leaves, stem bases and roots of F. paniculata during one growth cycle in mown and unmown fields and related them to the dynamics of soil mineral N concentration and soil moisture. Short-term results suggest that the regrowth of F. paniculata following mowing might be N-limited, first because of N dilution by C increments in the plant tissue, and second, due to low soil mineral N and soil moisture at this time of year. However, despite short-term effects of mowing on plant growth, C and N content and concentration at the beginning of the following growing season were not affected. Nevertheless, total biomass accumulation at peak standing biomass was largely reduced compared to unmown fields. Moreover, lower C storage capacity at the end of the growing season impacted C allocation to vegetative reproduction during winter, thereby dramatically limiting the horizontal growth of F. paniculata tussocks in the long term. We conclude that mowing reduces the growth of F. paniculata tussocks through both C and N limitation. Such results will help understanding how plant responses to defoliation regulate competitive interactions within plant communities.

Nitrogen isotope composition (δ(15) N) in plant organic matter is currently used as a natural tracer of nitrogen acquisition efficiency. However, the δ(15) N value of whole leaf material does not properly reflect the way in which N is assimilated because isotope fractionations along metabolic reactions may cause substantial differences among leaf compounds. In other words, any change in metabolic composition or allocation pattern may cause undesirable variability in leaf δ(15) N. Here, we investigated the δ(15) N in different leaf fractions and individual metabolites from rapeseed (Brassica napus) leaves. We show that there were substantial differences in δ(15) N between nitrogenous compounds (up to 30‰) and the content in ((15) N enriched) nitrate had a clear influence on leaf δ(15) N. Using a simple steady-state model of day metabolism, we suggest that the δ(15) N value in major amino acids was mostly explained by isotope fractionation associated with isotope effects on enzyme-catalysed reactions in primary nitrogen metabolism. δ(15) N values were further influenced by light versus dark conditions and the probable occurrence of alternative biosynthetic pathways. We conclude that both biochemical pathways (that fractionate between isotopes) and nitrogen sources (used for amino acid production) should be considered when interpreting the δ(15) N value of leaf nitrogenous compounds.

Further knowledge of the processes conditioning nitrogen use efficiency (NUE) is of great relevance to crop productivity. The aim of this paper was characterise C and N partitioning during grain filling and their implications for NUE. Cereals such as bread wheat (Triticum aestivum L. cv Califa sur), triticale (· Triticosecale Wittmack cv. Imperioso) and tritordeum (· Tritordeum Asch. & Graebn line HT 621) were grown under low (LN, 5 mm NH4NO3) and high (HN, 15 mm NH4NO3) N conditions. We conducted simultaneous double labelling ( 12 CO2 and 15 NH4 15 NO3 )i n order to characterise C and N partitioning during grain filling. Although triticale plants showed the largest total and ear dry matter values in HN conditions, the large investment in shoot and root biomass negatively affected ear NUE. Tritordeum was the only genotype that increased NUE in both N treatments (NUEtotal), whereas in wheat, no significant effect was detected. N labelling revealed that N fertilisation during post-anthesis was more relevant for wheat and tritordeum grain filling than for triticale. The study also revealed that the investments of C and N in flag leaves and shoots, together with the ‘waste’ of photoassimilates in respiration, conditioned the NUE of plants, and especially under LN. These results suggest that C and N use by these plants needs to be improved in order to increase ear C and N sinks, especially under LN. It is also remarkable that even though tritordeum shows the largest increase in NUE, the low yield of this cereal limits its agronomic value.

This study was supported in part by the European research project PERMED (INCO-CT-2004-509140), AGL2009-13539-C02-01 and CGL2009-13079-C02-02. G. Molero was the recipient of a research grant (AP2005-2916) from the Spanish MEC. We would like to thank M. Beltran, C. Caldelas, R. Roca and L. Berdie for their technical assistance. Dr L. Cabrera-Bosquet and F. Baptist are also acknowledged for their helpful comments on the manuscript.

Increasing atmospheric CO2 concentrations are expected to enhance plant photosynthesis and yield. Nevertheless, after long-term exposure, plants acclimate and show a reduction in photosynthetic activity (called down-regulation), which may cause a reduction in potential yield. Some authors suggest that down-regulation is related to nutrient availability, and more specifically, to an insufficient plant C sink strength caused by limited N supply. In this paper, we tested whether or not N availability prevents down-regulation of photosynthesis in nodulated alfalfa plants (Medicago sativa L.). To do so, we examined the effect of the addition of different levels of NH¿NO¿ (0, 10, and 15 mM) to 30-day-old nodulated alfalfa plants exposed to ambient (approximately 400 ¿mol mol¿¹) or elevated CO¿ (700 ¿mol mol¿¹) during a period of 1 month in growth chambers. After 2 weeks of exposure to elevated CO¿, no significant differences were observed in plant growth or photosynthesis rates. After 4 weeks of treatment, exclusively N¿ fixing alfalfa plants (0 mM NH¿NO¿) showed significant decreases in photosynthesis and Vc(max). Photosynthetic down-regulation of these plants was caused by the C/N imbalance as reflected by the carbohydrate and N data. On the other hand, plants supplied with 15 mM NH¿NO¿ grown under elevated CO¿ maintained high photosynthetic rates owing to their superior C/N adjustment. The intermediate N treatment, 10 mM NH¿NO¿, also showed photosynthetic down-regulation, but to a lesser degree than with 0 mM treatment. The present study clearly shows that external N supply can reduce or even avoid acclimation of photosynthesis to elevated CO¿ as a consequence of the increase in C sink strength associated with N availability., This work was supported by Ministerio de Ciencia e Innovación (MICINN BFU2008-01405), Fundación Universitaria de Navarra (PIUNA-2008) and Fundación Caja Navarra. A. Sanz-Sáez was the recipient of a research grant from Asociación de Amigos de la Universidad de Navarra and Iker Aranjuelo was the recipient of a Juan de la Cierva grant from Ministerio de Ciencia e Innovación. The assistance of A. Urdiain and M. Oyarzun is also appreciated.

Starch and soluble sugars are the major photosynthetic products, and their carbon isotope signatures reflect external versus internal limitations of CO2 fixation. There has been recent renewed interest in the isotope composition of carbohydrates, mainly for use in CO2 flux partitioning studies at the ecosystem level. The major obstacle to the use of carbohydrates in such studies has been the lack of an acknowledged method to isolate starch and soluble sugars for isotopic measurements. We here report on the comparison and evaluation of existing methods (acid and enzymatic hydrolysis for starch; ion-exchange purification and compound-specific analysis for sugars). The selectivity and reproducibility of the methods were tested using three approaches: (i) an artificial leaf composed of a mixture of isotopically defined compounds, (ii) a C4 leaf spiked with C3 starch, and (iii) two natural plant samples (root, leaf). Starch preparation methods based on enzymatic or acid hydrolysis did not yield similar results and exhibited contaminations by non-starch compounds. The specificity of the acidic hydrolysis method was especially low, and we therefore suggest terming these preparations as HCl-hydrolysable carbon, rather than starch. Despite being more specific, enzyme-based methods to isolate starch also need to be further optimized to increase specificity. The analysis of sugars by ion-exchange methods (bulk preparations) was fast but produced more variable isotope compositions than compound-specific methods. Compound-specific approaches did not in all cases correctly reproduce the target values, mainly due to unsatisfactory separation of sugars and background contamination. Our study demonstrates that, despite their wide application, methods for the preparation of starch and soluble sugars for the analysis of carbon isotope composition are not (yet) reliable enough to be routinely applied and further research is urgently needed to resolve the identified problems. Copyright © 2009 John Wiley & Sons, Ltd.

Recent advances in understanding the metabolic origin and the temporal dynamics in delta(13)C of dark-respired CO(2) (delta(13)C(res)) have led to an increasing awareness of the importance of plant isotopic fractionation in respiratory processes. Pronounced dynamics in delta(13)C(res) have been observed in a number of species and three main hypotheses have been proposed: first, diurnal changes in delta(13)C of respiratory substrates; second, post-photosynthetic discrimination in respiratory pathways; and third, dynamic decarboxylation of enriched carbon pools during the post-illumination respiration period. Since different functional groups exhibit distinct diurnal patterns in delta(13)C(res) (ranging from 0 to 10 per thousand diurnal increase), we explored these hypotheses for different ecotypes and environmental (i.e. growth light) conditions. Mass balance calculations revealed that the effect of respiratory substrates on diurnal changes in delta(13)C(res) was negligible in all investigated species. Further, rapid post-illumination changes in delta(13)C(res) (30 min), which increased from 2.6 per thousand to 5 per thousand over the course of the day, were examined by positional (13)C-labelling to quantify changes in pyruvate dehydrogenase (PDH) and Krebs cycle (KC) activity. We investigated the origin of these dynamics with Rayleigh mass balance calculations based on theoretical assumptions on fractionation processes. Neither the estimated changes of PDH and KC, nor decarboxylation of a malate pool entirely explained the observed pattern in delta(13)C(res). However, a Rayleigh fractionation of (12)C-discriminating enzymes and/or a rapid decline in the decarboxylation rate of an enriched substrate pool may explain the post-illumination peak in delta(13)C(res). These results are highly relevant since delta(13)C(res) is used in large-scale carbon cycle studies. 2009 John Wiley & Sons, Ltd.

Despite the relevance of carbon (C) loss through respiration processes (with its consequent effect on the lower C availability for grain filling), little attention has been given to this topic. Literature data concerning the role of respiration in cereals are scarce and these have been produced using indirect methods based on gas-exchange estimations. We have developed a new method based on the capture of respired CO(2) samples and their analysis by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). In order to analyse the main processes involved in the C balance during grain filling (photosynthesis, respiration, allocation and partitioning) the ambient isotopic (13)C/(12)C composition (delta(13)C) of the growth chamber was modified during this period (delta(13)C ca. -12.8 +/- 0.3 per thousand to ca. -20.0 +/- 0.2 per thousand). The physiological performance, together with the C allocation on total organic matter (TOM) and respiration of wheat (Triticum aestivum L., var. Califa sur) and two hybrids, tritordeum (X Tritordeum Asch. & Graebn line HT 621) and triticale (X Triticosecale Wittmack var. Imperioso), were compared during post-anthesis water stress. In spite of the larger ear DM/total ratio, especially under drought conditions, the grain filling of triticale and wheat was mainly carried out with pre-anthesis C, while the majority of C assimilated during post-anthesis was invested in respiration processes. In the case of wheat and tritordeum, the C balance data suggested a reallocation during grain filling of photoassimilates stored prior to anthesis from shoot to ear. Furthermore, the lower percentage of labeled C on respired CO(2) of droughted tritordeum plants, together with the lower plant biomass, explained the fact that those plants had more C available for grain filling.

Intense efforts are currently devoted to disentangling the relationships between plant carbon (C) allocation patterns and soil nitrogen (N) availability because of their consequences for growth and more generally for C sequestration. In cold ecosystems, only a few studies have addressed whole-plant C and/or N allocation along natural elevational or topographical gradients. (12)C/(13)C and (14)N/(15)N isotope techniques have been used to elucidate C and N partitioning in two alpine graminoids characterized by contrasted nutrient economies: a slow-growing species, Kobresia myosuroides (KM), and a fast-growing species, Carex foetida (CF), located in early and late snowmelt habitats, respectively, within the alpine tundra (French Alps). CF allocated higher labelling-related (13)C content belowground and produced more root biomass. Furthermore, assimilates transferred to the roots were preferentially used for growth rather than respiration and tended to favour N reduction in this compartment. Accordingly, this species had higher (15)N uptake efficiency than KM and a higher translocation of reduced (15)N to aboveground organs. These results suggest that at the whole-plant level, there is a compromise between N acquisition/reduction and C allocation patterns for optimized growth.

We examined the responses of the photosynthetic and respiratory electron transport and antioxidant systems in cell organelles of cucumber (Cucumis sativus L.) and tomato (Lycopersicon esculentum Mill.) leaves to infection of cucumber mosaic virus (CMV) by comparing the gas exchange, Chl fluorescence, respiratory electron transport, superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate-glutathione (AsA-GSH) cycle enzymes and the production of H(2)O(2) in chloroplasts, mitochondria and soluble fraction in virus-infected and non-infected leaves. Long-term CMV infection resulted in decreased photosynthesis and respiration rates. Photosynthetic electron flux to carbon reduction, respiratory electron transport via both complex I and complex II and also the Cyt respiration rate all significantly decreased, while photosynthetic alternative electron flux and alternative respiration significantly increased. These changes in electron transport were accompanied by a general increase in the activities of SOD/AsA-GSH cycle enzymes followed by an increased H(2)O(2) accumulation in chloroplasts and mitochondria. These results demonstrated that disturbance of photosynthetic and respiratory electron transport by CMV also affected the antioxidative systems, thereby leading to oxidative stress in various organelles.