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In terrestrial environments, water and nutrient availabilities and temperature conditions are highly variable, and especially in extreme environments limit survival, growth, and reproduction of plants. To sustain growth and maintain cell integrity under unfavourable environmental conditions, plants have developed a variety of biochemical and physiological mechanisms, orchestrated by a large set of stress-responsive genes and a complex network of transcription factors. Recently, cycling DOF factors (CDFs), a group of plant-specific transcription factors (TFs), were identified as components of the transcriptional regulatory networks involved in the control of abiotic stress responses. The majority of the members of this TF family are activated in response to a wide range of adverse environmental conditions in different plant species. CDFs regulate different aspects of plant growth and development such as photoperiodic flowering-time control and root and shoot growth. While most of the functional characterization of CDFs has been reported in Arabidopsis, recent data suggest that their diverse roles extend to other plant species. In this review, we integrate information related to structure and functions of CDFs in plants, with special emphasis on their role in plant responses to adverse environmental conditions.

Saffron is the world highest-priced spice because its production requires intensive hand labour. Reduce saffron production costs require containerised plant production under controlled conditions and expand the flowering period. Controlling the flowering process and identify the factors involved in saffron flowering is crucial to introduce technical improvements. The research carried out so far in saffron has allowed an extensive knowledge of the influence of temperature on the flower induction, but the molecular mechanisms controlling flowering induction processes are largely unknown. The present study is the first conducted to isolate and characterize a regulator gene of saffron floral induction the Short Vegetative Phase (SVP) gene, which represses the floral initiation genes in the temperature response pathway, which involved in saffron flower induction. The results obtained from both phylogenetic analysis and T-coffee alignment confirms that the isolated sequence belongs to the SVP gene clades of MADS-box gene family. Gene expression analysis in different developmental stages revealed the highest expression of SVP transcript (CsSVP) during the dormancy and the vegetative stages, but decrease when flower development initiated and it was the least in late September when flower primordia are developed. Furthermore, its expression increased in the apical bud when corms are storage at 9-10 ºC, thus inhibiting flower induction. Additionally, comparison of the CsSVP transcript in apical buds from big and small corms, differing in their flowering capacity, indicates that the CsSVP transcript is present only in vegetative buds. Taken together, these results suggested inhibitory role of the SVP gene.

26 Pág. Centro de Biotecnología y Genómica de Plantas, The development of high nitrogen use efficiency (NUE) cultivars under low N inputs is required for sustainable agriculture. To this end, in this study, we analyzed the impact of longterm suboptimal N conditions on the metabolome and transcriptome of tomato to identify specific molecular processes and regulators at the organ scale. Physiological and metabolic analysis revealed specific responses to maintain glutamate, asparagine, and sucrose synthesis in leaves for partition to sustain growth, while assimilated C surplus is stored in the roots. The transcriptomic analyses allowed us to identify root and leaf sets of genes whose expression depends on N availability. GO analyses of the identified genes revealed conserved biological functions involved in C and N metabolism and remobilization as well as other specifics such as the mitochondrial alternative respiration and chloroplastic cyclic electron flux. In addition, integrative analyses uncovered N regulated genes in root and leaf clusters, which are positively correlated with changes in the levels of different metabolites such as organic acids, amino acids, and formate. Interestingly, we identified transcription factors with high identity to TGA4, ARF8, HAT22, NF-YA5, and NLP9, which play key roles in N responses in Arabidopsis. Together, this study provides a set of nitrogen-responsive genes in tomato and new putative targets for tomato NUE and fruit quality improvement under limited N supply., This study was supported by grants from The National Institute for Agriculture and Food Research and Technology (CSIC-INIA) (RTA2015-00014-c02-00 to JMA and RTA2015-00014-c02-01 to SGN) and the Community of Madrid (AGRISOST-CM S2018/BAA-4330 to JMA). We also want to acknowledge the “Severo Ochoa Program for Centers of Excellence in R&D” from the Agencia Estatal de Investigación of Spain (Grant SEV-2016-0672) for supporting the scientific services used in this study. J. Canales was supported by the Agencia Nacional de Investigación y Desarrollo de Chile (ANID, FONDECYT 1190812) and ANID-Millennium Science Initiative Program (ICN17-022).

[EN] The demand for saffron is expected to rise in the coming years due to its nutraceutical and medicinal properties. To cope with this, it will be necessary to develop a mechanised production of saffron. Upgrading the production methods requires accurate control of the flowering time in this species. Nevertheless, little is known about the control of flowering time in Crocus sativus L. The aim of this study is to gain insight into the floral induction regulatory networks operating in this species. A transcriptomic analysis was performed from saffron main buds in different stages of development. The identification of putative integrators of flowering time signals, like FT, as well as meristem identity genes, such as LFY and TFL1, permitted the definition of the time of flowering induction of the buds, being able to use them as molecular markers. The identification of the transcripts encoded by a DROOPING LEAF-like (DL) gene is of particular relevance because this gene might be a novel factor for carpel specification in saffron. To elucidate the hormonal signalling networks working during flower induction, transcriptomic data were used, and the content of IAA, ABA and gibberellins was determined in competent and non-competent buds to flower, during the saffron life cycle. Our results suggested that ABA might be negatively regulating corm dormancy release, but its involvement in flower induction cannot be ruled out. ABI5 and the mediator of ABA regulated dormancy gene MARD1, could be key players of this pathway. In addition, a drop in GA4 levels may also be a necessary, but insufficient, condition for floral induction and development. DELLA, TFL1 and PIF3 genes might be involved in the gibberellin pathway. Notably, IAA seems to be a positive regulator of the process, involving MP/ARF5 and ANT genes in the pathway. Taken together, these results pave the way to the unveiling of the regulatory networks controlling the vegetative-to-reproductive phase change in saffron., The activities of this study have been supported by a project funded by the "Ministerio de Ciencia, Innovacion y Universidades de Espana" [AGL2016-77078-R].

The characterization of C. sativus ecotypes is of great interest for preserving them from a possible genetic erosion due to the decrease of European cultivation surface. In this study, we evaluated four ecotypes from Italy (Sardinia and Abruzzo), Spain (Castilla-La Mancha), and Greece (Kozani) in order to detect the existence of variability and promote the biodiversity of this crop. Thirty-one traits related to saffron flowering, flower morphology, production of spice and daughter corms, vegetative development (leaf and corm traits), and spice quality, were evaluated. In addition, a genetic analysis through three PCR-based approaches, SSRs, RAPD, and SRAP was assessed. Results highlighted a phenotypic variation among ecotypes during two consecutive years. All the studied parameters were influenced by the ecotype except for the stamen length, color coordinates of tepals, leaf length, and leaf number per plant. Sardinia had a longer flowering interval, earlier flowering, and higher spice yield and quality than the other corm origins. The maximum values of morphological traits, such as stigma length, dry weight of stigmas, tepals, flowers and leaves, leaf area, and daughter corm weight were observed in the Abruzzo ecotype. Principal component analysis (PCA) showed a clear separation among ecotypes, in which Sardinia and Spain showed more similarities than Abruzzo and Kozani. Significant negative correlation was found between days to flower with stigma yield and quality. However, we could not find molecular markers discriminating among corm origins. In conclusion, this study suggests the importance of C. sativus ecotypes as precious source of biodiversity and bioactive compounds, and of their enhancement as fundamental prerequisite for a sustainable development strategy and as an agricultural diversification opportunity for growers.

[EN] Unlike animals, plants possess diverse L-serine (Ser) biosynthetic pathways. One of them, the Phosphorylated Pathway of Serine Biosynthesis (PPSB) has been recently described as essential for embryo, pollen and root development, and required for ammonium and sulfur assimilation. The first and rate limiting step of PPSB is the reaction catalyzed by the enzyme phosphoglycerate dehydrogenase (PGDH). In Arabidopsis, the PGDH family consists of three genes, PGDH1, PGDH2 and PGDH3. PGDH1 is characterized as being the essential gene of the family. However, the biological significance of PGDH2 and PGDH3 remains unknown. In this manuscript, we have functionally characterized PGDH2 and PGDH3. Phenotypic, metabolomic and gene expression analysis in PGDH single, double and triple mutants indicate that both PGDH2 and PGDH3 are functional, affecting plant metabolism and development. PGDH2 has a stronger effect on plant growth than PGDH3, having a partial redundant role with PGDH1. PGDH3, however, could have additional functions in photosynthetic cells unrelated to Ser biosynthesis., We thank SCIE of the Universitat de Valencia for technical assistance. We thank Saleh Alseekh for assistance in the analysis of GC-MS samples. This research was supported by the Spanish Government and the European Union (FEDER/BFU2015-64204R, PID2019-107174GB-I00 to R. R. and BES-2016-077943 to R. C-A.), the Valencian Regional Government to S.R-T. (APOSTD/2017/132).

Tomato is one of the most widely cultivated vegetable crops and a model for studying fruit biology. Although several genes involved in the traits of fruit quality, development and size have been identified, little is known about the regulatory genes controlling its growth. In this study, we characterized the role of the tomato SlCDF4 gene in fruit development, a cycling DOF-type transcription factor highly expressed in fruits. The targeted overexpression of SlCDF4 gene in the fruit induced an increased yield based on a higher amount of both water and dry matter accumulated in the fruits. Accordingly, transcript levels of genes involved in water transport and cell division and expansion during the fruit enlargement phase also increased. Furthermore, the larger amount of biomass partitioned to the fruit relied on the greater sink strength of the fruits induced by the increased activity of sucrose-metabolising enzymes. Additionally, our results suggest a positive role of SlCDF4 in the gibberellin-signalling pathway through the modulation of GA4 biosynthesis. Finally, the overexpression of SlCDF4 also promoted changes in the profile of carbon and nitrogen compounds related to fruit quality. Overall, our results unveil SlCDF4 as a new key factor controlling tomato size and composition.

Under the global warming scenario, obtaining plant material with improved tolerance to abiotic stresses is a challenge for afforestation programs. In this work, maritime pine (Pinus pinaster Aiton) plants were produced from somatic embryos matured at different temperatures (18, 23, or 28 °C, named after M18, M23, and M28, respectively) and after 2 years in the greenhouse a heat stress treatment (45 °C for 3 h/day for 10 days) was applied. Temperature variation during embryo development resulted in altered phenotypes (leaf histology, proline content, photosynthetic rates, and hormone profile) before and after stress. The thickness of chlorenchyma was initially larger in M28 plants, but was significantly reduced after heat stress, while increased in M18 plants. Irrespective of their origin, when these plants were subjected to a heat treatment, relative water content (RWC) and photosynthetic carbon assimilation rates were not significantly affected, although M18 plants increased net photosynthesis rate after 10 days recovery (tR). M18 plants showed proline contents that increased dramatically (2.4-fold) when subjected to heat stress, while proline contents remained unaffected in M23 and M28 plants. Heat stress significantly increased abscisic acid (ABA) content in the needles of maritime pine plants (1.4-, 3.6- and 1.9-fold in M18, M23, and M28 plants, respectively), while indole-3-acetic acid content only increased in needles from M23 plants. After the heat treatment, the total cytokinin contents of needles decreased significantly, particularly in M18 and M28 plants, although levels of active forms (cytokinin bases) did not change in M18 plants. In conclusion, our results suggest that maturation of maritime pine somatic embryos at lower temperature resulted in plants with better performance when subjected to subsequent high temperature stress, as demonstrated by faster and higher proline increase, lower increases in ABA levels, no reduction in active cytokinin, and a better net photosynthesis rate recovery. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

[EN] Salinity and water shortages are two of the biggest environmental constraints that crops have to face in the climate change scenario. A fast and efficient way to overcome these stresses under the prism of a sustainable crop management is the use of grafting, combining the desired cultivar with the rootstock providing tolerances to abiotic stresses. Our aim was to validate three accessions previously selected for their tolerances to salt and water stresses (A25, B14 and C12) as rootstocks, in real field conditions. The physiological and productive behavior of the commercial pepper 'Adige' (A) grafted onto these accessions was compared along the growing cycle with this cultivar grafted onto the commercial rootstock 'Antinema' (ANT) and with the ungrafted pepper plant (A). Under water and salt stress, grafted plants onto the selected accessions, gave higher marketable yields than ungrafted plants or that plants grafted onto ANT, particularly the A25 accession. This rootstock was able to maintain high photosynthesis levels under stressing conditions through different adjustments made in the physiological processes, such as proline accumulation. The ANT rootstock showed comparable yields to A25 in control conditions. Under salt stress, Na+ and Cl- were equally accumulated in A/A25 plants and the ungrafted ones, but A/ANT, A/B14 and A/C12 were more restrictive in their absorption along the growing season. These results reinforce the idea that the use of tolerant pepper rootstocks is a good adaptation strategy for abiotic stressing conditions. The results also suggest that the abiotic stress was alleviated by the lack of negative effects mainly on photosynthesis, which maintained plant growth and the marketable yield., This work has been financed by INIA (Spain) through Project RTA2013-00022-C01 and the European Regional Development Fund (ERDF).

[EN] Species of the genus Crocus are found over a wide range of climatic areas. In natural habitats, these geophytes diverge in the flowering strategies. This variability was assessed by analyzing the flowering traits of the Spanish collection of wild crocuses, preserved in the Bank of Plant Germplasm of Cuenca. Plants of the seven Spanish species were analyzed both in their natural environments (58 native populations) and in common garden experiments (112 accessions). Differences among species observed in the native habitats were maintained under uniform environmental conditions, suggesting a genetic basis for flowering mechanisms. Two eco-morphological types, autumn- and spring-flowering species, share similar patterns of floral induction and differentiation period in summer. The optimal temperature for this process was 23 degrees C for both types. Unlike Irano-Turanian crocuses, spring-flowering Spanish species do not require low winter temperatures for flower elongation. Hysteranthous crocuses flower in autumn prior to leaf elongation. We conclude that the variability in flowering traits in crocuses is related to the genetic and environmental regulation of flower primordia differentiation and elongation prior to emergence above the soil surface. The elucidation of the physiological differences between eco-morphological types of crocuses: synanthous with cold requirements and synanthous and hysteranthous without cold requirements, unlocks a new approach to the flowering evolution of geophytes in Mediterranean regions. Crocus species can serve both as a new model in the study of the molecular basis of hysteranthy and for the purposes of developing the molecular markers for desirable flowering traits., The collection activities of plant materials included in this study were mainly supported by successive Spanish research projects funded by the "Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria" [INIA RF2004-00032-C03, INIA RF2008-00012-C03, INIA RF2011-00005-C03], co-funded by the European Regional Development Fund (ERDF-FEDER), and also by means of the European Action 018 Agri Gen Res (CrocusBank). The activities of PhD. TPF were supported by a pre-doctoral grant from the "Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria" within the framework of the project INIA RF2011-0005-C03-01. The preservation of these materials in the facilities of the Bank of Plant Germplasm of Cuenca (CIAF Albaladejito -IRIAF), as part of the Spanish Germplasm Collection of Saffron and other Crocus, is currently supported by the Spanish National Program for Conservation and Utilization of Plant Genetic Resources for Agriculture and Food (action INIA RFP2014-00012). Some activities have been also funded by the "Ministerio de Ciencia, Innovacion y Universidades de Espana" [AGL2016-77078-R].

[EN] In plants, phosphoglycerate kinase (PGK) converts 1,3-bisphosphoglycerate into 3-phosphoglycerate in glycolysis but also participates in the reverse reaction in gluconeogenesis and the Calvin-Benson cycle. In the databases, we found three genes that encode putative PGKs. Arabidopsis (Arabidopsis thaliana) PGK1 was localized exclusively in the chloroplasts of photosynthetic tissues, while PGK2 was expressed in the chloroplast/plastid of photosynthetic and nonphotosynthetic cells. PGK3 was expressed ubiquitously in the cytosol of all studied cell types. Measurements of carbohydrate content and photosynthetic activities in PGK mutants and silenced lines corroborated that PGK1 was the photosynthetic isoform, while PGK2 and PGK3 were the plastidial and cytosolic glycolytic isoforms, respectively. The pgk1.1 knockdown mutant displayed reduced growth, lower photosynthetic capacity, and starch content. The pgk3.2 knockout mutant was characterized by reduced growth but higher starch levels than the wild type. The pgk1.1 pgk3.2 double mutant was bigger than pgk3.2 and displayed an intermediate phenotype between the two single mutants in all measured biochemical and physiological parameters. Expression studies in PGK mutants showed that PGK1 and PGK3 were down-regulated in pgk3.2 and pgk1.1, respectively. These results indicate that the down-regulation of photosynthetic activity could be a plant strategy when glycolysis is impaired to achieve metabolic adjustment and optimize growth. The double mutants of PGK3 and the triose-phosphate transporter (pgk3.2 tpt3) displayed a drastic growth phenotype, but they were viable. This implies that other enzymes or nonspecific chloroplast transporters could provide 3-phosphoglycerate to the cytosol. Our results highlight both the complexity and the plasticity of the plant primary metabolic network., This work has been funded by the Spanish Government and the European Union: FEDER/ BFU2012-31519 and FEDER/ BFU2015-64204R, FPI fellowship to S.R.-T., and the Valencian Regional Government: PROMETEO II/2014/052.

[EN] Cycling Dof Factor (CDF) transcription factors (TFs) are involved in multiple processes related to plant growth and development. A member of this family, CDF3, has recently been linked in Arabidopsis to the regulation of primary metabolism and abiotic stress responses, but its role in crop production under stress is still unknown. In this study, we characterized tomato plants overexpressing the CDF3 genes from Arabidopsis and tomato and analyzed their effects on growth and yield under salinity, additionally gaining deeper insights into the molecular function of these TFs. Our results provide evidence for higher biomass production and yield in the 35S::AtCDF3 and 35S::SICDF3 plants, likely due to a higher photosynthetic capacity resulting in increased sucrose availability. Transcriptome analysis revealed that CDF3 genes regulate a set of genes involved in redox homeostasis, photosynthesis performance and primary metabolism that lead to enhanced biomass production. Consistently, metabolomic profiling revealed that CDF3 evokes changes in the primary metabolism triggering enhanced nitrogen assimilation, and disclosed that the amount of some protective metabolites including sucrose, GABA and asparagine were higher in vegetative tissues of CDF3 overexpressing plants. Altogether these changes improved performance of 35S::AtCDF3 and 35S::SICDF3 plants under salinity conditions. Moreover, the overexpression of CDF3 genes modified organic acid and sugar content in fruits, improving variables related to flavor perception and fruit quality. Overall, our results associate the CDF3 IF with a role in the control of growth and C/N metabolism, and highlight that overexpression of CDF3 genes can substantially improve plant yield., This work has been supported by grants from the Institute Nacional de Investigacion y Tecnologia Agraria y Alimentaria (Projects 2009-0004-C01 and 2012-0008-C01) and the Ministerio de Economia, Industria y Competitividad (Projects BFU2013-49665-EXP and BIO2014-53181-R). JD was supported by an INIA predoctoral fellowship.

Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121–158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of FV/FM values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge from different Chl a fluorescence analysis domains, yielding in several cases new insights. Fil: Kalaji, Hazem M.. Warsaw University of Life Sciences; Polonia Fil: Schansker, Gert. No especifica; Fil: Brestic, Marian. Slovak Agricultural University; Eslovaquia Fil: Bussotti, Filippo. University of Florence; Italia Fil: Calatayud, Angeles. Instituto Valenciano de Investigaciones Agrarias; España Fil: Ferroni, Lorenzo. University of Ferrara; Italia Fil: Goltsev, Vasilij. University of Sofia; Bulgaria Fil: Guidi, Lucia. Department of AgricultureFood and Environment; Italia Fil: Jajoo, Anjana. Devi Ahilya University; India Fil: Li, Pengmin. Northwest A&F University; China Fil: Losciale, Pasquale. Research Unit for Agriculture in Dry Environments; Italia Fil: Mishra, Vinod K.. College of Agriculture Science; India Fil: Misra, Amarendra N.. Central University of Jharkhand; India Fil: Nebauer, Sergio G.. Universidad Politécnica de Valencia; España Fil: Pancaldi, Simonetta. University of Ferrara; Italia Fil: Penella, Consuelo. Instituto Valenciano de Investigaciones Agrarias; España Fil: Pollastrini, Martina. University of Florence; Italia Fil: Suresh, Kancherla. Indian Institute of Oil Palm Research; India Fil: Tambussi, Eduardo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Yanniccari, Marcos Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Zivcak, Marek. Slovak Agricultural University; Eslovaquia Fil: Cetner, Magdalena D.. Warsaw University of Life Sciences; Polonia Fil: Samborska, Izabela A.. Warsaw University of Life Sciences; Polonia Fil: Stirbet, Alexandrina. Newport News; Estados Unidos Fil: Olsovska, Katarina. Slovak University of Agriculture; Eslovaquia Fil: Kunderlikova, Kristyna. Slovak University of Agriculture; Eslovaquia Fil: Shelonzek, Henry. University of Silesia; Polonia Fil: Rusinowski, Szymon. Institute for Ecology of Industrial Areas; Polonia Fil: Bąba, Wojciech. Jagiellonian University; Polonia

[EN] DNA-binding with one finger (DOF)-type transcription factors are involved in many fundamental processes in higher plants, from responses to light and phytohormones to flowering time and seed maturation, but their relation with abiotic stress tolerance is largely unknown. Here, we identify the roles of CDF3, an Arabidopsis DOF gene in abiotic stress responses and developmental processes like flowering time. CDF3 is highly induced by drought, extreme temperatures and abscisic acid treatment. The CDF3 T-DNA insertion mutant cdf3-1 is much more sensitive to drought and low temperature stress, whereas CDF3 overexpression enhances the tolerance of transgenic plants to drought, cold and osmotic stress and promotes late flowering. Transcriptome analysis revealed that CDF3 regulates a set of genes involved in cellular osmoprotection and oxidative stress, including the stress tolerance transcription factors CBFs, DREB2A and ZAT12, which involve both gigantea-dependent and independent pathways. Consistently, metabolite profiling disclosed that the total amount of some protective metabolites including -aminobutyric acid, proline, glutamine and sucrose were higher in CDF3-overexpressing plants. Taken together, these results indicate that CDF3 plays a multifaceted role acting on both flowering time and abiotic stress tolerance, in part by controlling the CBF/DREB2A-CRT/DRE and ZAT10/12 modules., We thank Dr Pablo Gonzalez-Melendi and Dr Jan Zouhar for technical handling of the confocal microscope and Dr Rafael Catala for the assistance with the low temperature stress assays. This work was supported by grants from Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA; projects 2009-0004-C01, 2012-0008-C01), Spanish Ministry of Science and Innovation (projects BIO2010-1487, BFU2013-49665-EXP). A.R.C. and J.D.F. were supported by INIA pre-doctoral fellowships

Previous works into photosynthesis regulation under salt stress have focused on the effect of NaCl, although other salts may significantly contribute to the toxicity of saline soils. In this paper, the effects of different salt sources (NaCl, Na2SO4, MgCl2 and MgSO4) on photosynthesis and vegetative growth in three tomato (Solanum lycopersicum L.) cultivars (Marmande RAF, Leader and Daniela) are presented. Differences were found in the net photosynthetic rate and vegetative growth among the studied cultivars and salinity treatments. Cultivar photosynthetic performance related not only to capability for toxic ion exclusion, but also to the maintenance of appropriate essential macronutrient concentrations in leaves. In addition, the role of metabolic and diffusion limitations in regulating photosynthesis varied depending on the studied genotypes. These data, along with variation in biomass and ion distribution in leaves and roots, show that distinct tomato cultivars can address salt tolerance differently, which should be considered when designing strategies to overcome plant sensitivity to salt stress.

Cycling Dof Factor (CDF) transcription factors (TFs) are involved in multiple processes related to plant growth and development. A member of this family, CDF3, has recently been linked in Arabidopsis to the regulation of primary metabolism and abiotic stress responses, but its role in crop production under stress is still unknown. In this study, we characterized tomato plants overexpressing the CDF3 genes from Arabidopsis and tomato and analyzed their effects on growth and yield under salinity, additionally gaining deeper insights into the molecular function of these TFs. Our results provide evidence for higher biomass production and yield in the 35S::AtCDF3 and 35S::SlCDF3 plants, likely due to a higher photosynthetic capacity resulting in increased sucrose availability. Transcriptome analysis revealed that CDF3 genes regulate a set of genes involved in redox homeostasis, photosynthesis performance and primary metabolism that lead to enhanced biomass production. Consistently, metabolomic profiling revealed that CDF3 evokes changes in the primary metabolism triggering enhanced nitrogen assimilation, and disclosed that the amount of some protective metabolites including sucrose, GABA and asparagine were higher in vegetative tissues of CDF3 overexpressing plants. Altogether these changes improved performance of 35S::AtCDF3 and 35S::SlCDF3 plants under salinity conditions. Moreover, the overexpression of CDF3 genes modified organic acid and sugar content in fruits, improving variables related to flavor perception and fruit quality. Overall, our results associate the CDF3 TF with a role in the control of growth and C/N metabolism, and highlight that overexpression of CDF3 genes can substantially improve plant yield.

[EN] The performance of a salt-tolerant pepper (Capsicum annuum L.) accession (A25) utilized as a rootstock was assessed in two experiments. In a first field experiment under natural salinity conditions, we observed a larger amount of marketable fruit (+75%) and lower Blossom-end Root incidence (-31%) in commercial pepper cultivar Adige (A) grafted onto A25 (A/A25) when compared with ungrafted plants. In order to understand this behavior a second greenhouse experiment was conducted to determine growth, mineral partitioning, gas exchange and chlorophyll a fluorescence parameters, antioxidant systems and proline content in A and A/A25 plants under salinity conditions (80 mM NaCl for 14 days). Salt stress induced significantly stunted growth of A plants (-40.6% of leaf dry weight) compared to the control conditions, while no alterations were observed in A/A25 at the end of the experiment. Accumulation of Na+ and Cl- in leaves and roots was similar in either grafted or ungrafted plants. Despite the activation of protective mechanisms (increment of superoxide dismutase, catalase, ascorbate peroxidase activity and non-photochemical quenching), A plants showed severely reduced photosynthetic CO2 assimilation (-45.6% of A(N390)) and substantial buildup of malondialdehyde (MDA) by-product, suggesting the inability to counteract salt-triggered damage. In contrast, A/A25 plants, which had a constitutive enhanced root apparatus, were able to maintain the shoot and root growth under salinity conditions by supporting the maintained photosynthetic performance. No increases in catalase and ascorbate peroxidase activities were observed in response to salinity, and MDA levels increased only slightly; indicating that alleviation of oxidative stress did not occur in A/A25 plants. In these plants the increased proline levels could protect enzymatic stability from salt-triggered damage, preserving the photosynthetic performance. The results could indicate that salt stress was vanished by the lack of negative effects on photosynthesis that support the maintained plant growth and increased marketable yield of the grafted plants., The authors are thankful to Miss Luciana Gaccione and Mr Omar Almolla for their assistance in the experiments. This work was financed by INIA (Spain) through Project RTA2013-00022-C02-01 and the European Regional Development Fund (ERDF). C.P. was a beneficiary of a doctoral fellowship (FPI-INIA).

A major constraint limiting saffron cultivation is the difficulty of obtaining high quality corms for propagation. There is limited knowledge about the physiology of vegetative development of saffron. The aim of this work was to analyse the relative contribution of different carbon sources (leaf photosynthetic activity and mother corm reserves) to the growth of vegetative organs and to analyse the influence of internal and external factors on the photosynthetic processes sustaining corm growth. The photosynthetic rate and changes in biomass were measured over the course of the vegetative growing season. The influence of the size of the mother corm, the cultivation method used (greenhouse versus field) and water availability on saffron production was assessed. Most of the remaining mother corm reserves after flowering were depleted during root and leaf development. After these organs reached their maximum size, the growth of replacement corms was initiated, and this development primarily relied on photosynthesis. Saffron mother corm reserves only contributed 10% to the biomass during vegetative development. The photosynthetic rate was consistently very high (26 mu mol m(-2) s(-1)) throughout the year but was reduced in plants from the largest corms. A sink capacity limitation arose during growth of replacement corms since the increase of biomass was limited before total leaf senescence. Saffron grown under greenhouse conditions exhibited traits characteristic of plants grown under low-irradiance, even after the saffron plants were transferred to the field. This fact limits the production of replacement corms in this environment. A decrease in the photosynthetic rate was observed under water stress, but the supply of water to the leaves from the corms and roots was found to maintain a still high photosynthetic rate (12 mu mol m(-2) s(-1)) even at highly negative soil water potentials., We thank the staff of the ANECOOP experimental station in Museros (Valencia, Spain) for their support and assistance in the field cultivation of saffron. This work was supported by the Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria, MEC, Spain (PET 2007-014-C05-02). We thank Elsevier Language Editing Services for language revision of the manuscript.

Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121–158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F V/F M values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge from different Chl a fluorescence analysis domains, yielding in several cases new insights.

The photosynthetic responses to a flowering-inductive water-stress period and recovery were studied and compared in two Citrus species. Under greenhouse conditions, Fino lemon and Owari satsuma trees were subjected to moderate (-2 MPa at predawn) and severe (-3 MPa) water stress levels and were re-watered after 60 days. Vegetative growth was inhibited during the stress assays, and strong defoliation levels were reported, especially in Fino lemon. In both species, bud sprouting was induced after re-watering. Flowers and vegetative shoots developed in Owari satsuma after a drought period, and the development was independent of the stress level. In Fino lemon, vegetative shoots and flowers were primarily formed after moderate and severe stress, respectively. The photosynthetic rate and stomatal conductance were reduced by water stress, and a marked increase in water-use efficiency at the moderate water deficit level was observed. Nevertheless, the photosynthetic apparatus was not damaged, since the maximum quantum yield, photosynthetic pigment concentrations and Rubisco level and activity did not change. Furthermore, the measured malonyldialdehyde (MDA) and peroxidase activity indicated that oxidative stress was not specifically triggered by water stress in our study. Therefore, the gas exchange, fluorescence and biochemical parameters suggested that diffusional limitations to photosynthesis predominated in both of the studied Citrus species, and explained the rapid recovery of the photosynthetic parameters after rehydration. The net CO 2 fixation rate and stomatal conductance were recovered within 24 h in Fino lemon, whereas 3 days were required in Owari satsuma. This suggests the presence of some metabolic limitations in the latter species. Furthermore, the sensibility of the defoliation rates, the accumulation of proline and the stomatal behaviour in response to water stress indicated a higher drought tolerance of Fino lemon, according to its better acclimation to hot climates. © 2011 Springer-Verlag., The authors thank Dr. J. Moreno and co-workers from the Departamento de Bioquimica of the Universidad de Valencia for his help and support in the Rubisco assays, and Dr. F. Fornes, Dr. A. Calatayud and Dr. E. Primo-Millo for the critical review of the manuscript. This work was funded by the Universitat Politecnica de Valencia, Spain (Ayudas para primeros proyectos de investigacion PAID06-06).

[EN] The Spanish type cultivar 'Piel de Sapo' (Cucumis melo L var. saccharinus), has a limited compatibility with the Cucurbita maxima x Cucurbita moschata hybrids currently used as rootstocks. Double grafting can be used to improve compatibility between rootstock and scion by means of an intermediate rootstock compatible with both. Non-grafted, single, and double grafted melon plants of the cultivar Piel de Sapo were evaluated for water, nutrient absorption, photosynthesis activity, biomass production in early phases, as well as for yields and fruit quality in a long term trial. The hybrid 'Shintoza' (C. maxima x C. moschata) was used as rootstock, and the cantaloupe type melon cultivar 'Sienne' as an intermediate scion. Grafting did not affect net photosynthetic values, yet increased water use efficiency (35%). Double grafted plants increased aerial dry weights (66% and 31% with respect to non-grafted and simple graft plants, respectively), and also increased capacity for uptaking beneficial minerals (between 61% and 13% and particularly for NO(3)(-), P, K, Ca, Mn, and Zn) with respect to non-grafted and single grafted plants. The quantum efficiency PSII photochemistry values increased in double grafted plants (12%) with respect to the control plants. Consequently, double grafting on a vigorous rootstock such as 'Shintoza', with an intermediate scion, results in improved mineral and water absorption and achieves an increase in ion influx to the scion - so enabling an increase in light photosynthetic reaction and biomass. Double grafted plants increased fruit yield when compared to simple grafted and non-grafted plants (12% and 56%, respectively) and did not affect fruit quality in terms of degrees Brix and colour. In conclusion, double grafting presents several beneficial aspects that are counter-balanced by the extra cost of the technique. The difference in yields reflects compatibility problems., The authors gratefully acknowledge Conselleria de Agricultura, Pesca y Alimentacion (Generalitat Valenciana, Spain) for its financial support especially for the contract for A. Calatayud. The revision of this paper was funded by the Universidad Politecnica de Valencia, Spain.

[EN] The aim of this work was to assess the relation between carbohydrate levels and flower and fruit production, as well as the role of carbohydrates on CO(2) fixation activity, by analysis of leaves, twigs and roots from the alternate bearing 'Salustiana' cultivar of sweet orange (Citrus sinensis [L.] Osbeck). A heavy crop load (on year) did not affect photosynthesis activity when compared to non-fruiting trees (off year). Fruiting trees accumulated most of the fixed carbon in mature fruits, whilst no accumulation was observed in roots before harvest. Non-fruiting trees transported part of the fixed carbon to the roots and mobilize it for growth processes and, at the end of the season (December), store it as reserves. Reserve carbohydrates accumulation in leaves started by early December for both tree types, showing the same levels in on and off trees until spring bud sprouting. A heavy flowering after an off year caused the rapid mobilization of the stored reserves, which were exhausted at full bloom. We found no evidence on carbon fixation regulation by either fruit demand or carbohydrate levels in leaves. Carbohydrate reserves played little or no role over fruit set, which actually relied on current photosynthesis, We thank Ing. Agr. J.M. Torres (ANECOOP, Valencia, Spain) for providing the orchard facilities and logistic help, the R + D + i Linguistic Assistance Office at the Universidad Politecnica de Valencia for their help in revising this article and Y. Bordon for her cooperation in some experiments. Thanks are due also to Dr. Olivares for the critical review of the manuscript. This research was funded by grants from the Conselleria de Agricultura, Pesca y Alimentacion (GV-CAPA00-11) and the Conselleria diEmpresa, Universitat i Ciencia, Generalitat Valenciana (Grupos 04/059).

The physiology of flower initiation and early flower development of the saffron plant (Crocus sativus L.) is well characterised. However, the optimal conditions for anthesis have not been ascertained. In this work we determined the influence of temperature after flower initiation on flowering and saffron spice production, as well as the influence of the environmental conditions in the greenhouse on plant growth. To this aim, saffron corms which had been stored at 25°C during 105 days, were sprouted at different temperature regimes in growth chambers in the light, with an 16/8 h photoperiod and a photon fluence rate of 20-40 μmol m -2 s -1 . One month after the completion of flowering the plants were transplanted to the open field, and their CO 2 assimilation rates were compared to that of plants coming from corms planted directly in the open. In plants grown at a constant temperature, this parameter, in the range 13-20°C, had no significant effect on the number of sprouts nor that of flowers formed per corm. Flowering occurred earlier at 17°C than at higher and lower temperatures. The flowers formed at 20°C had a wilt aspect. The size of the styles was directly related to temperature and therefore was highest at 20°C. in these plants, however, the styles were difficult to separate from the flower. Growing the plants at a 20/10°C term period hastened flowering and resulted in the formation of normal flowers, but also reduced style size. Growth in the chambers resulted in a 90% reduction of the maximum photosynthetic CO 2 fixation as compared with plants grown in the open. This loss was recovered in part (about 50%) several weeks after transferring the plants to the open field.

[EN] Soft skills have become part of the curriculum of content courses at the Universities. Evaluating those skills requires working in small groups run by various lecturers and those groups might become biased by the lecturer’s perception of the activities. In this study our hypothesis was that lecturers’ prejudices influence student perception of the course activities and that misguidance from lecturers can be amended with motivating coordination of the lecturers. Two team-based activities were assessed for lab evaluation. Besides, self- and peer-evaluation were carried out to mark students’ soft skills related to teamwork and leadership in relation to these activities. Perception of the activities and their assessment by students and lecturers were analysed. We concluded that lecturer prejudices bias students’ perception of the activities they carry out and that lecturers need to work as a close team for the course to become a successful project., [ES] Las competencias transversales se han convertido en parte del programa de las asignaturas en las Universidades. La evaluación de competencias requiere que los grupos de estudiantes sean reducidos, con lo que intervienen varios profesores en una misma actividad programada. Estas actividades pueden resultar influidas por la percepción que de ellas tenga cada profesor. Partimos de la hipótesis de que los prejuicios de los profesores influyen en la percepción que tiene el alumno de las actividades y que las desviaciones debidas a esos prejuicios pueden ser corregidas mediante una coordinación motivadora entre profesores. Se valoró la adecuación como evaluación de prácticas de laboratorio de dos actividades que los alumnos realizaron en equipos de tres. Además, se llevó a cabo un sistema de autoevaluación y evaluación entre pares para evaluar a los alumnos en la competencia “trabajo en equipo y liderazgo” en relación con esas dos actividades. Analizamos la valoración de alumnos y profesores respecto a las actividades propuestas y al sistema de evaluación en una encuesta abierta. Se concluye que los profesores influimos en la apreciación que los alumnos tienen de las actividades y que los profesores necesitaríamos trabajar estrechamente en equipo para que la asignatura sea un proyecto con éxito., Este estudio ha sido financiado por la Universitat Politècnica de València mediante el proyecto ref. PIME/2014/A/001/A. Los autores desean agradecer a los alumnos de la asignatura de Biología Celular (ref. 11112) del curso 2014-15 su inestimable colaboración en la realización del proyecto.

[EN] A fungal gene encoding a transcription factor is expressed from its own promoter in Arabidopsis phloem and improves drought tolerance by reducing transpiration and increasing osmotic potential. Horizontal gene transfer from unrelated organisms has occurred in the course of plant evolution, suggesting that some foreign genes may be useful to plants. The CtHSR1 gene, previously isolated from the halophytic yeast Candida tropicalis, encodes a heat-shock transcription factor-related protein. CtHSR1, with expression driven by its own promoter or by the Arabidopsis UBQ10 promoter, was introduced into the model plant Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation and the resulting transgenic plants were more tolerant to drought than controls. Fusions of the CtHSR1 promoter with beta-glucuronidase reporter gene indicated that this fungal promoter drives expression to phloem tissues. A chimera of CtHSR1 and green fluorescence protein is localized at the cell nucleus. The physiological mechanism of drought tolerance in transgenic plants is based on reduced transpiration (which correlates with decreased opening of stomata and increased levels of jasmonic acid) and increased osmotic potential (which correlates with increased proline accumulation). Transcriptomic analysis indicates that the CtHSR1 transgenic plants overexpressed a hundred of genes, including many relevant to stress defense such as LOX4 (involved in jasmonic acid synthesis) and P5CS1 (involved in proline biosynthesis). The promoters of the induced genes were enriched in upstream activating sequences for water stress induction. These results demonstrate that genes from unrelated organisms can have functional expression in plants from its own promoter and expand the possibilities of useful transgenes for plant biotechnology., We acknowledge support by Grants BFU2011-22526 of the Spanish MICINN (Madrid, Spain) and PROMETEO II 2014-041 of Generalitat Valenciana (Valencia, Spain). J. M.-B. was supported by a Juan de la Cierva contract of the Spanish MICINN. A. A. was supported by a short-term EMBO fellowship to visit the laboratory of R. Serrano. We thank Dr. Jose Maria Belles (IBMCP, Valencia, Spain) for assistance in the determination of sugars, Dr. Isabel Lopez-Diaz and Dr. Esther Carrera for the hormone analysis carried out at the Plant Hormone Quantification Service of IBMCP and Prof. Jorg Kudla (Westfalische Wilhelms-Universitat, Munster, Germany) for the pGPTVII.Hyg.PUBQ10::MCS plasmid.

[EN] The present state of knowledge concerning developmental processes and the secondary metabolism of saffron, Crocus sativus L. (Iridaceae), along with the genes involved in these processes so far known, is reviewed. Flowers and corms constitute the most valuable parts of saffron. Corm and flower development are two key aspects to be studied in saffron to increase the yield and quality of the spice, to raise its reproductive rate, and to implement new production systems. Important knowledge about the physiology of flowering and vegetative growth has been acquired in recent years, but there is still only limited information on molecular mechanisms controlling these processes. Although some genes involved in flower formation and meristem transition in other species have been isolated in saffron, the role of these genes in this species awaits further progress. Also, genes related with the synthesis pathway of abscisic acid and strigolactones, growth regulators related with bud endodormancy and apical dominance (paradormancy), have been isolated. However, the in-depth understanding of these processes as well as of corm development is far from being achieved. By contrast, saffron phytochemicals have been widely studied. The different flower tissues and the corm have been proved to be an important source of phytochemicals with pharmacological properties. The biotechnological prospects for saffron are here reviewed on the basis of the discovery of the enzymes involved in key aspects of saffron secondary metabolism, and we also analyze the possibility of transferring current knowledge about flowering and vegetative propagation in model species to the Crocus genus., This work was supported by the Spanish Ministerio de Economia y Competitividad (BIO2013-44239-R).

[EN] Grafting has been proposed as an interesting strategy that improves the responses of crops under salinity. In pepper, we reported increased fruit yield of the commercial 'Adige' cultivar under salinity when grafted onto accessions Capsicum chinense Jacq. 'ECU-973' (12) and Capsicum baccatum L. var. pendulum 'BOL-58' (14), whereas no,effect was observed when grafted onto accession Capsicum annuum L var. 'Serrano' (5). We also analysed the physiological and biochemical mechanisms related to the tolerance conferred by these rootstocks. Responses to salinity (40 mM NaCl) were studied in the different plant combinations for 30 days by determining water relations, mineral content, proline accumulation, photosynthetic parameters, nitrate reductase activity and antioxidant capacity. Higher salt tolerance was achieved when the 'Adige' cultivar was grafted onto the 12 genotype, which allowed not only lower Na+ and Cl- accumulation in the scion, but also ion selectivity maintenance, particularly Na+/K+ discrimination. These traits led to a minor negative impact on photosynthesis, nitrate reductase activity and lipid peroxidation in grafted scion leaves. This work suggests that using tolerant pepper rootstocks that maintain the scion's ion homeostasis is a promising strategy to provide salinity tolerance and can consequently improve crop yield., This work has been financed by INIA (Spain) through Project RTA2010-00038-C03-01 and the European Regional Development Fund (ERDF). C.P. is beneficiary of a doctoral fellowship (FPI-INIA) associate with this project.

Sweet pepper is one of the most important vegetable crops in arid and semiarid regions. Grafting has been proposed as an interesting strategy that improves the responses of crops under the salinity and hydric deficits occurring in these areas. In a previous work, we selected Capsicum spp accessions with different degrees of salinity and hydric stress tolerance to be used as rootstocks (the highest to lowest): Capsicum chinense Jacq. ‘ECU-973’ (code 12), Capsicum baccatum L. var. pendulum ‘BOL-58’ (code 14) and Capsicum annuum L var. ‘Serrano’ (code 5). The behavior of commercial cultivar seedlings grafted onto these rootstocks was compared during 14 days under water stress (5% polyethylene glycol) and salinity (40 mM NaCl) in hydroponic culture. Different physiological parameters were measured to test the hypothesis that tolerance might be related to the role of rootstock in altering the stress perception by the scion and to identify differences in pepper-grafted plants adaptation mechanisms in response to salt and osmotic stresses. At a similar osmotic pressure of the solution, grafted plants onto the 12 and 14 rootstocks activated tolerance mechanisms based on ion specific responses under salinity, whereas osmotic adjustment based on proline accumulation was performed under water stress. The maintenance of the scion's homeostasis under salinity was achieved through the restriction of Cl- transport to leaves and to diminished Na+ loading in roots and leaves, thus favouring K+ uptake. Under both stresses, a minor negative impact on photosynthesis, nitrate reductase activity and lipid peroxidation in scion leaves grafted onto 12 and 14 rootstocks was observed. In conclusion, the results of these works reinforce that the use of tolerant pepper rootstocks is a promising strategy to provide salinity and water stress tolerance and can consequently improve crop yield.

[Background] The increased selection pressure of the herbicide glyphosate has played a role in the evolution of glyphosate-resistance in weedy species, an issue that is becoming a threat to global agriculture. The molecular components involved in the cellular toxicity response to this herbicide at the expression level are still unidentified. [Results] In this study, we identify the protein kinase GCN2 as a cellular component that fosters the action of glyphosate in the model plant Arabidopsis thaliana. Comparative studies using wild-type and gcn2 knock-out mutant seedlings show that the molecular programme that the plant deploys after the treatment with the herbicide, is compromised in gcn2. Moreover, gcn2 adult plants show a lower inhibition of photosynthesis, and both seedlings and adult gcn2 plants accumulate less shikimic acid than wild-type after treatment with glyphosate. [Conclusions] These results points to an unknown GCN2-dependent factor involved in the cascade of events triggered by glyphosate in plants. Data suggest either that the herbicide does not equally reach the target-enzyme in a gcn2 background, or that a decreased flux in the shikimate pathway in a gcn2 plants minimize the impact of enzyme inhibition. © 2015 Faus et al.; licensee BioMed Central., This work was mainly supported by the Universidad Politecnica de Valencia (PAID2011-16) and the Ministerio Español de Ciencia y Tecnología (BFU2011-22526). The work was partially supported through a grant from the Ministerio Español de Ciencia y Tecnología (AGL-2010-18621). We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

[EN] Non-structural carbohydrates (NSC) in plant tissue are frequently quantified to make inferences about plant responses to environmental conditions. Laboratories publishing estimates of NSC of woody plants use many different methods to evaluate NSC. We asked whether NSC estimates in the recent literature could be quantitatively compared among studies. We also asked whether any differences among laboratories were related to the extraction and quantification methods used to determine starch and sugar concentrations. These questions were addressed by sending sub-samples collected from five woody plant tissues, which varied in NSC content and chemical composition, to 29 laboratories. Each laboratory analyzed the samples with their laboratory-specific protocols, based on recent publications, to determine concentrations of soluble sugars, starch and their sum, total NSC. Laboratory estimates differed substantially for all samples. For example, estimates for Eucalyptus globulus leaves (EGL) varied from 23 to 116 (mean = 56) mg g(-1) for soluble sugars, 6-533 (mean = 94) mg g-1 for starch and 53-649 (mean = 153) mg g-1 for total NSC. Mixed model analysis of variance showed that much of the variability among laboratories was unrelated to the categories we used for extraction and quantification methods (method category R-2 = 0.05-0.12 for soluble sugars, 0.10-0.33 for starch and 0.01-0.09 for total NSC). For EGL, the difference between the highest and lowest least squares means for categories in the mixed model analysis was 33 mg g-1 for total NSC, compared with the range of laboratory estimates of 596 mg g-1. Laboratories were reasonably consistent in their ranks of estimates among tissues for starch (r = 0.41-0.91), but less so for total NSC (r = 0.45-0.84) and soluble sugars (r = 0.11-0.83). Our results show that NSC estimates for woody plant tissues cannot be compared among laboratories. The relative changes in NSC between treatments measured within a laboratory may be comparable within and between laboratories, especially for starch. To obtain comparable NSC estimates, we suggest that users can either adopt the reference method given in this publication, or report estimates for a portion of samples using the reference method, and report estimates for a standard reference material. Researchers interested in NSC estimates should work to identify and adopt standard methods., M.G.R. was funded by McMaster fellowship (1158.C). S.P. was funded by Juan de la Cierva contract (MCI project) and project ARBALMONT/786-2012 (OPAN, MAAMA, Spain). F.P. was funded by Fondecyt 11121175. U.N. and M.T. were funded by the Estonian Ministry of Education and Science, grant IUT-8-3. N.G.M. and L.T.D. were funded by DOE-BER. H.D.A. was funded by LANL-LDRD. J.M.-V. was funded by the Spanish Government (CGL 2010-16376). S.H. was funded by the Montana Institute on Ecosystems' Graduate Enhancement Award from NSF EPSCoR Track-1 NSF-IIA-1443108. Valuable comments from Dr Mauricio Mencuccini (University of Edinburgh), Dan Binkley (Colorado State University) and two anonymous reviewers were also greatly appreciated.

The role of bud competence in the determination of flowering seasonality was studied in three Citrus cultivars, Bearss lime (Citrus latifolia Tan.), Fino lemon (C. limon [L.] Burm. f.) and Owari satsuma (C. unshiu (Mak.) Marc.), which differ in their adaptation to hot climates and their propensity to produce off-season blooms. Potted plants were kept in a greenhouse under non-inductive conditions (minimum temperature higher than 20°C), and periodically the flowering response was determined of a group of trees exposed for 30 days to an inductive temperature regime (15/8°C). A seasonal change in bud competence was demonstrated, and both bud sprouting and flower formation were highest when the low temperature regime was imposed during February and March. During the summer months, the low temperature regime resulted in a small increase in bud sprouting as compared to non-chilled trees, but only vegetative buds developed and no flowers were formed. The influence of environmental factors on the determination of bud competence was further studied. No effect of photoperiod was found, but raising the minimum air temperature above 25°C during 60 days, eliminated bud competence in Owari satsuma. In Bearss lime trees, the buds reacquired the competence after 4 months at 25/20°C, a temperature regime that does not induce flower formation. The reacquisition of competence was much faster at a lower temperature (15/8°C). A consistent relationship between the flowering response and DNA methylation in buds could not be demonstrated in all cultivars.

Cedrus atlantica (Pinaceae) is a large and exceptionally long-lived conifer native to the Rif and Atlas Mountains of North Africa. To assess levels and patterns of genetic diversity of this species, samples were obtained throughout the natural range in Morocco and from a forest plantation in Arbúcies, Girona (Spain) and analyzed using RAPD markers. Within-population genetic diversity was high and comparable to that revealed by isozymes. Managed populations harbored levels of genetic variation similar to those found in their natural counterparts. Genotypic analyses of molecular variance (AMOVA) found that most variation was within populations, but significant differentiation was also found between populations, particularly in Morocco. Bayesian estimates of F(ST) corroborated the AMOVA partitioning and provided evidence for population differentiation in C. atlantica. Both distance- and Bayesian-based clustering methods revealed that Moroccan populations comprise two genetically distinct groups. Within each group, estimates of population differentiation were close to those previously reported in other gymnosperms. These results are interpreted in the context of the postglacial history of the species and human impact. The high degree of among-group differentiation recorded here highlights the need for additional conservation measures for some Moroccan populations of C. atlantica.

Reversed-phase HPLC analysis and random amplified polymorphic DNA (RAPD) markers were used to monitor DNA methylation status and genetic stability of C. atlantica and C. libani shoots generated through axillary bud proliferation. Average DNA methylation in C. atlantica or C. libani seedlings and mature 200-year-old trees of C. libani was 19.8, 19.5 and 22.3%, respectively. These global amounts showed no significant variation after the in vitro establishment of seedling-originated shoot stocks. In contrast, in vitro culture caused a significant decrease in the amount of 5-methylcytosine in genomic DNA of the tissue culture (TC) progenies of one of the adult C. libani genotypes. This DNA demethylation event accompanied an enhancement of the regrowth capacity of this genotype. Detected RAPD variation between mother plants and their TC progenies was species-related, with C. libani TC progenies being genetically more stable than those of C. atlantica. Nevertheless, similarity indices ranged from 0.97 to 1 among mother plants and their TC progenies. Furthermore, the analyses of molecular variance (AMOVA) suggest that RAPD variation among the mother plants and their TC progenies might be considered as not significant. The application of various statistical approaches, including cluster-based genetic distance methods and AMOVA, demonstrates that RAPD markers discriminate C. atlantica and C. libani appropriately.

Summary Shoot formation in explants of Digitalis minor was achieved, through axillary bud proliferation and adventitious bud differentiation, by varying the amount and source of plant hormones. Shoot regeneration was also obtained after infection of D. minor with the wild-type Agrobacterium tumefaciens strain 82.139, which induced shooty tumours. These shoots were not transgenic, as revealed by nopaline assays and the use of a C58pMP90/T139GUS-INT strain harbouring the intron inactivated gusA gene. Plants were easily rooted and transplanted into the greenhouse. Shoot cultures of D. minor accumulated up to 226 μg cardenolides per g dry mass when cultured on agar-solidified medium. Cardenolide content was significantly lower in submerged shoot cultures, the effect being related to a high incidence of hyperhydricity. Greenhouse-grown plants presented the highest amounts of these metabolites. This is the first report on micropropagation of the Balearic endemic medicinal plant species D. minor . The induction of caulogenic galls by A. tumefaciens wild strain 82.139 opens new possibilities for the biotechnological improvement of Digitalis spp.

[EN] Water stress is a major environmental factor that limits crop production and it is important to develop crop varieties with higher yield under water scarcity. Increased pepper tolerance to water stress through grafting onto robust rootstocks could be an optimal alternative in the context of environmentally friendly agriculture. Our work evaluated the behaviour of 18 pepper genotypes during vegetative and reproductive stages under water stress in order to select tolerant genotypes to be used as rootstocks for pepper cultivation. The pepper tolerance screening was based on photosynthetic parameters. The genotypes Atlante, C-40, Serrano, PI-152225, ECU-973, BOL-58 and NuMex Conquistador were revealed as the most tolerant genotypes to water stress because they maintained net photosynthetic rate levels under water stress conditions. The selected genotypes were validated as rootstocks on a pepper cultivar in terms of productivity under severe water stress. Plants grafted onto cvs Atlante, PI-152225 and ECU-973 showed higher marketable yields when compared with ungrafted cultivar., Supported by Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Project RTA2010-00038-C03-01-03 and the European Regional Development Fund.

Tomato is one of the most important horticultural crops worldwide, and is cultivated in semi-arid regions in which soil and groundwater salinity is an increasing limitation to yield. The assessment of the responses of new cultivars to salt and the comparisons among cultivars and wild species are of great interest in tomato breeding. This assay provides a reproducible and reliable method for screening tomato responses to NaCl salinity under hydroponic conditions in growth chambers. Although NaCl is the most commonly used salt in salinity studies, other salts such as Na2SO4, MgCl2 or MgSO4, usually found in saline soils, can also be assayed (Nebauer et al., 2013). Plants can be maintained for 30-45 days under the described conditions, although significant effects on growth can be observed after 10 days, depending on the salt and concentration used., We gratefully acknowledge funding through grants from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA; project numbers: 2009-0004-C01, 2012-0008-C01) and the Spanish Ministry of Science and Innovation (project numbers: BIO2010-14871 and ERA-NET GEN2006-27772-C2-2).

Salinity is an important environmental constraint to crop productivity in arid and semiarid regions of the world. The evaluation of the responses to salinity of different Arabidopsis ecotypes or transgenic lines is important to identify and investigate the role of different key genes. These new characterized genes involved in the response to salinity stress are of great interest to be incorporated in crops breeding programs. Here we provide a reproducible method to evaluate the performance of Arabidopsis lines to salinity stress by analysing primary and lateral root growth and fresh weight of plants grown under in vitro conditions in growth chambers. Even though NaCl is the most frequent used salinity tests, other salts (e.g. KCl, MgCl2) can be also evaluated by this method. Arabidopsis plants can be maintained for 15-20 days in these conditions, although effects on growth and biomass can be observed, depending on the used salt and concentration, within the first 10 days., We gratefully acknowledge funding through grants from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA; project numbers: 2009-0004-C01, 2012-0008-C01) and the Spanish Ministry of Science and Innovation (project number: BIO2010-14871 and ERA-NET GEN2006-27772-C2-2).

The aim of this educational review is to providepractical information on the hardware, methodology, andthe hands on application of chlorophyll (Chl)afluores-cence technology. We present the paper in a question andanswer format like frequently asked questions. Althoughnearly all information on the application of Chlafluores-cence can be found in the literature, it is not always easilyaccessible. This paper is primarily aimed at scientists whohave some experience with the application of Chlafluo-rescence but are still in the process of discovering what itall means and how it can be used. Topics discussed are(among other things) the kind of information that can beobtained using different fluorescence techniques, theinterpretation of Chlafluorescence signals, specificapplications of these techniques, and practical advice ondifferent subjects, such as on the length of dark adaptationbefore measurement of the Chlafluorescence transient. Fil: Kalaji, Hazem. Warsaw University of Life Sciences. Varsovia; Polonia Fil: Schansker, Gert. Chêne-Bougeries; Suiza Fil: Ladle, Richard. Universidade Federal de Alagoas; Brasil Fil: Goltsev, Vasilij. St. Kliment Ohridski University of Sofia. Sofia; Bulgaria Fil: Bosa, Karolina. Warsaw University of Life Sciences. Varsovia; Polonia Fil: Allakhverdiev, Suleyman. Russian Academy of Sciences. Moscu; Rusia Fil: Brestic, Marian. Agricultural University. Nitra; Eslovaquia Fil: Bussotti, Filippo. University of Florence. Florencia; Italia Fil: Calatayud, Angeles. Instituto Valenciano de Investigaciones Agrarias. Valencia; España Fil: Dabrowski, Piotr. Warsaw University of Life Sciences. Varsovia; Polonia Fil: Elsheery, Nabil. Tanta University. Tanta; Egipto Fil: Ferroni, Lorenzo. Universita Di Ferrara; Italia Fil: Guidi, Lucia. Food and Environment. Pisa; Italia Fil: Hogewoning, Ssander. Plant Lighting BV. Bunnik; Países Bajos Fil: Jajoo, Anjama. Devi Ahilya University. Indore; India Fil: Misra, Amarendra. Central University of Jharkhand. Ranchi; India Fil: Nebauer, Sergio G. Universidad Politécnica de Valencia; España Fil: Pancaldi, Simonetta. Universita Di Ferrara; Italia Fil: Penella, Consuelo. Instituto Valenciano de Investigaciones Agrarias. Valencia; España Fil: Poli, Dorothy Belle. Roanoke College. Salem; Estados Unidos Fil: Pollastrini, M. University of Florence. Florencia; Italia Fil: Romanowska Duda, Zdzislawa. University of Lodz. Lodz; Polonia Fil: Rutkowska, Beata. Warsaw University of Life Sciences. Varsovia; Polonia Fil: Serodio, Joao. Universidade de Aveiro. Aveiro; Portugal Fil: Suresh, Kancherla. Directorate of Oil Palm Research. Pedavegi; India Fil: Szulc, Wieslaw. Warsaw University of Life Sciences. Varsovia; Polonia Fil: Tambussi, Eduardo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Yanniccari, Marcos Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Zivcak, Marek. Agricultural University. Nitra; Eslovaquia

DNA binding with One Finger (DOF) transcription factors are involved in multiple aspects of plant growth and development but their precise roles in abiotic stress tolerance are largely unknown. Here we report a group of five tomato DOF genes, homologous to Arabidopsis Cycling DOF Factors (CDFs), that function as transcriptional regulators involved in responses to drought and salt stress and flowering-time control in a gene-specific manner. SlCDF1–5 are nuclear proteins that display specific binding with different affinities to canonical DNA target sequences and present diverse transcriptional activation capacities in vivo. SlCDF1–5 genes exhibited distinct diurnal expression patterns and were differentially induced in response to osmotic, salt, heat, and low-temperature stresses. Arabidopsis plants overexpressing SlCDF1 or SlCDF3 showed increased drought and salt tolerance. In addition, the expression of various stress-responsive genes, such as COR15, RD29A, and RD10, were differentially activated in the overexpressing lines. Interestingly, overexpression in Arabidopsis of SlCDF3 but not SlCDF1 promotes late flowering through modulation of the expression of flowering control genes such as CO and FT. Overall, our data connect SlCDFs to undescribed functions related to abiotic stress tolerance and flowering time through the regulation of specific target genes and an increase in particular metabolites., This work was supported by grants from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA; project numbers: 2009-0004-C01, 2012-0008-C01), the Spanish Ministry of Science and Innovation (project number: BIO2010-14871), and the MERIT Project (FP7 ITN2010-264474). ARC was supported by a pre-doctoral fellowship from the INIA.

Digitalis minor (Scrophulariaceae) is a cardenolide-producing plant endemic to the eastern Balearic Islands (Mallorca, Menorca, and Cabrera) that occurs in two morphologically distinct varieties: D. minor var. minor (pubescent) and D. minor var. palaui (glabrous). Levels and patterns of genetic diversity in 162 individuals from 17 D. minor populations across the entire geographic range were assessed using random amplified polymorphic DNA (RAPD) markers. Comigrating RAPD fragments tested were found to be homologous by Southern hybridization in both var. minor and var. palaui. To avoid bias in parameter estimation, analyses of population genetic structure were restricted to those RAPD bands that fulfilled the 3/N criterion (observed frequencies were less than 1 - [3/N] in each population) either among or within each island. Analyses of molecular variance (AMOVAs) with distances among individuals corrected for the dominant nature of RAPD (genotypic analysis) showed low values (1.57-17.55%) of between-population variability, indicating a relatively restricted population differentiation as expected for an outcrossing species such as D. minor. Nested AMOVAs demonstrated, however, a not significant partitioning of genetic diversity among Mallorca, Menorca, and Cabrera islands. Estimates of the Wright, Weir, and Cockerham and the Lynch and Milligan F(ST) from null allele frequencies corroborated AMOVA partitioning and provided evidence for population differentiation in D. minor. Our RAPD data did not show significant differences between pubescent and glabrous populations of D. minor, suggesting a failure to find a correlation between the RAPD loci and this morphological trait.

RAPD markers were used to study inter-specific variation among six species of the genus Digitalis: D. obscura, D. lanata, D. grandiflora, D. purpurea, D. thapsi and D. dubia, and the hybrid D. excelsior (D. purpurea×D. grandiflora). A total of 91 highly reproducible bands amplified with four arbitrarily chosen decamer primers were obtained. Homology of the co-emigrating RAPD markers was tested by blot hybridisation and sequencing of selected bands. The application of a range of statistical approaches for RAPD data analysis, including distance and parsimony methods, family clustering and the analysis of molecular variance (AMOVA), indicated that these molecular markers were taxonomically informative in Digitalis. The species relationships revealed were fully consistent with those previously obtained using morphological affinities. The hybrid D. excelsior seems to have stronger affinity to the section Digitalis than to Grandiflorae. This is the first known report of the application of RAPD markers for the study of genetic relationships among species of the genus Digitalis.