Heat-priming during somatic embryogenesis increased resilience to drought stress in the generated maritime pine (Pinus pinaster) plants

Drought stress is becoming the most important factor of global warming in forests, hampering the production of reproductive material with improved resilience. Previously, we reported that heat-priming maritime pine (Pinus pinaster) megagametophytes during SE produced epigenetic changes that generated plants better adapted to subsequent heat stress. In this work, we tested, in an experiment performed under greenhouse conditions, whether heat-priming will produce cross-tolerance to mild drought stress (30 days) in 3-year-old priming-derived plants. We found that they maintain constitutive physiological differences as compared to controls, such as higher proline, abscisic acid, starch, and reduced glutathione and total protein contents, as well as higher ΦPSII yield. Primed plants also displayed a constitutive upregulation of the WRKY transcription factor and the Responsive to Dehydration 22 (RD22) genes, as well as of those coding for antioxidant enzymes (APX, SOD, and GST) and for proteins that avoid cell damage (HSP70 and DHNs). Furthermore, osmoprotectants as total soluble sugars and proteins were early accumulated in primed plants during the stress. Prolongated water withdrawal increased ABA accumulation and negatively affected photosynthesis in all plants but primed-derived plants recovered faster than controls. We concluded that high temperature pulses during somatic embryogenesis resulted in transcriptomic and physiological changes in maritime pine plants that can increase their resilience to drought stress, since heat-primed plants exhibit permanent activation of mechanisms for cell protection and overexpression of stress pathways that pre-adapt them to respond more efficiently to soil water deficit

The targeted overexpression of SlCDF4 in the fruit enhances tomato size and yield involving gibberellin signalling

[EN] 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 GA(4) 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.Renau-Morata, B.; Carrillo, L.; Cebolla Cornejo, J.; Molina Romero, RV.; Martí-Renau, R.; Domínguez-Figueroa, J.; Vicente-Carbajosa, J.... (2020). The targeted overexpression of SlCDF4 in the fruit enhances tomato size and yield involving gibberellin signalling. Scientific Reports. 10(1):1-14. https://doi.org/10.1038/s41598-020-67537-x1141011FAO. Crops production database. FAOSTAT. Latest update: 04/03/2020. Food and Agriculture Organization of the United Nations. Rome https://www.fao.org/faostat (2018).Willcox, J. K., Catignani, G. L. & Lazarus, S. Tomatoes and cardiovascular health. Crit. Rev. Food Sci. 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Crocus sativus L. Ecotypes from Mediterranean Countries: Phenological, Morpho-Productive, Qualitative and Genetic Traits

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

PsPMEP, a pollen specific pectin methylesterase of pea (Pisum sativum L.)

[EN] Pectin methylesterases (PMEs) are a family of enzymes involved in plant reproductive processes such as pollen development and pollen tube growth. We have isolated and characterized PsPMEP, a pea (Pisum sativum L.) pollen-specific gene that encodes a protein with homology to PMEs. Sequence analysis showed that PsPMEP belongs to group 2 PMEs, which are characterized by the presence of a processable amino-terminal PME inhibitor domain followed by the catalytic PME domain. Moreover, PsPMEP contains several motifs highly conserved among PMEs with the essential amino acid residues involved in enzyme substrate binding and catalysis. Northern blot and in situ hybridization analyses showed that PsPMEP is expressed in pollen grains from 4 days before anthesis till anther dehiscence and in pollinated carpels. In the PsPMEP promoter region, we have identified several conserved cis-regulatory elements that have been associated with gene pollen-specific expression. Expression analysis of PsPMEP promoter fused to the uidA reporter gene in Arabidopsis thaliana plants showed a similar expression pattern when compared with pea, indicating that this promoter is also functional in a non-leguminous plant. GUS expression was detected in mature pollen grains, during pollen germination, during pollen tube elongation along the transmitting tract, and when the pollen tube reaches the embryo sac in the ovule.This work was funded by grants BIO2006-09374 and BIO2009-08134 from the Spanish Ministry of Science and Innovation (MICINN). The collaboration and assistance of Julia Marin-Navarro in the catalytic activity assays of PsPMEP in yeast and Rafael Martinez-Pardo in the greenhouse is gratefully acknowledged. 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Flower induction and development in saffron: Timing and hormone signalling pathways

[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].Renau-Morata, B.; Nebauer, SG.; García-Carpintero, V.; Cañizares Sales, J.; Minguet, E.; De Los Mozos, M.; Molina Romero, RV. (2021). Flower induction and development in saffron: Timing and hormone signalling pathways. Industrial Crops and Products. 164:1-19. https://doi.org/10.1016/j.indcrop.2021.113370S11916

Integrative Transcriptomic and Metabolomic Analysis at Organ Scale Reveals Gene Modules Involved in the Responses to Suboptimal Nitrogen Supply in Tomato

[EN] 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 long-term 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 Investigacion of Spain (Grant SEV-2016-0672) for supporting the scientific services used in this study. J. Canales was supported by the Agencia Nacional de Investigacion y Desarrollo de Chile (ANID, FONDECYT 1190812) and ANID-Millennium Science Initiative Program (ICN17-022).Renau-Morata, B.; Molina Romero, RV.; Minguet, E.; Cebolla Cornejo, J.; Carrillo, L.; Martí-Renau, R.; García-Carpintero, V.... (2021). Integrative Transcriptomic and Metabolomic Analysis at Organ Scale Reveals Gene Modules Involved in the Responses to Suboptimal Nitrogen Supply in Tomato. Agronomy. 11(7):1-26. https://doi.org/10.3390/agronomy11071320S12611

Ongoing Evolution in the Genus Crocus: Diversityof Flowering Strategies on the Way to Hysteranthy

[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].Pastor-Férriz, T.; De-Los-Mozos-Pascual, M.; Renau-Morata, B.; Nebauer, SG.; Sanchís, E.; Busconi, M.; Fernández, J.... (2021). Ongoing Evolution in the Genus Crocus: Diversityof Flowering Strategies on the Way to Hysteranthy. Plants. 10(3):1-18. https://doi.org/10.3390/plants1003047711810

Multifaceted role of cycling DOF factor 3 (CDF3) in the regulation of flowering time and abiotic stress responses in Arabidopsis

[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 fellowshipsCorrales, AR.; Carrillo, L.; Lasierra, P.; Nebauer, SG.; Dominguez-Figueroa, J.; Renau-Morata, B.; Pollmann, S.... (2017). Multifaceted role of cycling DOF factor 3 (CDF3) in the regulation of flowering time and abiotic stress responses in Arabidopsis. Plant Cell & Environment. 40(5):748-764. https://doi.org/10.1111/pce.12894S748764405Achard, P., Gong, F., Cheminant, S., Alioua, M., Hedden, P., & Genschik, P. (2008). The Cold-Inducible CBF1 Factor–Dependent Signaling Pathway Modulates the Accumulation of the Growth-Repressing DELLA Proteins via Its Effect on Gibberellin Metabolism. The Plant Cell, 20(8), 2117-2129. doi:10.1105/tpc.108.058941Ahuja, I., de Vos, R. C. H., Bones, A. 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Ectopic expression of the AtCDF1 transcription factor in potato enhances tuber starch and amino acid contents and yield under open field conditions

Introduction Cycling Dof transcription factors (CDFs) have been involved in different aspects of plant growth and development. In Arabidopsis and tomato, one member of this family (CDF1) has recently been associated with the regulation of primary metabolism and abiotic stress responses, but their roles in crop production under open field conditions remain unknown. Methods In this study, we compared the growth, and tuber yield and composition of plants ectopically expressing the CDF1 gene from Arabidopsis under the control of the 35S promoter with wild-type (WT) potato plants cultured in growth chamber and open field conditions. Results In growth chambers, the 35S::AtCDF1 plants showed a greater tuber yield than the WT by increasing the biomass partition for tuber development. Under field conditions, the ectopic expression of CDF1 also promoted the sink strength of the tubers, since 35S::AtCDF1 plants exhibited significant increases in tuber size and weight resulting in higher tuber yield. A metabolomic analysis revealed that tubers of 35S::AtCDF1 plants cultured under open field conditions accumulated higher levels of glucose, starch and amino acids than WT tubers. A comparative proteomic analysis of tubers of 35S::AtCDF1 and WT plants cultured under open field conditions revealed that these changes can be accounted for changes in the expression of proteins involved in energy production and different aspects of C and N metabolism. Discussion The results from this study advance our collective understanding of the role of CDFs and are of great interest for the purposes of improving the yield and breeding of crop plants

Identification of Solanum pimpinellifolium genome regions for increased resilience to nitrogen deficiency in cultivated tomato

14 Pág.High-quality crop production with minimal fertilizer inputs is a key goal for the agriculture of the future. Globally, tomato is one of the most important vegetable crops and its intensive production and breeding has been based on the application of large quantities of nitrogen (N) fertilizers. Therefore, the development of N use efficient (NUE) cultivars with low N inputs needs to be addressed. Some variability in plant growth, fruit quality and NUE traits among tomato (Solanum lycopersicum L.) varieties under low N supply has been reported, however, the relevance of wild relatives of tomato has not yet been assessed. In this study, we found that S. pimpinellifolium accession To-937 (SP) may be a suitable resource to increase NUE in tomato. We studied a set of 29 introgression lines (IL) from SP into the Moneymaker cultivar (MM) in different seasons to investigate the potential of SP introgressions to maintain the tomato plant performance during the growth cycle under low N input in greenhouse conditions. We identified specific regions in the SP genome, on chromosomes 1, 3 and 10, involved in the responses to N inputs of fruit production and fruit quality. Notably, the line SP_10-4 maintained vegetative biomass and fruit yield production under limiting N supply. The introgressed region contained putative candidate genes as sucrose phosphate phosphatase (SPP), invertases (INV) and glutamine synthase 1 (GS1) genes, implicated in C and N metabolism. Genomic and expression analyses revealed differences in coding and non-coding sequences as well as in mRNA levels in SP_10-4, suggesting that these genes might well contribute to the reported biomass responses to N. Additionally, line SP_1-4 showed stable fruit amino acid contents under both sufficient and limiting N supplies, indicating that assimilated N partitioning to the fruit is maintained in response to N. Altogether, our results confirmed the suitability of SP as a source of NUE related traits and the interest in the studied ILs for developing new tomato cultivars with improved NUE under sustainable fertilization conditions.This work was supported by the Ministerio de Ciencia e Innovación (MCIN) and the Agencia Estatal de Investigación (grants RTA2015-00014-c02-00 to SGN and JM, PID2020-114165RR-C21 to JM, and PID2022-136541OB-I00 to SGN), the Agroalnext program (MCIN with funding of NextGenEU-PRTR-C17.I1 Generalitat Valenciana AGROALNEXT/2022/056 to SGN), Vicerrectorado de Investigación de la Universitat Politècnica de València (PAID-11-21 to SGN; PAID-10-20 and PAID-PD-22 to RM), Ministerio de Ciencia e Innovación (TED2021-129296B-I00 to SGN) and European Commission H2020 research and innovation program through HARNESSTOM grant agreement no. 101000716 (to AG) and RoxyCOST CA18210 for networking activities (to AG). We also want to acknowledge the "Severo Ochoa Program for Centers of Excellence in R&D" (CEX2020-000999-S) supported by MCIN/AEI/10.13039/501100011033. We thank Mike Bennett for revising the English language and Javier Forment from the IBMCP Bioinformatics service for support on genomic analyses.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2020‐000999‐S)Peer reviewe