DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques

Background High throughput applications of the reverse transcriptase quantitative PCR (RT-qPCR) for quantification of gene expression demand straightforward procedures to isolate and analyze a considerable number of DNA-free RNA samples. Published protocols are labour intensive, use toxic organic chemicals and need a DNase digestion once pure RNAs have been isolated. In addition, for some tissues, the amount of starting material may be limiting. The convenience of commercial kits is often prohibitive when handling large number of samples. Findings We have established protocols to isolate DNA-free RNA from Arabidopsis thaliana tissues ready for RT-qPCR applications. Simple non-toxic buffers were used for RNA isolation from Arabidopsis tissues with the exception of seeds and siliques, which required the use of organic extractions. The protocols were designed to minimize the number of steps, labour time and the amount of starting tissue to as little as 10–20 mg without affecting RNA quality. In both protocols genomic DNA (gDNA) can be efficiently removed from RNA samples before the final alcohol precipitation step, saving extra purification steps before cDNA synthesis. The expression kinetics of previously characterized genes confirmed the robustness of the procedures. Conclusion Here, we present two protocols to isolate DNA-free RNA from Arabidopsis tissues ready for RT-qPCR applications that significantly improve existing ones by reducing labour time and the use of organic extractions. Accessibility to these protocols is ensured by its simplicity and the low cost of the materials used

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. 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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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The Root-Colonizing Endophyte Piriformospora indica Supports Nitrogen-Starved Arabidopsis thaliana Seedlings with Nitrogen Metabolites

16 Pág.The root-colonizing endophytic fungus Piriformospora indica promotes the root and shoot growth of its host plants. We show that the growth promotion of Arabidopsis thaliana leaves is abolished when the seedlings are grown on media with nitrogen (N) limitation. The fungus neither stimulated the total N content nor did it promote 15NO3- uptake from agar plates to the leaves of the host under N-sufficient or N-limiting conditions. However, when the roots were co-cultivated with 15N-labelled P. indica, more labels were detected in the leaves of N-starved host plants but not in plants supplied with sufficient N. Amino acid and primary metabolite profiles, as well as the expression analyses of N metabolite transporter genes suggest that the fungus alleviates the adaptation of its host from the N limitation condition. P. indica alters the expression of transporter genes, which participate in the relocation of NO3-, NH4+ and N metabolites from the roots to the leaves under N limitation. We propose that P. indica participates in the plant's metabolomic adaptation against N limitation by delivering reduced N metabolites to the host, thus alleviating metabolic N starvation responses and reprogramming the expression of N metabolism-related genes.This project was financially supported by the collaborative ICPS research project executed in the framework of the EIG CONCERT-Japan joint call on Food Crops and Biomass Production Technologies and the related national funding agencies: grants 01DR17007A and 01DR17007B from the Federal Ministry of Education and Research (BMBF), Germany, to R.O.; grant EIG_JC1JAPAN-045 from the Centre National de la Recherche Scientifique (CNRS), France, to A.K.; grant PCIN-2016–037 from the Ministry of Economy and Competitiveness (MINECO), Spain, to J.V.-C. and S.P.; and grant JPMJSC16C3 from the Japan Science and Technology Agency (JST) to H.S. This work was further supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) CRC1127 ChemBioSys (project ID: 239748522) for R.O. This work benefited from the support of IJPB’s Plant Observatory technological platforms. The IJPB benefits from additional support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007).Peer reviewe

Harnessing symbiotic plant-fungus interactions to unleash hidden forces from extreme plant ecosystems

13 Pág.Global climate change is arguably one of the biggest threats of modern times and has already led to a wide range of impacts on the environment, economy, and society. Owing to past emissions and climate system inertia, global climate change is predicted to continue for decades even if anthropogenic greenhouse gas emissions were to stop immediately. In many regions, such as central Europe and the Mediterranean region, the temperature is likely to rise by 2-5 °C and annual precipitation is predicted to decrease. Expected heat and drought periods followed by floods, and unpredictable growing seasons, are predicted to have detrimental effects on agricultural production systems, causing immense economic losses and food supply problems. To mitigate the risks of climate change, agricultural innovations counteracting these effects need to be embraced and accelerated. To achieve maximum improvement, the required agricultural innovations should not focus only on crops but rather pursue a holistic approach including the entire ecosystem. Over millions of years, plants have evolved in close association with other organisms, particularly soil microbes that have shaped their evolution and contemporary ecology. Many studies have already highlighted beneficial interactions among plants and the communities of microorganisms with which they coexist. Questions arising from these discoveries are whether it will be possible to decipher a common molecular pattern and the underlying biochemical framework of interspecies communication, and whether such knowledge can be used to improve agricultural performance under environmental stress conditions. In this review, we summarize the current knowledge of plant interactions with fungal endosymbionts found in extreme ecosystems. Special attention will be paid to the interaction of plants with the symbiotic root-colonizing endophytic fungus Serendipita indica, which has been developed as a model system for beneficial plant-fungus interactions.The authors are grateful for financial assistance received from the collaborative IPSC research project realized in the framework of the EIG CONCERT-Japan joint call on Food Crops and Biomass Production Technologies and the related national funding agencies: grant PCIN-2016–037 from the Ministry of Economy and Competitiveness (MINECO), Spain, to SP and JVC; grants 01DR17007A and 01DR17007B from the Federal Ministry of Education and Research (BMBF), Germany, to JL-M and RO, respectively; grant JPMJSC16C3 from the Japan Science and Technology Agency (JST) to HS; and grant EIG_JC1JAPAN-045 from the Centre National de la Recherche Scientifique (CNRS), France, to AK. CGG was supported by the Severo Ochoa Program for Centers of Excellence in R&D from the Agencia Estatal de Investigación, Spain, grant SEV-2016-0672 (2017–2021) to the Centro de Biotecnología y Genómica de Plantas.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditationPeer reviewe

Local Changes in Chromatin Accessibility and Transcriptional Networks Underlying the Nitrate Response in Arabidopsis Roots

Transcriptional regulation, determined by the chromatin structure and regulatory elements interacting at promoter regions, is a key step in plant responses to environmental cues. Nitrate (NO3-) is a nutrient signal that regulates the expression of hundreds of genes in Arabidopsis thaliana. Here, we integrate mRNA sequencing, genome-wide RNA polymerase II (RNPII), chromatin immunoprecipitation sequencing, and DNase sequencing datasets to establish the relationship between RNPII occupancy and chromatin accessibility in response to NO3- treatments in Arabidopsis roots. Genomic footprinting allowed us to identify in vivo regulatory elements controlling gene expression in response to NO3- treatments. NO3--modulated transcription factor (TF) footprints are important for a rapid increase in RNPII occupancy and transcript accumulation over time. We mapped key TF regulatory interactions and functionally validated the role of NAP, an NAC-domain containing TF, as a new regulatory factor in NO3- transport. Taken together, our study provides a comprehensive view of transcriptional networks in response to a nutrient signal in Arabidopsis roots.This work is funded by Instituto Milenio iBio - Iniciativa Científica Milenio MINECON, Chile; by grants from the Fondo de Desarrollo de Areas Prioritarias (FONDAP) Center for Genome Regulation (15090007), Chile; and Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) (1180759), Chile; to R.A.G. J.M.A. is supported by postdoctoral grant FONDECYT (3140336), Chile.J.J. is funded by grant MCB-1412948 from the National Science Foundation, United States; and J.M. by funding from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Spain (RTA2015-00014-c02-01). We also want to acknowledge the “Severo Ochoa Program for Centers of Excellence in R&D” from the Agencia Estatal de Investigación of Spain (SEV-2016-0672 (2017–2021)) for supporting the scientific services used in this work.Peer reviewe

Salinity Assay in Tomato

[EN] 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).Renau Morata, B.; Sánchez Perales, M.; Medina, J.; Molina Romero, RV.; Corrales, AR.; Carrillo, L.; Fernández Nohales, P.... (2014). Salinity Assay in Tomato. Bio-protocol. 4:1-8. https://doi.org/10.21769/BioProtoc.1215S18

Characterization of tomato Cycling Dof Factors reveals conserved and new functions in the control of flowering time and abiotic stress responses

[EN] 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. SlCDF15 are nuclear proteins that display specific binding with different affinities to canonical DNA target sequences and present diverse transcriptional activation capacities in vivo. SlCDF15 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 Investigacion y Tecnologia 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. 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The endophytic fungus Serendipita indica affects auxin distribution in Arabidopsis thaliana roots through alteration of auxin transport and conjugation to promote plant growth

21 Pág.Plants share their habitats with a multitude of different microbes. This close vicinity promoted the evolution of interorganismic interactions between plants and many different microorganisms that provide mutual growth benefits both to the plant and the microbial partner. The symbiosis of Arabidopsis thaliana with the beneficial root colonizing endophyte Serendipita indica represents a well-studied system. Colonization of Arabidopsis roots with S. indica promotes plant growth and stress tolerance of the host plant. However, until now, the molecular mechanism by which S. indica reprograms plant growth remains largely unknown. This study used comprehensive transcriptomics, metabolomics, reverse genetics, and life cell imaging to reveal the intricacies of auxin-related processes that affect root growth in the symbiosis between A. thaliana and S. indica. Our experiments revealed the sustained stimulation of auxin signalling in fungus infected Arabidopsis roots and disclosed the essential role of tightly controlled auxin conjugation in the plant-fungus interaction. It particularly highlighted the importance of two GRETCHEN HAGEN 3 (GH3) genes, GH3.5 and GH3.17, for the fungus infection-triggered stimulation of biomass production, thus broadening our knowledge about the function of GH3s in plants. Furthermore, we provide evidence for the transcriptional alteration of the PIN2 auxin transporter gene in roots of Arabidopsis seedlings infected with S. indica and demonstrate that this transcriptional adjustment affects auxin signalling in roots, which results in increased plant growth.We acknowledge financial support by the collaborative IPSC research project executed in the framework of the EIG CONCERT-Japan joint call on Food Crops and Biomass Production Technologies and the related national funding agencies: grant PCIN-2016–037 from the Ministry of Economy and Competitiveness (MINECO), Spain, to Stephan Pollmann and Jesús Vicente-Carbajosa; grants 01DR17007A and 01DR17007B from the Federal Ministry of Education and Research (BMBF), Germany, to Jutta Ludwig-Müller and Ralf Oelmüller, respectively; grant JPMJSC16C3 from the Japan Science and Technology Agency (JST) to Hitoshi Sakakibara; and grant EIG_JC1JAPAN-045 from the Centre National de la Recherche Scientifique (CNRS), France, to Anne Krapp. In addition, the project obtained financial support by grant PID2020-119441RB-I00 funded by MCIN/AEI/10.13039/501100011033 and, as appropriate, by “ERDF A way of making Europe,” by the “European Union” or by the “European Union NextGenerationEU/PRTR” to Stephan Pollmann. Eoghan King and Manish K. Patel were supported by the ‘Severo Ochoa Program for Centers of Excellence in R&D' from the Agencia Estatal de Investigación of Spain, grant CEX2020-000999-S (2022-2025) to the CBGP.Peer reviewe