Regulation of DELLA proteins by post-translational modifications

[EN] DELLA proteins are the negative regulators of the gibberellin (GA) signaling pathway. GAs have a pervasive effect on plant physiology, influencing processes that span the entire life cycle of the plant. All the information encoded by GAs, either environmental or developmental in origin, is canalized through DELLAs, which modulate the activity of many transcription factors and transcriptional regulators. GAs unlock the signaling pathway by triggering DELLA polyubiquitination and degradation by the 26S proteasome. Recent reports indicate, however, that there are other pathways that trigger DELLA polyubiquitination and degradation independently of GAs. Moreover, results gathered during recent years indicate that other post-translational modifications (PTMs), namely phosphorylation, SUMOylation and glycosylation, modulate DELLA function. The convergence of several PTMs in DELLA therefore highlights the strict regulation to which these proteins are subject. In this review, we summarize these discoveries and discuss DELLA PTMs from an evolutionary perspective and examine the possibilities these and other post-translational regulations offer to improve DELLA-dependent agronomic traits.The Spanish Ministry of Science and Innovation (PID2019-109925GB-I00 to D.A.) and the European Union (H2020-MSCA-IF-2016-746396 to A.S.-M.). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Blanco-Touriñán, N.; Serrano-Mislata, A.; Alabadí Diego, D. (2020). Regulation of DELLA proteins by post-translational modifications. Plant and Cell Physiology. 61(11):1891-1901. https://doi.org/10.1093/pcp/pcaa113S18911901611

Differential growth at the apical hook: all roads lead to auxin

[EN] The apical hook is a developmentally regulated structure that appears in dicotyledonous seedlings when seeds germinate buried in the soil. It protects the shoot apical meristem and cotyledons from damage while the seedling is pushing upwards seeking for light, and it is formed by differential cell expansion between both sides of the upper part of the hypocotyl. Its apparent simplicity and the fact that it is dispensable when seedlings are grown in vitro have converted the apical hook in one of the favorite experimental models to study the regulation of differential growth. The involvement of hormones especially auxin in this process was manifested already in the early studies. Remarkably, a gradient of this hormone across the hook curvature is instrumental to complete its development, similar to what has been proposed for other processes involving the bending of an organ, such as tropic responses. In agreement with this, other hormones-mainly gibberellins and ethylene-and the light, regulate in a timely and interconnected manner the auxin gradient to promote hook development and its opening, respectively. Here, we review the latest findings obtained mainly with the apical hook of Arabidopsis thaliana, paying special attention to the molecular mechanisms for the cross-regulation between the different hormone signaling pathways that underlie this developmental process.This work was supported by grants from the Spanish Ministry of Science and Innovation (BIO2010-15071 and CSD2007-00057) and the Generalitat Valenciana (ACOMP/2011/288 and PROMETEO/2010/020).Abbas, M.; Alabadí Diego, D.; Blazquez Rodriguez, MA. (2013). Differential growth at the apical hook: all roads lead to auxin. Frontiers in Plant Science. 4:441-1-441-9. https://doi.org/10.3389/fpls.2013.00441S441-1441-9

Induction of auxin biosynthesis and WOX5 repression mediate changes in root development in Arabidopsis exposed to chitosan

[EN] Chitosan is a natural polymer with applications in agriculture, which causes plasma membrane permeabilisation and induction of intracellular reactive oxygen species (ROS) in plants. Chitosan has been mostly applied in the phylloplane to control plant diseases and to enhance plant defences, but has also been considered for controlling root pests. However, the effect of chitosan on roots is virtually unknown. In this work, we show that chitosan interfered with auxin homeostasis in Arabidopsis roots, promoting a 2-3 fold accumulation of indole acetic acid (IAA). We observed chitosan dose-dependent alterations of auxin synthesis, transport and signalling in Arabidopsis roots. As a consequence, high doses of chitosan reduce WOX5 expression in the root apical meristem and arrest root growth. Chitosan also propitiates accumulation of salicylic (SA) and jasmonic (JA) acids in Arabidopsis roots by induction of genes involved in their biosynthesis and signalling. In addition, high-dose chitosan irrigation of tomato and barley plants also arrests root development. Tomato root apices treated with chitosan showed isodiametric cells respect to rectangular cells in the controls. We found that chitosan causes strong alterations in root cell morphology. Our results highlight the importance of considering chitosan dose during agronomical applications to the rhizosphere.This work was supported by AGL 2015 66833-R Grant from the Spanish Ministry of Economy and Competitiveness Grant AGL 2015. We would like to thank Drs Isabel Lopez-Diaz and Esther Carrera for plant hormone quantitation (IBMCP, Valencia, Spain). Part of this work was filed for a patent (P201431399) by L. V. Lopez-Llorca, F. Lopez-Moya and N. Escudero as inventors. We would like to thank Dr Michael Kershaw (University of Exeter) for his English revision and critical comments of the manuscript. We also thank Ms Marta Suarez-Fernandez (University of Alicante) and Mr Alfonso Prieto for their technical support. All the authors reviewed and approved the manuscript.Lopez-Moya, F.; Escudero, N.; Zavala-Gonzalez, EA.; Esteve-Bruna, D.; Blazquez Rodriguez, MA.; Alabadí Diego, D.; Lopez-Llorca, LV. (2017). Induction of auxin biosynthesis and WOX5 repression mediate changes in root development in Arabidopsis exposed to chitosan. Scientific Reports. 7:1-14. https://doi.org/10.1038/s41598-017-16874-5S1147Savary, S., Ficke, A., Aubertot, J.-N. & Hollier, C. Crop losses due to diseases and their implications for global food production losses and food security. Food Sec. 4(4), 519–37 (2012).Pimentel, D., Zuniga, R. & Morrison, D. Update on the environmental and economic costs associated with alien-invasive species in the United States. Ecol. Econ. 52(3), 273–88 (2005).El-Hadrami, A. & Adam, L. R. Hadrami El, I. & Daayf, F. Chitosan in plant protection. Mar. 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Identification of Transgene-Free CRISPR-Edited Plants of Rice, Tomato, and Arabidopsis by Monitoring DsRED Fluorescence in Dry Seeds

Efficient elimination of the editing machinery remains a challenge in plant biotechnology after genome editing to minimize the probability of off-target mutations, but it is also important to deliver end users with edited plants free of foreign DNA. Using the modular cloning system Golden Braid, we have included a fluorescence-dependent transgene monitoring module to the genome-editing tool box. We have tested this approach in Solanum lycopersicum, Oryza sativa, and Arabidopsis thaliana. We demonstrate that DsRED fluorescence visualization works efficiently in dry seeds as marker for the detection of the transgene in the three species allowing an efficient method for selecting transgene-free dry seeds. In the first generation of DsRED-free CRISPR/Cas9 null segregants, we detected gene editing of selected targets including homozygous mutants for the plant species tested. We demonstrate that this strategy allows rapid selection of transgene-free homozygous edited crop plants in a single generation after in vitro transformation

Prefoldins contribute to maintaining the levels of the spliceosome LSM2–8 complex through Hsp90 in Arabidopsis

Although originally identified as the components of the complex aiding the cytosolic chaperonin CCT in the folding of actins and tubulins in the cytosol, prefoldins (PFDs) are emerging as novel regulators influencing gene expression in the nucleus. Work conducted mainly in yeast and animals showed that PFDs act as transcriptional regulators and participate in the nuclear proteostasis. To investigate new functions of PFDs, we performed a co-expression analysis in Arabidopsis thaliana. Results revealed co-expression between PFD and the Sm-like (LSM) genes, which encode the LSM2–8 spliceosome core complex, in this model organism. Here, we show that PFDs interact with and are required to maintain adequate levels of the LSM2–8 complex. Our data indicate that levels of the LSM8 protein, which defines and confers the functional specificity of the complex, are reduced in pfd mutants and in response to the Hsp90 inhibitor geldanamycin. We provide biochemical evidence showing that LSM8 is a client of Hsp90 and that PFD4 mediates the interaction between both proteins. Consistent with our results and with the role of the LSM2–8 complex in splicing through the stabilization of the U6 snRNA, pfd mutants showed reduced levels of this snRNA and altered pre-mRNA splicing patterns.Fil: Esteve Bruna, David. Universidad Politécnica de Valencia; EspañaFil: Carrasco López, Cristian. Consejo Superior de Investigaciones Científicas; EspañaFil: Blanco Touriñán, Noel. Universidad Politécnica de Valencia; EspañaFil: Iserte, Javier Alonso. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Calleja Cabrera, Julián. Universidad Politécnica de Valencia; EspañaFil: Perea Resa, Carlos. Consejo Superior de Investigaciones Científicas; EspañaFil: Úrbez, Cristina. Universidad Politécnica de Valencia; EspañaFil: Carrasco, Pedro. Universidad Politécnica de Valencia; EspañaFil: Yanovsky, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Blázquez, Miguel A.. Universidad Politécnica de Valencia; EspañaFil: Salinas, Julio. Consejo Superior de Investigaciones Científicas; EspañaFil: Alabadí, David. Universidad Politécnica de Valencia; Españ

Long-Day Photoperiod Enhances Jasmonic Acid-Related Plant Defense

[EN] Agricultural crops are exposed to a range of daylengths, which act as important environmental cues for the control of developmental processes such as flowering. To explore the additional effects of daylength on plant function, we investigated the transcriptome of Arabidopsis (Arabidopsis thaliana) plants grown under short days (SD) and transferred to long days (LD). Compared with that under SD, the LD transcriptome was enriched in genes involved in jasmonic acid-dependent systemic resistance. Many of these genes exhibited impaired expression induction under LD in the phytochrome A (phyA), cryptochrome 1 (cry1), and cry2 triple photoreceptor mutant. Compared with that under SD, LD enhanced plant resistance to the necrotrophic fungus Bottytis cinerea. This response was reduced in the phyA cry1 cry2 triple mutant, in the constitutive photomorphogenicl (cop1) mutant, in the myc2 mutant, and in mutants impaired in DELLA function. Plants grown under SD had an increased nuclear abundance of COP1 and decreased DELLA abundance, the latter of which was dependent on COP1. We conclude that growth under LD enhances plant defense by reducing COP1 activity and enhancing DELLA abundance and MYC2 expression.This study was supported by a Guggenheim Foundation fellowship (to J.J.C), by Agencia Nacional de Promocion Cientifica y Tecnologica (PICT-2015-1796), by the University of Buenos Aires (20020100100437, to J.J.C.), by the Howard Hughes Medical Institute (J.I.C.), and by the SIGNAT-Research and Innovation Staff Exchange (H2020-MSCA-RISE-2014, to P.D.C., M.A.B., D.A., and J.J.C.).Cagnola, J.; Cerdan, P.; Pacín, M.; Andrade, A.; Rodríguez, V.; Zurbriggen, M.; Legris, M.... (2018). Long-Day Photoperiod Enhances Jasmonic Acid-Related Plant Defense. PLANT PHYSIOLOGY. 178(1):163-173. https://doi.org/10.1104/pp.18.00443S163173178

Hormonal regulation of temperature-induced growth in Arabidopsis

[EN] Successful plant survival depends upon the proper integration of information from the environment with endogenous cues to regulate growth and development. We have investigated the interplay between ambient temperature and hormone action during the regulation of hypocotyl elongation, and we have found that gibberellins (GAs) and auxin are quickly and independently recruited by temperature to modulate growth rate, whereas activity of brassinosteroids (BRs) seems to be required later on. Impairment of GA biosynthesis blocked the increased elongation caused at higher temperatures, but hypocotyls of pentuple DELLA knockout mutants still reduced their response to higher temperatures when BR synthesis or auxin polar transport were blocked. The expression of several key genes involved in the biosynthesis of GAs and auxin was regulated by temperature, which indirectly resulted in coherent variations in the levels of accumulation of nuclear GFP-RGA (repressor of GA1) and in the activity of the DR5 reporter. DNA microarray and genetic analyses allowed the identification of the transcription factor PIF4 (phytochrome-interacting factor 4) as a major target in the promotion of growth at higher temperature. These results suggest that temperature regulates hypocotyl growth by individually impinging on several elements of a pre-existing network of signaling pathways involving auxin, BRs, GAs, and PIF4.We thank G. Choi (KAIST, Daejeon, South Korea), C. Fankhauser (University of Lausanne, Lausanne, Switzerland), T. Guilfoyle (Department of Biochemistry, University of Missouri, MO, USA), N. P. Harberd (Department of Plant Sciences, University of Oxford, Oxford, UK), E. Huq (University of Texas, Austin, TX, USA), T-p Sun (Department of Biology, Duke University, Durham, USA), S. G. Thomas (Rothamsted Research, Hertfordshire, UK), G. Vert (Institut de Biologie Integrative des Plantes, Montpellier, France), Z. Y. Wang (Department of Plant Biology, Carnegie Institution, Stanford, USA), Y. Yin (Plant Science Institute, Iowa State University, Ames, IA, USA), and the Arabidopsis Biological Resource Center for seeds; and X. W. Deng (Yale University, New Haven, CT, USA) for antibodies against RPT5. We also thank Dr Jorge Casal (Universidad de Buenos Aires, Buenos Aires, Argentina) for helpful suggestions on this work. Work in the authors' laboratories is funded by grant BIO2007-60923 from the Spanish Ministry of Science and Innovation and by grant 167890/110 from the Norwegian Research Council. JG-B was supported by a JAE pre-doctoral fellowship from CSIC.Stavang, JA.; Gallego-Bartolomé, J.; Gómez Jiménez, MD.; Yoshida, S.; Asami, T.; Olsen, JE.; García-Martínez, JL.... (2009). Hormonal regulation of temperature-induced growth in Arabidopsis. The Plant Journal. 60(4):589-601. https://doi.org/10.1111/j.1365-313X.2009.03983.x58960160

A genetic approach reveals different modes of action of prefoldins

[EN] The prefoldin complex (PFDc) was identified in humans as a co-chaperone of the cytosolic chaperonin T-COMPLEX PROTEIN RING COMPLEX (TRiC)/CHAPERONIN CONTAINING TCP-1 (CCT). PFDc is conserved in eukaryotes and is composed of subunits PFD1-6, and PFDc-TRiC/CCT folds actin and tubulins. PFDs also participate in a wide range of cellular processes, both in the cytoplasm and in the nucleus, and their malfunction causes developmental alterations and disease in animals and altered growth and environmental responses in yeast and plants. Genetic analyses in yeast indicate that not all of their functions require the canonical complex. The lack of systematic genetic analyses in plants and animals, however, makes it difficult to discern whether PFDs participate in a process as the canonical complex or in alternative configurations, which is necessary to understand their mode of action. To tackle this question, and on the premise that the canonical complex cannot be formed if one subunit is missing, we generated an Arabidopsis (Arabidopsis thaliana) mutant deficient in the six PFDs and compared various growth and environmental responses with those of the individual mutants. In this way, we demonstrate that the PFDc is required for seed germination, to delay flowering, or to respond to high salt stress or low temperature, whereas at least two PFDs redundantly attenuate the response to osmotic stress. A coexpression analysis of differentially expressed genes in the sextuple mutant identified several transcription factors, including ABA INSENSITIVE 5 (ABI5) and PHYTOCHROME-INTERACTING FACTOR 4, acting downstream of PFDs. Furthermore, the transcriptomic analysis allowed assigning additional roles for PFDs, for instance, in response to higher temperature.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness and "Agencia Estatal de Investigacion"/FEDER/European Union (BIO2013-43184-P to D.A. and M.A.B., and BIO2016-79133-P and PID2019-109925GB-I00 to D.A.). N.B.-T., A.S.-M., and A.P.-A. were recipient of Ministerio de Economia y Competitividad (BES-2014-068868), EU MSCA-IF (H2020-MSCA-IF-2016746396) and Ministerio de Educacion (FPU17/05186) fellowships, respectively.Esteve-Bruna, D.; Blanco-Touriñán, N.; Serrano-Mislata, A.; Esquinas-Ariza, RM.; Resentini, F.; Forment Millet, JJ.; Carrasco-López, C.... (2021). A genetic approach reveals different modes of action of prefoldins. Plant Physiology. 187(3):1534-1550. https://doi.org/10.1093/plphys/kiab348S15341550187

DELLA-Induced Early Transcriptional Changes during Etiolated Development in Arabidopsis thaliana

The hormones gibberellins (GAs) control a wide variety of processes in plants, including stress and developmental responses. This task largely relies on the activity of the DELLA proteins, nuclear-localized transcriptional regulators that do not seem to have DNA binding capacity. The identification of early target genes of DELLA action is key not only to understand how GAs regulate physiological responses, but also to get clues about the molecular mechanisms by which DELLAs regulate gene expression. Here, we have investigated the global, early transcriptional response triggered by the Arabidopsis DELLA protein GAI during skotomorphogenesis, a developmental program tightly regulated by GAs. Our results show that the induction of GAI activity has an almost immediate effect on gene expression. Although this transcriptional regulation is largely mediated by the PIFs and HY5 transcription factors based on target meta-analysis, additional evidence points to other transcription factors that would be directly involved in DELLA regulation of gene expression. First, we have identified cis elements recognized by Dofs and type-B ARRs among the sequences enriched in the promoters of GAI targets; and second, an enrichment in additional cis elements appeared when this analysis was extended to a dataset of early targets of the DELLA protein RGA: CArG boxes, bound by MADS-box proteins, and the E-box CACATG that links the activity of DELLAs to circadian transcriptional regulation. Finally, Gene Ontology analysis highlights the impact of DELLA regulation upon the homeostasis of the GA, auxin, and ethylene pathways, as well as upon pre-existing transcriptional networks