La régulation transcriptionnelle de l'expression génique dans le fruit de tomate : caractérisation fonctionnelle de promoteurs fruit-spécifiques et d'un co-facteur de la transcription de type MBF1

Les travaux présentés dans ce manuscrit s'intéressent à la régulation transcriptionnelle de l'expression des gènes au cours du développement du fruit de tomate à travers l'étude des cis-régulations et des trans-régulations. Dans un premier temps, une tentative de caractérisation de promoteur dirigeant l'expression génique spécifiquement dans le fruit a été menée. Pour cela, nous nous sommes focalisés sur l'étude de régions promotrices d'un gène codant une expansine et d'un autre codant une alcool acyl transférase. Ces études ont été menées en évaluant la capacité de ces régions promotrices à diriger l'expression du gène rapporteur codant pour la β-glucuronidase dans des plants de tomate et d'Arabidopsis. Néanmoins, ces approches fonctionnelles n'ont pas permis de dégager un motif consensus capable à lui seul de conférer une expression fruit-spécifique. C'est pourquoi, les travaux de thèse se sont orientés vers la caractérisation d'un co-activateur de transcription de type MBF1 (Multiprotein Bridging Factor1), SlER24 de tomate. Nous avons montré qu'une fusion de SlER24 à un motif répresseur EAR (Ethylene-responsive element-binding associated Amphiphilic Repression) est capable, dans un système cellulaire, de réduire l'expression du gène rapporteur GFP dirigée par un promoteur synthétique de réponse à l'éthylène. Cette validation fonctionnelle par une méthode transitoire, nous a conduit à surexprimer cette construction, de façon stable, dans la tomate MicroTom. Cette surexpression de la construction chimérique SlER24-EAR, induit un retard de germination mais n'a pas d'effet notable sur le développement de la plante. Une fusion similaire de son orthologue AtMBF1c au motif EAR entraîne chez Arabidopsis thaliana une réduction du pourcentage de germination et un nanisme de la plante. Au-delà de l'information sur le rôle des gènes MBF1 dans le développement des plantes, cette étude démontre que la stratégie utilisant le domaine répresseur EAR n'est pas seulement efficace pour l'étude des facteurs de transcription comme cela a été démontré jusque là, mais également pour les co-activateurs ne se liant pas directement à l'ADN. ABSTRACT : The work presented in this manuscript is focused on the study in tomato fruit of gene expression regulation at the transcriptional level and comprises two parts. The first part concerns the isolation and characterization of promoters capable of specifically driving gene expression in tomato fruit. For this purpose, the capacity of native promoter regions and various deletions from two tomato genes, one encoding an expansin and a second encoding an alcohol acyl transferase to drive the expression of the GUS reporter gene, has been studied after stable transformation. This approach does not allow us to define a consensus motif able to drive the activity of a minimal specifically to the fruit. This is why the second part of the thesis was redirected to the characterization of a transcriptional co-activator of the MBF1 family, SlER24, in tomato. It is shown here that a chimerical fusion of SlER24 with the EAR (Ethylene-responsive element-binding associated Amphiphilic Repression) motif is capable of reducing the expression of the GFP reporter gene, driven by a synthetic ethylene-responsive promoter, in a transient cell system. This functional validation led us to express stably the construct in tomato plant using MicroTom for practical reasons. The overexpression of the SlER24/EAR motif fusion caused a germination delay but it had no significant effect on the tomato plant growth. A similar fusion using AtMBF1c from Arabidopsis, caused a reduction in the percentage of seed germination and dwarfism of the plant. Considering the role of the MBF1 genes in plant development, this study demonstrates that the EAR strategy is efficient not only for transcription factors, as demonstrated so far, but also in the case of co-activators known to not bind directly to DN

La régulation transcriptionnelle dépendant de l'éthylène. Caractérisation fonctionnelle d'un cofacteur transcriptionnel du type MBF1 et d'un facteur de transcription de la famille des ERF chez la tomate.

La phytohormone éthylène contrôle des étapes importantes de la vie de la plante comme la germination, la maturation des fruits et la réponse aux stress biotiques et abiotiques. L'éthylène exerce ses effets physiologiques en modulant l'expression de gènes cibles par l'intermédiaire des facteurs de transcription. De ce fait, la caractérisation des facteurs de transcription associés à la réponse à l'éthylène est une étape fondamentale pour la compréhension des processus régulés physiologiques régulés par cette hormone. Ce travail décrit l'isolement et la caractérisation fonctionnelle de la famille de coactivateurs transcriptionnels de type MBF1 (Multiproteine Bridging Factor 1) et d'un facteur de transcription de la famille des ERF (Ethylene Response Factor) chez la tomate. Notre étude montre que la famille des MBF1 chez la tomate comprend quatre gènes (LeMBF1a, LeMBF1b, LeMBF1c et LeMBF1/ER24) qui sont tous capables de complémenter un mutant de la levure indiquant qu'il y a une parfaite conservation fonctionnelle de ces gènes de la levure aux plantes supérieures. Les études d'expression révèlent un profil spécifique pour ER24 qui est fortement exprimé en réponse à l'éthylène, au cours de la maturation des fruits et en réponse à différents stress abiotiques. Le criblage par double-hybride de la levure a permis d'identifier plusieurs partenaires des protéines MBF1 chez la tomate parmi lesquels des protéines associées à la maturation et aux stress abiotiques. La surexpression du gène LeMBF1/ER24 chez Arabidopsis confère une tolérance au stress thermique. Par ailleurs, nous avons isolé et caractérisé le gène LeERF2 de la tomate qui appartient à la grande famille des ERF (Ethylene Response Factor) codant pour des facteurs de transcription spécifiques aux plantes. L'expression du gène LeERF2 est induite durant la maturation des fruits et en réponse à la blessure. La surexpression de ce gène dans la tomate induit une germination précoce des graines et affecte la formation du crochet apical, un processus de développement régulé par l'éthylène. L'expression du gène de la mannanase2, un marqueur de la germination, est fortement induite dans les graines transgéniques sur-exprimant le gène LeERF2 suggèrant que LeERF2 stimule la germination à travers l'induction de ce gène. Ce travail a permis d'identifier de nouveaux acteurs de la régulation transcriptionnelle associée à l'action de l'éthylène et d'apporter un nouvel éclairage sur le mode d'action de cette hormone

In silico Transcriptional Regulatory Networks Involved in Tomato Fruit Ripening

ABSTRACTTomato fruit ripening is a complex developmental programme partly mediated by transcriptional regulatory networks. Several transcription factors (TFs) which are members of gene families such as MADS-box and ERF were shown to play a significant role in ripening through interconnections into an intricate network. The accumulation of large datasets of expression profiles corresponding to different stages of tomato fruit ripening and the availability of bioinformatics tools for their analysis provide an opportunity to identify TFs which might regulate gene clusters with similar co-expression patterns. We identified two TFs, a SlWRKY22-like and a SlER24 transcriptional activator which were shown to regulate modules by using the LeMoNe algorithm for the analysis of our microarray datasets representing four stages of fruit ripening, breaker, turning, pink and red ripe. The WRKY22-like module comprised a subgroup of six various calcium sensing transcripts with similar to the TF expression patterns according to real time PCR validation. A promoter motif search identified a cis acting element, the W-box, recognized by WRKY TFs that was present in the promoter region of all six calcium sensing genes. Moreover, publicly available microarray datasets of similar ripening stages were also analyzed with LeMoNe resulting in TFs such as SlERF.E1, SlERF.C1, SlERF.B2, SLERF.A2, SlWRKY24, SLWRKY37 and MADS-box/TM29 which might also play an important role in regulation of ripening. These results suggest that the SlWRKY22-like might be involved in the coordinated regulation of expression of the six calcium sensing genes. Conclusively the LeMoNe tool might lead to the identification of putative TF targets for further physiological analysis as regulators of tomato fruit ripening

Natural variations of HSFA2 enhance thermotolerance in grapevine

Heat stress limits growth and development of crops including grapevine which is a popular fruit in the world. Genetic variability in crops thermotolerance is not well understood. We identified and characterized heat stress transcription factor HSFA2 in heat sensitive Vitis vinifera 'Jingxiu' (named as VvHSFA2) and heat tolerant Vitis davidii 'Tangwei' (named as VdHSFA2). The transcriptional activation activities of VdHSFA2 are higher than VvHSFA2, the variation of single amino acid (Thr315Ile) in AHA1 motif leads to the difference of transcription activities between VdHSFA2 and VvHSFA2. Based on 41 Vitis germplasms, we found that HSFA2 is differentiated at coding region among heat sensitive V. vinifera, and heat tolerant Vitis davidii and Vitis quinquangularis. Genetic evidence demonstrates VdHSFA2 and VvHSFA2 are positive regulators in grape thermotolerance, and the former can confer higher thermotolerance than the latter. Moreover, VdHSFA2 can regulate more target genes than VvHSFA2. As a target gene of both VdHSFA2 and VvHSFA2, overexpression of MBF1c enhanced the grape thermotolerance whereas dysfunction of MBF1c resulted in thermosensitive phenotype. Together, our results revealed that VdHSFA2 confers higher thermotolerance than VvHSFA2, and MBF1c acts as their target gene to induce thermotolerance. The VdHSFA2 may be adopted for molecular breeding in grape thermotolerance. © 2023 The Author(s)

Patellins 3 and 6, two members of the Plant Patellin family, interact with the movement protein of Alfalfa mosaic virus and interfere with viral movement

This is the accepted version of the following article: Peiró Morell, A.; Izquierdo Garcia, AC.; Sanchez Navarro, JA.; Pallás Benet, V.; Mulet Salort, JM.; Aparicio Herrero, F. (2014). Patellins 3 and 6, two members of the Plant Patellin family, interact with the movement protein of Alfalfa mosaic virus and interfere with viral movement. Molecular Plant Pathology. 15(9):881-891. doi:10.1111/mpp.12146., which has been published in final form at http://dx.doi.org/10.1111/mpp.12146.[EN] Movement proteins (MPs) encoded by plant viruses interact with host proteins to facilitate or interfere with intra- and/or intercellular viral movement. Using yeast two-hybrid and bimolecular fluorescence complementation assays, we herein present invivo evidence for the interaction between Alfalfa mosaic virus (AMV) MP and Arabidopsis Patellin 3 (atPATL3) and Patellin 6 (atPATL6), two proteins containing a Sec14 domain. Proteins with Sec14 domains are implicated in membrane trafficking, cytoskeleton dynamics, lipid metabolism and lipid-mediated regulatory functions. Interestingly, the overexpression of atPATL3 and/or atPATL6 interfered with the plasmodesmata targeting of AMV MP and correlated with reduced infection foci size. Consistently, the viral RNA levels increased in the single and double Arabidopsis knockout mutants for atPATL3 and atPATL6. Our results indicate that, in general, MP-PATL interactions interfere with the correct subcellular targeting of MP, thus rendering the intracellular transport of viral MP-containing complexes less efficient and diminishing cell-to-cell movement.AP was a recipient of a Pre-Doctoral Fellowship from the program JAE Pre-Doc of Consejo superior de Investigaciones Cientificas. ACI-G was a recipient of a Pre-Doctoral Fellowship associated with the project BFU2008-00604. FA was a recipient of a contract Ramon y Cajal (RYC-2010-06169) Program of the Ministerio de Educacion y Ciencia of Spain. We thank L. Corachan for excellent technical assistance. This work was supported by grants BIO2011-25018 from the Direccion General de Investigacion Cientifica y Tecnica, the Prometeo Program GV2011/003 from the Generalitat Valenciana and PAID-06-10-1496 from the Universitat Politecnica de Valencia (Spain).Peiró Morell, A.; Izquierdo García, AC.; Sanchez Navarro, JA.; Pallás Benet, V.; Mulet Salort, JM.; Aparicio Herrero, F. (2014). Patellins 3 and 6, two members of the Plant Patellin family, interact with the movement protein of Alfalfa mosaic virus and interfere with viral movement. Molecular Plant Pathology. 15(9):881-891. https://doi.org/10.1111/mpp.12146S88189115

Transcriptomic profiling of the extremophile eutrema salsugineum response to environmental stressors

Plants are sessile organisms that are constantly exposed to a variety of abiotic and biotic environmental stresses. Some plants are known to be more tolerant to those environmental stressors than others; those are the extremophilic plants. Studying the stress response pathways in such plants is extremely important in developing transgenic crop plants with enhanced tolerance to environmental stresses. Eutrema salsugineum is an extremophilic plant that is known to be resistant to many abiotic stress factors such as drought, cold, salt, and nitrogen deficiency. Experiments were carried out in KAUST by exposing the extremophilic plant to heat stress and exogenous ABA stress. RNA sequencing was done in order to get the transcriptome profile of the plant in response to the stresses. De novo transcriptome assembly was done followed by transcript abundance quantification and normalization using Kallisto. Differential expression analaysis was done to identify the differentially expressed transcripts in response to the different treatments in the shoot and root using the R bioconductor package EdgeR. The transcripts were annotated using EggNOG. The protein coding transcripts were identified by aligning them to the nr protein database using tblastx. Functional analysis of the DE transcripts to get the enriched terms was carried out using DAVID. Trinity de novo assembly produced 49857 genes and 134493 transcripts. Out of the 134493 transcripts, 114692 (85.28%) transcripts had tblastx hits (protein coding). Thus, 19801 potentially non coding or novel transcripts have been identified. A large variety of proteins were found to be differentially expressed depending on the pair-wise comparison. The genes were mainly involved in plant heat and ABA stress, ROS signaling pathway, ROS scavenging, secondary metabolite production, and lipid transfer. Further investigation of the role of secondary metabolites such as flavonoids, and nitrogen and sulfur containing compounds in the abiotic stress response of E. salsugineum is needed since it appears to be a major mechanism used by the plant. The results of this research offer a wide variety of stress related protein in E.salsugineum. Investigation of the over-expression of some of these genes in stress sensitive plants will help in further understanding their functions and mechanisms of action

Heat Shock Proteins (Hsps) Mediated Signalling Pathways During Abiotic Stress Conditions

The perception of abiotic stress and signal transduction to switch on adaptive innate responses are crucial steps in plant persistence under adverse environmental conditions. The ability of plants to respond to different stresses by maintaining protein in functional active form is of high importance and essential for cell survival. This is possible with the functional activity of heat shock proteins (Hsps), which play a role in maintaining the cellular homeostasis, and preserving the nonnative protein in a competent state for further remodeling as well as in signal transduction. Hsps mediate the stress signals either directly or indirectly, and play an important role in maintaining the homeostasis of plant cells under adverse stress conditions. Much research has been conducted to understand the complex mechanisms of the Hsps including Hsp70/90, ATPase-coupled conformational modifications, and interactions with cochaperones. In addition to this, several signaling proteins including MAP kinases, Ca2+ regulated proteins, Hsfs, reactive oxygen species are seen to be interacting with Hsps. In this study, we discuss the signal transduction of different Hsps, and their role in different signaling mechanisms

ROLE FOR HYDROGEN PEROXIDE DURING ABIOTIC AND BIOTIC STRESS SIGNALING IN PLANTS

Plants must adapt to negative environmental conditions that limit their growth, development and yield. Common for such stress conditions is that they induce the accumulation of harmful reactive oxygen species (ROS). ROS, such as hydrogen peroxide (H2O2), are now also considered to be important signal molecules that regulate the defense response of plants to stress. In this thesis, different strategies were pursued to identify genes that are involved in the stress response of plants

Regulation of the floral transition and inflorescence development by the bZIP transcription factor FD in Arabidopsis thaliana

The integration of signals in response to endogenous and exogenous stimuli at the shoot apical meristem (SAM) determines the timing of the transition from vegetative to reproductive development in Arabidopsis. Under inductive long-day (LD) photoperiods, FLOWERING LOCUS T (FT) and the bZIP transcription factor FD form the large transcriptional complex FD–(14-3-3)–FT/TSF at the SAM. Within this complex, FD is the DNA-binding component and some target genes of FD have been well characterized. The fd mutant is late flowering under LD conditions due to the improper regulation of its targets. Despite increased knowledge on the regulatory pathways that act through the FD–(14-3-3)–FT/TSF complex, the cis-regulatory elements that are required for the binding of FD to its targets remain poorly defined, and it is unclear how different targets show distinct spatiotemporal expression patterns. For example, although FD enhances FRUITFULL (FUL) transcription within the SAM, APETALA1 (AP1) transcription is promoted by FD later in development and AP1 transcripts are specific to floral primordia. Furthermore, the subset of direct targets that are involved in the floral transition before the upregulation of FUL and AP1 remain uncharacterized. During this PhD, I generated a transgenic fd mutant line in which the translocation of FD into the nucleus can be induced at different developmental time points. Induction of FD in this line promoted flowering, and showed that FD activity was required for several days to complete floral transition. I performed RNA-seq on apices of these plants following FD induction and identified putative additional components of the FD transcriptional network. The earliest targets of FD from this whole-transcriptome analysis could not be linked to floral transition, although FUL and AP1 were upregulated at later stages, confirming that floral transition occurred following FD induction. Much evidence exists to support that FUL is regulated by FD and I identified two putative conserved binding sites in the proximal promoter of FUL. However, mutation of these cis-elements did not affect flowering time nor the accumulation or pattern of FUL protein at the SAM. Under non-inductive short-day (SD) conditions, the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15 (SPL15) protein also binds to FUL and regulates its expression. Although FD and SPL15 regulate FUL through the LD and SD pathways, respectively, I hypothesise that they do not compete at the promoter level and that activation of FUL by FD can occur indirectly through FD-mediated activation of other transcription factors or that FD binds to redundant sites at the FUL locus under LDs. Abscisic acid (ABA) regulates stress responses such as the drought-escape response, and aspects of plant development, including axillary meristem growth and meristem arrest. The FD protein is phylogenetically closely related to bZIP transcription factors involved in ABA signalling; however, evidence for the involvement of FD in ABA signalling is weak. I disrupted ABA signalling specifically within the FD expression domain, which resulted in defects in plant shoot architecture under LDs. In legumes, FD paralogues mediate the floral transition but also determine inflorescence architecture. I identified HOMEOBOX PROTEIN 21 (HB-21), which is involved in ABA signalling, to be a direct downstream target of FD. The level of HB-21 mRNA was lower in fd than in wild-type inflorescences. The hb21 and fd mutants produced taller shoots with more siliques on the main shoot compared with wild type; thus, the regulation of HB-21 by FD links FD with inflorescence development in Arabidopsis potentially through ABA signalling. This PhD focuses on the bZIP transcription factor FD and how it regulates flowering time and inflorescence development in Arabidopsis. Collectively, the results show that FD functions throughout the Arabidopsis life cycle, and provide insight into the temporal FD-mediated transcriptional network at the SAM

RNA-seq analysis of cold and drought responsive transcriptomes of Zea mays ssp. mexicana L

The annual Zea mays ssp. mexicana L. is a member of teosinte, a wild relative of the Zea mays spp. mays L. This subspecies has strong growth and regeneration ability, high tiller numbers, high protein and lysine content as well as resistance to many fungal diseases, and it can be effectively used in maize improvement. In this study, we reported a Zea mays ssp. mexicana L. transcriptome by merging data from untreated control (CK), cold (4°C) and drought (PEG2000, 20%) treated plant samples. A total of 251,145 transcripts (N50 = 1,269 bp) and 184,280 unigenes (N50 = 923 bp) were predicted, which code for homologs of near 47% of the published maize proteome. Under cold conditions, 2,232 and 817 genes were up-regulated and down-regulated, respectively, while fewer genes were up-regulated (532) and down-regulated (82) under drought stress, indicating that Zea mays ssp. mexicana L. is more sensitive to the applied cold rather than to the applied drought stresses. Functional enrichment analyses identified many common or specific biological processes and gene sets in response to drought and cold stresses. The ABA dependent pathway, trehalose synthetic pathway and the ICE1-CBF pathway were up-regulated by both stresses. GA associated genes have been shown to differentially regulate the responses to cold in close subspecies in Zea mays. These findings and the identified functional genes can provide useful clues for improving abiotic stress tolerance of maize