TRANSCRIPTIONAL REGULATION OF STOMATAL RESPONSES TO STRESS: FROM A MODEL SYSTEM TO VITIS VINIFERA

Grapevine (Vitis spp.) is a fruit crop traditionally subjected to moderate or severe water stress. Vitis species adapt well to drought conditions due to good osmotic adjustment, but the strength and timing of these responses varies between different cultivars and major differences in water stress tolerance can be found among species or hybrids from the Vitis genus. These genotype-related variations involve different aspects of the physiology of the plant, including differences in stomatal conductance (gs, mmol H2O m-2s-1). Stomatal conductance is a key trait in grapevine, as it directly determines the isohydric/anisohydric behaviour displayed by different genotypes. Stomata are small pores on the surfaces of leaves and stems, surrounded by a pair of guard cells that control the exchange of gases between the atmosphere and the interior of the leaf. This allows the plant to cope with the conflicting needs of ensuring adequate uptake of CO2 for photosynthesis and preventing water loss by transpiration. Moreover stomata play an active role in plant defence, restricting bacterial invasion as part of the plant innate immune system. Goal of this work is to investigate the molecular basis of stomatal responses, through the analysis of the transcriptional changes occurring in guard cells (GCs) in response to biotic and abiotic stress. Among GC related genes, the transcription factor AtMYB60 has been shown to play a pivotal role in stomata opening. Arabidopsis loss of function atmyb60-1 lines are characterised by closer stomata and enhanced survival when subjected to lethal drought conditions. Most importantly, AtMYB60 from Arabidopsis and its grape counterpart, VvMYB60, display an exceptionally high degree of sequence identity and a conserved function in GCs. We focused on the plant model system Arabidopsis in order to gain more insights into the transcriptional mechanisms that regulate stomatal activity. Next, we analysed the transcriptional changes occurring in GCs in response to water stress in different grapevine genotypes and different grafted combination. In the first part of this thesis we focused on technological aspects of stomata analysis. In order to improve the current methods employed to investigate stomata activity, usually performed with epidermal peel, we developed a semi-automatic confocal microscopy technique that allows measuring stomatal opening in intact leaf samples over extended periods of time. We successfully confirmed the sensibility of this approach in Arabidopsis, testing light induced stomatal aperture and ABA induced stomatal closure. The same approach was used to investigate the role of AtMYB60 in response to pathogen associated molecular pattern (PAMPs). The treatment with flg22 and LPS on WT and atmyb60-1 revealed that AtMYB60 is not involved in stomatal closure in response to these PAMPs. In order to improve the accuracy of transcriptional analyses in GCs, two different approaches have been employed for the purification of RNAs specifically from GCs of both Arabidopsis and grapevine. Laser micro-dissection (LMD) is the most accurate technique to obtain RNA samples from pure preparation of single cell types. This approach allowed us to compare GCs transcript with mesophyll cells transcript. As an alternative to LMD, we have adopted a mechanical disruption protocol of the leaf tissues to obtain epidermal preparations enriched in GCs. Both these approaches revealed enhanced expression of GC marker genes proving that a GC-enrichment occurred. LMD guaranteed the high purity of GCs sample, whereas the blender method allowed obtaining intact GCs in a short period of time and in relatively large amounts. We first focused on the analysis of the Arabidopsis atmyb60-1 mutant to gain more insights into the mechanisms by which AtMYB60 mediates stomatal activity. Metabolomic analysis of lipids accumulation in whole leaves or in purified GCs, revealed an increased accumulation of oxylipins in atmyb60-1 mutant plants compared with the WT. Moreover the accumulation was higher in GCs of the mutant compared to WT GCs. Interestingly, oxylipins have been recently shown to directly promote stomata closure in response to both drought and pathogen attack. qPCR analysis of the expression of genes involved in oxylipin biosynthesis did not uncovered significant differences in transcript levels between WT and mutant plants. This suggests a possible indirect and more complex role of AtMYB60 transcription factor in regulating oxylipins accumulation in GCs. Transcriptomic analysis suggested a possible involvement of AtMYB60 in the salicylic acid (SA)-mediated innate immune response in GC. Indeed we demonstrated that SA-induced stomatal closure was impaired in the atmyb60-1 mutant. Consistently, analysis of gene expression in laser micro-dissected GCs, revealed that SA-regulated genes, as Pathoghenesis Related Proteins 1, were less activated in the mutant compared to WT. VvMYB60 was already shown to represent a true ortholog of AtMYB60, mainly based on experiments performed in an heterologous system. Expression profiling of LMD-purified samples confirmed its guard cell specificity in grapevine as well. Analysis of VvMYB60 expression in different rootstocks highlighted a positive correlation between the level of gene expression and the regulation of stomatal conductance in response to water stress. Comparative analysis of M4 and 101.14 disclosed interestingly results; data showed a strong down-regulation of VvMYB60 expression in 101.14 leaves compared to M4. This correlated with an increased stomatal closure of 101.14 under drought stress. To get more insight into guard cell gene regulation, the genes involved in the regulation of the ABA pathway have been analyzed. More into details, genes involved in ABA-synthesis and ABA-mediated responses to drought have been assessed, including the PYL/RCAR receptor gene family, the PP2C protein phosphatases, the SnRK protein kinases and guard cell-related downstream targets, in different rootstocks under normal and water stress conditions. Analysis of the ABA synthesis marker gene VvNCED1, showed a general up-regulation in response to water stress. Extensive analysis of ABA receptor genes in grape revealed a high degree of variability among VvRCARs under drought stress. However, the VvRCAR family showed a general down-regulation in most genotypes analysed, with the exception of VvRCAR3, whose expression resulted mostly up-regulated. Most PP2C genes were generally up-regulated under drought stress. VvPP2C24, putative ortholog of AtABI1, was strongly up-regulated in almost all the genotypes. We found a positive correlation with ABA synthesis, through the analysis of VvNCED1, and ABA perception, in particular with VvPP2C24 gene expression. Contrary to the expected, VvERA1, a positive regulator of ABA signalling, showed little variations under drought stress. In particular, VvERA1 resulted more up-regulated in K5BB and in M4 compared to 1103P and 101.14. This together to VvMYB60 expression, VvERA1 behaviour explains the enhanced stomatal responses disclosed by 1103P and 101.14 compared to K5BB and M4. The positive correlation of the guard cell-specific genes (VvMYB60 and VvSIRK) with ABA related genes could contribute to the different phenotypic responses shown by different genotypes under water stress. Moreover, some genotypes disclosed an ABA independent response. However these genotypes showed a down-regulation of VvMYB60 and decrease of gs. Thus, like in Arabidopsis, the expression of VvMYB60 in grape could be modulated by both ABA-dependent and ABA-independent pathways. Grapevine rootstocks can confer resistance to various pathogens and tolerance to abiotic stresses. The transport from the root to the scion through the xylem of chemical signals (including ABA) in the early stages of water-deficit reduces leaf transpiration and restrains leaf growth. Little is known about the influence of the rootstocks on the regulation of the expression of GC-related genes in the scion. Both M4 and 101.14 showed a constitutive up-regulation of ABA biosynthetic and signalling genes in leaves from grafted plants (M4/M4; 101.14/101.14) compared with ungrafted plants. This could suggest a possible stress condition induced by the graft itself. Moreover the comparison of CS grafted on M4 and 101.14 showed that the regulation of ABA synthesis (VvNCED1) is independent by the rootstocks, in agreement with the existence of a cell-autonomous ABA biosynthetic pathway in GC. However, ABA perception was affected by the genotype of the rootstock. VvPP2C24 was more up-regulated in GC from CS/M4 and M4/M4 compared to CS/101.14 and 101.14/101.14. Moreover VvMYB60 displayed a similar rootstock-dependent response. Finally, in order to analyze the role of VvMYB60 in biotic response we performed a SA treatment on grapevines plants. Preliminary analysis showed a down-regulation of VvMYB60 suggesting a similar behavior for both the Arabidopsis and grape MYB60 genes.