TRANSCRIPTIONAL REGULATION OF STOMATAL RESPONSES TO STRESS: FROM A MODEL SYSTEM TO VITIS VINIFERA
Tesi di Dottorato
Data di Pubblicazione:
2015
Citazione:
TRANSCRIPTIONAL REGULATION OF STOMATAL RESPONSES TO STRESS: FROM A MODEL SYSTEM TO VITIS VINIFERA / F. Simeoni ; supervisor: O. Failla ; cosupervisor: M. Galbiati. DIPARTIMENTO DI SCIENZE AGRARIE E AMBIENTALI - PRODUZIONE, TERRITORIO, AGROENERGIA, 2015 Jan 23. 27. ciclo, Anno Accademico 2014. [10.13130/simeoni-fabio_phd2015-01-23].
Abstract:
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 m
Tipologia IRIS:
Tesi di dottorato
Keywords:
stomata; drought stress; biotic stress; Vitis; Arabidopsis
Elenco autori:
F. Simeoni
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