Novel mechanisms and transcription factors involved in the control of stomatal behaviour in Arabidopsis thaliana / Nuevos mecanismos y factores de transcripción involucrados en el control del comportamiento estomático en Arabidopsis thaliana

Abstract

The increase in water use efficiency in order to maintain or improve crop yields in times of global warming and progressive reduction of water resources is one of the major challenges faced by agricultural sciences in the last decades. Our work is focused on the mechanisms plants use to cope with water deficit. The functional center of drought response is located into the guard cells: the regulation of their turgor determines the stomatal aperture levels and, therefore, the rates of CO2 assimilation and water loss, representing a key point in the whole plant physiological state. It is known that stomata respond to abscisic acid (ABA) as the main closure inductor, nevertheless, stomatal behavior is regulated through the integration of a huge number of physiological and external signals into a complex network capable of responding to a wide spectrum of environmental conditions. The comprehension of these mechanisms is therefore considered a critical step towards the achievement of an improvement in plants water use efficiency

Despite our increasing knowledge on the transcriptional networks connecting abscisic acid (ABA) signalling with the circadian clock, the molecular nodes in which both pathways converge to translate the environmental information into a physiological response are not known. In the first chapter of this work, we provide evidence of a feedback mechanism linking the circadian clock with plant responses to drought in Arabidopsis thaliana. A key clock component (TOC1, timing of CAB expression 1) binds to the promoter of the ABA-related gene (ABAR/CHLH/GUN5) and controls its circadian expression. TOC1 is in turn acutely induced by ABA and this induction advances the phase of TOC1 binding and modulates ABAR circadian expression. Moreover, the gated induction of TOC1 by ABA is abolished in ABAR RNAi plants suggesting that the reciprocal regulation between ABAR and TOC1 expression is important for sensitized ABA activity. Genetic studies with TOC1 and ABAR overexpressing and RNAi plants showed defective responses to drought, which support the notion that clock-dependent gating of ABA function is important for cellular homeostasis under dry environments.

In the second chapter we have identified six transcription factors whose transient over-expression influenced water retain in Arabidopsis thaliana seedlings through a screening approach. The screening was performed on a collection of Arabidopsis thaliana lines expressing hundreds of different transcription factors under the control of an inducible promoter. The differences in water content of the seedlings were detected through infrared thermal imaging. We verified that three of the candidates displayed altered stomatal behaviour that were consistent with the thermal phenotype. The over-expression of DEAR4 and NAC87, produce stomatal closure, while induced expression of HSFA8 results in a stronger stomatal opening. The mRNA levels of HSFA8 present slight changes in response to ABA, dehydration, osmotic stress or heat shock. The transcription of this gene seems in turn to be influenced by the circadian clock, reaching higher levels of transcript around the subjective dusk compared to dawn. Proteomics and transcriptomics analysis of HSFA8 overexpressing seedlings suggest that this transcription factor might influence the concentrations of reactive oxygen species, free iron and ABA. Moreover HSFA8 seems to be slightly induced by flagellin, while its over-expression produces stomatal insensitivity to the same elicitor suggesting that our candidate gene may function as a modulator of pathogen response. The overall picture suggested by our data suggests that HSFA8 produces a general relaxation of the cell wall accompanied by an enhanced stomatal aperture in order to favour photosynthesis and growth in a time window of decreasing pathogen risk and increasing air humidity.

Nuestro trabajo se centra en los mecanismos que las plantas utilizan para lidiar con la falta de agua. A nivel fisiológico, la respuesta a sequía se centra en los estomas. Estos órganos regulan el intercambio de gases con el exterior controlando la perdida de agua y la asimilación de CO2. La fitohormona ácido abscísico (ABA) es el principal inductor del cierre estomático, sin embargo, el comportamiento de los estomas es regulado por varias señales fisiológicas y externas que constituyen una red capaz de responder a un amplio espectro de condiciones medioambientales.

En la primera parte de este trabajo se descubrió una conexión molecular entre las respuestas a sequía y el reloj circadiano en Arabidopsis thaliana. La proteína circadiana TOC1, une el promotor del gen ABAR/CHLH/GUN5, que está implicado en la respuesta a ABA, y controla su expresión circadiana. TOC1 es, en cambio, inducido por ABA en ciertos momentos del día. La inducción, que produce un adelanto en la represión de ABAR mediada por TOC1, se observa solo en presencia de ABAR. Este mecanismo de retroacción negativa contribuye a modular la sensibilidad a ABA a lo largo del día, como evidencian los cambios en las respuestas a ABA y sequía observados en plantas con niveles alterados de TOC1 y ABAR.

En la segunda parte se identifican 6 factores de transcripción que alteran la capacidad de retención de agua en plántulas de Arabidopsis por medio de un screening de temperatura foliar. La sobre-expresión de DEAR4 y NAC87 favorece el cierre estomático, mientras HSFA8 induce la abertura. Ulteriores estudios sobre HSFA8 revelan que su transcripción está levemente regulada por estreses abióticos y oscila ampliamente a lo largo del día. Experimentos de proteómica y transcriptómica sugieren que HSFA8 podría regular las concentraciones fisiológicas de especies reactivas del oxígeno, hierro y ABA; además de relajar la pared celular. HSFA8 es inducido por flagelina y produce insensibilidad estomática a dicho elicitor, lo que sugiere que actúa como modulador de la respuesta a patógeno. HSFA8 podría inducir la abertura de los estomas favoreciendo fotosíntesis y crecimiento en una ventana temporal de menor riesgo de patógenos y sequía.