Clima laboral percibido por el personal docente

Decades of work in the long day-plant, vernalization-responsive model species

Arabidopsis thaliana has shown that environmental cues controlling flowering are signalled

through multiple genetic pathways and integrated with endogenous signals by a few genes normally referred to as floral integrator genes. The interaction between these integrators will determine the expression level of a mobile signal (florigen) that will lead to the activation of floral identity genes and initiation of floral development (Lifschitz 2014). Multiple lines of evidence all indicate that the identity of this mobile signal in A. thaliana is the FT protein, a member of a small family of plant proteins related to phosphatidyl ethanolamine binding protein

Chapter 1 General introduction

19 (PEBP) signalling proteins in animals (Kobayashi et al. 1999). FT expression is induced in the vascular tissue, from where the FT protein migrates to the shoot apex. Within the shoot apical meristem, FT binds the bZIP transcription factor FD to form the ‘Florigen Activation Complex’ (FAC), which may also include a 14-3-3 protein (Taoka et al. 2011; Corbesier et al. 2007; Abe et al. 2005). Once active, this complex activates floral meristem identity genes and other floral promoters such as APETALA1 (AP1), FRUITFULL (FUL) and SUPPRESSOR OF

OVEREXPRESSION OF CONSTANS 1 (SOC1), which specify the differentiation of cells in the

flank of the apical meristem into floral meristems (Wigge et al. 2005). A close FT paralog TWIN

SISTER OF FT (TSF) acts redundantly to promote flowering using the same molecular

mechanism (Yamaguchi et al. 2005).

Hundreds of genes interact in a very complex network to control FT expression in Arabidopsis (Andrés and Coupland 2012; Song et al. 2015), and are traditionally grouped in different pathways. The most important of these are the photoperiod and the vernalization pathways. The molecular signalling in response these two environmental cues converges in each case on an integrator gene that functions upstream of FT to directly modulate its expression according to the environment. In the case of the response to photoperiod, this gene is CONSTANS (CO), a zinc- finger transcription factor that serves to integrate information from photoreceptors and the circadian clock for measurement of photoperiod (Greenham and McClung 2015; Huang and Nusinow 2016; Nakamichi 2011). The Arabidopsis clock is currently understood to involve the co-action of over 25 genes that form several interconnected transcriptional feedback loops and additional associated genes. The clock helps generate a diurnal rhythm in CO transcript levels such that CO peak expression occurs late in the day in long days but after dusk in short days (Suárez-López et al. 2001). CO protein levels are also adjusted according to information about light quality and intensity perceived through different photoreceptors, including phytochromes and cryptochromes (Somers et al. 1998); CO protein is stabilized by light but degraded in darkness, so the interactions between light and the clock ensure that CO protein only accumulates and CO-dependent FT induction occurs only under LD (Valverde et al. 2004).

Temperature is another key environmental variable limiting the distribution of plant species (Wigge 2013). Studies on how temperature affects flowering time have been mostly focused on the vernalization response, which can be defined as the promotion of the competence for

Chapter 1 General introduction

20 flowering by long periods of low temperatures (Kim et al. 2009). In Arabidopsis, this response is mostly mediated through the previously mentioned MADS-box FLOWERING LOCUS C (FLC) (Michaels and Amasino 1999, 2001), a floral repressor that delays flowering by preventing the expression of floral integrators such as FT and SOC1 (Searle et al. 2006). Multiple pathways regulate FLC expression; FRIGIDA (FRI) and FRIGIDA-LIKE genes are the main transcriptional activators, responsible for establishing the high FLC expression that maintains the vegetative state of the plant during winter (Johanson et al. 2000; Michaels et al. 2004; Schlappi 2006). Vernalization promotes flowering through the stable epigenetic silencing of FLC, which is achieved through a complex mechanism involving a group of antisense transcripts produced at the FLC locus (collectively named COOLAIR) and the combined action of many other genes, among which those belonging to the Polycomb repressive complex 2 (PRC2; FIE, VRN2, MSI1 and SWN or CLF), and the plant homeodomain (PHD) family VRN5, VIN3 and VEL1 are especially relevant, since they form a complex responsible for placing the histone marks repressing FLC and also for the maintenance of this epigenetic inactive state (Berry and Dean 2015; Berry et al. 2015; Sung and Amasino 2006; Sung and Amasino 2004; Sung et al. 2006). Little is known about the cold sensing mechanism, but in Arabidopsis, the cellular signalling initiated by cold triggers the induction of VIN3. This induction is correlated with the duration of cold exposure and the strength of the vernalization response, so it is likely that this gene is essential in the molecular mechanism of vernalization in this species (Sung and Amasino 2004). Several genes included in the so called “autonomous pathway” also participate in the Arabidopsis vernalization response, but in addition they regulate FLC in a parallel and independent manner, through different processes such as RNA processing (FCA, FY, FPA and

FLOWERING LOCUS K) and chromatin remodelling by histone deacetylation (FVE and FLOWERING LOCUS D) (Feng and Michaels 2011; Schmitz and Amasino 2007; He 2012;

Swiezewski et al. 2009).

In contrast to the detailed understanding of the vernalization pathway, less is known about the molecular basis by which ambient temperature influences flowering time. Nevertheless, a number of genes involved in temperature sensing and response have been identified and their mechanism of action uncovered. One example is the well-studied interaction between

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PHASE (SVP). Two major FLM isoforms exist whose relative abundance depends on ambient

temperature. FLM-β is preferentially produced at low ambient temperature, forms a complex with SVP that down-regulates the expression of important flowering time genes such as SOC1 and FT. By contrast, the FLM-δ isoform is more abundant at higher temperatures and forms a complex with SVP that is unable to bind DNA, thus releasing the flowering repression exerted by SVP/FLM-β (Jeong et al. 2007; Lee et al. 2013; Posé et al. 2013). Genes initially located in other pathways also may have a role in the molecular signalling of temperature. For instance, fca,

fve, fy and the tfl1/elf3 double mutant are insensitive to temperature changes (Strasser et al. 2009;

Blázquez et al. 2003). Recent findings suggest that CO might also be implicated in the FT up- regulation in response to temperature (Fernandez et al. 2016). Finally, chromatin composition might be also important for temperature perception. An alternative histone (H2A.Z) is evicted from gene regulatory regions at high temperatures, allowing the binding of transcriptional regulators. The Arabidopsis flowering promotion by high temperatures uses this mechanism to allow the binding of the transcription factor PHYTOCHROME INTERACTING FACTOR to the

FT promoter to up-regulate its expression (Proveniers and van Zanten 2013; Kumar and Wigge

2010; Kumar et al. 2012).

In parallel with external signals, certain metabolic and hormonal signals also have an important influence on regulation of flowering in Arabidopsis (Galvão and Schmid 2014). Several hormones influence flowering by interacting with known floral pathways, and among these, gibberellic acid (GA) is perhaps the most dominant (Conti 2017; Campos-Rivero et al. 2017). The control of flowering by GA signalling is thought to involve multiple mechanisms that modulate the expression of key floral genes such as FT (Hisamatsu and King 2008; Porri et al. 2012), LFY (Gocal et al. 2001) and SOC1 (Moon et al. 2003a).

Also especially relevant is the age-dependent pathway, present throughout the plant kingdom (Bratzel and Turck 2015). In Arabidopsis, two major microRNAs (miRNAs) families, play a key role in this process; levels of miR156 decline as plants become older, while miR172 levels follow the opposite pattern. They also have antagonist roles in flowering; miR172 induces FT expression by targeting floral repressors like APETALA2 (AP2) and SCHLAFMÜTZE (SMZ) (Aukerman and Sakai 2003; Jung et al. 2007; Mathieu et al. 2009), while miR156 prevent precocious flowering by downregulation of miR172 and many members of the SQUAMOSA

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PROMOTER-BINDING PROTEIN-LIKE (SPL) family (Wu and Poethig 2006). The SPL genes

are essential to regulate the juvenile to adult phase transition and influence flowering through direct activation of the expression of FT and also of meristem identity genes such as LEAFY (LFY), FUL and AP1 (Blázquez et al. 2003).