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In the age‐dependent pathway, microRNA 156 (miR156) is essential for the correct timing of developmental transitions. miR156 negatively regulates several SPL genes, which promote the juvenile‐to‐adult and floral transitions in part through upregulation of miR172. The transcriptional repressors TEMPRANILLO1 (TEM1) and TEM2 delay flowering in Arabidopsis thaliana at least through direct repression of FLOWERING LOCUS T (FT) and gibberellin biosynthetic genes, and have also been reported to participate in the length of the juvenile phase. Levels of TEM mRNA and miR156 decrease gradually, allowing progression through developmental phases. Given these similarities, we hypothesized that TEMs and the miR156/SPL/miR172 module could act through a common genetic pathway. We analyzed the effect of TEMs on levels of miR156, SPL and miR172, tested binding of TEMs to these genes using chromatin immunoprecipitation and analyzed the genetic interaction between TEMs and miR172. We found that TEMs played a stronger role in the floral transition than in the juvenile‐to‐adult transition. TEM1 repressed MIR172A, MIR172B and MIR172C expressions and bound in vivo to at least MIR172C sequences. Genetic analyses indicated that TEMs affect the regulation of developmental timing through miR172.

The biological relevance of non-protein coding RNAs in the regulation of critical plant processes has been firmly established in recent years. This has been mostly achieved with the discovery and functional characterization of small non-coding RNAs, such as small interfering RNAs and microRNAs (miRNAs). However, recent next-generation sequencing techniques have widened our view of the non-coding RNA world, which now includes long non-coding RNAs (lncRNAs). Small and lncRNAs seem to diverge in their biogenesis and mode of action, but growing evidence highlights their relevance in developmental processes and in responses to particular environmental conditions. Light can affect MIRNA gene transcription, miRNA biogenesis, and RNA-induced silencing complex (RISC) activity, thus controlling not only miRNA accumulation but also their biological function. In addition, miRNAs can mediate several light-regulated processes. In the lncRNA world, few reports are available, but they already indicate a role in the regulation of photomorphogenesis, cotyledon greening, and photoperiod-regulated flowering. In this review, we will discuss how light controls MIRNA gene expression and the accumulation of their mature forms, with a particular emphasis on those miRNAs that respond to different light qualities and are conserved among species. We will also address the role of small non-coding RNAs, particularly miRNAs, and lncRNAs in the regulation of light-dependent pathways. We will mainly focus on the recent progress done in understanding the interconnection between these non-coding RNAs and photomorphogenesis, circadian clock function, and photoperiod-dependent flowering.

La floración es probablemente el proceso más importante en el desarrollo de la planta, ya que la perpetuación de las especies vegetales depende de ella. En Arabidopsis thaliana, la inducción floral está controlada por varias rutas genéticas que responden a estímulos ambientales y endógenos. En nuestro laboratorio se han identificado los genes TEMPRANILLO (TEM) como represores de la floración tanto en condiciones inductivas de día largo (LD, 16 horas de luz) como no inductivas de día corto (SD, 8 horas de luz). Las proteínas TEM pertenecen a una familia de factores de transcripción llamada RAV, que se caracterizan por contener dos dominios de unión al DNA, que son los dominios APETALA2 (AP2) y B3. En Arabidopsis esta familia está compuesta por 6 genes. En LD, la ruta del fotoperiodo induce la floración principalmente a través de la activación del gen FLOWERING LOCUS T (FT), mientras que en SD la floración depende principalmente de la acumulación de giberelinas (GAs). TEM1 y TEM2 retrasan la floración en ambas condiciones al reprimir directamente la expresión de los genes FT, GA3OXIDASE 1 (GA3OX1) y GA3OX2, siendo los dos últimos genes responsables de la biosíntesis de la GA bioactiva GA4. Por tanto, TEM1 y TEM2 controlan el tiempo de floración a través de al menos dos de las rutas genéticas que controlan la inducción floral: la del fotoperíodo (Castillejo & Pelaz, 2008) y la de las GAs (Osnato et al, 2012). El objetivo de este trabajo ha sido profundizar en el papel de los genes TEM en otras rutas genéticas implicadas en el control de la floración y en otros procesos de desarrollo en Arabidopsis thaliana. Existe una ruta genética que responde a la edad de la planta y que evita que ésta florezca en su etapa juvenil. Primero se produce la transición de la etapa vegetativa juvenil a la adulta y después la inducción floral. Los microRNAs miR156 y miR172 están implicados en la regulación de estas transiciones entre las fases del desarrollo de la planta (Huijser & Schmid, Flowering is probably the most important process in plant development since the perpetuation of the species depends on it. In Arabidopsis thaliana, floral induction is controlled by several genetic pathways that respond to environmental and endogenous stimuli. In our laboratory we have identified the TEMPRANILLO (TEM) genes as flowering repressors under both inductive long-day (LD, 16 hours of light) and noninductive short-day (SD, 8 hours of light) conditions. The TEM proteins belong to a family of transcription factors called RAV, characterized by the presence of two DNA binding domains, the APETALA2 (AP2) and B3 domains. In Arabidopsis this family is composed of 6 genes. Under LD the photoperiod pathway induces flowering mainly through activation of FLOWERING LOCUS T (FT), while under SD flowering depends mainly on the accumulation of gibberellins (GAs). TEM1 and TEM2 delay flowering under both conditions by directly repressing the expression of the FT, GA 3-OXIDASE 1 (GA3OX1) and GA3OX2 genes, the latter two genes being responsible for the biosynthesis of bioactive GA4. Therefore, TEM1 and TEM2 control flowering time through at least two of the genetic pathways that control floral induction: the photoperiod (Castillejo & Pelaz, 2008) and the GA pathway (Osnato et al, 2012). In this PhD thesis we aimed to deepen the role of TEM genes in other genetic pathways controlling flowering and other developmental processes in Arabidopsis thaliana. There is a genetic pathway that responds to the age of the plant and prevents flowering at the juvenile phase. First there is a transition from the juvenile to the adult vegetative stage and then floral induction occurs. The microRNAs miR156 and miR172 are involved in the regulation of these phase transitions of plant development (Huijser & Schmid, 2011). MiR156 maintains the juvenile phase and delays the floral transition (Wu & Poeting, 2006; Wu et al, 2009), while miR156-target SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene

Plant trichomes are defensive specialized epidermal cells. In all accepted models, the epidermis is the layer involved in trichome formation, a process controlled by gibberellins (GAs) in Arabidopsis rosette leaves. Indeed, GA activates a genetic cascade in the epidermis for trichome initiation. Here we report that TEMPRANILLO (TEM) genes negatively control trichome initiation not only from the epidermis but also from the leaf layer underneath the epidermis, the mesophyll. Plants over-expressing or reducing TEM specifically in the mesophyll, display lower or higher trichome numbers, respectively. We surprisingly found that fluorescently labeled GA 3 accumulates exclusively in the mesophyll of leaves, but not in the epidermis, and that TEM reduces its accumulation and the expression of several newly identified GA transporters. This strongly suggests that TEM plays an essential role, not only in GA biosynthesis, but also in regulating GA distribution in the mesophyll, which in turn directs epidermal trichome formation. Moreover, we show that TEM also acts as a link between GA and cytokinin signaling in the epidermis by negatively regulating downstream genes of both trichome formation pathways. Overall, these results call for a re-evaluation of the present theories of trichome formation as they reveal mesophyll essential during epidermal trichome initiation.

Plants integrate day length and ambient temperature to determine the optimal timing for developmental transitions. In Arabidopsis (Arabidopsis thaliana), the floral integrator FLOWERING LOCUS T (FT) and its closest homolog TWIN SISTER OF FT promote flowering in response to their activator CONSTANS under long-day inductive conditions. Low ambient temperature (16°C) delays flowering, even under inductive photoperiods, through repression of FT, revealing the importance of floral repressors acting at low temperatures. Previously, we have reported that the floral repressors TEMPRANILLO (TEM; TEM1 and TEM2) control flowering time through direct regulation of FT at 22°C. Here, we show that tem mutants are less sensitive than the wild type to changes in ambient growth temperature, indicating that TEM genes may play a role in floral repression at 16°C. Moreover, we have found that TEM2 directly represses the expression of FT and TWIN SISTER OF FT at 16°C. In addition, the floral repressor SHORT VEGETATIVE PHASE (SVP) directly regulates TEM2 but not TEM1 expression at 16°C. Flowering time analyses of svp tem mutants indicate that TEM may act in the same genetic pathway as SVP to repress flowering at 22°C but that SVP and TEM are partially independent at 16°C. Thus, TEM2 partially mediates the temperature-dependent function of SVP at low temperatures. Taken together, our results indicate that TEM genes are also able to repress flowering at low ambient temperatures under inductive long-day conditions.

[Background]: Transcription factors of the RAV (RELATED TO ABI3 AND VP1) family are plant-specific and possess two DNA-binding domains. In Arabidopsis thaliana, the family comprises six members, including TEMPRANILLO 1 (TEM1) and TEM2. Arabidopsis RAV1 and TEM1 have been shown to bind bipartite DNA sequences, with the consensus motif C(A/C/G)ACA(N)2–8(C/A/T)ACCTG. Through direct binding to DNA, RAV proteins act as transcriptional repressors, probably in complexes with other co-repressors., [Scope and Conclusions]: In this review, a summary is given of current knowledge of the regulation and function of RAV genes in diverse plant species, paying particular attention to their roles in the control of flowering in arabidopsis. TEM1 and TEM2 delay flowering by repressing the production of two florigenic molecules, FLOWERING LOCUS T (FT) and gibberellins. In this way, TEM1 and TEM2 prevent precocious flowering and postpone floral induction until the plant has accumulated enough reserves or has reached a growth stage that ensures survival of the progeny. Recent results indicate that TEM1 and TEM2 are regulated by genes acting in several flowering pathways, suggesting that TEMs may integrate information from diverse pathways. However, flowering is not the only process controlled by RAV proteins. Family members are involved in other aspects of plant development, such as bud outgrowth in trees and leaf senescence, and possibly in general growth regulation. In addition, they respond to pathogen infections and abiotic stresses, including cold, dehydration, high salinity and osmotic stress.

La inducción floral es un proceso de gran importancia en el desarrollo de las plantas, que determina el punto de inicio de la formación de las estructuras reproductoras. Es necesario un estricto control genético de este proceso para optimizar el momento en que la planta movilizará sus reservas e invertirá su energía en la producción de la descendencia. Por ello, la transición de la fase vegetativa a la reproductiva debe darse en el momento fisiológico óptimo para la planta y coincidir con unas condiciones ambientales favorables. En Arabidopsis thaliana, la inducción floral se produce, principalmente, por la activación del gen FLOWERING LOCUS T (FT), en condiciones inductoras de día largo ("ruta del fotoperiodo"), y por la acción de las hormonas giberelinas (GA) en condiciones no-inductoras de día corto. La existencia de represores de la floración es esencial para garantizar una fase vegetativa suficientemente larga para asegurar el éxito del proceso reproductivo. En el laboratorio hemos trabajado con los genes TEMPRANILLO1 (TEM1) y TEMPRANILLO2 (TEM2), represores directos de FT y de los genes de síntesis de GA. El conocimiento generado en el laboratorio nos permite postular que los TEM conectan ambas rutas de control de la floración (ruta de fotoperiodo y ruta hormonal), controlando directamente la acumulación de sustancias inductoras de la floración y determinando cuándo debe desencadenarse la floración. Esta tesis doctoral parte del conocimiento previo generado en el laboratorio y explora la función de los TEM en otras rutas genéticas de control de la inducción floral. Basándonos en el amplio conocimiento del efecto de los factores ambientales en la regulación de la floración, decidimos explorar si los genes TEM están regulados por algunas de las rutas genéticas mejor conocidas: calidad de la luz (mediada por phyB y factores dependientes de phyB), temperatura ambiental (con SVP/FLC mediando la respuesta a bajas temperaturas) y el control de las transiciones de fas, Floral induction is probably the most important process in plant development, since it determines the formation of reproductive structures. So, it is necessary to establish a strict genetic control of this process to optimize the moment in which plants should mobilize their reserves and invest their energy in producing the offspring. Therefore, the transition from vegetative to reproductive phase must happen in the best physiological moment and coincide with favorable environmental conditions. In Arabidopsis thaliana, floral induction occurs mainly by the activation of FLOWERING LOCUS T (FT) in inducing long-day conditions ("photoperiodic pathway"), and by the action of the hormone gibberellin (GA) in non-inducing short day conditions. The existence of floral repressors is essential to ensure a vegetative phase long enough to ensure the success of the reproductive process. In the laboratory we have worked with TEMPRANILLO1 and TEMPRANILLO2 genes (TEM1 and TEM2, respectively), direct repressors of FT and GA synthesis genes. The knowledge generated in the laboratory allows us to postulate that TEM connect both routes of flowering (photoperiod and hormone pathways), directly controlling the accumulation of substances that induce flowering and determining when flowering should be triggered. This thesis begins from the previous knowledge generated in the laboratory and it explores the role of TEM in other genetic pathways of floral induction. Based on the extensive knowledge of the environmental factors effect in the regulation of flowering, we decided to explore whether TEM genes are regulated by some of the best known genetic pathways: quality of light (mediated by phyB and phyB dependent factors), temperature (SVP/FLC mediating the response to low temperatures) and the control of phase transitions (adult and juvenile phase, and the age-dependent flowering, both controlled mainly by the miRNA miR156/miR172 and the transcription factor SPL9). Thus, our main objective wa

The identification of FLOWERING LOCUS T (FT) and several FT homologs as phloem-mobile proteins that regulate flowering has sparked the search for additional homologs involved in the long-distance regulation of other developmental processes. Given that flowering and tuber induction share regulatory pathways, the quest for long-distance tuberization signals has been further stimulated. Several tuberization regulators have been proposed as mobile molecules, including the FT family protein StSP6A, the plant growth regulators gibberellins and the microRNA miR172. Although some of these hypotheses are attractive and plausible, evidence that these molecules are transmissible in potato has yet to be obtained. Two mRNAs encoding transcription factors, StBEL5 and POTATO HOMEOBOX 1 (POTH1), are mobile and correlate with tuber induction. However, evidence that StBEL5 or POTH1 are required for tuberization is not available yet. Therefore, there are several good candidates for long-distance molecules in the tuberization process. Further research should test their role as systemic tuberization signals.

CONSTANS (CO) is involved in the photoperiodic control of plant developmental processes, including flowering in several species and seasonal growth cessation and bud set in trees. It has been proposed that CO could also affect the day-length regulation of tuber induction in Solanum tuberosum (potato), a plant of great agricultural relevance. To address this question, we examined the role of CO in potato. A potato CO-like gene, StCO, was identified and found to be highly similar to a previously reported potato gene of unknown function. Potato plants overexpressing StCO tuberized later than wild-type plants under a weakly inductive photoperiod. StCO silencing promoted tuberization under both repressive and weakly inductive photoperiods, but did not have any effect under strongly inductive short days, demonstrating that StCO represses tuberization in a photoperiod-dependent manner. The effect of StCO on tuber induction was transmitted through grafts. In addition, StCO affected the mRNA levels of StBEL5 – a tuberization promoter, the mRNA of which moves long distances in potato plants – and StFT/StSP6A, a protein highly similar to FLOWERING LOCUS T (FT), which is a key component of systemic flowering signals in other species. We also found that StFT/StSP6A transcript levels correlate with the induction of tuber formation in wild-type plants. These results show that StCO plays an important role in photoperiodic tuberization and, together with the recent demonstration that StFT/StSP6A promotes tuberization, indicate that the CO/FT module participates in controlling this process. Moreover, they support the notion that StCO is involved in the expression of long-distance regulatory signals in potato, as CO does in other species.

MicroRNAs (miRNAs) are key regulators of numerous genes in many eukaryotes. Some plant miRNAs are involved in developmental and physiological processes that require intercellular or inter-organ signaling. Movement of other small RNAs within plants has been established. Recent findings also demonstrate intercellular signaling by miRNAs and strongly support that a subset of these regulatory molecules move from one cell to another or over long distances. Phloem exudates contain diverse miRNAs and at least two of them, involved in responses to nutrient availability, are transmitted through grafts, indicating long-distance movement. Two miRNAs that regulate developmental processes are present in cells outside their domains of expression. Several results strongly support that one of them moves from cell to cell. Research on a mutant affected in plasmodesmata trafficking indicates that these intercellular channels are required for transmission of miRNA activity to adjacent cells. Moreover, ARGONAUTE proteins might be involved in the regulation of miRNA trafficking. Hypothesis on the features and mechanisms that may determine miRNA mobility are presented. Future challenges include identifying other mobile miRNAs; demonstrating that miRNA movement is required for non-cell autonomous action; and characterizing the mechanisms of translocation and genetic pathways that regulate miRNA movement.

The photoreceptor phytochrome B (PHYB) and the homeodomain protein BEL5 are involved in the response of potato tuber induction to the photoperiod. However, whether they act in the same tuberization pathway is unknown. Here we show the effect of a microRNA, miR172, on this developmental event. miR172 levels are higher under tuber-inducing short days than under non-inductive long days and are upregulated in stolons at the onset of tuberization. Overexpression of this microRNA in potato promotes flowering,accelerates tuberization under moderately inductive photoperiods and triggers tuber formation under long days. In plants with a reduced abundance of PHYB,which tuberize under long days, both BEL5 mRNA and miR172levels are reduced in leaves and increased in stolons. This, together with the presence of miR172 in vascular bundles and the graft transmissibility of its effect on tuberization, indicates that either miR172 might be mobile or it regulates long-distance signals to induce tuberization. Consistent with this, plants overexpressing miR172 show increased levels of BEL5 mRNA, which has been reported to be transmissible through grafts. Furthermore, we identify an APETALA2-like mRNA containing a miR172 binding site, which is downregulated in plants overexpressing miR172 and plants in which PHYB is silenced. Altogether, our results suggest that miR172 probably acts downstream of the tuberization repressor PHYB and upstream of the tuberization promoter BEL5 and allow us to propose a model for the control of tuberization by PHYB, miR172 and BEL5.

Flower development at the shoot apex is initiated in response to environmental cues. Day length is one of the most important of these and is perceived in the leaves. A systemic signal, called the floral stimulus or florigen, is then transmitted from the leaves through the phloem and induces floral development at the shoot apex. Genetic analysis in Arabidopsis identified a pathway of genes required for the initiation of flowering in response to day length. The nuclear zinc-finger protein CONSTANS (CO) plays a central role in this pathway, and in response to long days activates the transcription of FT, which encodes a RAF-kinase-inhibitor-like protein. We show using grafting approaches that CO acts non-cell autonomously to trigger flowering. Although CO is expressed widely, its misexpression from phloem-specific promoters, but not from meristem-specific promoters, is sufficient to induce early flowering and complement the co mutation. The mechanism by which CO triggers flowering from the phloem involves the cell-autonomous activation of FT expression. Genetic approaches indicate that CO activates flowering through both FT-dependent and FT-independent processes, whereas FT acts both in the phloem and the meristem to trigger flowering. We propose that, partly through the activation of FT, CO regulates the synthesis or transport of a systemic flowering signal, thereby positioning this signal within the established hierarchy of regulatory proteins that controls flowering.