SUMMARYFlowering is controlled by a network of pathways that converge to regulate a small number of floral integrator genes. We studied the interactions in Arabidopsis between three of these integrators, FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), as well as their repression by the MADS box transcription factor SHORT VEGETATIVE PHASE (SVP). FT is a mobile signal transmitted from the leaf to the meristem to initiate flowering. Using mRNA null alleles, we show that FT and the closely related TSF are not essential for flowering, but that the double mutant is photoperiod-insensitive. Inactivation of both genes also fully suppresses the early-flowering phenotype caused by over-expression of CONSTANS (CO), a transcriptional regulator in the photoperiod pathway. In addition, we demonstrate that TSF and FT have similar biochemical functions by showing that they interact in yeast with the same bZIP transcription factors. Expression of FT or TSF from promoters specific for phloem companion cells drives early flowering of the double mutant, so no expression of either gene is required in the meristem. Furthermore, TSF, like FT, is repressed by SVP, but the triple mutant svp-41 ft-10 tsf-1 expresses SOC1 in the meristem sooner and flowers earlier than ft-10 tsf-1. Thus we distinguish the functions of SVP in repressing FT and TSF in the leaf and SOC1 in the meristem. In addition, a time course of in situ hybridizations suggested that repression of SVP and activation of SOC1 proceed simultaneously in the meristem. These observations clarify the relationships between these early regulators of the floral transition, and further emphasize the relatedness of mechanisms acting in the leaf and meristem to control flowering time.

SUMMARYThe plant growth regulator gibberellin (GA) contributes to many developmental processes, including the transition to flowering. In Arabidopsis, GA promotes this transition most strongly under environmental conditions such as short days (SDs) when other regulatory pathways that promote flowering are not active. Under SDs, GAs activate transcription of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and LEAFY (LFY) at the shoot meristem, two genes encoding transcription factors involved in flowering. Here, the tissues in which GAs act to promote flowering were tested under different environmental conditions. The enzyme GIBBERELLIN 2 OXIDASE 7 (GA2ox7), which catabolizes active GAs, was overexpressed in most tissues from the viral CaMV 35S promoter, specifically in the vascular tissue from the SUCROSE TRANSPORTER 2 (SUC2) promoter or in the shoot apical meristem from the KNAT1 promoter. We find that under inductive long days (LDs), GAs are required in the vascular tissue to increase the levels of FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) mRNAs, which encode a systemic signal transported from the leaves to the meristem during floral induction. Similarly, impairing GA signalling in the vascular tissue reduces FT and TSF mRNA levels and delays flowering. In the meristem under inductive LDs, GAs are not required to activate SOC1, as reported under SDs, but for subsequent steps in floral induction, including transcription of genes encoding SQUAMOSA PROMOTER BINDING PROMOTER LIKE (SPL) transcription factors. Thus, GA has important roles in promoting transcription of FT, TSF and SPL genes during floral induction in response to LDs, and these functions are spatially separated between the leaves and shoot meristem.

Flowering of Arabidopsis thaliana is induced by exposure to long days (LDs). During this process, the shoot apical meristem is converted to an inflorescence meristem that forms flowers, and this transition is maintained even if plants are returned to short days (SDs). We show that exposure to five LDs is sufficient to commit the meristem of SD-grown plants to flower as if they were exposed to continuous LDs. The MADS box proteins SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and FRUITFULL (FUL) play essential roles in this commitment process and in the induction of flowering downstream of the transmissible FLOWERING LOCUS T (FT) signal. We exploited laser microdissection and Solexa sequencing to identify 202 genes whose transcripts increase in the meristem during floral commitment. Expression of six of these transcripts was tested in different mutants, allowing them to be assigned to FT-dependent or FT-independent pathways. Most, but not all, of those dependent on FT and its paralog TWIN SISTER OF FT (TSF) also relied on SOC1 and FUL. However, this dependency on FT and TSF or SOC1 and FUL was often bypassed in the presence of the short vegetative phase mutation. FLOR1, which encodes a leucine-rich repeat protein, was induced in the early inflorescence meristem, and flor1 mutations delayed flowering. Our data contribute to the definition of LD-dependent pathways downstream and in parallel to FT.

BackgroundMADS-domain transcription factors play important roles during plant development. The Arabidopsis MADS-box gene SHORT VEGETATIVE PHASE (SVP) is a key regulator of two developmental phases. It functions as a repressor of the floral transition during the vegetative phase and later it contributes to the specification of floral meristems. How these distinct activities are conferred by a single transcription factor is unclear, but interactions with other MADS domain proteins which specify binding to different genomic regions is likely one mechanism.ResultsTo compare the genome-wide DNA binding profile of SVP during vegetative and reproductive development we performed ChIP-seq analyses. These ChIP-seq data were combined with tiling array expression analysis, induction experiments and qRT-PCR to identify biologically relevant binding sites. In addition, we compared genome-wide target genes of SVP with those published for the MADS domain transcription factors FLC and AP1, which interact with SVP during the vegetative and reproductive phases, respectively.ConclusionsOur analyses resulted in the identification of pathways that are regulated by SVP including those controlling meristem development during vegetative growth and flower development whereas floral transition pathways and hormonal signaling were regulated predominantly during the vegetative phase. Thus, SVP regulates many developmental pathways, some of which are common to both of its developmental roles whereas others are specific to only one of them.

SUMMARYCytokinins are involved in many aspects of plant growth and development, and physiological evidence also indicates that they have a role in floral transition. In order to integrate these phytohormones into the current knowledge of genetically defined molecular pathways to flowering, we performed exogenous treatments of adult wild type and mutant Arabidopsis plants, and analysed the expression of candidate genes. We used a hydroponic system that enables synchronous growth and flowering of Arabidopsis, and allows the precise application of chemicals to the roots for defined periods of time. We show that the application of N 6 -benzylaminopurine (BAP) promotes flowering of plants grown in non-inductive short days. The response to cytokinin treatment does not require FLOWERING LOCUS T (FT), but activates its paralogue TWIN SISTER OF FT (TSF), as well as FD, which encodes a partner protein of TSF, and the downstream gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). Treatment of selected mutants confirmed that TSF and SOC1 are necessary for the flowering response to BAP, whereas the activation cascade might partially act independently of FD. These experiments provide a mechanistic basis for the role of cytokinins in flowering, and demonstrate that the redundant genes FT and TSF are differently regulated by distinct floral-inducing signals.

In Arabidopsis thaliana environmental and endogenous cues promote flowering by activating expression of a small number of integrator genes. The MADS box transcription factor SHORT VEG-ETATIVE PHASE (SVP) is a critical inhibitor of flowering that directly represses transcription of these genes. However, we show by genetic analysis that the effect of SVP cannot be fully explained by repressing known floral integrator genes. To identify additional SVP functions, we analyzed genome-wide transcriptome data and show that GIBBERELLIN 20 OXIDASE 2, which encodes an enzyme required for biosynthesis of the growth regulator gibberellin (GA), is upregulated in svp mutants. GA is known to promote flowering, and we find that svp mutants contain elevated levels of GA that correlate with GA-related phenotypes such as early flowering and organ elongation. The ga20ox2 mutation suppresses the elevated GA levels and partially suppresses the growth and early flowering phenotypes of svp mutants. In wild-type plants, SVP expression in the shoot apical meristem falls when plants are exposed to photoperiods that induce flowering, and this correlates with increased expression of GA20ox2. Mutations that impair the photoperiodic flowering pathway prevent this downregulation of SVP and the strong increase in expression of GA20ox2. We conclude that SVP delays flowering by repressing GA biosynthesis as well as integrator gene expression and that, in response to inductive photoperiods, repression of SVP contributes to the rise in GA at the shoot apex, promoting rapid induction of flowering.

The development of a new crop variety is a time-consuming and costly process due to plant breeding's reliance on gene shuffling to introduce desired genes into elite germplasm followed by backcrossing. We propose alternative technology that transiently targets various regulatory circuits within a plant, leading to operator-specified alterations of agronomic traits, such as time of flowering, vernalization requirement, plant height or drought tolerance. We redesigned techniques of gene delivery, amplification and expression around RNA viral transfection methods that can be implemented on an industrial scale and with multiple crop plants. The process does not involve genetic modification of the plant genome and is thus limited to a single plant generation, is broadly applicable, fast, tunable, versatile, and can be used throughout much of the crop cultivation cycle. The RNA-based reprogramming may be especially useful in case of major plant pathogen pandemics, but also for commercial seed production and for rapid adaptation of orphan crops.3 Modern plant breeding relies on recombination to introduce novel useful genes/alleles into elite germplasm. Development of a new variety is time-consuming and expensive, even with a use of most advanced technologies such as genome editing. We sought to design a flexible, rapid and industrially scalable alternative platform to alter hormonal and other regulatory circuits within a plant, by rebuilding the known techniques of transient gene expression around gene delivery methods that can be performed on an industrial scale, and that can be practiced with multiple crop plants. Our approach focused on two types of vectors commonly used in laboratory science; namely, Agrobacterium as the primary DNA vector, and RNA viral amplicons as secondary/primary vectors and amplifiers of information molecules. We and others have successfully used Agrobacterium-based transfection to design industrial-scale manufacturing processes for producing recombinant proteins in plants [1][2][3][4] , including biopharmaceuticals, vaccines and biomaterials 5 . This earlier-generation transient reprogramming focused on a single plant species, Nicotiana benthamiana. The method required vacuum-assisted infiltration of bacteria into the intercellular leaf space and, by design, ignored the general agronomic performance of the plant other than the high-level expression of heterologous recombinant proteins that were almost exclusively of non-plant origin. A few attempts to modify agronomic traits, namely viral induction of flowering, were also previously reported, but were limited to research-scale experiments [6][7][8][9][10][11] .We report here that multiple economically important crop plants can be induced to exhibit desirable agronomic performance traits, by simply spraying them with agrobacteria carrying viral replicons to express plant genes. Moreover, we also demonstrate that most of the agronomic traits can also be engineered by spraying plants with packaged RNA viral vectors thus 4 eliminating DNA release...

During the floral transition the shoot apical meristem changes its identity from a vegetative to an inflorescence state. This change in identity can be promoted by external signals, such as inductive photoperiod conditions or vernalization, and is accompanied by changes in expression of key developmental genes. The change in meristem identity is usually not reversible, even if the inductive signal occurs only transiently. This implies that at least some of the key genes must possess an intrinsic memory of the newly acquired expression state that ensures irreversibility of the process. In this review, we discuss different molecular scenarios that may underlie a molecular memory of gene expression.