Frédéric Bouché

Cytokinin promotes flowering of Arabidopsis via transcriptional activation of the FT paralogue TSF

Cytokinins 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 N⁶-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.

FLOR-ID: an interactive database of flowering-time gene networks in Arabidopsis thaliana

Flowering is a hot topic in Plant Biology and important progress has been made in Arabidopsis thaliana toward unraveling the genetic networks involved. The increasing complexity and the explosion of literature however require development of new tools for information management and update. We therefore created an evolutive and interactive database of flowering time genes, named FLOR-ID (Flowering-Interactive Database), which is freely accessible at http://www.flor-id.org. The hand-curated database contains information on 306 genes and links to 1595 publications gathering the work of >4500 authors. Gene/protein functions and interactions within the flowering pathways were inferred from the analysis of related publications, included in the database and translated into interactive manually drawn snapshots.

Winter Memory throughout the Plant Kingdom: Different Paths to Flowering

Molecular mechanisms contribute to the memory of winter in different plant groups.

Extracellular peptidase hunting for improvement of protein production in plant cells and roots

Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion), in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes) were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA) and human serum immunoglobulins G (hIgGs). Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine, and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing.

Genetic Architecture of Flowering-Time Variation in Brachypodium distachyon

QTL associated with VERNALIZATION1/PHYC and VERNALIZATION2 account for much of the natural variation in flowering time between the Brachypodium distachyon lines Bd21 and Bd1-1. The transition to reproductive development is a crucial step in the plant life cycle, and the timing of this transition is an important factor in crop yields. Here, we report new insights into the genetic control of natural variation in flowering time in Brachypodium distachyon, a nondomesticated pooid grass closely related to cereals such as wheat (Triticum spp.) and barley (Hordeum vulgare L.). A recombinant inbred line population derived from a cross between the rapid-flowering accession Bd21 and the delayed-flowering accession Bd1-1 were grown in a variety of environmental conditions to enable exploration of the genetic architecture of flowering time. A genotyping-by-sequencing approach was used to develop SNP markers for genetic map construction, and quantitative trait loci (QTLs) that control differences in flowering time were identified. Many of the flowering-time QTLs are detected across a range of photoperiod and vernalization conditions, suggesting that the genetic control of flowering within this population is robust. The two major QTLs identified in undomesticated B. distachyon colocalize with VERNALIZATION1/PHYTOCHROME C and VERNALIZATION2, loci identified as flowering regulators in the domesticated crops wheat and barley. This suggests that variation in flowering time is controlled in part by a set of genes broadly conserved within pooid grasses.

Integrating roots into a whole plant network of flowering time genes in Arabidopsis thaliana.

Molecular data concerning the involvement of roots in the genetic pathways regulating floral transition are lacking. In this study, we performed global analyses of the root transcriptome in Arabidopsis in order to identify flowering time genes that are expressed in the roots and genes that are differentially expressed in the roots during the induction of flowering. Data mining of public microarray experiments uncovered that about 200 genes whose mutations are reported to alter flowering time are expressed in the roots (i.e. were detected in more than 50% of the microarrays). However, only a few flowering integrator genes passed the analysis cutoff. Comparison of root transcriptome in short days and during synchronized induction of flowering by a single 22-h long day revealed that 595 genes were differentially expressed. Enrichment analyses of differentially expressed genes in root tissues, gene ontology categories, and cis-regulatory elements converged towards sugar signaling. We concluded that roots are integrated in systemic signaling, whereby carbon supply coordinates growth at the whole plant level during the induction of flowering. This coordination could involve the root circadian clock and cytokinin biosynthesis as a feed forward loop towards the shoot.

Heat can erase epigenetic marks of vernalization in Arabidopsis

Vernalization establishes a memory of winter that must be maintained for weeks or months in order to promote flowering the following spring. The stability of the vernalized state varies among plant species and depends on the duration of cold exposure. In Arabidopsis thaliana, winter leads to epigenetic silencing of the floral repressor gene FLOWERING LOCUS C (FLC) and the duration of cold is measured through the dynamics of chromatin modifications during and after cold. The growing conditions encountered post-vernalization are thus critical for the maintenance of the vernalized state. We reported that high temperature leads to devernalization and, consistently, to FLC reactivation in Arabidopsis seedlings. Here we show that the repressive epigenetic mark H3K27me3 decreases at the FLC locus when vernalized seedlings are grown at 30°C, unless they were first exposed to a stabilizing period at 20°C. Ambient temperature thus controls the epigenetic memory of winter.

Establishment of a vernalization requirement in Brachypodium distachyon requires REPRESSOR OF VERNALIZATION1

Significance A key feature in the evolution of all vernalization systems is a cold-regulated component. In pooid grasses, up-regulation of the flowering promoter VERNALIZATION1 (VRN1) by prolonged cold is a key feature of vernalization, although little is known about the genes that repress VRN1 prior to cold exposure or activate it afterward. Here, we report the identification of REPRESSOR OF VERNALIZATION1 (RVR1), a repressor of VRN1 that is involved in creating a vernalization requirement in Brachypodium distachyon. RVR1 is present in all sequenced flowering plant genomes but is not found outside the plant kingdom. This report describes a role for the RVR1 class of genes in plants and an upstream component of the VRN1 regulatory system. A requirement for vernalization, the process by which prolonged cold exposure provides competence to flower, is an important adaptation to temperate climates that ensures flowering does not occur before the onset of winter. In temperate grasses, vernalization results in the up-regulation of VERNALIZATION1 (VRN1) to establish competence to flower; however, little is known about the mechanism underlying repression of VRN1 in the fall season, which is necessary to establish a vernalization requirement. Here, we report that a plant-specific gene containing a bromo-adjacent homology and transcriptional elongation factor S-II domain, which we named REPRESSOR OF VERNALIZATION1 (RVR1), represses VRN1 before vernalization in Brachypodium distachyon. That RVR1 is upstream of VRN1 is supported by the observations that VRN1 is precociously elevated in an rvr1 mutant, resulting in rapid flowering without cold exposure, and the rapid-flowering rvr1 phenotype is dependent on VRN1. The precocious VRN1 expression in rvr1 is associated with reduced levels of the repressive chromatin modification H3K27me3 at VRN1, which is similar to the reduced VRN1 H3K27me3 in vernalized plants. Furthermore, the transcriptome of vernalized wild-type plants overlaps with that of nonvernalized rvr1 plants, indicating loss of rvr1 is similar to the vernalized state at a molecular level. However, loss of rvr1 results in more differentially expressed genes than does vernalization, indicating that RVR1 may be involved in processes other than vernalization despite a lack of any obvious pleiotropy in the rvr1 mutant. This study provides an example of a role for this class of plant-specific genes.

A florigen paralog is required for short-day vernalization in a pooid grass

Perception of seasonal cues is critical for reproductive success in many plants. Exposure to winter cold is a cue that can confer competence to flower in the spring via a process known as vernalization. In certain grasses, exposure to short days is another winter cue that can lead to a vernalized state. In Brachypodium distachyon, we find that natural variation for the ability of short days to confer competence to flower is due to allelic variation of the florigen paralog FT-like9 (FTL9). An active FTL9 allele is required for the acquisition of floral competence, demonstrating a novel role for a member of the florigen family of genes. Loss of the short-day vernalization response appears to have arisen once in B. distachyon and spread through diverse lineages indicating that this loss has adaptive value, perhaps by delaying spring flowering until the danger of cold damage to flowers has subsided.