Martin Kieffer

Molecular and Phylogenetic Analyses of the Complete MADS-Box Transcription Factor Family in Arabidopsis: New Openings to the MADS World

MADS-box transcription factors are key regulators of several plant development processes. Analysis of the complete Arabidopsis genome sequence revealed 107 genes encoding MADS-box proteins, of which 84% are of unknown function. Here, we provide a complete overview of this family, describing the gene structure, gene expression, genome localization, protein motif organization, and phylogenetic relationship of each member. We have divided this transcription factor family into five groups (named MIKC, Mα, Mβ, Mγ, and Mδ) based on the phylogenetic relationships of the conserved MADS-box domain. This study provides a solid base for functional genomics studies into this important family of plant regulatory genes, including the poorly characterized group of M-type MADS-box proteins. MADS-box genes also constitute an excellent system with which to study the evolution of complex gene families in higher plants.

Comprehensive Interaction Map of the Arabidopsis MADS Box Transcription Factors

Interactions between proteins are essential for their functioning and the biological processes they control. The elucidation of interaction maps based on yeast studies is a first step toward the understanding of molecular networks and provides a framework of proteins that possess the capacity and specificity to interact. Here, we present a comprehensive plant protein–protein interactome map of nearly all members of the Arabidopsis thaliana MADS box transcription factor family. A matrix-based yeast two-hybrid screen of >100 members of this family revealed a collection of specific heterodimers and a few homodimers. Clustering of proteins with similar interaction patterns pinpoints proteins involved in the same developmental program and provides valuable information about the participation of uncharacterized proteins in these programs. Furthermore, a model is proposed that integrates the floral induction and floral organ formation networks based on the interactions between the proteins involved. Heterodimers between flower induction and floral organ identity proteins were observed, which point to (auto)regulatory mechanisms that prevent the activity of flower induction proteins in the flower.

Analysis of the Transcription Factor WUSCHEL and Its Functional Homologue in Antirrhinum Reveals a Potential Mechanism for Their Roles in Meristem Maintenance

One of the most significant features of plant development is the way in which it can be elaborated and modulated throughout the life of the plant, an ability that is conferred by meristems. The Arabidopsis thaliana WUSCHEL gene (WUS), which encodes a homeodomain transcription factor, is required to maintain the stem cells in the shoot apical meristem in an undifferentiated state. The mechanism by which WUS prevents the differentiation of stem cells is unknown. We have characterized a meristem maintenance mutant in Antirrhinum majus and shown that it arises from a defect in the WUS orthologue ROSULATA (ROA). Detailed characterization of a semidominant roa allele revealed an essential role for the conserved C-terminal domain. Expression of either ROA or WUS lacking this domain causes a failure of meristem maintenance. The conserved domain mediates an interaction between WUS and two members of a small family of corepressor-like proteins in Arabidopsis. Our results suggest that WUS functions by recruiting transcriptional corepressors to repress target genes that promote differentiation, thereby ensuring stem cell maintenance.

TCP14 and TCP15 affect internode length and leaf shape in Arabidopsis

TCP transcription factors constitute a small family of plant-specific bHLH-containing, DNA-binding proteins that have been implicated in the control of cell proliferation in plants. Despite the significant role that is likely to be played by genes that control cell division in the elaboration of plant architecture, functional analysis of this family by forward and reverse genetics has been hampered by genetic redundancy. Here we show that mutants in two related class I TCP genes display a range of growth-related phenotypes, consistent with their dynamic expression patterns; these phenotypes are enhanced in the double mutant. Together, the two genes influence plant stature by promoting cell division in young internodes. Reporter gene analysis and use of SRDX fusions suggested that TCP14 and TCP15 modulate cell proliferation in the developing leaf blade and specific floral tissues; a role that was not apparent in our phenotypic analysis of single or double mutants. However, when the relevant mutants were subjected to computer-aided morphological analysis of the leaves, the consequences of loss of either or both genes became obvious. The effects on cell proliferation of perturbing the function of TCP14 and TCP15 vary with tissue, as has been suggested for other TCP factors. These findings indicate that the precise elaboration of plant form is dependent on the cumulative influence of many TCP factors acting in a context-dependent fashion. The study highlights the need for advanced methods of phenotypic analysis in order to characterize phenotypes and to construct a dynamic model for TCP gene function.

Defining auxin response contexts in plant development

The apparent domination of so much of plant development by the hormone auxin raises important questions about how this simple, generic signalling molecule can give rise to such an enormous range of very specific developmental outputs. What is becoming increasingly clear is that alongside the tight control of auxin distribution within the plant, the cellular and developmental context in which the auxin signal is received is of utmost importance. Recent work has highlighted that these distinct auxin response contexts are defined by complex and inter-dependent relationships between auxin metabolism, transport and response that can be modulated at many levels by inputs from both endogenous and environmental signals.

The Arabidopsis O-Linked N-Acetylglucosamine Transferase SPINDLY Interacts with Class I TCPs to Facilitate Cytokinin Responses in Leaves and Flowers

The Arabidopsis O-linked N-acetylglucosamine transferase SPINDLY (SPY) promotes cytokinin responses by unknown mechanisms. This work shows that SPY interacts with and is required for the activity of two closely related class I TCP transcription factors, TCP14 and TCP15, that promote cytokinin responses in leaves and flowers, affecting leaf shape and trichome development. O-linked N-acetylglucosamine (O-GlcNAc) modifications regulate the posttranslational fate of target proteins. The Arabidopsis thaliana O-GlcNAc transferase (OGT) SPINDLY (SPY) suppresses gibberellin signaling and promotes cytokinin (CK) responses by unknown mechanisms. Here, we present evidence that two closely related class I TCP transcription factors, TCP14 and TCP15, act with SPY to promote CK responses. TCP14 and TCP15 interacted with SPY in yeast two-hybrid and in vitro pull-down assays and were O-GlcNAc modified in Escherichia coli by the Arabidopsis OGT, SECRET AGENT. Overexpression of TCP14 severely affected plant development in a SPY-dependent manner and stimulated typical CK morphological responses, as well as the expression of the CK-regulated gene RESPONSE REGULATOR5. TCP14 also promoted the transcriptional activity of the CK-induced mitotic factor CYCLIN B1;2. Whereas TCP14-overexpressing plants were hypersensitive to CK, spy and tcp14 tcp15 double mutant leaves and flowers were hyposensitive to the hormone. Reducing CK levels by overexpressing CK OXIDASE/DEHYDROGENASE3 suppressed the TCP14 overexpression phenotypes, and this suppression was reversed when the plants were treated with exogenous CK. Taken together, we suggest that responses of leaves and flowers to CK are mediated by SPY-dependent TCP14 and TCP15 activities.

Corrigendum: HSP90 regulates temperature-dependent seedling growth in Arabidopsis by stabilizing the auxin co-receptor F-box protein TIR1

Recent studies have revealed that a mild increase in environmental temperature stimulates the growth of Arabidopsis seedlings by promoting biosynthesis of the plant hormone auxin. However, little is known about the role of other factors in this process. In this report, we show that increased temperature promotes rapid accumulation of the TIR1 auxin co-receptor, an effect that is dependent on the molecular chaperone HSP90. In addition, we show that HSP90 and the co-chaperone SGT1 each interact with TIR1, confirming that TIR1 is an HSP90 client. Inhibition of HSP90 activity results in degradation of TIR1 and interestingly, defects in a range of auxin-mediated growth processes at lower as well as higher temperatures. Our results indicate that HSP90 and SGT1 integrate temperature and auxin signalling in order to regulate plant growth in a changing environment.

Rapid mass production of cauliflower propagules from fractionated and graded curd

Abstract A new system of cauliflower micropropagation is described. The meristematic layer of the curd is removed and partially homogenised to disrupt the meristem clusters and then graded through precision sieves to produce homogenous size-classes. For a single curd, over 400 000 explants of size-class 0.1–0.3 mm can be produced. The explants are cultured following a two-step protocol. Shoot production uses an agitated liquid medium, explant size influences the number of shoots produced per explant with the optimal explant size-class of 0.1–0.3 mm allowing the production of one to three shoots per explant. Accurate control of the explant density in culture is required to produce good quality shoots and to control hyperhydricity. The number of ‘microshoots’ produced from one curd within 2 weeks is over 10 000. The rooting step takes place on a semi-solid medium in the presence of IBA, within 2 weeks over 80% of shoots are rooted. Plantlets are waned within 40 days following culture initiation. This protocol has the qualities of simplicity, large scale propagation, and high quality progagules, making it superior to conventional methods and a cost effective candidate for an industrial semi-automated system of propagule production.

A cost effective protocol for in vitro mass propagation of cauliflower

An improved and cost effective protocol for in vitro mass propagation of cauliflower from fractionated and graded curd is presented. The protocol is optimised for the production of clones of 2000 plants from one mother curd. Microshoots are produced en masse in 12 days, selected by flotation on a sucrose pad and transferred to rooting medium after suspension in a viscous medium. Fully rooted propagules are transferred to the glasshouse in 4-5 weeks and ready for transfer to the field 3-5 weeks later. The propagule unit cost was drastically reduced and is now close to that of a seed derived module grown plantlet. The strengths and limitations of this protocol are discussed. Clones produced highly homogeneous curds in the field with a short cutting period. Their overall quality depended essentially on the quality of the selected mother curd. The screening of populations to select elite genotypes combining high quality curd and good response in vitro is central to the use of this protocol on an industrial scale.

The developmental and environmental regulation of gravitropic setpoint angle in Arabidopsis and bean

Root and shoot branches are major determinants of plant form and critical for the effective capture of resources below and above ground. These branches are often maintained at specific angles with respect to gravity, known as gravitropic set point angles (GSAs). We have previously shown that the mechanism permitting the maintenance of non-vertical GSAs is highly auxin-dependent and here we investigate the developmental and environmental regulation of root and shoot branch GSA. We show that nitrogen and phosphorous deficiency have opposing, auxin signalling-dependent effects on lateral root GSA in Arabidopsis: while low nitrate induces less vertical lateral root GSA, phosphate deficiency results in a more vertical lateral root growth angle, a finding that contrasts with the previously reported growth angle response of bean adventitious roots. We find that this root-class-specific discrepancy in GSA response to low phosphorus is mirrored by similar differences in growth angle response to auxin treatment between these root types. Finally we show that both shaded, low red/far-red light conditions and high temperature induce more vertical growth in Arabidopsis shoot branches. We discuss the significance of these findings in the context of efforts to improve crop performance via the manipulation of root and shoot branch growth angle.