Pierre Hilson

Receptor-Like Kinase ACR4 Restricts Formative Cell Divisions in the Arabidopsis Root

During the development of multicellular organisms, organogenesis and pattern formation depend on formative divisions to specify and maintain pools of stem cells. In higher plants, these activities are essential to shape the final root architecture because the functioning of root apical meristems and the de novo formation of lateral roots entirely rely on it. We used transcript profiling on sorted pericycle cells undergoing lateral root initiation to identify the receptor-like kinase ACR4 of Arabidopsis as a key factor both in promoting formative cell divisions in the pericycle and in constraining the number of these divisions once organogenesis has been started. In the root tip meristem, ACR4 shows a similar action by controlling cell proliferation activity in the columella cell lineage. Thus, ACR4 function reveals a common mechanism of formative cell division control in the main root tip meristem and during lateral root initiation.

Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana.

Cell proliferation is the main driving force for plant growth. Although genome sequence analysis revealed a high number of cell cycle genes in plants, little is known about the molecular complexes steering cell division. In a targeted proteomics approach, we mapped the core complex machinery at the heart of the Arabidopsis thaliana cell cycle control. Besides a central regulatory network of core complexes, we distinguished a peripheral network that links the core machinery to up‐ and downstream pathways. Over 100 new candidate cell cycle proteins were predicted and an in‐depth biological interpretation demonstrated the hypothesis‐generating power of the interaction data. The data set provided a comprehensive view on heterodimeric cyclin‐dependent kinase (CDK)–cyclin complexes in plants. For the first time, inhibitory proteins of plant‐specific B‐type CDKs were discovered and the anaphase‐promoting complex was characterized and extended. Important conclusions were that mitotic A‐ and B‐type cyclins form complexes with the plant‐specific B‐type CDKs and not with CDKA;1, and that D‐type cyclins and S‐phase‐specific A‐type cyclins seem to be associated exclusively with CDKA;1. Furthermore, we could show that plants have evolved a combinatorial toolkit consisting of at least 92 different CDK–cyclin complex variants, which strongly underscores the functional diversification among the large family of cyclins and reflects the pivotal role of cell cycle regulation in the developmental plasticity of plants.

Recombinational Cloning with Plant Gateway Vectors

The study of biological systems relies to a large extent on DNA cloning technologies enabling the analysis of recombinant genes through transgenic research. In this context, the advent of recombinational cloning methods was a significant progress because DNA fragments can now be assembled regardless

Plant CLE peptides from two distinct functional classes synergistically induce division of vascular cells

The Clavata3 (CLV3)/endosperm surrounding region (CLE) signaling peptides are encoded in large plant gene families. CLV3 and the other A-type CLE peptides promote cell differentiation in root and shoot apical meristems, whereas the B-type peptides (CLE41–CLE44) do not. Instead, CLE41 inhibits the differentiation of Zinnia elegans tracheary elements. To test whether CLE genes might code for antagonistic or synergistic functions, peptides from both types were combined through overexpression within or application onto Arabidopsis thaliana seedlings. The CLE41 peptide (CLE41p) promoted proliferation of vascular cells, although delaying differentiation into phloem and xylem cell lineages. Application of CLE41p or overexpression of CLE41 did not suppress the terminal differentiation of the root and shoot apices triggered by A-type CLE peptides. However, in combination, A-type peptides enhanced all of the phenotypes associated with CLE41 gain-of-function, leading to massive proliferation of vascular cells. This proliferation relied on auxin signaling because it was enhanced by exogenous application of a synthetic auxin, decreased by an auxin polar transport inhibitor, and abolished by a mutation in the Monopteros auxin response factor. These findings highlight that vascular patterning is a process controlled in time and space by different CLE peptides in conjunction with hormonal signaling.

Building Blocks for Plant Gene Assembly

The MultiSite Gateway cloning system, based on site-specific recombination, enables the assembly of multiple DNA fragments in predefined order, orientation, and frame register. To streamline the construction of recombinant genes for functional analysis in plants, we have built a collection of 36 reference Gateway entry clones carrying promoters, terminators, and reporter genes, as well as elements of the LhG4/LhGR two-component system. This collection obeys simple engineering rules. The genetic elements (parts) are designed in a standard format. They are interchangeable, fully documented, and can be combined at will according to the desired output. We also took advantage of the MultiSite Gateway recombination sites to create vectors in which two or three genes can be cloned simultaneously in separate expression cassettes. To illustrate the flexibility of these core resources for the construction of a wide variety of plant transformation vectors, we generated various transgenes encoding fluorescent proteins and tested their activity in plant cells. The structure and sequence of all described plasmids are accessible online at http://www.psb.ugent.be/gateway/. All accessions can be requested via the same Web site.

Systems-based analysis of Arabidopsis leaf growth reveals adaptation to water deficit

Leaves have a central role in plant energy capture and carbon conversion and therefore must continuously adapt their development to prevailing environmental conditions. To reveal the dynamic systems behaviour of leaf development, we profiled Arabidopsis leaf number six in depth at four different growth stages, at both the end‐of‐day and end‐of‐night, in plants growing in two controlled experimental conditions: short‐day conditions with optimal soil water content and constant reduced soil water conditions. We found that the lower soil water potential led to reduced, but prolonged, growth and an adaptation at the molecular level without a drought stress response. Clustering of the protein and transcript data using a decision tree revealed different patterns in abundance changes across the growth stages and between end‐of‐day and end‐of‐night that are linked to specific biological functions. Correlations between protein and transcript levels depend on the time‐of‐day and also on protein localisation and function. Surprisingly, only very few of >1700 quantified proteins showed diurnal abundance fluctuations, despite strong fluctuations at the transcript level.

APETALA2/ETHYLENE RESPONSE FACTOR and basic helix–loop–helix tobacco transcription factors cooperatively mediate jasmonate‐elicited nicotine biosynthesis

Transcription factors of the plant-specific apetala2/ethylene response factor (AP2/ERF) family control plant secondary metabolism, often as part of signalling cascades induced by jasmonate (JA) or other elicitors. Here, we functionally characterized the JA-inducible tobacco (Nicotiana tabacum) AP2/ERF factor ORC1, one of the members of the NIC2-locus ERFs that control nicotine biosynthesis and a close homologue of ORCA3, a transcriptional activator of alkaloid biosynthesis in Catharanthus roseus. ORC1 positively regulated the transcription of several structural genes coding for the enzymes involved in nicotine biosynthesis. Accordingly, overexpression of ORC1 was sufficient to stimulate alkaloid biosynthesis in tobacco plants and tree tobacco (Nicotiana glauca) root cultures. In contrast to ORCA3 in C. roseus, which needs only the GCC motif in the promoters of the alkaloid synthesis genes to induce their expression, ORC1 required the presence of both GCC-motif and G-box elements in the promoters of the tobacco nicotine biosynthesis genes for maximum transactivation. Correspondingly, combined application with the JA-inducible Nicotiana basic helix-loop-helix (bHLH) factors that bind the G-box element in these promoters enhanced ORC1 action. Conversely, overaccumulation of JAZ repressor proteins that block bHLH activity reduced ORC1 functionality. Finally, the activity of both ORC1 and bHLH proteins was post-translationally upregulated by a JA-modulated phosphorylation cascade, in which a specific mitogen-activated protein kinase kinase, JA-factor stimulating MAPKK1 (JAM1), was identified. This study highlights the complexity of the molecular machinery involved in the regulation of tobacco alkaloid biosynthesis and provides mechanistic insights about its transcriptional regulators.

GOLVEN Secretory Peptides Regulate Auxin Carrier Turnover during Plant Gravitropic Responses

Growth and development are coordinated by an array of intercellular communications. Known plant signaling molecules include phytohormones and hormone peptides. Although both classes can be implicated in the same developmental processes, little is known about the interplay between phytohormone action and peptide signaling within the cellular microenvironment. We show that genes coding for small secretory peptides, designated GOLVEN (GLV), modulate the distribution of the phytohormone auxin. The deregulation of the GLV function impairs the formation of auxin gradients and alters the reorientation of shoots and roots after a gravity stimulus. Specifically, the GLV signal modulates the trafficking dynamics of the auxin efflux carrier PIN-FORMED2 involved in root tropic responses and meristem organization. Our work links the local action of secretory peptides with phytohormone transport.

Probing the Reproducibility of Leaf Growth and Molecular Phenotypes: A Comparison of Three Arabidopsis Accessions Cultivated in Ten Laboratories

A major goal of the life sciences is to understand how molecular processes control phenotypes. Because understanding biological systems relies on the work of multiple laboratories, biologists implicitly assume that organisms with the same genotype will display similar phenotypes when grown in comparable conditions. We investigated to what extent this holds true for leaf growth variables and metabolite and transcriptome profiles of three Arabidopsis (Arabidopsis thaliana) genotypes grown in 10 laboratories using a standardized and detailed protocol. A core group of four laboratories generated similar leaf growth phenotypes, demonstrating that standardization is possible. But some laboratories presented significant differences in some leaf growth variables, sometimes changing the genotype ranking. Metabolite profiles derived from the same leaf displayed a strong genotype × environment (laboratory) component. Genotypes could be separated on the basis of their metabolic signature, but only when the analysis was limited to samples derived from one laboratory. Transcriptome data revealed considerable plant-to-plant variation, but the standardization ensured that interlaboratory variation was not considerably larger than intralaboratory variation. The different impacts of the standardization on phenotypes and molecular profiles could result from differences of temporal scale between processes involved at these organizational levels. Our findings underscore the challenge of describing, monitoring, and precisely controlling environmental conditions but also demonstrate that dedicated efforts can result in reproducible data across multiple laboratories. Finally, our comparative analysis revealed that small variations in growing conditions (light quality principally) and handling of plants can account for significant differences in phenotypes and molecular profiles obtained in independent laboratories.

Functional Modules in the Arabidopsis Core Cell Cycle Binary Protein–Protein Interaction Network

This study describes the creation of a binary protein–protein interaction map of core cell cycle proteins of Arabidopsis thaliana using two complementary interaction assays, yeast two-hybrid and bimolecular fluorescence complementation. It integrates this map with expression data and describes 357 protein–protein interactions, of which 293 are previously unreported. As in other eukaryotes, cell division in plants is highly conserved and regulated by cyclin-dependent kinases (CDKs) that are themselves predominantly regulated at the posttranscriptional level by their association with proteins such as cyclins. Although over the last years the knowledge of the plant cell cycle has considerably increased, little is known on the assembly and regulation of the different CDK complexes. To map protein–protein interactions between core cell cycle proteins of Arabidopsis thaliana, a binary protein–protein interactome network was generated using two complementary high-throughput interaction assays, yeast two-hybrid and bimolecular fluorescence complementation. Pairwise interactions among 58 core cell cycle proteins were tested, resulting in 357 interactions, of which 293 have not been reported before. Integration of the binary interaction results with cell cycle phase-dependent expression information and localization data allowed the construction of a dynamic interaction network. The obtained interaction map constitutes a framework for further in-depth analysis of the cell cycle machinery.