Geert De Jaeger

NINJA connects the co-repressor TOPLESS to jasmonate signalling.

Jasmonoyl-isoleucine (JA-Ile) is a plant hormone that regulates a broad array of plant defence and developmental processes. JA-Ile-responsive gene expression is regulated by the transcriptional activator MYC2 that interacts physically with the jasmonate ZIM-domain (JAZ) repressor proteins. On perception of JA-Ile, JAZ proteins are degraded and JA-Ile-dependent gene expression is activated. The molecular mechanisms by which JAZ proteins repress gene expression remain unknown. Here we show that the Arabidopsis JAZ proteins recruit the Groucho/Tup1-type co-repressor TOPLESS (TPL) and TPL-related proteins (TPRs) through a previously uncharacterized adaptor protein, designated Novel Interactor of JAZ (NINJA). NINJA acts as a transcriptional repressor whose activity is mediated by a functional TPL-binding EAR repression motif. Accordingly, both NINJA and TPL proteins function as negative regulators of jasmonate responses. Our results point to TPL proteins as general co-repressors that affect multiple signalling pathways through the interaction with specific adaptor proteins. This new insight reveals how stress-related and growth-related signalling cascades use common molecular mechanisms to regulate gene expression in plants.

The Arabidopsis bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses

This work identifies two transcription factors, MYC3 and MYC4, as targets of JAZ repressors and regulators of responses to jasmonate. It finds a specificity of transcription factor activity that could be a clue to understanding the diversity of JA-regulated responses. Jasmonates (JAs) trigger an important transcriptional reprogramming of plant cells to modulate both basal development and stress responses. In spite of the importance of transcriptional regulation, only one transcription factor (TF), the Arabidopsis thaliana basic helix-loop-helix MYC2, has been described so far as a direct target of JAZ repressors. By means of yeast two-hybrid screening and tandem affinity purification strategies, we identified two previously unknown targets of JAZ repressors, the TFs MYC3 and MYC4, phylogenetically closely related to MYC2. We show that MYC3 and MYC4 interact in vitro and in vivo with JAZ repressors and also form homo- and heterodimers with MYC2 and among themselves. They both are nuclear proteins that bind DNA with sequence specificity similar to that of MYC2. Loss-of-function mutations in any of these two TFs impair full responsiveness to JA and enhance the JA insensitivity of myc2 mutants. Moreover, the triple mutant myc2 myc3 myc4 is as impaired as coi1-1 in the activation of several, but not all, JA-mediated responses such as the defense against bacterial pathogens and insect herbivory. Our results show that MYC3 and MYC4 are activators of JA-regulated programs that act additively with MYC2 to regulate specifically different subsets of the JA-dependent transcriptional response.

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.

A Tandem Affinity Purification-based Technology Platform to Study the Cell Cycle Interactome in Arabidopsis thaliana

Defining protein complexes is critical to virtually all aspects of cell biology because many cellular processes are regulated by stable protein complexes, and their identification often provides insights into their function. We describe the development and application of a high throughput tandem affinity purification/mass spectrometry platform for cell suspension cultures to analyze cell cycle-related protein complexes in Arabidopsis thaliana. Elucidation of this protein-protein interaction network is essential to fully understand the functional differences between the highly redundant cyclin-dependent kinase/cyclin modules, which are generally accepted to play a central role in cell cycle control, in all eukaryotes. Cell suspension cultures were chosen because they provide an unlimited supply of protein extracts of actively dividing and undifferentiated cells, which is crucial for a systematic study of the cell cycle interactome in the absence of plant development. Here we report the mapping of a protein interaction network around six known core cell cycle proteins by an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, tandem affinity purification adapted for plant cells, matrix-assisted laser desorption ionization tandem mass spectrometry, data analysis, and functional assays. We identified 28 new molecular associations and confirmed 14 previously described interactions. This systemic approach provides new insights into the basic cell cycle control mechanisms and is generally applicable to other pathways in plants.

Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences

Over the past decade, several high value proteins have been produced in different transgenic plant tissues such as leaves, tubers, and seeds. Despite recent advances, many heterologous proteins accumulate to low concentrations, and the optimization of expression cassettes to make in planta production and purification economically feasible remains critical. Here, the regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) of common bean (Phaseolus vulgaris) were evaluated for producing heterologous proteins in dicotyledonous seeds. The murine single chain variable fragment (scFv) G4 (ref. 4) was chosen as model protein because of the current industrial interest in producing antibodies and derived fragments in crops. In transgenic Arabidopsis thaliana seed stocks, the scFv under control of the 35S promoter of the cauliflower mosaic virus (CaMV) accumulated to approximately 1% of total soluble protein (TSP). However, a set of seed storage promoter constructs boosted the scFv accumulation to exceptionally high concentrations, reaching no less than 36.5% of TSP in homozygous seeds. Even at these high concentrations, the scFv proteins had antigen-binding activity and affinity similar to those produced in Escherichia coli. The feasibility of heterologous protein production under control of arc5-I regulatory sequences was also demonstrated in Phaseolus acutifolius, a promising crop for large scale production.

CDKB1;1 Forms a Functional Complex with CYCA2;3 to Suppress Endocycle Onset

The mitosis-to-endocycle transition requires the controlled inactivation of M phase-associated cyclin-dependent kinase (CDK) activity. Previously, the B-type CDKB1;1 was identified as an important negative regulator of endocycle onset. Here, we demonstrate that CDKB1;1 copurifies and associates with the A2-type cyclin CYCA2;3. Coexpression of CYCA2;3 with CDKB1;1 triggered ectopic cell divisions and inhibited endoreduplication. Moreover, the enhanced endoreduplication phenotype observed after overexpression of a dominant-negative allele of CDKB1;1 could be partially complemented by CYCA2;3 co-overexpression, illustrating that both subunits unite in vivo to form a functional complex. CYCA2;3 protein stability was found to be controlled by CCS52A1, an activator of the anaphase-promoting complex. We conclude that CCS52A1 participates in endocycle onset by down-regulating CDKB1;1 activity through the destruction of CYCA2;3.

Plants as bioreactors for protein production: avoiding the problem of transgene silencing.

Plants are particularly attractive as large-scale production systems for proteins intended for therapeutical or industrial applications: they can be grown easily and inexpensively in large quantities that can be harvested and processed with the available agronomic infrastructures. The effective use of plants as bioreactors depends on the possibility of obtaining high protein accumulation levels that are stable during the life cycle of the transgenic plant and in subsequent generations. Silencing of the introduced transgenes has frequently been observed in plants, constituting a major commercial risk and hampering the general economic exploitation of plants as protein factories. Until now, the most efficient strategy to avoid transgene silencing involves careful design of the transgene construct and thorough analysis of transformants at the molecular level. Here, we focus on different aspects of the generation of transgenic plants intended for protein production and on their influence on the stability of heterologous gene expression.

PYRABACTIN RESISTANCE1-LIKE8 Plays an Important Role for the Regulation of Abscisic Acid Signaling in Root

Summary: The abscisic acid receptor PYL8 plays an important role for regulation of root abscisic acid sensitivity, and abscisic acid-dependent inhibition of PP2Cs by PYR/PYLs is required for root hydrotropism. Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABA-INSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches. We also discovered that PYR/PYL receptors and clade A PP2Cs are crucial for the hydrotropic response that takes place to guide root growth far from regions with low water potential. Thus, an ABA-hypersensitive pp2c quadruple mutant showed enhanced hydrotropism, whereas an ABA-insensitive sextuple pyr/pyl mutant showed reduced hydrotropic response, indicating that ABA-dependent inhibition of PP2Cs by PYR/PYLs is required for the proper perception of a moisture gradient.

Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds

Production of high-value recombinant proteins in transgenic seeds is an attractive and economically feasible alternative to conventional systems based on mammalian cells and bacteria. In contrast to leaves, seeds allow high-level accumulation of recombinant proteins in a relatively small volume and a stable environment. We demonstrate that single-chain variable fragment (scFv)-Fc antibodies, with N-terminal signal sequence and C-terminal KDEL tag, can accumulate to very high levels as bivalent IgG-like antibodies in Arabidopsis thaliana seeds and illustrate that a plant-produced anti-hepatitis A virus scFv-Fc has similar antigen-binding and in vitro neutralizing activities as the corresponding full-length IgG. As expected, most scFv-Fc produced in seeds contained only oligomannose-type N-glycans, but, unexpectedly, 35–40% was never glycosylated. A portion of the scFv-Fc was found in endoplasmic reticulum (ER)-derived compartments delimited by ribosome-associated membranes. Additionally, consistent with the glycosylation data, large amounts of the recombinant protein were deposited in the periplasmic space, implying a direct transport from the ER to the periplasmic space between the plasma membrane and the cell wall. Aberrant localization of the ER chaperones calreticulin and binding protein (BiP) and the endogenous seed storage protein cruciferin in the periplasmic space suggests that overproduction of recombinant scFv-Fc disturbs normal ER retention and protein-sorting mechanisms in the secretory pathway.

Boosting tandem affinity purification of plant protein complexes.

Protein-interaction mapping based on the tandem affinity purification (TAP) approach has been successfully established for several systems, such as yeast and mammalian cells. However, relatively few protein complex purifications have been reported for plants. Here, we highlight solutions for the pitfalls and propose a major breakthrough in the quest for a better TAP tag in plants.