Interplay between Jasmonic Acid, Phosphate Signaling and the Regulation of Glycerolipid Homeostasis in Arabidopsis.

Jasmonic acid (JA) biosynthesis and signaling are activated in Arabidopsis cultivated in phosphate (Pi) deprived conditions. This activation occurs mainly in photosynthetic tissues and is less important in roots. In leaves, the enhanced biosynthesis of JA coincides with membrane glycerolipid remodeling triggered by the lack of Pi. We addressed the possible role of JA on the dynamics and magnitude of glycerolipid remodeling in response to Pi deprivation and resupply. Based on combined analyses of gene expression, JA biosynthesis and glycerolipid remodeling in wild-type Arabidopsis and in the coi1-16 mutant, JA signaling seems important in the determination of the basal levels of phosphatidylcholine, phosphatidic acid (PA), monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol. JA impact on MGDG steady state level and fluctuations seem contradictory. In the coi1-16 mutant, the steady state level of MGDG is higher, possibly due to a higher level of PA in the mutant, activating MGD1, and to an increased expression of MGD3. These results support a possible impact of JA in limiting the overall content of this lipid. Concerning lipid variations, upon Pi deprivation, JA seems rather associated with a specific MGDG increase. Following Pi resupply, whereas the expression of glycerolipid remodeling genes returns to basal level, JA biosynthesis and signaling genes are still upregulated, likely due to a JA-induced positive feedback remaining active. Distinct impacts on enzymes synthesizing MGDG, that is, downregulating MGD3, possibly activating MGD1 expression and limiting the activation of MGD1 via PA, might allow JA playing a role in a sophisticated fine tuning of galactolipid variations.

Strigolactone promotes degradation of DWARF14, an α/ß hydrolase essential for strigolactone signaling in Arabidopsis.

Strigolactones (SLs) are phytohormones that play a central role in regulating shoot branching. SL perception and signaling involves the F-box protein MAX2 and the hydrolase DWARF14 (D14), proposed to act as an SL receptor. We used strong loss-of-function alleles of the Arabidopsis thaliana D14 gene to characterize D14 function from early axillary bud development through to lateral shoot outgrowth and demonstrated a role of this gene in the control of flowering time. Our data show that D14 distribution in vivo overlaps with that reported for MAX2 at both the tissue and subcellular levels, allowing physical interactions between these proteins. Our grafting studies indicate that neither D14 mRNA nor the protein move over a long range upwards in the plant. Like MAX2, D14 is required locally in the aerial part of the plant to suppress shoot branching. We also identified a mechanism of SL-induced, MAX2-dependent proteasome-mediated degradation of D14. This negative feedback loop would cause a substantial drop in SL perception, which would effectively limit SL signaling duration and intensity.

Chemical Genetics in Dissecting Membrane Glycerolipid Functions.

Chemical genetics has emerged as a powerful approach to dissect biological processes, based on the utilization of small molecules disturbing the function of specific target proteins. By analogy with classical genetics, 'reverse chemical genetics' refers to the utilization of drugs acting on a known target, enabling its functional characterization at the levels of the cells, tissues and organisms. Likewise, 'direct chemical genetics' refers to the utilization of a drug of unknown mode of action, but triggering a phenotype of interest. In that case, one has to identify the target(s) possibly blocked (or possibly activated) by the small molecule. This chapter illustrates both approaches, like the analysis of the elongation of fatty acids, the biosynthesis of galactoglycerolipids or the catabolism of phosphoglycerolipids by reverse chemical genetics or the study of the membrane glycerolipid remodeling triggered upon phosphate starvation, by direct chemical genetics.

[How independent pharmacological screenings in plants and humans led to the discovery of a new family of lipid metabolism inhibitors]. / Identification par deux criblages simultanés indépendants d'une famille d'inhibiteurs du métabolisme des glycérolipides.

In eukaryotic cells, phosphatidic acid (PA) and diacylglycerol (DAG), are at the origin of all membrane glycerolipids. Their interconversion is achieved by dephosphorylation of PA and phosphorylation of DAG: they form therefore a metabolic hub. PA and DAG are also known to be versatile signaling molecules. Two independent pharmacological screenings conducted on plant and human targets, led to the discovery of a new family of compounds acting on enzymes binding to either PA or DAG, in biological contexts that seemed initially independent. On the one hand, in plants, monogalactosyldiacylglycerol synthases (MGDG synthases or MGD) are responsible for the synthesis of MGDG, which is the most profuse lipid of photosynthetic membranes, and thus essential for metabolism and development. MGD use DAG as substrate. On the other hand, in mammals, phospholipases D (PLD), that produce PA, are involved in a variety of signaling cascades that control a broad spectrum of cellular functions, and play a role in the development of cancers. The two independent pharmacological screenings described in this review aimed to identify inhibitory molecules of either MGD of the plant model Arabidopsis, or human PLD. In both cases, the obtained molecules are piperidinyl-benzimidazolone derivatives, thereby allowing to propose this family of molecules as a novel source of inspiration for the search of compounds interfering with glycerolipid metabolism, that could be useful for other biological and therapeutics contexts.

GeBP/GPL transcription factors regulate a subset of CPR5-dependent processes.

The CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (CPR5) gene of Arabidopsis (Arabidopsis thaliana) encodes a putative membrane protein of unknown biochemical function and displays highly pleiotropic functions, particularly in pathogen responses, cell proliferation, cell expansion, and cell death. Here, we demonstrate a link between CPR5 and the GLABRA1 ENHANCER BINDING PROTEIN (GeBP) family of transcription factors. We investigated the primary role of the GeBP/GeBP-like (GPL) genes using transcriptomic analysis of the quadruple gebp gpl1,2,3 mutant and one overexpressing line that displays several cpr5-like phenotypes including dwarfism, spontaneous necrotic lesions, and increased pathogen resistance. We found that GeBP/GPLs regulate a set of genes that represents a subset of the CPR5 pathway. This subset includes genes involved in response to stress as well as cell wall metabolism. Analysis of the quintuple gebp gpl1,2,3 cpr5 mutant indicates that GeBP/GPLs are involved in the control of cell expansion in a CPR5-dependent manner but not in the control of cell proliferation. In addition, to our knowledge, we provide the first evidence that the CPR5 protein is localized in the nucleus of plant cells and that a truncated version of the protein with no transmembrane domain can trigger cpr5-like processes when fused to the VP16 constitutive transcriptional activation domain. Our results provide clues on how CPR5 and GeBP/GPLs play opposite roles in the control of cell expansion and suggest that the CPR5 protein is involved in transcription.

New Insights on Thylakoid Biogenesis in Plant Cells.

Photosynthetic membranes, or thylakoids, are the most extensive membrane system found in the biosphere. They form flattened membrane cisternae in the cytosol of cyanobacteria and in the stroma of chloroplasts. The efficiency of light energy capture and conversion, critical for primary production in ecosystems, relies on the rapid expansion of thylakoids and their versatile reorganization in response to light changes. Thylakoid biogenesis results from the assembly of a lipid matrix combined with the incorporation of protein components. Four lipid classes are conserved from cyanobacteria to chloroplasts: mono- and digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, and phosphatidyldiacylglycerol. This review focuses on the production and biophysical properties of galactolipids, making them determinant factors for the nonvesicular/nonlamellar biogenesis and for the three-dimensional architecture of nascent thylakoids. The regulation of MGD1, the committing enzyme of galactolipid biosynthesis in Arabidopsis, via feedback regulatory loops and control of protein binding to membranes, is also detailed.

GeBP and GeBP-like proteins are noncanonical leucine-zipper transcription factors that regulate cytokinin response in Arabidopsis.

Understanding the role of transcription factors (TFs) is essential in reconstructing developmental regulatory networks. The plant-specific GeBP TF family of Arabidopsis thaliana (Arabidopsis) comprises 21 members, all of unknown function. A subset of four members, the founding member GeBP and GeBP-like proteins (GPL) 1, 2, and 3, shares a conserved C-terminal domain. Here we report that GeBP/GPL genes represent a newly defined class of leucine-zipper (Leu-zipper) TFs and that they play a redundant role in cytokinin hormone pathway regulation. Specifically, we demonstrate using yeast, in vitro, and split-yellow fluorescent protein in planta assays that GeBP/GPL proteins form homo- and heterodimers through a noncanonical Leu-zipper motif located in the C-terminal domain. A triple loss-of-function mutant of the three most closely related genes gebp gpl1 gpl2 shows a reduced sensitivity to exogenous cytokinins in a subset of cytokinin responses such as senescence and growth, whereas root inhibition is not affected. We find that transcript levels of type-A cytokinin response genes, which are involved in the negative feedback regulation of cytokinin signaling, are higher in the triple mutant. Using a GPL version that acts as a constitutive transcriptional activator, we show that the regulation of Arabidopsis response regulators (ARRs) is mediated by at least one additional, as yet unknown, repressor acting genetically downstream in the GeBP/GPL pathway. Our results indicate that GeBP/GPL genes encode a new class of unconventional Leu-zipper TF proteins and suggest that their role in the cytokinin pathway is to antagonize the negative feedback regulation on ARR genes to trigger the cytokinin response.