Analysis of exocyst function in endodermis reveals its widespread contribution and specificity of action

The exocyst is the main plasma membrane vesicle-tethering complex in eukaryotes and is composed of eight different subunits. Yet, in plant genomes, many subunits display multiple copies, thought to reflect evolution of complex subtypes with divergent functions. In Arabidopsis thaliana root endodermal cells, the isoform EXO70A1 is required for positioning of CASP1 at the Casparian Strip Domain, but not for its non-targeted secretion to the plasma membrane. Here, we show that exo84b resembles exo70a1 mutants regarding CASP1 mistargeting and secretion of apoplastic proteins, but exo84b additionally affects secretion of other integral plasma membrane proteins. Moreover, conditional, cell-type-specific gene editing of the single-copy core component SEC6 allows visualization of secretion defects in plant cells with a complete lack of exocyst complex function. Our approach opens avenues for deciphering the complexity/diversity of exocyst functions in plant cells and enables analysis of central trafficking components with lethal phenotypes. Genetic analysis of exocyst isoforms reveals their distinct roles in cargo secretion.Peer reviewe

Mutations in UDP-Glucose:Sterol Glucosyltransferase in Arabidopsis Cause Transparent Testa Phenotype and Suberization Defect in Seeds

In higher plants, the most abundant sterol derivatives are steryl glycosides (SGs) and acyl SGs. Arabidopsis (Arabidopsis thaliana) contains two genes, UGT80A2 and UGT80B1, that encode UDP-Glc:sterol glycosyltransferases, enzymes that catalyze the synthesis of SGs. Lines having mutations in UGT80A2, UGT80B1, or both UGT80A2 and UGT8B1 were identified and characterized. The ugt80A2 lines were viable and exhibited relatively minor effects on plant growth. Conversely, ugt80B1 mutants displayed an array of phenotypes that were pronounced in the embryo and seed. Most notable was the finding that ugt80B1 was allelic to transparent testa15 and displayed a transparent testa phenotype and a reduction in seed size. In addition to the role of UGT80B1 in the deposition of flavanoids, a loss of suberization of the seed was apparent in ugt80B1 by the lack of autofluorescence at the hilum region. Moreover, in ugt80B1, scanning and transmission electron microscopy reveals that the outer integument of the seed coat lost the electron-dense cuticle layer at its surface and displayed altered cell morphology. Gas chromatography coupled with mass spectrometry of lipid polyester monomers confirmed a drastic decrease in aliphatic suberin and cutin-like polymers that was associated with an inability to limit tetrazolium salt uptake. The findings suggest a membrane function for SGs and acyl SGs in trafficking of lipid polyester precursors. An ancillary observation was that cellulose biosynthesis was unaffected in the double mutant, inconsistent with a predicted role for SGs in priming cellulose synthesis

Characterization of THESEUS1, a membrane anchored receptor-like kinase, involved in cell wall integrity signaling in Arabidopsis thaliana

La croissance des végétaux nécessite une coordination entre la synthèse de la paroi et le remodelage des polysaccharides. Cette coordination semble impliquer un retro-signal de la paroi vers le cytoplasme grâce à des senseurs pariétaux restant encore inconnus. Afin d étudier ces phénomènes, nous utilisons comme modèle la croissance de l hypocotyle à l obscurité chez Arabidopsis. L inhibition de synthèse de cellulose chez des mutants ou après traitement par des herbicides entraîne une inhibition de croissance. Ce phénomène semble être une inhibition active car des mutations peuvent restaurer le défaut de croissance du mutant nain procuste1 sans restauration de sa déficience en cellulose. Un locus (THE1), identifié par trois allèles, a été cloné et code un récepteur kinase. Deux allèles the1 causent une substitution d acide aminé dans le domaine extracellulaire de la protéine alors que le troisième allèle (the1-3) est causé par l insertion d un ADN-T tronquant plus de la moitié de la protéine. La mutation the1-3 ne révèle son phénotype qu en contexte de perturbation pariétale, suggérant un rôle de senseur d intégrité pariétale. Une fusion THE1-GFP montre que THE1 est ancré dans la membrane plasmique. La surexpression de cette construction entraîne une réponse accrue à une altération pariétale. THE1 possède un domaine kinase actif possédant une activité d autophosphorylation in vitro. L analyse du transcriptome des simples et doubles mutants prc1 et the1 apporte des données sur la voie de signalisation de THE1. L ensemble de nos données converge vers un rôle de THE1 comme senseur d intégrité pariétale contrôlant le dépôt de la paroi pendant l élongation.Growth in plants requires the coordination between wall synthesis and cell expansion-promoting polysaccharide remodeling. This coordination presumably requires feedback signaling from the wall to the cytoplasm through putative cell wall sensors. To study these processes, we are using the dark-grown hypocotyl of Arabidopsis as a model system. The inhibition of cellulose synthesis in mutants or by herbicides, leads to a rapid inhibition of cell elongation. This inhibition involves an active process since second site mutants were found that attenuate the growth defect of the cellulose-deficient mutant prc1 without restoring the cellulose defect. One locus (THE1) identified by three second-site suppressor mutations was cloned and encodes a novel membrane-bound ser/thr receptor-like kinase (RLK). Two the1 alleles cause amino acid changes in the extracellular domain of the RLK and the third (the1-3) carries a T-DNA insertion eliminating two thirds of this protein. the1-3 separated from the prc1 mutation only showed a phenotype in the context of particular cell wall perturbations, suggesting a role for THE1 in sensing the integrity of the cell wall. THE1 transcripts are present in growing zones of many organs. THE1 is present in the plasma membrane, as shown by a THE1:GFP localization. Overexpression of a THE1 enhanced the response to cell wall damage. THE1 possess a functional kinase domain with in vitro autophosphorylation activity. Microarray analysis of single and double mutants (prc1/the1) provided insights into THE1 signaling. Together, these results suggest a role for THE1 as a cell wall integrity sensor in the coordination between cell wall deposition and cell elongation.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

<i>COBRA</i> transcript level increase in the suppressed <i>cobra</i>.

<p>(<b>A</b>) qPCR determined <i>COBRA</i> transcript level in etiolated seedlings grown on ½ MS medium. Genotypes, mean and SE are indicated, n = 3 pools of seedlings. Asterisks indicate <i>P</i> values for comparison with <i>cob-6</i>: * <i>P</i><0.05; ** <i>P</i><0.001 (Student’s <i>t</i>-test). (<b>B</b>) Phenotype comparison of etiolated <i>srf6-1cob-6</i> and <i>srf6-1cob-4</i> seedlings. Pictures are representative of multiple plants for each genotype.</p

The effect of epigenome modification on <i>srf6</i> SALK T-DNA caused <i>cob-6</i> suppression.

<p>Etiolated seedlings grown on ½ MS medium with (<b>A</b>) 30 µM of DNA methylation inhibitor 5-Azacytidine (5Aza) (<b>B</b>) 30 µM of DNA methylation inhibitor zebularine (Zeb). (<b>C</b>) 1.6 µM of histone deacetylase inhibitor Trichostatin A (TSA). Genotypes are indicated. Pictures are representative of multiple plants for each genotype. (<b>D</b>) qPCR determined <i>COBRA</i> transcript level in etiolated seedlings grown on ½ MS medium with 30 µM 5-azacytidine (5-Aza) normalised against Col-0 without 5-Aza. Genotypes, mean and SE are indicated, n = 3 pools of seedlings. (<b>E</b>) The effect of DNA methylation mutants on <i>srf6</i> SALK T-DNA caused <i>cob-6</i> suppression. Genotypes are indicated. Pictures are representative of multiple plants for each genotype.</p

Effect of the different DNA methylation mutants on the <i>trans cob-6</i> SALK T-DNA interaction.

*<p>A, B,C,D,E indicate ranking by Duncan test at <i>P</i>≤0.01;</p><p>a,b,c,d,e indicate ranking by Duncan test at <i>P</i>≤0.05.</p

Hypocotyl length of the randomly selected SALK T-DNA <i>r1, r2</i> and <i>r3cob-6</i> and the <i>srf4-1</i> SAIL T-DNA <i>cob-6</i> double mutants.

*<p>A, B and C indicate ranking by Duncan test at <i>P</i>≤0.01;</p><p>a, b and c indicate ranking by Duncan test at <i>P</i>≤0.05.</p

Inhibition of very long acyl chain sphingolipid synthesis modifies membrane dynamics during plant cytokinesis

Plant cytokinesis requires intense membrane trafficking and remodeling to form a specific membrane structure, the cell plate that will ultimately separate the daughter cells. The nature and the role of lipids involved in the formation of the cell plate remain unclear. Plant membranes are particularly rich in sphingolipids such as glucosylceramides with long (16 carbons) or very long (24 carbons) acyl chains. We reveal here that inhibition of the synthesis of sphingolipids with very long acyl chains induces defective cell plates with persistent vesicular structures and large gaps. Golgi-derived vesicles carrying material toward the cell plate display longer vesicle-vesicle contact time and their cargos accumulate at the cell plate, suggesting membrane fusion and/or recycling defects. In vitro fusion experiments between artificial vesicles show that glycosphingolipids with very long acyl chains stimulate lipid bilayer fusion. Therefore we propose that the very long acyl chains of sphingolipids are essential structural determinants for vesicle dynamics and membrane fusion during cytokinesis

<i>srf6</i> SALK T-DNA triggered suppression of <i>cobra</i> phenotype and inheritance of <i>epicob-6</i>.

<p>(<b>A</b>) Quantification of hypocotyl length in four-day-old dark grown seedlings. Genotypes, mean and SE are indicated, n = 30–40. (<b>B</b>) Four-day-old dark grown seedlings of Col-0, <i>srf6-1</i>, <i>srf6-1cob-6</i>, <i>epicob-6</i> and <i>cob-6</i>. (<b>C</b>) Phenotype comparison of etiolated <i>epicob-6</i> for four generations.</p