The MtSNF4b subunit of the sucrose non-fermenting-related kinase complex connects after-ripening and constitutive defense responses in seeds of Medicago truncatula

Dormant seeds are capable of remaining alive in the hydrated state for extended periods of time without losing vigor, until environmental cues or after-ripening result in the release of dormancy. Here, we investigated the possible role of the regulatory subunit of the sucrose non-fermenting-related kinase complex, MtSNF4b, in dormancy of Medicago truncatula seeds. Expression of MtSNF4b and its involvement in a high-molecular-weight complex are found in dormant seeds, whereas imbibition of fully after-ripened, non-dormant seeds leads to dissociation of the complex. MtSNF4b is capable of complementing the yeast Δsnf4 mutant and of interacting with the MtSnRK1 α-subunit in a double hybrid system. Transcriptome analyses on freshly harvested and after-ripened RNAi Mtsnf4b and wild-type embryos implicate MtSNF4b in the defense response in hydrated dormant embryonic tissues, affecting the expression of genes encoding enzymes of flavonoid and phenylpropanoid metabolism, WRKY transcription factors and pathogenesis-related proteins. Silencing MtSNF4b also increased the speed of after-ripening during dry storage, an effect that appears to be related to a change in base water potential. No significant difference in ABA content or sensitivity was detected between mutant and wild-type seeds. Pharmacological studies using hexoses and sugar analogs revealed that mannose restored germination behavior and expression of the genes PAL, CHR and IFR in RNAi Mtsnf4b seeds towards that of the wild-type, suggesting that MtSNF4b might act upstream of sugar-sensing pathways. Overall, the results suggest that MtSNF4b participates in regulation of a constitutively activated defense response in hydrated, dormant seeds

D'une revue de la littérature sur l'évaluation de la durabilité en agriculture à la méthode IDEA comme outil opérationnel de mise en oeuvre d'une politique agricole durable

International audienceThe agreement on the Health Check of the Common Agricultural Policy (CAP, 2008) is a key step towards a more sustainable agriculture. Henceforth, member states shall be authorized to transfer a part of the direct payments from conventional agriculture to different types of agriculture more sustainable. The difficulty for Member States is now to give a concrete meaning of this concept which has also to be accepted by European Commission and farmers too. That is why, in France, the scientific community is asked by authorities to evaluate the ability of existing farm sustainability methods to assess farming systems in an operational way. The aim of this paper is to illustrate how the French method called IDEA (Indicateurs de Durabilité des Exploitations Agricoles or Farm Sustainability Indicators) should support the implementation of this new challenge. After briefly going back to the main economic measures promoting sustainable agriculture, we present from a large state of the art, (1) a synthesis of the sustainable agriculture concept and (2) results on main features of farm sustainability assessment methods developed since 1990. Among all of them, the scientific approach of the IDEA method is developed and then illustrated by tests of the IDEA method on French case studies. The last part is dedicated to the presentation of two recently emerged opportunities regarding the use of the IDEA method. The first opportunity concerns the application of the IDEA method on a national scale. We focus on the capacity of this method, developed on a farm level, to be used on an aggregated level (the national one) with data issuing from national agricultural data surveys. The second opportunity is related to the use of two systemic IDEA indicators (economic efficiency / rate of ecological infrastructures in the cultivated area) to be integrated into official rules for certifying a High Environmental Value agriculture

Storage of tissue-type plasminogen activator in Weibel-Palade bodies of human endothelial cells

Tissue-type plasminogen activator (t-PA) is acutely released by endothelial cells. Although its endothelial storage compartment is still not well defined, t-PA release is often accompanied by release of von Willebrand factor (vWf), a protein stored in Weibel-Palade bodies. We investigated, therefore, whether t-PA is stored in these secretory organelles. Under basal culture conditions, a minority of human umbilical vein endothelial cells (HUVEC) exhibited immunofluorescent staining for t-PA, which was observed only in Weibel-Palade bodies. To increase t-PA expression, HUVEC were infected with a t-PA recombinant adenovirus (AdCMVt-PA). Overexpressed t-PA was detected in Weibel-Palade bodies and acutely released together with endogenous vWf by thrombin or calcium ionophore stimulation. In contrast, plasminogen activator inhibitor type 1 and urokinase were not detected in Weibel-Palade bodies after adenovirus-mediated overexpression. Infection of HUVEC with proinsulin recombinant adenovirus resulted in the storage of insulin in Weibel-Palade bodies, indicating that these organelles can also store nonendothelial proteins that show regulated secretion. Infection of AtT-20 pituitary cells, a cell type with regulated secretion, with AdCMVt-PA resulted in the localization of t-PA in adrenocorticotropic hormone-containing granules, indicating that t-PA can be diverted to secretory granules independently of vWf. Coinfection of AtT-20 cells with AdCMVt-PA and proinsulin recombinant adenovirus resulted in the colocalization of t-PA and insulin in the same granules. Taken together, these results suggest that HUVEC have protein sorting mechanisms similar to those of other regulated secretory cells. Although the results did not exclude an alternative storage site for t-PA in HUVEC, they established that t-PA can be stored in Weibel-Palade bodies. This finding may explain the acute coordinate secretion of t-PA and vWf

Interaction of anti-phospholipid antibodies with late endosomes of human endothelial cells

Anti-phospholipid antibodies (APLAs) are associated with thrombosis and/or recurrent pregnancy loss. APLAs bind to anionic phospholipids directly or indirectly via a cofactor such as β2-glycoprotein 1 (β2GPI). The lipid target of APLA is not yet established. Recently, we observed that APLAs in vitro can bind lysobisphosphatidic acid (LBPA). The internal membranes of late endosomes are enriched in this phospholipid. The current study was undertaken to determine to what extent binding of APLA to LBPA is correlated with binding to cardiolipin and to β2GPI and to determine whether patient antibodies interact with late endosomes of human umbilical vein endothelial cells (HUVECs) and thus modify the intracellular trafficking of proteins. Binding of patient immunoglobulin G (n=37) to LBPA was correlated significantly with binding to cardiolipin. Although LBPA binding was correlated to a lesser extent with β2GPI binding, we observed that β2GPI binds with high affinity to LBPA. Immunofluorescence studies showed that late endosomes of HUVECs contain LBPA. Patient but not control antibodies recognized late endosomes, but not cardiolipin-rich mitochondria, even when we used antibodies that were immunopurified on cardiolipin. Incubation of HUVECs with patient plasma samples immunoreactive toward LBPA resulted in an accumulation of the antibodies in late endosomes and led to a redistribution of the insulinlike growth factor 2/mannose-6-phosphate receptor from the Golgi apparatus to late endosomes. Our results suggest that LBPA is an important lipid target of APLA in HUVECs. These antibodies are internalized by the cells and accumulate in late endosomes. By modifying the intracellular trafficking of proteins, APLA could contribute to several of the proposed pathogenic mechanisms leading to the antiphospholipid syndrome

Rab3D and annexin A2 play a role in regulated secretion of vWF, but not tPA, from endothelial cells

von-Willebrand factor (vWF) and tissue-type plasminogen activator (tPA) are products of endothelial cells acutely released into the vasculature following cell activation. Both factors are secreted after intraendothelial Ca(2+) mobilization, but exhibit opposing physiological effects with vWF inducing coagulation and tPA triggering fibrinolysis. To identify components that could regulate differentially the release of pro- and antithrombogenic factors, we analyzed the contribution of Rab3D and the annexin A2/S100A10 complex, proteins implicated in exocytotic events in other systems. We show that mutant Rab3D proteins interfere with the formation of bona fide Weibel–Palade bodies (WPbs), the principal storage granules of multimeric vWF, and consequently the acute, histamine-induced release of vWF. In contrast, neither appearance nor exocytosis of tPA storage granules is affected. siRNA-mediated downregulation of annexin A2/S100A10 and disruption of the complex by microinjection of peptide competitors result in a marked reduction in vWF but not tPA secretion, without affecting the appearance of WPbs. This indicates that distinct mechanisms underlie the acute secretion of vWF and tPA, enabling endothelial cells to fine-regulate the release of thrombogenic and fibrinolytic factors