Interplay between Rab5 and PtdIns(4,5)P2 controls early endocytosis in the Drosophila germline.

International audiencePhosphoinositides have emerged as key regulators of membrane traffic through their control of the localization and activity of several effector proteins. Both Rab5 and phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] are involved in the early steps of the clathrin-dependent endocytic pathway, but little is known about how their functions are coordinated. We have studied the role of PtdIns(4,5)P(2) and Rab5 in the Drosophila germline during oogenesis. We found that Rab5 is required for the maturation of early endocytic vesicles. We show that PtdIns(4,5)P(2) is required for endocytic-vesicle formation, for Rab5 recruitment to endosomes and, consistently, for endocytosis. Furthermore, we reveal a previously undescribed role of Rab5 in releasing PtdIns(4,5)P(2), PtdIns(4,5)P(2)-binding budding factors and F-actin from early endocytic vesicles. Finally, we show that overexpressing the PtdIns(4,5)P(2)-synthesizing enzyme Skittles leads to an endocytic defect that is similar to that seen in rab5 loss-of-function mutants. Hence, our results argue strongly in favor of the hypothesis that the Rab5-dependant release of PtdIns(4,5)P(2) from endosomes that we discovered in this study is crucial for endocytosis to proceed

The Endosperm-Derived Embryo Sheath Is an Anti-adhesive Structure that Facilitates Cotyledon Emergence during Germination in Arabidopsis

International audienceGermination sensu stricto in Arabidopsis involves seed-coat and endosperm rupture by the emerging seedling root. Subsequently, the cotyledons emerge rapidly from the extra-embryonic tissues of the seed, allowing autotrophic seedling establishment [1, 2]. Seedling survival depends upon the presence of an intact seedling cuticle that prevents dehydration, which has hitherto been assumed to form the interface between the newly germinated seedling and its environment [- ]. Here, we show that in Arabidopsis, this is not the case. The primary interface between the emerging seedling and its environment is formed by an extra-cuticular endosperm-derived glycoprotein-rich structure called the sheath, which is maintained as a continuous layer at seedling surfaces during germination and becomes fragmented as cotyledons expand. Mutants lacking an endosperm-specific cysteine-rich peptide (KERBEROS [KRS]) show a complete loss of sheath production [6]. Although krs mutants have no defects in germination sensu stricto, they show delayed cotyledon emergence, a defect not observed in seedlings with defects in cuticle biosynthesis. Biophysical analyses reveal that the surfaces of wild-type cotyledons show minimal adhesion to silica beads in an aqueous environment at cotyledon emergence but that adhesion increases as cotyledons expand. In contrast, krs mutant cotyledons show enhanced adhesion at germination. Mutants with defects in cuticle biosynthesis, but no sheath defects, show a similar adhesion profile to wild-type seedlings at germination. We propose that the sheath reduces the adhesiveness of the cotyledon surface under the humid conditions necessary for seed germination and thus promotes seed-coat shedding and rapid seedling establishment

ZHOUPI and KERBEROS mediate embryo/endosperm separation by promoting the formation of an extracuticular sheath at the embryo surface

Arabidopsis thaliana seed development requires the concomitant development of two zygotic compartments, the embryo and the endosperm. Following fertilization, the endosperm expands and the embryo grows invasively through the endosperm, which breaks down. Here, we describe a structure we refer to as the embryo sheath that forms on the surface of the embryo as it starts to elongate. The sheath is deposited outside the embryonic cuticle and incorporates endosperm-derived material rich in extensin-like molecules. Sheath production is dependent upon the activity of ZHOUPI, an endosperm-specific transcription factor necessary for endosperm degradation, embryo growth, embryo-endosperm separation, and normal embryo cuticle formation. We show that the peptide KERBEROS, whose expression is ZHOUPI dependent, is necessary both for the formation of a normal embryo sheath and for embryo-endosperm separation. Finally, we show that the receptor-like kinases GSO1 and GSO2 are required for sheath deposition at the embryo surface but not for production of sheath material in the endosperm. We present a model in which sheath formation depends on the coordinated production of material in the endosperm and signaling within the embryo, highlighting the complex molecular interaction between these two tissues during early seed development.International Mobility Programme to Strengthen Skills and Excellence in Research for Agricultur

Divergent Functional Diversification Patterns in the SEP/AGL6/AP1 MADS-box Transcription Factor Superclade

International audienceMembers of SEPALLATA (SEP) and APETALA1 (AP1)/SQUAMOSA (SQUA) MADS-box transcriptionfactor subfamilies play key roles in floral organ identity determination and floral meristem determinacyin the Rosid species Arabidopsis. Here, we present a functional characterization of the seven SEP/AGL6and four AP1/SQUA genes in the distant Asterid species Petunia x hybrida petunia. Based on the analysisof single and higher order mutants, we report that the petunia SEP1/SEP2/SEP3 orthologs together withAGL6 encode classical SEP floral organ identity and floral termination functions, with a master role forthe petunia SEP3 ortholog FLORAL BINDING PROTEIN 2 (FBP2). By contrast, the FBP9 subclademembers FBP9 and FBP23, for which no clear ortholog is present in Arabidopsis, play a major role indetermining floral meristem identity together with FBP4, while contributing only moderately to floralorgan identity. In turn, the four members of the petunia AP1/SQUA subfamily redundantly are requiredfor inflorescence meristem identity, and act as B-function repressors in the first floral whorl, togetherwith BEN/ROB genes. Overall, these data together with studies in other species suggest majordifferences in the functional diversification of the SEP/AGL6 and AP1/SQUA MADS-box subfamiliesduring angiosperm evolution

Divergence of the floral a-function between an asterid and a rosid species

The ABC model is widely used as a genetic framework for understanding floral development and evolution. In this model, the A-function is required for the development of sepals and petals and to antagonize the C-function in the outer floral whorls. In the rosid species Arabidopsis thaliana, the AP2-type AP2 transcription factor represents a major A-function protein, but how the A-function is encoded in other species is not well understood. Here, we show that in the asterid species petunia (Petunia hybrida), AP2B/BLIND ENHANCER (BEN) confines the C-function to the inner petunia floral whorls, in parallel with the microRNA BLIND. BEN belongs to the TOE-type AP2 gene family, members of which control flowering time in Arabidopsis. In turn, we demonstrate that the petunia AP2-type REPRESSOR OF B-FUNCTION (ROB) genes repress the B-function (but not the C-function) in the first floral whorl, together with BEN. We propose a combinatorial model for patterning the B-and C-functions, leading to the homeotic conversion of sepals into petals, carpels, or stamens, depending on the genetic context. Combined with earlier results, our findings suggest that the molecular mechanisms controlling the spatial restriction of the floral organ identity genes are more diverse than the well-conserved B and C floral organ identity functions

ZmZHOUPI, an endosperm-specific basic helix-loop-helix transcription factor involved in maize seed development

In angiosperm seeds the embryo is embedded within the endosperm, which is in turn enveloped by the seed coat, making inter-compartmental communication essential for coordinated seed growth. In this context the basic helix-loop-helix domain transcription factor AtZHOUPI (AtZOU) fulfils a key role in both the lysis of the transient endosperm and in embryo cuticle formation in Arabidopsis thaliana. In maize (Zea mays), a cereal with a persistent endosperm, a single gene, ZmZOU, falls into the same phylogenetic clade as AtZOU. Its expression is limited to the endosperm where it peaks during the filling stage. In Zm-ZOURNA interference knock-down lines embryo size is slightly reduced and the embryonic suspensor and the adjacent embryo surrounding region show retarded breakdown. Ectopic expression of ZmZOU reduces stomatal number, possibly due to inappropriate protein interactions. ZmZOU forms functional heterodimers with AtICE/AtSCREAM and the closely related maize proteins ZmICEb and ZmICEc, but its interaction is more efficient with the ZmICEa protein, which shows sequence divergence and only has close homologues in other monocotyledonous species. Consistent with the observation that these complexes can trans-activate target gene promoters from Arabidopsis, ZmZOU partially complements the Atzou-4 mutant. However, structural, trans-activation and gene expression data support the hypothesis that ZmZOU and ZmICEa may have coevolved to form a functional complex unique to monocot seeds. This divergence may explain the reduced functionality of ZmZOU in Arabidopsis, and reflect functional specificities which are unique to the monocotyledon lineage