Rab-dependent vesicular traffic affects female gametophyte development in Arabidopsis

Eukaryotic cells rely on the accuracy and efficiency of vesicular traffic. In plants, disturbances in vesicular trafficking are well studied in quickly dividing root meristem cells or polar growing root hairs and pollen tubes. The development of the female gametophyte, a unique haploid reproductive structure located in the ovule, has received far less attention in studies of vesicular transport. Key molecules providing the specificity of vesicle formation and its subsequent recognition and fusion with the acceptor membrane are Rab proteins. Rabs are anchored to membranes by covalently linked geranylgeranyl group(s) that are added by the Rab geranylgeranyl transferase (RGT) enzyme. Here we show that Arabidopsis plants carrying mutations in the gene encoding the beta subunit of RGT (rgtb1) exhibit severely disrupted female gametogenesis and this effect is of sporophytic origin. Mutations in rgtb1 lead to internalization of the PIN1 and PIN3 proteins from the basal membranes to vesicles in pro-vascular cells of the funiculus. Decreased transport of auxin out of the ovule is accompanied by auxin accumulation in a tissue surrounding the growing gametophyte. In addition, female gametophyte development arrests at the uni- or binuclear stage in a significant portion of the rgtb1 ovules. These observations suggest that communication between the sporophyte and the developing female gametophyte relies on Rab dependent vesicular traffic of the PIN1 and PIN3 transporters and auxin efflux out of the ovule.Joanna Rojek, Matthew R Tucker, Sara C Pinto, Michał Rychłowski, Małgorzata Lichocka, Hana Soukupova ... et al

Two tomato andoglucanases have a function during syncytium development

Globodera rostochiensis, as well as other cyst nematodes, induces formation of a multinucleate feeding site, called syncytium, in host roots. In tomato roots infected with a potato cyst nematode, the syncytium is initiated in the cortex or pericycle. Progressive cell wall dissolution and subsequent fusion of protoplasts of newly incorporated cells lead to syncytium formation. Expansion and development of a syncytium strongly depends on modifications of a cell wall, including its degradation, elongation, thickening, and formation of ingrowths within it in close contact with tracheary elements. Recent reports have demonstrated that during formation of syncytium, numerous genes of plant origin, coding for cell wall-modifying enzymes are up-re-gulated. In this research, we studied a detailed distribution and function of two tomato 1,4-β-endoglucanases in developing feeding sites induced by G. rostochiensis. In situ localization of tomato LeCel7 and LeCel8 transcripts and proteins demonstrated that these enzymes were specifically up-regulated within syncytium and in the cells adjacent to the syncytium. In non-infected roots an expression of LeCel7 and LeCel8 was observed in the root cap and lateral root primordia. Our data confirm that cell wall-modifying enzymes of plant origin have a role in a modification of cell wall within syncytia, and demonstrate that plant endoglucanases are involved in syncytia formation

Rab geranylgeranyl transferase β subunit is essential for male fertility and tip growth in Arabidopsis

Rab proteins, key players in vesicular transport in all eukaryotic cells, are post-translationally modified by lipid moieties. Two geranylgeranyl groups are attached to the Rab protein by the heterodimeric enzyme Rab geranylgeranyl transferase (RGT) αβ. Partial impairment in this enzyme activity in Arabidopsis, by disruption of the AtRGTB1 gene, is known to influence plant stature and disturb gravitropic and light responses. Here it is shown that mutations in each of the RGTB genes cause a tip growth defect, visible as root hair and pollen tube deformations. Moreover, FM 1–43 styryl dye endocytosis and recycling are affected in the mutant root hairs. Finally, it is demonstrated that the double mutant, with both AtRGTB genes disrupted, is non-viable due to absolute male sterility. Doubly mutated pollen is shrunken, has an abnormal exine structure, and shows strong disorganization of internal membranes, particularly of the endoplasmic reticulum system

Expression of Two Functionally Distinct Plant Endo-ß-1,4-Glucanases Is Essential for the Compatible Interaction Between Potato Cyst Nematode and Its Hosts

For the proliferation of their feeding sites (syncytia), the potato cyst nematode Globodera rostochiensis is thought to recruit plant endo-ß-1,4-glucanases (EGases, EC. 3.2.1.4). Reverse-transcription polymerase chain reaction experiments on tomato (Solanum lycopersicum) indicated that the expression of two out of the at least eight EGases, namely Sl-cel7 and Sl-cel9C1, is specifically upregulated during syncytium formation. In situ hybridization and immunodetection studies demonstrated that both EGases are specifically expressed inside and adjacent to proliferating syncytia. To assess the importance of Sl-cel7 and Sl-cel9C1 for nematode development, we decided to knock them out individually. Sl-cel9C1 probably is the only class C EGase in tomato, and we were unable to regenerate Sl-cel9C1¿silenced plants. Potato (S. tuberosum), a close relative of tomato, harbors at least two class C EGases, and St-cel7-or St-cel9C1¿silenced potato plants showed no obvious aberrant phenotype. Infection with potato cyst nematodes resulted in a severe reduction of the number of adult females (up to 60%) and a sharp increase in the fraction of females without eggs (up to 89%). Hence, the recruitment of CEL7, an enzyme that uses xyloglucan and noncrystalline cellulose as natural substrates, and CEL9C1, an enzyme that uses crystalline cellulose, is essential for growth and development of potato cyst nematodes

Quantitative analysis of subcellular distribution of the SUMO conjugation system by confocal microscopy imaging

Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.This work was supported by the European Research Council (grant ERC-2007-StG-205927) and Departament d’Innovació, Universitats i Empresa from the Generalitat de Catalunya (Xarxa de Referència en Biotecnologia and 2014SGR447). A.M. was supported by predoctoral fellowships FPU12/05292. This article is based upon work from COST Action (PROTEOSTASIS BM1307), supported by COST (European Cooperation in Science and Technology).Peer reviewe

Diorganotin(IV) and triorganotin(IV complexes of meso tetra (4 sulfonatophenyl) porphine: do they bind DNA?

It was observed that organometallic porphyrin systems, where the Sn(IV) residue is in side chains, coordinated via sulphonatophenyl groups of porphyrin, show interesting and peculiar in vitro activity, in agreement with the anti-tumour activity of organotin complexes