Poutací komplex exocyst a polarizovaný transport auxinu

The polarization of exocytosis in yeast and animals is assisted by the exocyst - an octameric vesicle tethering complex and an effector of Rab and Rho GTPases. Recently, the exocyst was described as a functional complex involved in morphogenesis also in plants. Hála et al. (2008) described involvement of exocyst complex in pollen tube growth and hypocotyls elongation in dark grown seedlings, Fendrych et al. (2010) uncovered key role of exocyst in cell plate formation, Kulich et al. (2010) emphasized the participation of exocyst in seed coat generation and Pečenková et al. (2011) described the contribution of exocyst subunits in plant defense towards the pathogens. All these processes are intimately linked to polarized secretion. Here we show involvement of exocyst in auxin efflux carriers PINs recycling. Using direct auxin transport measurement and GFP-tagged proteins, we showed that the exocyst is involved in recycling and polarization of PIN proteins and polar auxin transport regulation. Rootward polar auxin transport is compromised in loss-of-function mutants in exocyst subunits EXO70A1. On the cellular level we have detected small portion of PIN2:GFP in the "BFA-like" FM4-64 labelled compartments distinct from VHAa1 labeled endosoms. Moreover recycling of PIN1 and PIN2 is retarded in roots of...U kvasinek a živočišných buněk se na polarizaci exocytózy významně podílí exocyst - oktamerní proteinový komplex, který je řízen pomocí Rab a Rho GTPáz. Nedávno byl exocyst popsán také jako funkční komplex podílející se na morfogenezi rostlin. Hála et al., (2008) objasnil zapojení tohoto komplexu do procesů růstu pylové láčky a elongace hypokotylu etiolovaných semenáčků, Fendrych et al., (2010) odhalil klíčovou roli exocystu při tvorbě nové buněčné přepážky, Kulich et al., (2010) podtrhl význam exocystu při tvorbě semenných obalů a Pečenková et al., (2011) popsala účast podjednotek exocystu v odpovědi rostliny k napadení patogenem. Všechny tyto procesy jsou úzce spjaty s polarizovanou sekrecí. Naše práce ukazuje zapojení exocystu v recyklaci auxinových přenašečů PIN. Přímým měřením auxinového transportu jsme ukázali, že transport auxinu ke špičce kořene mutanta exo70A1 je výrazně zpomalen. Na buněčné úrovni jsme odhalili akumulaci malého množství fúzního proteinu PIN2:GFP do kompartmentů odlišných od endosomů nesoucích VHAa1. Navíc byla v kořenech mutantů exo70A1 a sec8-m1 po působení brefeldinu A výrazně zpomalena recyklace fúzních proteinů PIN1:GFP a PIN2:GFP. Ještě výraznější recyklační defekt byl patrný po prodlouženém působení BFA. Tento přístup vyvolává transcytózu - tj. relokalizaci PIN proteinů z...Department of Experimental Plant BiologyKatedra experimentální biologie rostlinFaculty of SciencePřírodovědecká fakult

Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana

The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering

Developmental plasticity of Arabidopsis hypocotyl is dependent on exocyst complex function

The collet (root–hypocotyl junction) region is an important plant transition zone between soil and atmospheric environments. Despite its crucial importance for plant development, little is known about how this transition zone is specified. Here we document the involvement of the exocyst complex in this process. The exocyst, an octameric tethering complex, participates in secretion and membrane recycling and is central to numerous cellular and developmental processes, such as growth of root hairs, cell expansion, recycling of PIN auxin efflux carriers and many others. We show that dark-grown Arabidopsis mutants deficient in exocyst subunits can form a hair-bearing ectopic collet-like structure above the true collet, morphologically resembling the true collet but also retaining some characteristics of the hypocotyl. The penetrance of this phenotypic defect is significantly influenced by cultivation temperature and carbon source, and is related to a defect in auxin regulation. These observations provide new insights into the regulation of collet region formation and developmental plasticity of the hypocotyl

Genome fractionation and loss of heterozygosity in hybrids and polyploids: Mechanisms, consequences for selection, and link to gene function

Hybridization and genome duplication have played crucial roles in the evolution of many animal and plant taxa. The subgenomes of parental species undergo considerable changes in hybrids and polyploids, which often selectively eliminate segments of one subgenome. However, the mechanisms underlying these changes are not well understood, particularly when the hybridization is linked with asexual reproduction that opens up unexpected evolutionary pathways. To elucidate this problem, we compared published cytogenetic and RNAseq data with exome sequences of asexual diploid and polyploid hybrids between three fish species; Cobitis elongatoides, C taenia, and C tanaitica. Clonal genomes remained generally static at chromosome-scale levels but their heterozygosity gradually deteriorated at the level of individual genes owing to allelic deletions and conversions. Interestingly, the impact of both processes varies among animals and genomic regions depending on ploidy level and the properties of affected genes. Namely, polyploids were more tolerant to deletions than diploid asexuals where conversions prevailed, and genomic restructuring events accumulated preferentially in genes characterized by high transcription levels and GC-content, strong purifying selection and specific functions like interacting with intracellular membranes. Although hybrids were phenotypically more similar to C taenia, we found that they preferentially retained C elongatoides alleles. This demonstrates that favored subgenome is not necessarily the transcriptionally dominant one. This study demonstrated that subgenomes in asexual hybrids and polyploids evolve under a complex interplay of selection and several molecular mechanisms whose efficiency depends on the organism's ploidy level, as well as functional properties and parental ancestry of the genomic region.Web of Science38125274525

The secretory vesicles tethering complex exocyst and the auxin transport polarization

The polarization of exocytosis in yeast and animals is assisted by the exocyst - an octameric vesicle tethering complex and an effector of Rab and Rho GTPases. Recently, the exocyst was described as a functional complex involved in morphogenesis also in plants. Hála et al. (2008) described involvement of exocyst complex in pollen tube growth and hypocotyls elongation in dark grown seedlings, Fendrych et al. (2010) uncovered key role of exocyst in cell plate formation, Kulich et al. (2010) emphasized the participation of exocyst in seed coat generation and Pečenková et al. (2011) described the contribution of exocyst subunits in plant defense towards the pathogens. All these processes are intimately linked to polarized secretion. Here we show involvement of exocyst in auxin efflux carriers PINs recycling. Using direct auxin transport measurement and GFP-tagged proteins, we showed that the exocyst is involved in recycling and polarization of PIN proteins and polar auxin transport regulation. Rootward polar auxin transport is compromised in loss-of-function mutants in exocyst subunits EXO70A1. On the cellular level we have detected small portion of PIN2:GFP in the "BFA-like" FM4-64 labelled compartments distinct from VHAa1 labeled endosoms. Moreover recycling of PIN1 and PIN2 is retarded in roots of..

Demographic population structure and fungal associations of plants colonizing High Arctic glacier forelands, Petuniabukta, Svalbard

The development of vegetation in Arctic glacier forelands has been described as unidirectional, non-replacement succession characterized by the gradual establishment of species typical for mature tundra with no species turnover. Our study focused on two early colonizers of High Arctic glacier forelands: Saxifraga oppositifolia (Saxifragaceae) and Braya purpurascens (Brassicaceae). While the first species is a common generalist also found in mature old growth tundra communities, the second specializes on disturbed substrate. The demographic population structures of the two study species were investigated along four glacier forelands in Petuniabukta, north Billefjorden, in central Spitsbergen, Svalbard. Young plants of both species occurred exclusively on young substrate, implying that soil conditions are favourable for establishment only before soil crusts develop. We show that while S. oppositifolia persists from pioneer successional stages and is characterized by increased size and flowering, B. purpurascens specializes on disturbed young substrate and does not follow the typical unidirectional, non-replacement succession pattern. Plants at two of the forelands were examined for the presence of root-associated fungi. Fungal genus Olpidium (Fungus incertae sedis) was found along a whole successional gradient in one of the forelands