YODA and MPK6 have critical roles in postembryonic root development

Abstracts / New Biotechnology 33S (2016) S1–S213

for prevention of losses of biologically valuable components during the processing and storage of northern berries. http://dx.doi.org/10.1016/j.nbt.2016.06.1283

P18-32 Analysis of cold-regulated microRNAs in soybean 2,∗ , Joanna Gracz 1 , Agata Tyczewska 1 ´ Jakub Kuczynski 1 2

Institute of Bioorganic Chemistry, Poland PAS, Poland

Soybean is a legume species of great economic importance due to its extensive use as animal feed and oil source. It is a crop plant originating from South Asia and therefore requires high temperatures and humidity for growth and development. Poland, as well as majority of European area, due to its temperate climate was excluded until recently from soybean cultivation. Nowadays farmers can grow soybean in a Polish climate as long as they use varieties that are day-neutral, but also, if not primarily, cold-resistant. Therefore breeders made efforts to create cold resistant varieties. In our laboratory we are trying to elucidate molecular basis of cold stress resistance of chosen soybean varieties. First, we focused on Augusta, bred by Prof. Jerzy Nawracała in the Department of Genetics and Plant Breeding, Poznan´ University of Life Sciences. Small regulatory RNAs have been shown to play crucial roles in precise and timely regulation of stress-responsive gene networks. Therefore we chose few miRNA, such as miR167 or miR398, which have previously been associated with plant abiotic stress response and we verified their expression levels after cold stress treatment as well as in control conditions, in three developmental stages of soybean. To assess the microRNA expression patterns we used Northern-blot method and for estimation of expression levels of transcripts targeted by selected miRNAs – Real Time PCR. http://dx.doi.org/10.1016/j.nbt.2016.06.1284

P18-33 YODA and MPK6 have critical roles in postembryonic root development Despina Samakovli 2,∗ , Veronika Zapletalová 1 , George Komis 2 , 1 , Miroslav Oveˇ ˇ Olga Samajová cka 2 , Anna Kuchaˇrová 1 , Jozef 2 ˇ Samaj 1 2

Palacky University Olomouc, Czech Republic Faculty of Natural Science – CRH, Czech Republic

The YODA signalling pathway plays a major role in establishing different cell fates in embryogenesis and stomata development. The role of YODA, a mitogen activated protein kinase kinase kinase 4 (MAPKKK4) upstream of mitogen activated protein kinase 6 (MPK6) was studied during post-embryonic root development of Arabidopsis thaliana. Loss- and gain-of-function YODA mutants (yda1 and DNyda1) were characterized in terms of root patterning. Disoriented cell divisions in primary and lateral roots were observed in yda1 and DNyda1, while both mutants displayed abortive cytokinesis and differential subcellular localization of MPK6 and microtubule associated protein 65-1 (MAP 65-1). It was accompanied by deregulated expression of tangled 1 (TAN 1), phragmoplast orienting kinesin 1 (POK 1) and gamma tubulin complex protein 4 (GCP 4). Our findings showed that MPK6 is localized at preprophase bands (PPBs) and phragmoplasts. The mpk6-4 mutant trans-

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formed with MPK6AEF (inactive version of MPK6) showed a root phenotype similar to that of yda1. These data indicate that YODA and MPK6 are important players in the regulation of postembryonic root development and mitotic microtubule (PPB and phragmoplast) organization. This work was supported by the Grant No. LO1204 (Sustainable development of research in the Centre of the Region Haná) from the National Program of Sustainability I, MEYS. http://dx.doi.org/10.1016/j.nbt.2016.06.1285

P18-34 Super-resolution microscopy for dynamic imaging of plant cortical microtubules 1,∗ , George Komis 2 , Martin Mistrík 1 , Miroslav ˇ Olga Samajová 3 ˇ Oveˇcka 2 , Jiri Bartek 1 , Jozef Samaj 1

Palack´ y University Olomouc, Czech Republic Centre of the Region Haná for Biotechnological and Agricultural Research, Czech Republic 3 Institute of Molecular and Translational Medicine, Czech Republic 2

Although the plant-oriented applications of super-resolution microscopy remain limited, different super-resolution applications can be employed depending on the type of the sample under examination, the desired resolution in all directions, or the temporal frame rate of image acquisition. Using a commercially available structured illumination microscopy (SIM) we provide a protocol that may be widely applied to the imaging of plant cells expressing appropriate fluorescent markers. Highnumerical aperture objectives were combined with SIM adopted for the optimal documentation of Arabidopsis epidermal cells in order to elucidate microtubule size, bundling and branch formation. We also characterize quantitative parameters of plus end associated dynamic instability events at subdiffraction resolution in seedlings of wild-type and mitogen-activated protein kinase mutant mpk4 expressing a GFP-MBD and a GFP-TUA6 microtubular markers. SIM imaging revealed nano-sized defects in microtubule bundles, spatially resolved nascent microtubule branching and release, and finally allowed the quantification of individual microtubules within cortical bundles of wild-type and mutant mpk4 plants. Dynamic live SIM imaging allowed the visualization of subdiffraction, short-lived excursions of the microtubule plus end, and dynamic instability behavior of both ends during free, intrabundle, or microtubule-templated microtubule growth and shrinkage. Thus, the present work demonstrates the potential of SIM for superresolution time-lapse imaging of plant cells, showing unprecedented details accompanying microtubule dynamic organization. This work was supported by the Grant No. LO1204 (Sustainable development of research in the Centre of the Region Haná) from the National Program of Sustainability I, MEYS. http://dx.doi.org/10.1016/j.nbt.2016.06.1286