Collection of Phaseolus genetic resources in the Slovene Plant Gene Bank

Transcription

1 Collection of Phaseolus genetic resources in the Slovene Plant Gene Bank SMARTLEG Meeting April 2017, Ljubljana, Slovenia Jelka ŠUŠTAR VOZLIČ

2 Slovene Plant Gene Bank Agricultural Institute of Slovenia, Ljubljana Slovenian Institute for Hop Research and and Brewing, Žalec SLOVENE PLANT GENE BANK Central Seed Bank (AIS LJ) PSARS (MAE) - Central Database Biotechnical Faculty University of Ljubljana MEDPLANT Database NGO Urban Furrows, botanical gardens, schools, Faculty of Agriculture and Life Sciences University of Maribor

3 Gene Bank at the Agricultural Institute of Slovenia Group of species Species Number of accessions Grain legumes Phaseolus spp Vicia faba 37 Skupaj 1162 Fodder crops Trifolium sp. 199 Medicago sp. 39 Vicia sp. 69 Lotus sp. 33 Lolium sp. 30 Festuca sp. 83 Dactylis 110 Phleum 39 Travniške zeli 65 others 202 Skupaj 869 Field crops Zea mays 11 Triticum 76 Secale 12 Papaver 26 Panicum 15 ostale 60 Skupaj 200 Vegetables Lactuca sp. 227 Small fruites Allium cepa 56 Brassica oleracea 11 Diplotaxis spp. 18 ostale(cichorium, Valerianella, Solanum Lyc.) 14 Allium sativum 8 Skupaj 334 Vaccinium corymbosum, Rubus, Ribes, Fragaria 230 Apple trees M. domestica, M. floribunda 256 Grapewine Vitis 90 Potato Solanum tuberosum L. 34 Other potato species Solanum sp diploid populations Total 642 Total 3207 > 3400 accessions

4 Gene Bank at the Agricultural Institute of Slovenia Collecting Documentation and regeneration Characterization and evaluation Conservation (storage at + 4 C, -20 C) Exchange, research, breeding

5 Beans in Slovenia Collection of Phaseolus accessions in the Slovene Plant Gene Bank: >1030 accessions collected in Slovenia: - >800 Phaseolus vulgaris L. ssp. vulgaris var. vulgaris, - >180 Phaseolus vulgaris L. ssp. vulgaris var. nanus, - 53 Phaseolus coccineus L. >60 accessions from international expeditions, >40 accessions from other gene banks worldwide

6 Characterization

7 Evaluation of common bean Diversity studies (morphological, biochemical, molecular markers) Temporal changes in genetic diversity and studies of dissemination pathways in Central Europe and Balkans Disease resistance (Colletotrichum lindemuthianum)

8 Evaluation of common bean Drought tolerance: Physiological response : RWC, water potential, antioxidant activities, photosynthetic parameters,.. Identification of genes whose expression is altered under conditions of drought Proteases under drought: Identification of novel subtilases from leaves Proteomics: Leaf proteome Stem proteome Subcellular proteomics

9 Drought tolerance - gene expression: Phaseolus sp. P. coccineus, P. lunatus, P. acutifolius 13 transcripts identified as up- or down-regulated in leaves of P. vulgaris were compared under drought stress, in order to ascertain whether changes in their expression in Phaseolus spp. are part of a general or a species specific response to drought. The mode of expression was found consistent within Phaseolus spp., despite the fact that the species differ in their responses to drought at the physiological and morphological levels. The results suggested that this was a common feature of the response of Phaseolus spp. Kavar et al JCEA. 12 (4): ;

10 Plant proteases and drought The activity of proteases is responsible for protein turnover, a basis for changes in protein content. Proteases not only provide the amino acids necessary for the synthesis of proteins needed in the response to stress but are also very important in posttranslational modifications of proteins. We have shown that there are several serine endopeptidases and aminopeptidases in leaves of common bean which are differently affected by drought. Hieng et al J. Plant Physiol. 51: Budič et al Annal Biochem ph profiles of phenylalanine (A) and alanine aminopeptidase (B) activities in an extract of common bean leaves after native PAGE

11 Plant proteases and drought Two novel subtilases, PvSLP1 and PvSLP2, have been identified from leaves of common bean cv. Zorin. PvSLP1 was identified at the gene level only and is thus a putative protease. Expression of its transcript did not change on water withdrawal. PvSLP2 has been characterized at the gene, mrna transcript and protein levels. Its proteolytic activity demonstrated to be higher in leaves of plants exposed to withdrawal of water. Budič et al Plant Physiol Biochem 62: PvSLP2 ph profile was analyzed by zymography using Z-Arg-AMC as substrate following native PAGE (A). Fluorescence intensity of individual bands was determined by image analysis using ImageJ program and relative fluorescence values were calculated (B).

12 Drought stress the leaf proteome - Starozagorski čern sensitive, Tiber tolerant - Greenhouse experiment, different levels of drought stress applied - 2D-DIGE analysis of leaf protein extracts (4 biological replicates, 2 treatments / cultivar) - Protein spots that differed significantly in abundance were analysed on LC-MS/MS A total of 543 protein spots were matched across all the gels in cultivar Starozagorski and 400 spots in cultivar Tiber Starozagorski : Tiber: 68 protein spots changed significantly in abundance between stressed and control plants (36 more abundant and 32 less abundant under drought) 64 proteins successfully identified 62 protein spots changed significantly in abundance between stressed and control plants (42 spots more abundant and 20 less abundant in drought) 58 proteins were successfully identified

13 Functional classification of identified proteins Tiber (left) and Starozagorski (right) Zadražnik et al J Proteomics

14 Analysis of protein-protein interaction of identified proteins Cultivar Tiber Zadražnik et al J Proteomics

15 Biological pathways and molecular function network A B Biological pathway (A) and molecular function (B) networks generated by BiNGO. GO categories of TAIR homologous proteins for cultivar Tiber. Zadražnik et al J Proteomics

16 Drought stress: stem proteome Cultivar Tiber, SDS-PAGE separation of isotope labeled protein extracts from stems. 16 protein slices of drought stress samples and 16 slices of control samples excised from the gel. Proteins were in-gel labeled, digested with trypsin and analyzed by LC-MS/MS. A total of 560 proteins identified, after filtering narrowed down to proteins changed in abundance between control and stressed samples. ZADRAŽNIK et al J Plant Physiol, 6: 1-20 Proteolysis and folding Transport 11% 5% Structure 2% Regulatory 7% ATP conversion 9% Storage 5% Protein synthesis 11% Signaling 2% Energy metabolism 24% ROS scavering, defence and stress 11% Photosynthesis 13% Functional classification of quantified proteins

17 Glycoproteins Posttranslational modifications play an important role in tolerance and response to abiotic stress. Glycosylation - a widespread protein posttranslational modification that effect protein structure and its function. Cultivar Tiber, Lectin affinity chromatography, SDS separation, LC-MS/MS analysis Drought stress has the most negative effect on proteins involved in the biochemical metabolism of the cell wall. The abundance of proteins involved in defence against stress, proteolysis, energy metabolism were also influenced by drought. These protein groups fit into the general concept of plant response to drought stress. Classification of proteins into functional groups.

18 Genetic mapping for QTL detection P1 cv. Tiber (tolerant) X P2 cv. Starozagorski (sensitive) F1, F2, F3, F4, F5,, F8 (82 RILs) Genetic characterization using molecular markers (SSR, AFLP,..) Construction of genetic map, QTL detection, marker identification

19 Re-introduction of traditional Slovenian common bean landraces 16 Češnjevec accessions and 23 accessions of landrace Lišček (traditionally grown common bean type) were selected from the gene bank at AIS. Three cultivars were included as outgroups. Field studies were conducted in three successive years; plant growth, resistance to diseases and pests were evaluated and the yield was determined. The chemical composition of raw seeds was analysed (macro and microelements, dry matter and starch content, crude proteins, crude fiber and polyphenols. Sensory analysis was performed.

20 New varieties inscribed in the List of varieties as conservation varieties Češnjevec pisani visoki Lišček rdeči marmorirani

21 Genetic variability, genetic erosion MARAS M, IBUSOSKA A, KRATOVALJEVA S, AGIĆ R, ŠUŠTAR VOZLIČ J, MEGLIČ V, VELIMIROVIĆ A Genetic diversity of common bean accessions from former Yugoslav republic of Macedonia as revealed by molecular and morphological markers Genetika, (Acta biologica Iugoslavica),48: MARAS M, PIPAN B, ŠUŠTAR VOZLIČ J, MEGLIČ V, et al Examination of genetic diversity of Common bean in Western Balkans. J Amer Soc Hort Sci, 140: MEGLIČ V, ŠUŠTAR VOZLIČ J, MARAS M South East European (SEE) autochtonous Phaseolus bean germplasm genetic diversity and reintroduction of traditional landraces. Legume perspectives,, 5: MARAS M, ŠUŠTAR VOZLIČ J, KAINZ W, MEGLIČ V Genetic diversity and dissemination pathways of Common bean in Central Europe. J Amer Soc Hort Sci, 138: ŠUŠTAR VOZLIČ J, MARAS M, MUNDA Al, ZADRAŽNIK T, MEGLIČ V Variability of common bean accessions in the gene bank of Agricultural institute of Slovenia. Acta agriculturae Slovenica, 99: RAZINGER J, DRINOVEC L, ŠUŠTAR VOZLIČ J, ČREMOŽNIK B, MEGLIČ V, ČERENA, A Physiological response of common bean (Phaseolus vulgaris L.) to drought stress, 17: MARAS M, SUŠNIK BAJEC S, MEGLIČ V, ŠUŠTAR VOZLIČ J Characterization and genetic diversity changes in the Slovenian common bean Češnjevec landrace. Acta Biologica Cracoviensia. Series Botanica, 48: ŠUŠTAR VOZLIČ J, MARAS M, JAVORNIK B, MEGLIČ V Genetic diversity and origin of Slovene common bean (Phaseolus vulgaris L.) germplasm as revealed by AFLP markers and phaseolin analysis. J Amer Soc Hort Sci, 131: MARAS M, SUŠNIK BAJEC S, ŠUŠTAR VOZLIČ J, MEGLIČ V Temporal changes in genetic diversity of common bean (Phaseolus vulgaris L.) accessions cultivated between 1800 and Russian journal of genetics, 42:

22 Drought tolerance ZUPIN M, SEDLAR A, KIDRIČ M, MEGLIČ V Drought-induced expression of aquaporin genes in leaves of two common bean cultivars differing in tolerance to drought stress. J Plant Res, 1: 11 BUDIČ M, CIGIĆ B, ŠOŠTARIČ M, SABOTIČ J, MEGLIČ V, KOS J, KIDRIČ M The response of aminopeptidases of Phaseolus vulgaris to drought depends on the developmental stage of the leaves. Plant physiology and biochemistry, 109: ZADRAŽNIK T, EGGE-JACOBSEN W, MEGLIČ V, ŠUŠTAR VOZLIČ J Proteomic analysis of common bean stem under drought stress using in-gel stable isotope labeling. J Plant Physiol, 1-20 ZADRAŽNIK T, HOLLUNG K, EGGE-JACOBSEN W, MEGLIČ V, ŠUŠTAR VOZLIČ J Differential proteomic analysis of drought stress response in leaves of common bean (Phaseolus vulgaris L.). J proteomics, 78: BUDIČ M, SABOTIČ J, MEGLIČ V, KOS J, KIDRIČ M Characterization of two novel subtilases from common bean (Phaseolus vulgaris L.) and their responses to drought. Plant physiol biochem, 62: KAVAR T, MARAS M, KIDRIČ M, ŠUŠTAR VOZLIČ J, MEGLIČ V The expression profiles of selected genes in different bean species (Phaseolus spp.) as response to water deficit. J Central Eur agric, 12: BUDIČ M, KIDRIČ M, MEGLIČ V, CIGIĆ B A quantitative technique for determining proteases and their substrate specificity and ph optima in crude enzyme extracts. Analytical biochemistry, 388: KAVAR T, MARAS M, KIDRIČ M, ŠUŠTAR VOZLIČ J, MEGLIČ V Identification of genes involved in the response of leaves of Phaseolus vulgaris to drought stress. Molecular breeding. 21: HIENG B, UGRINOVIĆ K, ŠUŠTAR VOZLIČ J, KIDRIČ M Different classes of proteases are involved in the response to drought of Phaseolus vulgaris L. cultivars differing in sensitivity. J Plant Physiol 161:

23 Resistance to biotic stress MUNDA, Alenka, RADIŠEK, Sebastjan, ŠUŠTAR VOZLIČ, Jelka, JAVORNIK, Branka Genetic variability of Colletotrichum lindemuthianum isolates from Slovenia and resistance of local Phaseolus vulgaris germplasm. Journal of plant diseases and protection, 116: MAVRIČ PLEŠKO, Irena, ŠUŠTAR VOZLIČ, Jelka Virus diseases and resistance to Bean common mosaic and Bean common mosaic necrosis potyvirus in common bean (Phaseolus vulgaris L.) Acta agriculturae Slovenica, 83: MUNDA, Alenka, ŽERJAV, Metka, ŠUŠTAR VOZLIČ, Jelka Identification of physiological races of Colletotrichum lindemuthianum occuring in Slovenia = Acta biologica slovenica, 45: 3-7

24 Agricultural Institute of Slovenia Vladimir Meglič Marko Maras Tanja Zadražnik Barbara Pipan Aleš Sedlar Alenka Munda Irena Mavrič Kristina Ugrinović Jaka Razinger Špela Velikonja Bolta Tatjana Kavar Jelka Šuštar Vozlič Boštjan Ogorevc, Mojca Polak Biotechnical Faculty, University of Ljubljana Dominik Vodnik University of Oslo, N Wolfgang Eege-Jacobsen Nofima, Aas, N Kristin Hollung Institute Jožef Stefan Marjetka Kidrič Maruška Budič Foto: J. Verbič

25 Photo: J. Verbič Thank you for your attention!