Collection of Phaseolus genetic resources in the Slovene

Plant Gene Bank

SMARTLEG Meeting

25 – 26 April 2017, Ljubljana, Slovenia

Jelka ŠUŠTAR VOZLIČ

SLOVENE PLANT

GENE BANK Central Seed Bank

(AIS LJ)

PSARS (MAE) - Central Database

MEDPLANT Database

Faculty of Agriculture and Life Sciences University of Maribor

NGO Urban Furrows, botanical gardens, schools, …

Agricultural Institute of Slovenia, Ljubljana

Slovenian Institute for Hop Research and and Brewing, Žalec

Biotechnical Faculty University of Ljubljana

Slovene Plant Gene Bank

Gene Bank at the Agricultural Institute of Slovenia

Group of species Species Number of accessions

Grain legumes Phaseolus spp. 1125

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

Allium cepa 56

Brassica oleracea 11

Diplotaxis spp. 18

ostale(Cichorium, Valerianella, Solanum Lyc.) 14

Allium sativum 8

Skupaj 334

Small fruites 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. 18 + 14 diploid populations

Total 642

Total 3207

> 3400 accessions

• Collecting

• Documentation and regeneration

• Characterization and evaluation

• Conservation (storage at + 4°C, -20 °C)

• Exchange, research, breeding

Gene Bank at the Agricultural Institute of Slovenia

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

Characterization

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)

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

• 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.

Drought tolerance - gene expression: Phaseolus sp.

Kavar et al. 2011. JCEA. 12 (4): 557-568;

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. 2004. J. Plant Physiol. 51:519-530 Budič et al. 2009. Annal Biochem 56-62

pH profiles of phenylalanine (A) and alanine aminopeptidase (B) activities in an extract of common bean leaves after native PAGE

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. 2013. Plant Physiol Biochem 62: 79-87

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).

- 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

Drought stress – the leaf proteome

A total of 543 protein spots were matched across all the gels in cultivar Starozagorski and 400 spots in cultivar Tiber

Starozagorski :

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

Tiber:

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

Tiber (left) and Starozagorski (right)

Functional classification of identified proteins

Zadražnik et al. 2013. J Proteomics 254-272

Analysis of protein-protein interaction of identified proteins

Cultivar Tiber

Zadražnik et al. 2013. J Proteomics 254-272

Biological pathways and molecular function network

Biological pathway (A) and molecular function (B) networks generated by BiNGO. GO categories of TAIR homologous proteins for cultivar Tiber.

A B

Zadražnik et al. 2013. J Proteomics 254-272

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.

Energy metabolism

24%

Photosynthesis13%

ROSscavering,

defence and stress11%

Protein synthesis

11%

ATP conversion

9%

Regulatory7%

Structure2%

Transport5%

Proteolysis and folding

11%

Storage5%

Signaling2%

A total of 560 proteins identified, after filtering narrowed down to 222.

45 proteins changed in abundance between control and stressed samples.

Functional classification of quantified proteins

ZADRAŽNIK et al. 2016. J Plant Physiol, 6: 1-20

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

Classification of proteins into functional groups.

• 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.

Genetic mapping for QTL detection

P1 cv. Tiber (tolerant)

P2 cv. Starozagorski (sensitive)

X

F1, F2, F3, F4, F5,…, F8 (82 RILs)

Genetic characterization using molecular markers (SSR, AFLP, ..)

Construction of genetic map, QTL detection, marker identification

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.

New varieties inscribed in the List of varieties as conservation varieties

Češnjevec pisani visoki

Lišček rdeči marmorirani

Genetic variability, genetic erosion

• MARAS M, IBUSOSKA A, KRATOVALJEVA S, AGIĆ R, ŠUŠTAR VOZLIČ J, MEGLIČ V, VELIMIROVIĆ A. 2016. Genetic diversity of common bean accessions from former Yugoslav republic of Macedonia as revealed by molecular and morphological markers Genetika, (Acta biologica Iugoslavica),48: 729-742

• MARAS M, PIPAN B, ŠUŠTAR VOZLIČ J, MEGLIČ V, et al. 2015. Examination of genetic diversity of Common bean in Western Balkans. J Amer Soc Hort Sci, 140: 308-316

• MEGLIČ V, ŠUŠTAR VOZLIČ J, MARAS M. 2014. South East European (SEE) autochtonous Phaseolus bean germplasm genetic diversity and reintroduction of traditional landraces. Legume perspectives,, 5: 28-29

• MARAS M, ŠUŠTAR VOZLIČ J, KAINZ W, MEGLIČ V. 2013. Genetic diversity and dissemination pathways of Common bean in Central Europe. J Amer Soc Hort Sci, 138: 297-305

• ŠUŠTAR VOZLIČ J, MARAS M, MUNDA Al, ZADRAŽNIK T, MEGLIČ V. 2012. Variability of common bean accessions in the gene bank of Agricultural institute of Slovenia. Acta agriculturae Slovenica, 99: 399-411

• RAZINGER J, DRINOVEC L, ŠUŠTAR VOZLIČ J, ČREMOŽNIK B, MEGLIČ V, ČERENA, A. 2010. Physiological response of common bean (Phaseolus vulgaris L.) to drought stress, 17: 44-56

• MARAS M, SUŠNIK BAJEC S, MEGLIČ V, ŠUŠTAR VOZLIČ J. 2006. Characterization and genetic diversity changes in the Slovenian common bean Češnjevec landrace. Acta Biologica Cracoviensia. Series Botanica, 48: 39-47

• ŠUŠTAR VOZLIČ J, MARAS M, JAVORNIK B, MEGLIČ V. 2006. 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: 242-249

• MARAS M, SUŠNIK BAJEC S, ŠUŠTAR VOZLIČ J, MEGLIČ V. 2006. Temporal changes in genetic diversity of common bean (Phaseolus vulgaris L.) accessions cultivated between 1800 and 2000. Russian journal of genetics, 42: 775-782

Drought tolerance

• ZUPIN M, SEDLAR A, KIDRIČ M, MEGLIČ V. 2017. 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. 2016. The response of aminopeptidases of Phaseolus vulgaris to drought depends on the developmental stage of the leaves. Plant physiology and biochemistry, 109: 326-336

• ZADRAŽNIK T, EGGE-JACOBSEN W, MEGLIČ V, ŠUŠTAR VOZLIČ J. 2016. 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. 2013. Differential proteomic analysis of drought stress response in leaves of common bean (Phaseolus vulgaris L.). J proteomics, 78: 254-272

• BUDIČ M, SABOTIČ J, MEGLIČ V, KOS J, KIDRIČ M. 2013. Characterization of two novel subtilases from common bean (Phaseolus vulgaris L.) and their responses to drought. Plant physiol biochem, 62: 79-87

• KAVAR T, MARAS M, KIDRIČ M, ŠUŠTAR VOZLIČ J, MEGLIČ V. 2011. The expression profiles of selected genes in different bean species (Phaseolus spp.) as response to water deficit. J Central Eur agric, 12: 557-568

• BUDIČ M, KIDRIČ M, MEGLIČ V, CIGIĆ B. 2009. A quantitative technique for determining proteases and their substrate specificity and pH optima in crude enzyme extracts. Analytical biochemistry, 388: 56-62

• KAVAR T, MARAS M, KIDRIČ M, ŠUŠTAR VOZLIČ J, MEGLIČ V. 2008. Identification of genes involved in the response of leaves of Phaseolus vulgaris to drought stress. Molecular breeding. 21: 159-172

• HIENG B, UGRINOVIĆ K, ŠUŠTAR VOZLIČ J, KIDRIČ M. 2004. Different classes of proteases are involved in the response to drought of Phaseolus vulgaris L. cultivars differing in sensitivity. J Plant Physiol 161: 519-530

Resistance to biotic stress

• MUNDA, Alenka, RADIŠEK, Sebastjan, ŠUŠTAR VOZLIČ, Jelka, JAVORNIK, Branka. 2009. Genetic variability of Colletotrichum lindemuthianum isolates from Slovenia and resistance of local Phaseolus vulgaris germplasm. Journal of plant diseases and protection, 116: 23-29

• MAVRIČ PLEŠKO, Irena, ŠUŠTAR VOZLIČ, Jelka. 2004. Virus diseases and resistance to Bean common mosaic and Bean common mosaic necrosis potyvirus in common bean (Phaseolus vulgaris L.) Acta agriculturae Slovenica, 83: 181-190

• MUNDA, Alenka, ŽERJAV, Metka, ŠUŠTAR VOZLIČ, Jelka. 2002. Identification of physiological races of Colletotrichum lindemuthianum occuring in Slovenia = Acta biologica slovenica, 45: 3-7

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

Institute Jožef Stefan • Marjetka Kidrič • Maruška Budič

Biotechnical Faculty, University of Ljubljana • Dominik Vodnik

University of Oslo, N • Wolfgang Eege-Jacobsen

Nofima, Aas, N • Kristin Hollung

Foto: J. Verbič

Photo: J. Verbič Thank you for your attention!