MOLECULAR CHARACTERIZATION AND PROTEOMICS OF PREHARVEST SPROUTING TOLERANCE IN WHEAT

Abstract

Wheat (Triticum aestivum L.) is a staple food crop for India and the world. Around the world wheat is cultivated in EU-25, China, India, America, Russia, Australia, Canada, Pakistan, Turkey and Argentina. Pre harvest sprouting (PHS) is a worldwide problem and is greater in white kennelled wheat than red kennelled wheat. PHS is a .condition when physiologically mature grain sprouts while still in the ear under moist and wet conditions. In damaged food grains a-amylase activity is upregulated which damages wheat grains. Hence, seed viability is lost and milled flour has inferior baking properties. Therefore, sprouted wheat looses market value and is rendered unfit for human consumption. Taking above problems into account the development of PHS tolerant lines is a long term solution to PI-IS as these cultivars will be able to tolerate damaging effects of rain. To address the problem of PHS the present study was undertaken using near isogenic lines (NILs) derived from a cross between amber and PHS susceptible cultivar PBW343 and red and PHS tolerant cultivar SPR8198. The NILs were developed at PAU Ludhiana. The two NILs used in the study were PHST9 (amber coloured and PHS susceptible) and PHSTO (red coloured and PHS tolerant). Using PHS test it was determined that NIL PHSTO did not germinate under moist and wet conditions till ten days. Sprouting tolerance in wheat is lost between two months after harvesting. Dormancy is dissipated by after ripening in dry storage at room temperature in a similar manner for the red and white lines. We preserved P1-IS tolerance after harvesting by storing seeds of NIL PHSTO at -86°C. It was interesting to observe that the (preharvest sprouting tolerance) PHST in NIL PHSTO was preserved for around two years. The preserved seeds of PHSTO were dormant and did not germinate till ten days after imbibition. To the best of my knowledge this is the first report of prolonged maintenance of PHST under deep freezer storage conditions. It is important to mention here, that the plant material stored at -86°C was used for phytohormone analysis in our study. The effect of exogenous application of growth regulators, abscisic acid (ABA) and gibberellic acid (GA) on PBW343 and two NILs was tested at different phytohormone concentrations. This test with growth regulators demonstrated that the susceptible parent cultivar PBW343 and NIL PHST9 were ABA insensitive while tolerant NIL PHSTO was ABA sensitive. Taking seed morphology into account seed anatomy was studied for the three experimental lines using scanning electron microscopy (SEM) which demonstrated that in PHSTO three layers i.e. epidermis, aleurone layer and intermediate layer between epidermis and aleurone were thicker than PB W343 and PHST9. These differences in the thickness of the three layers might add to seed dormancy. Moreover thousand grain weight of PHSTO was also greater than PBW343 and PI-IST9. Other than the above mentioned genes and transcription factors quantitative trait loci (QTL) studies have also been employed to localize agronomically important genes like PHS on wheat chromosomes. QTLs involved in PHS tolerance or seed dormancy have been mapped on 20 out of 21 chromosomes in wheat. The QTLs other than those on 3AL and 4AL are minor Q'1'Ls affecting PHS. QTL QPhs.ccsu-3A.1, on chromosome 3AL is the major QTL explaining 78% phenotypic variation. This major QTL was flanked by SSR markers Xwmc]53 and Xgwm155. In addition to 3AL QTL flanking markers we also used seven SSR markers lying between Xwmc153 and Xgwml55 on wheat genetic map. Similarly dormancy in white grained wheat has been linked to chromosome 4AL as indicated by QTL studies. The 4AL QTL is flanked by Xbarcl 70, Xgwm269 and Xgwm397. The marker zxql 18 has been mapped between the flanking markers reported for 4AL. In present study we tried to validate 3AL and 4AL QTL flanking markers. The lines used for marker validation included ten selected IIT Roorkee (11TR) landraces in addition to PBW343, NILs PHSTO and PHST9. In our study we could not validate chromosome 3AL QTL flanking markers wmcl53 and gwml55 but chromosome 4AL Q'l'I, flanking markers Xgwm269 and Xzxg118 were validated in our plant material. In our study gwm269, differentiated between amber tolerant and amber susceptible lines and can therefore be used for marker assisted breeding. The main aim of the present study was proteome analysis to identify a broad spectrum of proteins that are expressed in wheat during PHS. For proteome study two dimensional electrophoresis (2-DE) was coupled with mass spectrometric (MS) tool, LC-ESI-MS/MS and the information generated was utilized for protein identification. 2-DE was done for endosperm, mature embryo and immature embryo proteins. For proteome analysis seed material was collected and stored at -80°C at two developmental stages i.e stage87 (hard dough) and stage95 (dry caryopses). From endosperm LC-ESI-MS/MS was done for three proteins and from embryo LC-ESI-MS/MS was done for four proteins. The endosperm proteins designated as PH-ENDO-1, PH-ENDO-2 and PH-END 0-3 were identified as LMW proteins for spot 1 and 2 while protein disulfide isomerise (PDI) for spot3. PDI is responsible for protein folding and on consultation of literature it was found to have an alternate function as well. The enzyme is also responsible for production of hydrogen peroxide via oxalate oxidase during stress/ pathogen attack. This is important in our context as the protein spot for PDI was identified in tolerant NIL PHSTO. Therefore PDI was further used for transcriptomic studies using gene specific primers. For mature embryo LC-ESI-MS/MS was done for four protein spots which were designated as PH-EMB-3, PH-EMB-5, PH-EMB-6 and PH-EMB-7. PI-I-EMB-3 was identified as an unknown protein from gymnosperm, PH-EMB-5 was identified as serine carboxypeptidase, PH-EMB-6 was identified as unknown protein Os01g0749000 from Oryza sativa and PH-EMB-7 was identified as avenin like precursor from T. aestivuin. The results of LC-ESI-MS/MS for the two protein spots i.e PH-EMB-3 and PH-EMB-6 were further analyzed using blastp. The peptide for PH-EMB-3 blasted with Oryza sativa sequences and the sequences showing 100% coverage were selected for further study. Hence, nine Oryza sequences were selected for the study. The mRNA sequences of these nine selected protein sequences were further blasted with wheat mapped ESTs on graingenes and three mRNA sequences blasted on group 3 chromosomes of wheat. The second protein spot i.e PH-EMB-6 from immature embryo proteins 2-DE was analyzed using Blastp. Two peptides were identified for PI-I-EMB-6 i.e. Os01g0749000 and Os10g0125700 were unknown proteins. Os01g0749000 belongs to DUF1264 superfamily and is an embryo specific protein. Upon Blastp of OsO 1 g0749000 a wide range of hypothetical proteins from oryza were identified, while for Os 10g0125700, which is blast resistance protein in oryza, blastp identified RGA-1(repressor of gibberellic acid-1) like protein from T. aestivum along with hypothetical proteins from oryza and sorghum. For the same spot DNA binding / transcription factor from Arabidopsis thaliana was identified with a low probability which was identified as dehydration response element binding protein (DREB). Therefore for same protein spot taken from 2-DE gel of PHSTO, two physiologically important proteins i.e. RGA-1 and DREB were identified. Here it can be said that the above study has been helpful as with the help of NIL PHSTO we could identify two important genes. For transcriptome study gene specific primers were designed for myblO, dihydro flavonoid-4- reductase (DFR) and PDI (protein disulfide isomerase-enzyme responsible for protein folding). The plant material used for the analysis included parent PBW343 and NILs PHST9 and I'IISTO. Differences were observed among the three lines for PDI and myblO but in case of l)FR expression was observed only in PHSTO. The DFR transcription factor is present on ehromosome3A which is consistent with the observation of major PHS gene on chromosome 3AL as indicated by QTL study. These results suggest that PHS/ dormancy gene might be present on chromosome 3A. Chromosome bin maps of wheat are available for all the 21 chromosomes. With the help of chromosome bin mapping genes can be assigned to specific bins based on expressed sequence tags (ESTs). We also employed chromosome bin mapping for chromosome 3AL for markers wmcl53, gwml55, zxg118 and gwm269 to identify the chromosome bin carrying the iv PHS/dormancy gene. The same was also done for proteins. The results from both markers and proteins were same as in both the case two bins 3AL3-0.42-0.78 and 3AL5-0.78-1.00 were identified. In our study, bin mapping was done with an objective to identify either of the two above mentioned bins, but we could not narrow down on either bin as both had almost equal number of ESTs. Hence we would propose that the gene for P1-1ST might be present in either of the bins, 3AL3-0.42-0.78 or 3AL5-0.78-1.00, as indicated both by markers and proteins. This bin identification also raises the possibility of the gene being present in distal region of chromosome 3AL. Therefore, with above study it can also be said that dormancy in wheat might exist both on chromosome 3AL and 4AL and extensive studies are required at the proteome level for identification of the dormancy/PHST gene.