Final Report Summary - QUIPROD (Quercus Ilex proteomic studies on the germination and early seedling growth under drought stress) 1. FINAL PUBLISHABLE SUMMARY REPORTObjectives. The QUIPROD project aim is proteomic study of drought stress response of Holm oak (Quercus ilex L.), applied at earliest developmental stages. Holm oak is a dominant tree in the Mediterranean natural communities. In spite of the ecological and economic importance of this forest species, still very limited information could be found about the proteomic changes in it, especially at earliest developmental stages and under stress conditions frequently occurring in the Mediterranean area. Besides, holm oak acorns belong to the group of the recalcitrant seeds; very scarce information exists about them. Combining holistic and targeted approaches, the results are expected to provide comprehensive understanding of the response to drought in a recalcitrant seed - producing tree species at the earliest developmental stages. This project aims also, by training through research and attending specialized courses, to build a strong capacity for proteomic research for the researcher involved. Work performed under the QUIPROD project and the main results achieved Seed recalcitrance, maintaining seed viability for longer periods, synchronising germinationThe main problem with Q. ilex acorns is their very short life span (as reported, about 3 months). Orthodox seeds may be stored in dry state for years with small losses of seed viability. On the contrary, recalcitrant ones (only 10% of the seeds) are very unstable and cannot be stored for a long period. They remain metabolically active; show germination associated changes during storage and loss of viability upon drying. Up to 6 months storage of acorns was achieved by optimising their water content. Germination percentage was not affected even after prolonged storage. Germination was synchronized by half-cutting the acorns and omitting the coat as natural seed dormancy is a phenomenon induced mainly by the coat. Coat and half-cotyledon elimination did not impair the seedling development and had no effect on the size of plants. Experimental design of the drought stress and sampling plant material for analysesPeriodical germination tests did tot evidence diminution in acorn viability for all the storage period, the very large cotyledon size and the frequently observed spontaneous germination were the main reasons for shifting the drought stress beginning from germination sensu stricto to already germinated plants. Two stages were considered - a heterotrophic one containing only growing radicle and cotyledon (seedling establishment), as well as on plantlets with first developed leaves (autotrophic growth stage). Three different approaches of inducing drought stress were tested – quick desiccation using silica gel or dry perlite, slow drought stress imposition by water withholding and intermediate way of inducing stress by maintaining water limited conditions, all including a recovery phase from stress. Changes in water content, observations for visible changes in plants (photos); growth inhibition and biomass reduction were registered in the time course. Water limitation to 1/3 of optimal water supply was chosen as the most suitable treatment. At leaflet stage plants were more drought-resistant than at early growth stage. Sampling for proteomics and targeted analyses using one selected experimental schedule was in dynamics at regular time intervals. Root white parts (tips), root bases, and cotyledons/ leaves were collected separately, frozen in liquid nitrogen and stored at -80oC. Holm oak physiological and biochemical response to drought Water content in control plants was in the limits: root tips 84-89%, root bases 71-83%; cotyledons 48-61%. Drought stress induced slight change in water content at day 3rd of stress but this parameter did not diminish further with stress prolongation and was completely restored in recovery. More affected by the applied stress was the root biomass and root growth rate. At leaflet stage the same tendencies of were observed; a two-fold increase in relative electrolyte leakage (membrane instability parameter) was registered in roots under drought stress, but in leaves EL remained very low which was possibly linked with establishment of xeromorphic leaf structure. Proline accumulation was proportional to stress duration. Oxidative damage to lipids (TBRS assay) was high only for day 3rd of stress and diminished thereafter in roots, while in cotyledons TBRS were strongly increased at days 6th and 9th of stress and remained high in recovery. The content of phenolic compounds was higher in the roots compared to cotyledons; it increased in drought stress and decreased after recovery. Accumulation of soluble sugars in cotyledons was proportional to stress duration and was maintained high at recovery. These data highlight the importance of metabolite changes under drought stress. Proteome analysis – drought stress response at heterotrophic growth stageThe proteomic analysis followed the workflow: protein extraction, 1-DE/2-DE electrophoresis, gel images analysis, deciphering of drought-responsive proteins, MS analysis for protein identification. All variants were subjected to 1-DE analysis, whereas 2-DE analysis was applied to selected variants in which the stress-related metabolic and protein changes were most evident (9th day of stress and recovery from, with the respective age controls). Proteins were extracted from 3 biological replicas of 3 tissues (root tips, root bases and cotyledons) according to the protocol of Wang et al (2006) using TCA/acetone-phenol-methanol. Bio-Rad equipment and software were used for the analyses. For 1-DE, samples were run at 12% SDS-PAGE (Protean II cells) and the acquired images were analysed using Quantity 1 software. For 2-DE samples were isoelectrofocused in the range of pI 5-8 (Protean IEF Cell system, 17 cm IPG strips), load 250 µg protein load per strip, active rehydration for 16 h at 50 V, rapid voltage ramp to 10 000V, 50 000 Volt-hours in total, 500 V maintaining. The second dimension was run at 12% SDS (PROTEAN® Plus Dodeca Cell) and gels were stained with Sypro-ruby. Images of the gels were analysed with PDQuest software. Data were subjected to multivariate statistical analysis including sample clustering, ANOVA and Principal component analysis (NIA arrays analysis tools, http://lgsun.grc.nia.nih.gov/ANOVA/index.html). Selected bands/spots of interest (60 from 1-DE, 102 from 2-DE) were manually cut for MS analysis. The MALDI-TOF/TOF PMF, MS/MS and protein identification using was carried out in the UCO-SCAI proteomics facility, a member of Carlos III Networked Proteomics Platform, ProteoRed-ISCIII. Combined search (MS plus MS/MS) was performed using GPS Explorer software v 3.5 (Applied Biosystems) over NCBInr database Viridiplantae and the MASCOT search engine (http://www.matrixscience.com ). Identified proteins were classified into functional groups: metabolism, protein biosynthesis and proteolysis, defence against biotic stress, cellular protection against abiotic stress, intracellular transport. The work on identified proteins continues. Data are expected to enter in two publications. Proteome analysis - Drought stress response at leaflet stageAs the Host group has already results on drought-related changes in Quercus ilex leaves, only the roots have been subjected to 2-DE based proteomic analysis as follows: roots after 10 and 20 days of drought stress with the respective age controls and 10-days recovery after 10 days of drought stress. Conditions of protein extraction and 2-DE were the same as previously described, but the protein load on strips was 400 µg and staining of the gels was with Coomassie colloidal. Images were analysed with PDQuest software and evaluated with multivariate statistics. Selected spots (90 in total) were manually excised from 2-DE gels. Protein identification with MALDI-TOF/TOF PMF, MS/MS and database searching as described was carried out in the UCO-SCAI proteomics facility. The work on identified protein species continues. Data are expected be part of an article. Initial screening studies for some PTMs The following methods were used for PTM screening: proQ diamond staining for phosphoproteins, PAS staining for glycosylated protein, immunoblotting with anti-ubiquitin antibodies. As positive controls were used casein for phosphoproteins, peroxidase for glycoproteins, and ubiquitin. Root protein samples from both drought early stage and drought leaflet stage were tested. This work will continue in the Host laboratory as a part of a PhD thesis, applying also enrichment methods. Oxidative stress and antioxidative protection under drought stress Analysis of oxidative status included: content and oxidation state of the low-molecular antioxidants ascorbate and glutathione, in-gel staining for isoforms of catalase, peroxidase and superoxide dismutase, monitoring the oxidative damage to lipids and to proteins. Results obtained present some alterations in the antioxidative protection during stress and recovery and are in agreement with the changes in catalase and dehydroascorbate reductase protein bands established in 1-DE. Analyses at the transcript level of some proteins of interest Total RNA was isolated from samples of the experiment drought early stage root tips using Spin Plant RNA Mini Kit InviTrap (Invitek) according to the manufacturers’ instructions. The isolated RNA was quantified using Qbit and checked for quality by capillary electrophoresis. Synthesis of cDNA was performed using Quiagen QuantiTest reverse transcription protocol. This work will continue in the Host laboratory and will be included in a Master thesis on transcriptomics. Conclusions. The obtained results provide an insight on the metabolic adjustment in Quercus ilex as a response to drought stress and are a basis for at least 3 publications in plant stress proteomics. Socio-economic impact of the project. Results obtained in QUIPROD project will contribute in understanding the molecular bases of drought response in Holm oak. Generated knowledge will be published and used by the scientific community, as well as by breeders working with forest trees. It will enhance additionally the Host competence in forest tree –omics. Researcher`s new proteomic qualification will be beneficial for spreading advanced knowledge in her own country.
