As a first approach in establishing the holm oak leaf proteome, we have optimised a protocol for this plant and tissue which includes the following steps: trichloroacetic acid-acetone extraction, two-dimensional gel electrophoresis (2-DE) on pH 5 to 8 linear gradient immobilised pH gradient strips as the first dimension, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 13% polyacrylamide gels as the second one. Proteins were detected by Coomassie staining. Gel images were recorded and digitalized, and the protein spots quantified by using a linear regression equation of protein quantity on spot volume obtained against standard proteins. Analytical variance was calculated for one-hundred protein spots from three replicate 2-DE gels of the same protein extract. Biological variance was determined for the same protein spots from independent tissue extracts corresponding to leaves from different trees, or the same tree at different orientations or sampling times during a day. Values of 26% for the analytical variance and 58.6% for the biological variance among independent trees were obtained. These values provide a quantified and statistical basis for the evaluation of protein expression changes in comparative proteomic investigations with this species. A representative set of the major proteins, covering the isoelectric point range of 5 to 8 and the relative molecular mass(r) range of 14 to 78 kDa, were subjected to liquid chromatography-tandem mass spectrometry analysis. Due to the absence of Quercus DNA or protein sequence databases, a method based on the procedure reported by Liska and Shevchenko including de novo sequencing and BLAST similarity searching against other plant species databases was used for protein identification. Out of 43 analysed spots, 35 were positively identified. The identified proteins mainly corresponded to enzymes involved in photosynthesis and energetic metabolism, with a significant number corresponding to RubisCO.

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Major proteins of the holm oak leaf proteome have been previously identified using a combination of 2-DE, MS analysis and BLAST similarity search (Jorge et al., Proteomics 2005, 5, 222-234). That study, conducted with field samples from mature trees, revealed the existence of a great variability in the 2-DE protein map, with qualitative as well as quantitative changes, both analytical and biological. A similar study has been carried out with 2-year-old seedlings to analyze and study: (i) changes in the 2-DE protein profile at different tree developmental stages; (ii) the 2-DE protein map variability between three different Spanish provenances; and (iii) variations in the 2-DE protein profile in response to drought stress. Although the protein profile of leaves from seedlings and mature trees was fairly similar, the biological variance found was lower in the former. In the present study, new proteins have been identified. At least four different protein spots differentiated Spanish provenances, two of them identified as an ATP synthase alpha chain, and a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase. Fourteen different protein spots were qualitatively variable between well-watered and drought-stressed seedlings, with some of them corresponding to enzymes of carbohydrate and protein metabolism. Data presented indicated the mobilization of storage proteins and carbohydrates, as well as photosynthesis inhibition under drought conditions.

We have studied in this work the natural variability in Holm oak (Quercus ilex subsp. ballota) by analyzing acorn morphometry and chemical composition in 13 populations from the Andalusia region (southern Spain). Acorn morphometry showed statistic significant differences between the populations with a higher acorn weight, length, and diameter in the northern provenances (TJA, GSE, CTH, CHU, APS, and PCO), while the Southern provenances (RMA, SAA, CCO, BCA, and SCA) showed lower value that even decreased with the altitude. Acorn chemical composition also showed statistic significant differences between populations. Morphometry and chemical composition data were subjected to clustering analysis, and two major clusters were detected. This analysis showed that northern populations (CTH, TJA, SCA, CHU, PCO, GSE, and APS) showed a tendency to present values highest in acorn weight (5.42 g vs. 3.21 g); acorn length (37.21 mm vs. 28.73 mm); acorn diameter (16.12 mm vs. 13.79 mm); ash (1.88% vs. 1.55%); protein (4.82% vs. 4.38%); fat (12.55% vs. 9.92%); and oleic acid (66.93 vs. 66.4%). On the contrary, Southern populations (SSA, VJA, CCO, BCA, RG, and RMA) showed a tendency to present values lowest in sugar (10.28% vs. 11.25%); energy (20,076.71 kJ/kg vs. 20,149.26 kJ/kg); palmitic (16.11% vs. 16.39%) and linoleic acids (13.07% vs. 13.11%). The populations studied here seemed to integrate acorn morphology and chemical composition related to environmental conditions of the original provenances. These data have provided biochemical markers that help establish phylogenic associations between populations and also reveal potentially account as an alternative source for dietary nutrition.

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