Proteomic Analysis of Theobroma cacao Pod Husk

Abstract

Cocoa (Theobroma cacao L.) is an important crop for many tropical countries and is mainly cultivated for its beans (seeds) for making chocolate. In Malaysia and other Southeast Asian countries, cocoa production is plagued by the cocoa pod borer (CPB) moth, Conopomorpha cramer-ella Snellen. 1) Damage is caused by larvae that bore into the pod and feed on the placenta and pulp tissue surrounding the beans. This disrupts nutrient and water flow to developing beans and results in premature pod ripening. 2) The resulting immature beans clump together and become unextractable, and are low in cocoa butter (tri-acylglycerol) quality. 35) Current control efforts rely heavily on the use of insecticides, which are expensive and dangerous. 6) In the search for host plant resistance, many studies have been focused on the relationship of pod morphology and anatomy to CPB infestation. 1,3,7,8) It was found that the hardness and thickness of the sclerotic layer plays an important role in CPB resistance. 3) The sclerotic layer is a lignified layer of tissue in the middle of the pod husk. The harder and thicker the sclerotic layer, the more difficult it is for CPB larvae to penetrate into or out of the pod. Other nonphysical elements may also contribute to CPB resistance, but no studies have yet been conducted. Nevertheless, there is still very little knowledge on many aspects of pod development, particularly at the cellular and molecular level. Proteomics offers an alternative and powerful method to address biological problems that cannot be dealt with adequately by genomics. 9) It has been used extensively in many plants including important crops. 10,11) Currently a gelbased approach using 2DE coupled with MS is still widely used as the core technique in proteomics. 12,13) 2DE gives high resolution of intact proteins from large complex protein mixtures that reveal changes in isoforms, posttranslational modifications, and protein expression levels, and at a reasonable cost. 13,14) Furthermore, it is recommended as an excellent proteomic approach for non model plants, 15) such as cocoa. To date, only a few proteomic analyses have been reported for cocoa. Previous studies were done to investigate protein changes in beans during fruit ripening, fermentation and other processes in chocolate making. 16,17) Recently, protein extraction for 2DE has been established for leaves and meristem. 18) There is no proteomic study, however, for pod husk, and none for any cocoa tissue combining 2DE with MS. Pod husk is especially rich in polyphenols (particularly procyanidins) and gums (charged polysaccharides). 1921) Such compounds can hamper protein isolation, 2DE analysis, and protein identification by MS. 14,22) Thus, a suitable sample preparation for pod husk is needed. Here, we report on the extraction of total proteins from cocoa pod husk for 2DE analysis and also identification of the major proteins by MALDITOFTOF MS to analyzing the pod husk proteome. For protein identification, we used de novo sequence analysis and cross species ho-mology search, because the complete cocoa genome is not yet available. Sequencing was aided by simplified MALDITOFTOF MS spectra obtained by N terminal sulfonation of tryptic peptides. The same technique has also been used in other unsequenced organisms. 2326) To the best of our knowledge, this is the first extensive report Abstract: We performed two dimensional electrophoresis (2-DE) coupled with MS analysis on Theobroma ca-cao pod husk (fruit pericarp) to explore the proteome of this recalcitrant tissue. Using a phenol extraction methanol-ammonium acetate precipitation method, we have obtained 2-DE images with approximately 700 protein spots detected after colloidal CBB staining. Two hundred and forty-four protein spots were analyzed by de novo sequencing of SPITC-derivatized tryptic peptides by MALDI-TOFTOF MS. Applying this technique , 144 protein spots from cocoa pod husk were identified. The majority of the identified proteins were involved in metabolism and energy. Several of these proteins could be linked to pod growth and development processes.