Proteomic and histological analyses of endosperm development in Cyclamen persicum as a basis for optimization of somatic embryogenesis.

The endosperm plays an important role for the development of zygotic embryos, while somatic embryos lack a seed coat and endosperm and often show physiological disorders. This study aims at elucidating the cellular and physiological processes within the endosperm of the ornamental species Cyclamen persicum Mill. Histological analyses were performed from 0 to 11 weeks after pollination (WAP). At 3WAP, a syncytium was clearly visible with a globular zygotic embryo. From 4WAP, cellularization of the endosperm, at 5WAP a small torpedo shaped embryo, and from 7WAP cell expansion was observed. By 11WAP the endosperm appeared fully differentiated. Total soluble proteins were extracted from the endosperm at 4, 5, 7, 9 and 11WAP and resolved using two dimensional isoelectric focussing/sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2D IEF/SDS-PAGE). A shift from high-molecular-mass proteins to low-molecular-mass proteins during endosperm development was observed. A total of 1137proteinspots/gel were detected in the three protein fractions extracted at 7, 9 and 11WAP. Mass spectrometry analysis of the 48 predominant protein spots in endosperm at 7, 9 and 11WAP resulted in the identification of 62 proteins, ten of which were described for the first time in Cyclamen. Additionally, 186 proteins were identified using the C. persicum embryo proteome reference map. Proteins involved in abscisic acid signalling and oxidative stress responsive proteins were found to be important for seed development in Cyclamen. The new insights into endosperm physiology including storage compounds are discussed.

The native structure and composition of the cruciferin complex in Brassica napus.

Globulins are an important group of seed storage proteins in dicotyledonous plants. They are synthesized during seed development, assembled into very compact protein complexes, and finally stored in protein storage vacuoles (PSVs). Here, we report a proteomic investigation on the native composition and structure of cruciferin, the 12 S globulin of Brassica napus. PSVs were directly purified from mature seeds by differential centrifugations. Upon analyses by blue native (BN) PAGE, two major types of cruciferin complexes of ∼ 300-390 kDa and of ∼470 kDa are resolved. Analyses by two-dimensional BN/SDS-PAGE revealed that both types of complexes are composed of several copies of the cruciferin α and ß polypeptide chains, which are present in various isoforms. Protein analyses by two-dimensional isoelectric focusing (IEF)/SDS-PAGE not only revealed different α and ß isoforms but also several further versions of the two polypeptide chains that most likely differ with respect to posttranslational modifications. Overall, more than 30 distinct forms of cruciferin were identified by mass spectrometry. To obtain insights into the structure of the cruciferin holocomplex, a native PSV fraction was analyzed by single particle electron microscopy. More than 20,000 images were collected, classified, and used for the calculation of detailed projection maps of the complex. In contrast to previous reports on globulin structure in other plant species, the cruciferin complex of Brassica napus has an octameric barrel-like structure, which represents a very compact building block optimized for maximal storage of amino acids within minimal space.

Comparative proteomic analysis of early somatic and zygotic embryogenesis in Theobroma cacao L.

Somatic embryogenesis can efficiently foster the propagation of Theobroma cacao, but the poor quality of resulted plantlet hinders the use of this technique in the commercial scale. The current study has been initiated to systematically compare the physiological mechanisms underlying somatic and zygotic embryogenesis in T. cacao on the proteome level. About 1000 protein spots per fraction could be separated by two-dimensional isoelectric focusing/SDS PAGE. More than 50 of the protein spots clearly differed in abundance between zygotic and somatic embryos: 33 proteins spots were at least 3-fold higher in abundance in zygotic embryos and 20 in somatic embryos. Analyses of these protein spots differing in volume by mass spectrometry resulted in the identification of 68 distinct proteins. Many of the identified proteins are involved in genetic information processing (21 proteins), carbohydrate metabolism (11 proteins) and stress response (7 proteins). Somatic embryos especially displayed many stress related proteins, few enzymes involved in storage compound synthesis and an exceptional high abundance of endopeptidase inhibitors. Phosphoenolpyruvate carboxylase, which was accumulated more than 3-fold higher in zygotic embryos, represents a prominent enzyme in the storage compound metabolism in cacao seeds. Implications on the improvement of somatic embryogenesis in cacao are discussed.