Differential expression and modification of proteins during ontogenesis in Malus domestica

Abstract

Many morphological and physiological changes have been widely reported during ontogeny in higher plants. In order for the better understanding of the proteomic differences between ontogenetic phases, protein compositions between leaves of juvenile, adult vegetative and reproductive phases were compared in an apple (Malus domestica Borkh., Jonathan × Golden Delicious) seedling. Totally, 122 differentially expressed or modified protein spots were separated by DIGE. Of the 122 protein spots, 44, 17 and 29 were abundant in the leaf samples from the juvenile, adult vegetative and reproductive phases, respectively, two spots showed a lower level in the adult vegetative tissue, while the amount of protein increased in 21 spots during ontogeny and declined in nine spots. One hundred and fifteen spots were successfully picked and 95 spots were identified by MALDI-TOF-TOF high-resolution tandem mass spectrometry. Twenty-three juvenile phase abundant or down-regulated spots were photosynthesis-associated proteins, implying a juvenile phase-related photosynthesis enhancement. The expression of 10 enzymes and coenzymes involved in protein synthesis and catabolism was elevated in the adult reproductive phase or up-regulated during ontogeny, contributing a phase change-related activation in protein metabolism. Six proteins generated 30 differential gel spots via post-translational modifications. The differential expression of NADP-dependent D-sorbitol-6-phosphate dehydrogenase was confirmed by Western blotting in six seedlings derived from two hybrid populations. The results of semi-quantitative PCR indicate that some but not all of these proteomic changes were transcriptionally regulated.

Protein phosphorylation differs significantly among ontogenetic phases in Malus seedlings

“…In all seedlings in this study, Rubisco activase spots were in greatest abundance during the juvenile phase, and then decreased. Similar dynamic changes have been detected previously in apple, with five out of six Rubisco activase spots in an earlier study expressed more actively in the juvenile phase [16]. ADP to ATP physiological ratios can significantly inhibit Rubisco activation in species containing both α- and β-Rubisco activase (e.g., Arabidopsis and Camelina sativa ) or only β-Rubisco activase [e.g., tobacco ( Nicotiana tabacum )].…”

“…Additionally, Rubisco has been found with different molecular weights and isoelectric points on 2-DE gels [32,33]. In our previous study, 15 differentially expressed protein spots with varying molecular weights were identical to the Rubisco large subunit or its segments [16]. In the present experiment, the protein spots in group A with different molecular weights and isoelectric points were all identified as Rubisco large-subunit fragments.…”

“…The three groups of Rubisco large-subunit fragments (groups A6, A33 and A48) varied significantly with respect to protein abundance and degree of phosphorylation among ontogenetic phases. In our previous study, the abundance of 15 differentially expressed protein spots that were identical to the Rubisco large subunit or its segments differed drastically among developmental phases [16]. …”

“…All seedlings were planted at a density of 0.5 m × 2.5 m and were subjected to conventional field management and pest control. A basal sprout and a relay shoot (i.e., an axillary shoot on which node numbers partially overlapped with those of the basal sprout) were allowed to grow from each seedling in early spring [16,35,36]. Fully expanded young leaves of identical shapes and sizes were sampled from juvenile, adult vegetative and reproductive parts of each plant in May 2011.…”

“…Based on western blotting with a phosphotyrosine-specific antibody, three 25-kDa, 39-kDa and 54-kDa phosphorylated proteins were found to be more abundant in juvenile than in adult S. sempervirens tissues, whereas two tyrosine phosphorylated proteins of 25 kDa and 34 kDa were more highly expressed in adult than in juvenile or rejuvenated tissues [15]. PTM types and levels of some enzymes, i.e., transketolase, chloroplast protease, and heat shock proteins, are postulated to vary from one developmental phase to another [16]. Nevertheless, insufficient data are available with respect to the degree and extent of variation of protein phosphorylation during ontogenesis in woody perennials.…”