Analysis of desiccation‐induced candidate phosphoproteins from Craterostigma plantagineum isolated with a modified metal oxide affinity chromatography procedure

Abstract

Reversible protein phosphorylation/dephosphorylation is crucial for regulation of many cellular events, and increasing evidence indicates that this post-translational modification is also involved in the complex process of acquisition of desiccation tolerance. To analyze the phosphoproteome of the desiccation tolerant resurrection plant Craterostigma plantagineum, MOAC-enriched proteins from leaves at different stages of a de-/rehydration cycle were separated by 2-D PAGE and detected by phosphoprotein-specific staining. Using this strategy 20 putative phosphoproteins were identified by MALDI-TOF MS and MS/MS, which were not detected when total proteins were analyzed. The characterized desiccation-related phosphoproteins CDeT11-24 and CDeT6-19 were used as internal markers to validate the specificity of the analyses. For 16 of the identified proteins published evidence suggests that they are phosphoproteins. Comparative analysis of the 2-D gels showed that spot intensities of most identified putative phosphoproteins change during the de-/rehydration cycle. This suggests an involvement of these proteins in desiccation tolerance. Nearly all changes in the phosphoproteome of C. plantagineum, which are triggered by dehydration, are reversed within 4 days of rehydration, which is in agreement with physiological observations. Possible functions of selected proteins are discussed in the context of the de-/rehydration cycle.

Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro

“…In that study we applied Al(OH) 3 -metal oxide/hydroxide affinity chromatography for phosphoprotein enrichment (allowing the annotation of only one phosphorylation site), and TiO 2 phosphopeptide enrichment for the analysis of phosphorylation sites of selected candidate proteins from mature pollen. Such improvement in the number of phosphorylation sites after phosphopeptide enrichment in the actual study compared with the phosphoprotein enrichment in the previous study is in accordance with several previous studies where phosphoprotein enrichment revealed only a limited number of phosphorylation sites (6,38,39). Moreover, a tandem approach enriching first for phosphoproteins and then after trypsin digest also for phosphopeptides was shown beneficial (13,14).…”

“…Similar to pollen activation, the rehydration of African xerophyte Craterostigma plantagineum was accompanied by changes in protein phosphorylation (6). Attachment of a phosphate group to the polypeptide chain shifts the pI of a protein to more acidic range (7).…”

Molecular mechanisms of desiccation tolerance in the resurrection glacial relic Haberlea rhodopensis

“…Protein phosphorylation and dephosphorylation are essential signaling events leading to acquisition of drought/ desiccation tolerance, and changes in the phosphorylated status of a number of proteins have been shown for C. plantagineum [45]. The most drought-and desiccationupregulated gene in H. rhodopensis encoded a protein phosphatase, indicating that phosphorylation events are also an essential part of drought stress signaling in this resurrection species.…”

Response to biotic and oxidative stress in Arabidopsis thaliana: Analysis of variably phosphorylated proteins

“…Its molecular target, namely ribulose carboxylase/oxygenase large subunit (spot 12), was also downregulated. This phosphorylated protein has been already reported to be enriched by MOAC procedures [17]. Other enzymes involved in carbon metabolism, namely cysteine synthase (spot 16), mitochondrial NAD-dependent malate dehydrogenase (spot 15), a probable chloroplastic isoform of fructose bisphosphate aldolase (At2g21330) (spot 10) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) subunit A (spots 13 and 14), also resulted down-regulated.…”

“…After 8 h-treatments, total proteins were extracted and phosphoproteins enriched by using a MOAC procedure [17]. Input loads and eluted fractions were analysed by SDS-PAGE; gels were stained with the aspecific fluorescent dye SyproRuby ( Fig.…”