Phosphorylation of the 12 S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thale cress) seeds

Abstract

Cruciferin (a 12 S globulin) is the most abundant storage protein in the seeds of Arabidopsis thaliana (thale cress) and other crucifers, sharing structural similarity with the cupin superfamily of proteins. Cruciferin is synthesized as a precursor in the rough endoplasmic reticulum. Subunit assembly is accompanied by structural rearrangements involving proteolysis and disulfidebond formation prior to deposition in protein storage vacuoles. The A. thaliana cv. Columbia genome contains four cruciferin loci, two of which, on the basis of cDNA analysis, give rise to three alternatively spliced variants. Using MS, we confirmed the presence of four variants encoded by genes At4g28520.1, At5g44120.3, At1g03880.1 and At1g3890.1 in A. thaliana seeds. Two-dimensional gel electrophoresis, along with immunological detection using anti-cruciferin antiserum and antibodies against phosphorylated amino acid residues, revealed that cruciferin was the major phosphorylated protein in Arabidopsis seeds and that polymorphism far exceeded that predicted on the basis of known isoforms. The latter may be attributed, at least in part, to phosphorylation site heterogeneity. A total of 20 phosphorylation sites, comprising nine serine, eight threonine and three tyrosine residues, were identified by MS. Most of these are located on the IE (interchain disulfide-containing) face of the globulin trimer, which is involved in hexamer formation. The implications of these findings for cruciferin processing, assembly and mobilization are discussed. In addition, the protein phosphatase 2C-impaired mutant, abi1-1, was found to exhibit increased levels of cruciferin phosphorylation, suggesting either that cruciferin may be an in vivo target for this enzyme or that abi1-1 regulates the protein kinase/phosphatase system required for cruciferin phosphorylation.

Geomagnetic Field (Gmf) and Plant Evolution: Investigating the Effects of Gmf Reversal on <em>Arabidopsis thaliana </em>Development and Gene Expression

“…It is then transported to the protein storage vacuoles 13 . Seedling germination requires the breakdown of cruciferin, which is used as an initial source of nitrogen.…”

“…To test the hypothesis, we therefore built a triaxial octagonal Helmholtz coil-pairs magnetic field compensation system (triaxial coils), which is able to accurately reverse the normal GMF conditions. We used Arabidopsis thaliana as a model plant and we tested the effect of reversed GMF on gene expression of some important genes: CRUCIFERIN 3 (CRU3), that encodes a 12S seed storage protein that is tyrosine-phosphorylated and its phosphorylation state is modulated in response to ABA in Arabidopsis thaliana seeds 12,13 ; the Copper Transport Protein1 (COTP1), that encodes a heavy metal transport/ detoxification superfamily protein with the predominant function in soil Cu acquisition and pollen development 14 ; and Redox Responsive Transcription Factor1 (RRTF1), that encodes a member of the ERF (ethylene response factor) subfamily B-3 of ERF/AP2 transcription factor family that contains one AP2 domain that facilitate the synergistic co-activation of gene expression pathways and confer cross tolerance to abiotic and biotic stresses 15 . .…”

Signal processing by protein tyrosine phosphorylation in plants

“…23 Immunoblotting with anti-phospho-Tyr antibodies have permitted detection of plant proteins phosphorylated on Tyr in several other species. 17,19,[24][25][26][27][28][29][30][31][32][33][34][35][36][37] Involvement of PTKs and PTPs by pharmacological approaches. One way to probe specifically elements of signal transduction pathways is by perturbating these pathways through the loss of function of one of these elements.…”

Expression of seed storage product genes (CRA1 and OLEO4) in embryogenic cultures of somatic tissues of Arabidopsis

“…Many of the cupin functions are dependent on enzymatic activity, but SSPs have lost this feature (Dunwell et al 2000). The Arabidopsis Col-0 genome contains four cruciferin loci: At1g03880 (CRB), At1g03890 (CRU2), At4g28520 (CRC) and At5g44120 (CRA1), with the latter two contributing most to the cruciferin seed content (Wan et al 2007;Baud et al 2008). Oleosomes are spherical structures of 0.2-2.0 lm in diameter, and they consist of a TAG core surrounded by phospholipid monolayer, which in turn is encapsulated by unique proteins called oleosins.…”

“…Globulins constitute a large fraction of dicotyledon, monocotyledon and gymnosperm SSPs, and belong to a large protein superfamily called cupins, which include a diverse array of prokaryotic as well as eukaryotic proteins that play a variety of different roles (Dunwell et al 2000;Wan et al 2007). Many of the cupin functions are dependent on enzymatic activity, but SSPs have lost this feature (Dunwell et al 2000).…”