Protein kinase CK2 phosphorylates wheat eIF2, eIF3, eIF4B, eIF5 and three 60S ribosomal proteins. The substrate specificity of CK2[alpha] toward various plant initiation factor substrates was altered in vitro through holoenzyme formation in the presence of regulatory [beta]-subunits. This presents a potential mechanism through which the differential expression and sub-cellular distribution of CK2 [beta]-subunits could regulate phosphorylation of various CK2 substrates in plants. Our analysis of initiation factor phosphopeptides produced by in vitro phosphorylation identified 20 CK2 phosphorylation sites in eIF2[alpha], eIF2[beta], eIF3c, eIF4B, and eIF5. Native wheat eIF5 was prepared in the presence of phosphatase inhibitors and analyzed by mass spectrometry. Native wheat eIF5 was determined to be a phosphoprotein containing at least 3 phosphorylation sites. The C-terminal CK2 site (S451) of native eIF5 was completely phosphorylated, and tryptic fragments containing the other in vitro CK2 two sites (S209, T240) also appear to be partially phosphorylated. Many of the CK2 phosphorylation sites identified are in conserved binding domains of the yeast multifactor complex (eIF1/eIF3/eIF5/eIF2/GTP/Met-tRNAi[superscript Met). This observation lead to the hypothesis that CK2 phosphorylation may regulate the formation of plant multifactor complexes. The results presented here suggest that plant initiation factors are capable of forming complexes similar to those previously reported in yeast. The in vitro interaction of initiation factors within these complexes appears to be enhanced by phosphorylation of eIF2, eIF3c, and eIF5 by CK2. Site-directed mutagenesis of eIF5 to remove CK2 phosphorylation sites not only prevents the CK2 mediated increase in interaction with eIF1, but also resulted in reduced stimulation of translation initiation in vitro. / text

[Truncated abstract] The highly weathered, phosphorus-fixing soils of Western Australia require large amounts of P fertiliser to produce acceptable crop yields. Chickpea (Cicer arietinum L.) is an important leguminous crop that is increasingly used in rotations with wheat (Triticum aestivum L.), Western Australia’s major crop. Chickpea and a range of other species exude P-mobilising carboxylates into the rhizosphere. Plants that exude carboxylates may need less P fertiliser or may use P in the soil that is unavailable to other plants. There is a wealth of information about P mobilisation and carboxylate exudation by white lupin; in contrast, research on carboxylate exudation by chickpea is fairly limited. The major aim of this PhD research project was to investigate the relationships between exudation of carboxylates and soil and plant P status for chickpea ... In conclusion, whereas carboxylate exudation of plants such as white lupin is clearly targeted at P acquisition, chickpea showed constitutive carboxylate exudation mainly of malonate into the rhizosphere in a series of experiments, each with a different design. Unlike white lupin, chickpea forms associations with mycorrhizal fungi that may improve plant P status. Some of the functions of constitutive carboxylate exudation by chickpea may include P acquisition and deterring microorganisms, but the exact reasons and mechanisms remain unresolved.

[Truncated abstract] Native Australian soils contain very low amounts of phosphorus. The soils of southwestern Australia are ancient and highly weathered. Consequently, the availability of phosphorus in these soils is too low for cropping purposes, so the application of P is necessary to maintain productivity. When P is applied to soil, typically as soluble superphosphate, it tends to be transformed to increasingly less soluble forms over time. Sparingly soluble forms of soil P are relatively inaccessible to Triticum aestivum; however, many grain legumes have a higher P-acquisition efficiency, allowing them to access pools of soil P that T. aestivum cannot. The P-acquisition efficiency of some grain legumes has been attributed in part to their ability to release large quantities of carboxylates, coupled with the development of cluster roots for species such as Lupinus albus. There are a number of unexplained observations in terms of the P-acquisition efficiency of grain legume species and the way that those species respond to P fertilisation. This PhD project aimed to study carboxylate release from a range of crop species, and investigate its role in variation among species for acquisition of phosphorus from sparingly soluble forms (chapter 1). ... L. albus (chapter 5). There was considerable variation in P acquisition among accessions. The variation cannot be attributed to differences in carboxylate release, cluster-root development or whole root system rhizosphere extract pH as measured in this study. We hypothesise that the variation might be attributed to differences in the ratio of release of protons and other cations localised around cluster roots. In conclusion studies of carboxylate exudation and sparingly soluble forms should use more than a single form if the aim is to draw generalised conclusions on P-uptake efficiency from sparingly soluble forms. Comparative studies of a range of species are a useful tool for enhancing our understanding of root physiology. While the benefit of carboxylates for providing access to poorly soluble P has been demonstrated, questions remain as to potential other roles for carboxylates, particularly in species that do not form cluster roots. Variation in P uptake among accessions of L. albus is present, and more work on proton release and ion balance of root clusters is necessary to understand intraspecific variation.

Indiana University-Purdue University Indianapolis (IUPUI) / Drought and cold conditions are among the major factors affecting plant growth and crop production globally. Dehydrins are group II late embryogenesis abundant (LEA) proteins characterized by a conserved K-region (EKKGIMDKIKEKLPG consensus sequence) that accumulate in many plants during drought, low temperature, and high salinity to confer stress tolerance. While it has been demonstrated that overexpression of dehydrins improves cold tolerance in various crop plants, the mechanism leading to cold tolerance is still unclear. Previous studies reported phosphorylation of AtERD14 dehydrin by casein kinase II (CKII) led to an increase in calcium binding activity. Mass spectroscopy analysis determined that the phosphorylation was localized to a poly-serine (S) region. To further characterize the S-region, GST fused ERD14 mutants were created via site-directed mutagenesis and deletion of either the amino or carboxyl ends of ERD14 via the QuickChange® Multi Site-Directed Mutagenesis Kit. Phosphorylation of purified mutant proteins by CKII was analyzed via gel shift and direct phosphorylation assays. The effect of phosphorylation on calcium binding activity was also analyzed. Results showed the serine (S) residue at position 83 was crucial to phosphorylation-dependent molecular mass shift and Ca2+-binding activities followed by the serine residue at position 85 in importance. Mutation of serines at positions 83, 84, and 85 completely eliminated the phosphorylation-dependent gel shift and calcium binding. Examination of truncation mutants determined the N-terminal was an important region for protein structure modification and phosphorylation ability leading to Ca2+ activation. Calcium binding activity of the truncated mutants indicated the calcium binding site was localized in the region between the S-region and the K-region near the C-terminal end. To characterize the acidic dehydrins contribution to cold tolerance in vivo, three single (erd10, erd14, cor47) knockouts (KOs) were characterized. Single KOs produced no cold sensitive phenotype indicating the need for multiple dehydrin KOs in Arabidopsis in order to potentially produce a cold sensitive phenotype.