Autophosphorylation profiling of Arabidopsis protein kinases using the cell-free system

Protein phosphorylation is one of the main process in the signal transduction pathway. In recent years, there has been increasing attention to plant phosphorylation signaling and many laboratories are trying to elucidate pathways using various approaches. Although more than 1000 protein kinase (PK) genes have been annotated in the Arabidopsis genome, biochemical characterization of those PKs is limited. In this work, we demonstrate high-throughput profiling of serine/threonine autophosphorylation activity by a combination of the 759N-terminal biotinylated proteins library, produced using a wheat germ cell-free protein production system, and a commercially available luminescence system. Luminescent analysis revealed that 179 of the 759 PKs had autophosphorylation activity. From these 179 PKs, 67 of the most active PKs were analyzed to determine their function using the PlantP database. This analysis revealed that 35 (53%) of the proteins were classified as non-transmembrane protein kinases, and 15 (23%) were receptor-like protein kinases. Additionally, PKs from Group 4.4-MAP3K, Group 1.6, Group 4.5-MAPK/CDC/CK2/GSK kinases and Group 1.10-receptor like cytoplasmic kinases contained the highest percentage of autophosphorylated activity. Next, to get a better overview of the annotated 67 PKs, we used the gene ontology annotation search on the TAIR website to classify the 67 PKs into functional category. As a result, some of these PKs may be involved in phospho-signaling pathways such as signal transduction, stress response, and the regulation of cell division. Information from this study may shed light on many unknown plant PKs. This study will be a basis for understanding the function of PKs in phosphorylation network for future research.     

Molecular interactions between wheat and cereal aphid (Sitobion avenae): analysis of changes to the wheat proteome

Aphids are major insect pests of cereal crops, acting as virus vectors as well as causing direct damage. The responses of wheat to infestation by cereal aphid (Sitobion avenae) were investigated in a proteomic analysis. Approximately, 500 protein spots were reproducibly detected in the extracts from leaves of wheat seedlings after extraction and 2‐DE. Sixty‐seven spots differed significantly between control and infested plants following 24 h of aphid feeding, with 27 and 11 up‐regulated, and 8 and 21 down‐regulated, in local or systemic tissues, respectively. After 8 days, 80 protein spots differed significantly between control and aphid treatments with 13 and 18 up‐regulated and 27 and 22 down‐regulated in local or systemic tissues, respectively. As positive controls, plants were treated with salicylic acid or methyl jasmonate; 81 and 37 differentially expressed protein spots, respectively, were identified for these treatments. Approximately, 50% of differentially expressed protein spots were identified by PMF, revealing that the majority of proteins altered by aphid infestation were involved in metabolic processes and photosynthesis. Other proteins identified were involved in signal transduction, stress and defence, antioxidant activity, regulatory processes, and hormone responses. Responses to aphid attack at the proteome level were broadly similar to basal non‐specific defence and stress responses in wheat, with evidence of down‐regulation of insect‐specific defence mechanisms, in agreement with the observed lack of aphid resistance in commercial wheat lines.     

Differential expression of wheat aspartic proteinases, WAP1 and WAP2, in germinating and maturing seeds

Two aspartic proteinase (AP) cDNA clones, WAP1 and WAP2, were obtained from wheat seeds. Proteins encoded by these clones shared 61% amino acid sequence identity. RNA blotting analysis showed that WAP1 and WAP2 were expressed in both germinating and maturing seeds. The level of WAP2 mRNA expression was clearly weaker than that of WAP1 in all tissues of seeds during germination and maturation. APs purified from germinating seeds were enzymatically active and digested the wheat storage protein, gluten. To elucidate the physiological functions of WAP1 and WAP2 in seeds, we investigated the localisation of WAP1 and WAP2 by in situ hybridisation. In germinating seeds investigated 24h after imbibition, both WAP1 and WAP2 were expressed in embryos, especially in radicles and shoots, scutellum, and the aleurone layer. In maturing seeds, WAP1 was expressed in the whole embryo, with slightly stronger expression in radicles and shoots. WAP1 was also expressed in the aleurone layer 3 weeks after flowering. Strong signals of WAP1 mRNA were detected in the whole embryo and aleurone layer 6 weeks after flowering. On the other hand, WAP2 was scarcely detected in seeds 3 weeks after flowering, and thereafter weak signals began to appear in the whole embryo. WAP1 and WAP2 were expressed widely in germinating and maturing seeds. Such diversity in site- and stage-specific expression of the two enzymes suggests their differential functions in wheat seeds.     

Stress-induced synthesis of proline confers tolerance to water deficit in transgenic wheat

Water deficit is one of the main abiotic factors that affect spring wheat planted in subtropical regions. Accumulation of proline appears to be a promising approach to maintain the productivity of plants under stress condition. However, morphological alterations and growth reduction are observed in transgenic plants carrying genes coding for osmoprotectants controlled by constitutive promoters. We report here the effects of water deficit on wheat plants transformed with the Vigna aconitifolia Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) cDNA that encodes the key regulatory enzyme in proline biosynthesis, under the control of a stress-induced promoter complex-AIPC. Transgenic wheat plants submitted to 15 days of water shortage presented a distinct response. We have found that drought resulted in the accumulation of proline. The tolerance to water deficit observed in transgenic plants was mainly due to protection mechanisms against oxidative stress and not caused by osmotic adjustment.     

Chemical and biological properties of wheat soil in response to paddy straw incorporation and its biodegradation by fungal inoculants

A field experiment was conducted to evaluate the relative contribution of organic fertilizers (paddy straw, microbial inoculants and vermicompost) and inorganic fertilizers (urea and superphosphate) in improving pH, C, N, humus, microbial biomass, dehydrogenase, phosphatase, cellulase, β-glucosidase and xylanase activities of soil under wheat crop. Vermicompost fertilization resulted in highest microbial biomass, available phosphorus, and nitrogen content of wheat soil. It was also found effective in minimizing the alkalinity of soil compared to other treatments as indicated by pH change. However incorporation of paddy straw in conjunction with N₆₀P₆₀ and T. reesei inoculation resulted in maximum dehydrogenase, alkaline phosphatase and highest humus content of soil. Mixed inoculation of A. awamori and T. reesei did not prove effective in improving the soil biochemical properties in comparison to single inoculation of T. reesei. Results showed that in situ incorporation of paddy straw in combination with N₆₀P₆₀ and T. reesei inoculation can be used as an effective measure for valuable disposal of paddy straw and to improve the soil health by reducing mineral fertilization.     

Single-copy genes define a conserved order between rice and wheat for understanding differences caused by duplication, deletion, and transposition of genes

The high-quality rice genome sequence is serving as a reference for comparative genome analysis in crop plants, especially cereals. However, early comparisons with bread wheat showed complex patterns of conserved synteny (gene content) and colinearity (gene order). Here, we show the presence of ancient duplicated segments in the progenitor of wheat, which were first identified in the rice genome. We also show that single-copy (SC) rice genes, those representing unique matches with wheat expressed sequence tag (EST) unigene contigs in the whole rice genome, show more than twice the proportion of genes mapping to syntenic wheat chromosome as compared to the multicopy (MC) or duplicated rice genes. While 58.7% of the 1,244 mapped SC rice genes were located in single syntenic wheat chromosome groups, the remaining 41.3% were distributed randomly to the other six non-syntenic wheat groups. This could only be explained by a background dispersal of genes in the genome through transposition or other unknown mechanism. The breakdown of rice–wheat synteny due to such transpositions was much greater near the wheat centromeres. Furthermore, the SC rice genes revealed a conserved primordial gene order that gives clues to the origin of rice and wheat chromosomes from a common ancestor through polyploidy, aneuploidy, centromeric fusions, and translocations. Apart from the bin-mapped wheat EST contigs, we also compared 56,298 predicted rice genes with 39,813 wheat EST contigs assembled from 409,765 EST sequences and identified 7,241 SC rice gene homologs of wheat. Based on the conserved colinearity of 1,063 mapped SC rice genes across the bins of individual wheat chromosomes, we predicted the wheat bin location of 6,178 unmapped SC rice gene homologs and validated the location of 213 of these in the telomeric bins of 21 wheat chromosomes with 35.4% initial success. This opens up the possibility of directed mapping of a large number of conserved SC rice gene homologs in wheat. Overall, only 46.4% of these SC genes code for proteins with known functional domains; the remaining 53.6% have unknown function, and hence, represent an important, but yet, under explored category of genes. Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users.     

Changes in stomatal conductance and net photosynthesis during phenological development in spring wheat: implications for gas exchange modelling

Gas exchange was measured from 1 month before the onset of anthesis until the end of grain filling in field-grown spring wheat, Triticum aestivum L., cv. Vinjett, in southern Sweden. Two g _s models were parameterised using these data: one Jarvis-type multiplicative g _s model (J-model), and one combined stomatal-photosynthesis model (L-model). In addition, the multiplicative g _s model parameterisation for wheat used within the European Monitoring and Evaluation Programme (EMEP-model) was tested and evaluated. The J-model performed well (R ²=0.77), with no systematic pattern of the residuals plotted against the driving variables. The L-model explained a larger proportion of the variation in g _s data when observations of A _n were used as input data (R ²=0.71) compared to when A _n was modelled (R ²=0.53). In both cases there was a systematic model failure, with g _s being over- and underestimated before and after anthesis, respectively. This pattern was caused by the non-parallel changes in g _s and A _n during plant phenological development, with A _n both peaking and starting to decline earlier as compared to g _s . The EMEP-model accounted for 41% of the variation in g _s data, with g _s being underestimated after anthesis. We conclude that, under the climatic conditions prevailing in southern Scandinavia, the performance of the combined stomatal-photosynthesis approach is hampered by the non-parallel changes in g _s and A _n, and that the phenology function of the EMEP-model, having a sharp local maximum at anthesis, should be replaced by a function with a broad non-limiting period after anthesis.     

Force profiles of protein pulling with or without cytoskeletal links studied by AFM

To test the capability of the atomic force microscope for distinguishing membrane proteins with/without cytoskeletal associations, we studied the pull-out mechanics of lipid tethers from the red blood cell (RBC). When wheat germ agglutinin, a glycophorin A (GLA) specific lectin, was used to pull out tethers from RBC, characteristic force curves for tether elongation having a long plateau force were observed but without force peaks which are usually attributed to the forced unbinding of membrane components from the cytoskeleton. The result was in agreement with the reports that GLA is substantially free of cytoskeletal interactions. On the contrary, when the Band 3 specific lectin, concanavalin A, was used, the force peaks were indeed observed together with a plateau supporting its reported cytoskeletal association. Based on these observations, we postulate that the state of cytoskeletal association of particular membrane proteins can be identified from the force profiles of their pull-out mechanics.