Construction and evaluation of cDNA libraries for large-scale expressed sequence tag sequencing in wheat (Triticum aestivum L.).

A total of 37 original cDNA libraries and 9 derivative libraries enriched for rare sequences were produced from Chinese Spring wheat (Triticum aestivum L.), five other hexaploid wheat genotypes (Cheyenne, Brevor, TAM W101, BH1146, Butte 86), tetraploid durum wheat (T. turgidum L.), diploid wheat (T. monococcum L.), and two other diploid members of the grass tribe Triticeae (Aegilops speltoides Tausch and Secale cereale L.). The emphasis in the choice of plant materials for library construction was reproductive development subjected to environmental factors that ultimately affect grain quality and yield, but roots and other tissues were also included. Partial cDNA expressed sequence tags (ESTs) were examined by various measures to assess the quality of these libraries. All ESTs were processed to remove cloning system sequences and contaminants and then assembled using CAP3. Following these processing steps, this assembly yielded 101,107 sequences derived from 89,043 clones, which defined 16,740 contigs and 33,213 singletons, a total of 49,953 "unigenes." Analysis of the distribution of these unigenes among the libraries led to the conclusion that the enrichment methods were effective in reducing the most abundant unigenes and to the observation that the most diverse libraries were from tissues exposed to environmental stresses including heat, drought, salinity, or low temperature.

An abscisic acid-induced protein kinase, PKABA1, mediates abscisic acid-suppressed gene expression in barley aleurone layers.

The phytohormone abscisic acid (ABA) induces genes-encoding proteins involved in desiccation tolerance and dormancy in seeds, but ABA also suppresses gibberellin (GA)-responsive genes encoding hydrolytic enzymes essential for postgermination growth. A unique serine/threonine protein kinase, PKABA1 mRNA, up-regulated by ABA in seeds, has been identified. In this report, the effect of PKABA1 on the signal transduction pathway mediating ABA induction and suppression of genes has been determined in aleurone layers of barley seeds. Two groups of gene constructs were introduced to barley aleurone layers by using particle bombardment: the reporter constructs containing the coding sequence of beta-glucuronidase gene linked to hormone-responsive promoters and the effector constructs containing the coding region of protein kinases linked to a constitutive promoter. Constitutive expression of PKABA1 drastically suppressed expression of low- and high-pI alpha-amylase and protease genes induced by GA. However, the presence of PKABA1 had only a small effect on the ABA induction of a gene encoding a late embryogenesis abundant protein, HVA1. Our results indicate that PKABA1 acts as a key intermediate in the signal transduction pathway leading to the suppression of GA-inducible gene expression in cereal aleurone layers.

Protein Kinases in Zucchini (Characterization of Calcium-Requiring Plasma Membrane Kinases).

Using an in situ phosphorylation assay with zucchini (Cucurbita pepo L. cv Dark Green) seedling tissue, we have identified numerous polypeptides that are capable of acting as protein kinases. Total protein preparations from different organs contain different kinase profiles, but all are within the range of 55 to 70 kD. At least four kinases are associated with highly purified plasma membranes from etiolated zucchini hypocotyls. The major phosphorylated polypeptides from plasma membranes range in apparent molecular mass from 58 to 68 kD. The plasma membrane kinases are activated by micromolar concentrations of calcium and phosphorylate serine, and, to a lesser extent, threonine residues. These characteristics are similar to those of a soluble calcium-dependent protein kinase that has been purified to homogeneity from soybean suspension cultures. Three of the zucchini plasma membrane kinases share antigenic epitopes with the soluble soybean kinase. The presence of kinase activity at different apparent molecular masses may be indicative of separate kinases with similar characteristics. The zucchini hypocotyl protein kinases are not removed from plasma membrane vesicles by 0.5 M NaCl/5 mM ethylenediaminetetraacetate or by detergent concentrations below the critical micelle concentration of two types of detergent. This indicates that the plasma membrane protein kinases are tightly associated with the membrane in zucchini seedlings.