Direct immunological identification of full-length cDNA clones for plant protein without gene fusion to E. coli protein

AbstractBy immunological screening of a cDNA library constructed from potato tuber poly(A)+ RNA and Escherichia coli expression vector pUC8 by the vector-primer and linker procedure of Okayama and Berg [(1982) Mol. Cell Biol. 2, 161-170], nearly full-length cDNA clones for patatin, a major protein of potato tuber, were identified. The cDNA carrying part of the 5'-noncoding region of the patatin mRNA, in addition to entire coding and 3'-noncoding regions, expressed prepatatin in E. coli cells by translational initiation inside cDNA. These results suggest that nearly full-length cDNA clones with entire coding region can be identified directly by immunological screening without gene fusion to E. coli proteins at least for some plant mRNAs

Abscisic Acid–Induced Transcription Is Mediated by Phosphorylation of an Abscisic Acid Response Element Binding Factor, TRAB1

The rice basic domain/Leu zipper factor TRAB1 binds to abscisic acid (ABA) response elements and mediates ABA signals to activate transcription. We show that TRAB1 is phosphorylated rapidly in an in vivo labeling experiment and by phosphatase-sensitive mobility shifts on SDS–polyacrylamide gels. We had shown previously that a chimeric promoter containing GAL4 binding sites became ABA inducible when a GAL4 binding domain–TRAB1 fusion protein was present. This expression system allowed us to assay the ABA response function of TRAB1. Using this system, we show that Ser-102 of TRAB1 is critical for this function. Because no ABA-induced mobility shift was observed when Ser-102 was replaced by Ala, we suggest that this Ser residue is phosphorylated in response to ABA. Cell fractionation experiments, as well as fluorescence microscopy observations of transiently expressed green fluorescent protein–TRAB1 fusion protein, indicated that TRAB1 was localized in the nucleus independently of ABA. Our results suggest that the terminal or nearly terminal event of the primary ABA signal transduction pathway is the phosphorylation in the nucleus of preexisting TRAB1

Differential Activation of the Rice Sucrose Nonfermenting1–Related Protein Kinase2 Family by Hyperosmotic Stress and Abscisic Acid

To date, a large number of sequences of protein kinases that belong to the sucrose nonfermenting1–related protein kinase2 (SnRK2) family are found in databases. However, only limited numbers of the family members have been characterized and implicated in abscisic acid (ABA) and hyperosmotic stress signaling. We identified 10 SnRK2 protein kinases encoded by the rice (Oryza sativa) genome. Each of the 10 members was expressed in cultured cell protoplasts, and its regulation was analyzed. Here, we demonstrate that all family members are activated by hyperosmotic stress and that three of them are also activated by ABA. Surprisingly, there were no members that were activated only by ABA. The activation was found to be regulated via phosphorylation. In addition to the functional distinction with respect to ABA regulation, dependence of activation on the hyperosmotic strength was different among the members. We show that the relatively diverged C-terminal domain is mainly responsible for this functional distinction, although the kinase domain also contributes to these differences. The results indicated that the SnRK2 protein kinase family has evolved specifically for hyperosmotic stress signaling and that individual members have acquired distinct regulatory properties, including ABA responsiveness by modifying the C-terminal domain

Ectopic gene expression and organogenesis in Arabidopsis mutants missing BRU1 required for genome maintenance

Chromatin reconstitution after DNA replication and repair is essential for the inheritance of epigenetic information, but mechanisms underlying such a process are still poorly understood. Previously, we proposed that Arabidopsis BRU1 functions to ensure the chromatin reconstitution. Loss-of-function mutants of BRU1 are hypersensitive to genotoxic stresses and cause release of transcriptional gene silencing of heterochromatic genes. In this study, we show that BRU1 also plays roles in gene regulation in euchromatic regions. bru1 mutations caused sporadic ectopic expression of genes, including those that encode master regulators of developmental programs such as stem cell maintenance and embryogenesis. bru1 mutants exhibited adventitious organogenesis, probably due to the misexpression of such developmental regulators. The key regulatory genes misregulated in bru1 alleles were often targets of PcG SET-domain proteins, although the overlap between the bru1-misregulated and PcG SET-domain-regulated genes was limited at a genome-wide level. Surprisingly, a considerable fraction of the genes activated in bru1 were located in several subchromosomal regions ranging from 174 to 944 kb in size. Our results suggest that BRU1 has a function related to the stability of subchromosomal gene regulation in the euchromatic regions, in addition to the maintenance of chromatin states coupled with heritable epigenetic marks