The ARR4 gene from Saccharomyces cerevisiae was highly homological with E. coli arsA. The defective nucleotide-binding domain of ARR4[G30R] exhibited negative dominance from the growth curve experiment, showing that ARR4 protein might act as a dimer in vivo. On the nonreducing gel, the purified 40 kDa ARR4 presented as monomer, dimer and oligomer conformational states. Besides, the chemical cross-linking experiments confirmed the positional size of dimeric ARR4. The metal ions of Co2+, Sb3+, As3+ and As5+ have not effect on the ATPase activity and ARR4 oligomerization. ARR4 exhibit a low level of Mg2+-ATPase activity in the range of 50-70 and 100-120 nmol/min/mg respectively as estimated by phosphate released and NADH-coupled assay. The maximal activity was obtained at a ratio of ATP:Mg2+ of 2:1 and the ATPase activity of ARR4[G30R] was about 50% of that of the ARR4 from both ATPase assays. In the presence of ATP or Mg2+, ARR4 has similar ratios of dimer and monomer. When incubated with Mg2+-ATP, ARR4 tended to form more dimer than with ATP and Mg2+ individually. ARR4[G30R] exhibited less dimer formation. Mixing ARR4 and ARR4[G30R] at a constant total protein concentration with different molar ratios (1:1, 1:2 and 1:4), a near linear relationship of activity versus amount of the ARR4 protein was observed. This observation suggests that ATP hydrolysis takes places in one ATP binding site independent of the other site of NBD. The proportional ATPase activities were consistent with the ratios of ARR4-ARR4[G30R] dimerization on the nonreducing gels. In conclusion, the conformational changes induced by Mg2+-ATP is related to the dimerization of ARR4 and both its NBDs would hydrolyze ATP independently.

The ArsA homologue ARR4 in Saccharomyces cerevisiae, encoded by YDL100C. Homologues of the E. coli ArsA are found in S. cerevisiae about 29 % from Genetic Computer Group (GCG). The ARR4 gene product contains an ATP binding site that is similar to protein ArsA from E. coli Disruption of ARR4 in yeast is not lethal but the disrupted strain was unable to grow at 40¢J, suggesting that the possible cause of cell death in KO strain at 40¢J was investigated. The accumulation of trehalose and the in vivo molecular oxidation level are higher in KO strain than that in WT strain under heat stress condition. These suggest that the increased reactive oxygen species (ROS) but not the amount of thermoprotectant trehalose is most likely to be the reason for cell death in KO strain. In this report ROS scavenger system show that the activities of ROS scavenger system are lower in KO compared to that in WT strain at 30¢J or 40¢J. This suggests that ARR4 is involved in the heat stress ¡Boxidative stress and osmatic stress triggers activation of the STRE ( stress tolerance response element) regulon. Further studies involvement ARR4 of CTT1, SOD1, and TSL1 gene of STRE-drive gene by RT-PCR. Here the report that the KO strain exhibits a thermosensitivity phenotype in comparison to wild-type strain, indicating that ARR4 may act as a component of a stress tolerance network.

Cytokinins are plant hormones that affect the primary growth of shoots and roots. Application of the cytokinin N6-benzylaminopurine (BAP) to the shoot apical meristem of <i>Arabidopsis thaliana Landsberg erecta </i>(L.) Heynh induces aberrant flower development and a significant genetic response, and some of these phenotypes and expression patterns were carried to the next generation. Analysis of altered transcript levels with Affymetrix GeneChips® indicated significant changes in transcript levels of genes associated with shoot meristem activity, circadian rhythms, cytokinin metabolism, two-component systems, stress and defense responses, auxin regulation, ethylene and salicylic acid biosynthesis, and signal transduction. Specific genes were also mined from the data as potentially responsible for the BAP-induced aberrant floral phenotypes, increased floral organ number, buds in axils of sepals, and mosaic floral organs. Of particular note was a decrease in the transcript levels of CLAVATA1 (CLV1), a gene encoding a receptor kinase involved in organ differentiation and maintenance of shoot and floral meristems. Time course analysis by RT-PCR showed a decline and subsequent recovery of transcript levels of CLV1 and a coincident increase in WUSCHEL (WUS) transcript, consistent with the known suppression of WUS by CLV. WUS encodes a homeodomain protein associated with shoot meristem proliferation. The temporal coincidence of an increased floral organ phenotype with changes in transcript levels of CLV1 and WUS suggests that cytokinins regulate flower development by affecting the activity of genes controlling shoot meristem activity. Aberrant floral phenotypes in subsequent non-treated generations suggest epigenetic inheritance of some BAP-altered transcript patterns. Repressed expression of the majority of significant genes in the untreated T1 population suggests a mechanism of gene silencing, such as methylation, was involved in this epigenetic inheritance. Also, transcript levels of time-keeping genes, including CIRCADIAN CLOCK ASSOCIATED 1 / ELONGATED HYPOCOTYL, and associated genes with oscillating expression patterns, such as COLD-RESPONSIVE, were affected by BAP in treated plants and the subsequent generation, suggesting the capacity of cytokinins to affect the phase of the circadian clock. Hormonal regulation of heritably altered diurnal periodicity and environmental responses may provide a developmental and, therefore, evolutionary advantage to plants.

Cytokinins are plant hormones that affect the primary growth of shoots and roots. Application of the cytokinin N6-benzylaminopurine (BAP) to the shoot apical meristem of <i>Arabidopsis thaliana Landsberg erecta </i>(L.) Heynh induces aberrant flower development and a significant genetic response, and some of these phenotypes and expression patterns were carried to the next generation. Analysis of altered transcript levels with Affymetrix GeneChips® indicated significant changes in transcript levels of genes associated with shoot meristem activity, circadian rhythms, cytokinin metabolism, two-component systems, stress and defense responses, auxin regulation, ethylene and salicylic acid biosynthesis, and signal transduction. Specific genes were also mined from the data as potentially responsible for the BAP-induced aberrant floral phenotypes, increased floral organ number, buds in axils of sepals, and mosaic floral organs. Of particular note was a decrease in the transcript levels of CLAVATA1 (CLV1), a gene encoding a receptor kinase involved in organ differentiation and maintenance of shoot and floral meristems. Time course analysis by RT-PCR showed a decline and subsequent recovery of transcript levels of CLV1 and a coincident increase in WUSCHEL (WUS) transcript, consistent with the known suppression of WUS by CLV. WUS encodes a homeodomain protein associated with shoot meristem proliferation. The temporal coincidence of an increased floral organ phenotype with changes in transcript levels of CLV1 and WUS suggests that cytokinins regulate flower development by affecting the activity of genes controlling shoot meristem activity. Aberrant floral phenotypes in subsequent non-treated generations suggest epigenetic inheritance of some BAP-altered transcript patterns. Repressed expression of the majority of significant genes in the untreated T1 population suggests a mechanism of gene silencing, such as methylation, was involved in this epigenetic inheritance. Also, transcript levels of time-keeping genes, including CIRCADIAN CLOCK ASSOCIATED 1 / ELONGATED HYPOCOTYL, and associated genes with oscillating expression patterns, such as COLD-RESPONSIVE, were affected by BAP in treated plants and the subsequent generation, suggesting the capacity of cytokinins to affect the phase of the circadian clock. Hormonal regulation of heritably altered diurnal periodicity and environmental responses may provide a developmental and, therefore, evolutionary advantage to plants.