Ovate family protein 1 as a plant Ku70 interacting protein involving in DNA double-strand break repair.

The Ku heterodimer, a DNA repair protein complex consisting of 70- and 80-kDa subunits, is involved in the non-homologous end-joining (NHEJ) pathway. Plants are thought to use the NHEJ pathway primarily for the repair of DNA double-strand breaks (DSBs). The Ku70/80 protein has been identified in many plants and been shown to possess several similar functions to its counter protein complex in mammals. In the present study, ovate family protein 1 (AtOFP1) was demonstrated to be a plant Ku-interacting protein by yeast two-hybrid screening and the GST pull-down assay. Truncation analysis revealed that the C-terminal domain of AtKu70 contains interacting sites for AtOFP1. The electrophoretic mobility shift assay (EMSA) indicated that AtOFP1 is also a DNA binding protein with its binding domain at the N-terminus. In 3-week-old seedlings, expression of the AtOFP1 gene increased after exposure to DNA-damaging agents (such as methyl methanesulfonate (MMS) and menadione) in a time dependent manner. Seedlings lacking the AtOFP1 protein were more sensitive to MMS and menadione as compared with wild-type. Furthermore, similar to AtKu70(-/-) and AtKu80(-/-), the AtOFP1(-/-) mutant showed relatively lower NHEJ activity in vivo. Taken together, these results suggest that AtOFP1 may play a role in DNA repair through the NHEJ pathway accompanying with the AtKu protein.

Regulation of Arabidopsis thaliana Ku genes at different developmental stages under heat stress.

Ku, a heterodimeric protein consisting of 70- and 80-kDa subunits, is involved in many cellular processes, such as DNA replication, cell cycle regulation and heat shock response. Moreover, the expression of Arabidopsis thaliana Ku genes (AtKu) is modulated by certain plant hormones through several signal transduction pathways. This study investigated how AtKu are regulated by heat stress. AtKu expression in 3-week-old young seedlings was down-regulated by heat stress in a time-dependent manner, as examined using real-time quantitative PCR, GUS reporter systems, and western blotting analysis. Additionally, the heat-induced repression of AtKu was mediated through the abscisic acid (ABA) biosynthetic pathway, as shown by the reversal of AtKu suppression in the ABA biosynthesis mutant, aba3, and by an increase in the ABA level as analyzed by reverse-phase high performance liquid chromatography. Heat stress-induced regulation of AtKu repression also involved ethylene signaling, DNA repair pathways, and fatty acid synthesis. Furthermore, AtKu expression was repressed in stems, rosette leaves, and cauline leaves in 4-5-week-old plants under heat stress, whereas it remained unchanged in roots and primary inflorescence, indicating that heat differentially modulated AtKu expression in distinct tissues of Arabidopsis.

Signaling pathways mediating the suppression of Arabidopsis thaliana Ku gene expression by abscisic acid.

The Ku heterodimer facilitates the regulation of DNA repair, DNA replication, cell cycle, and telomere maintenance. The plant hormone abscisic acid (ABA) is a plant growth inhibitor. This study investigates how Arabidopsis thaliana Ku (AtKu) genes are regulated by ABA in 3-week-old seedlings. First, beta-Glucuronidase assay and real time quantitative PCR analysis results indicate that ABA represses the AtKu gene in a time- and concentration-dependent manner. However, adding of ABA biosynthesis inhibitors, fluride and tungate, did not eliminate AtKu suppression. Moreover, analysis of inhibitor treatments and ABA-responsive mutants suggested that AtKu repression by ABA was mediated through the pathway of extracellular Ca(2+), phospholipase D alpha, p38-type mitogen-activated protein kinase (MAPK), MAPK6 and ABA transcription factors, ABI3 and ABI5. Finally, no cross-talk in modulating AtKu gene expression existed between ABA and antagonist hormones (auxins and gibberellic acid).

Regulation of Ku gene promoters in Arabidopsis by hormones and stress.

The Ku70/Ku80 heterodimer plays a crucial role in non-homologous end-joining during DNA repair, and is also involved in multiple cellular processes such as telomere maintenance, transcription, and apoptosis. In this study, we investigate the regulation of AtKu genes in higher plants. Promoters of the AtKu70 and AtKu80 were isolated from Arabidopsis and their activities characterised using GUS reporter constructs. AtKu promoter activities were relatively higher in hypocotyls and cotyledons upon germination and in stigma and siliques as well at their early developing stages. Furthermore, AtKu promoter activities could be enhanced by gibberellic acid, auxins, and jasmonic acid, but repressed by abscisic acid, salicylic acid, heat, drought and cold, respectively. Deletion analysis demonstrates minimal lengths of ~400 bp and 600 bp upstream of transcription start site for functional promoters of AtKu70 and AtKu80, respectively. Taken together, expressions of Ku genes are regulated both by developmental programs as well as by plant hormones and environmental stresses.

Functional roles of arginine residues in mung bean vacuolar H+-pyrophosphatase.

Plant vacuolar H+-translocating inorganic pyrophosphatase (V-PPase EC 3.6.1.1) utilizes inorganic pyrophosphate (PPi) as an energy source to generate a H+ gradient potential for the secondary transport of ions and metabolites across the vacuole membrane. In this study, functional roles of arginine residues in mung bean V-PPase were determined by site-directed mutagenesis. Alignment of amino-acid sequence of K+-dependent V-PPases from several organisms showed that 11 of all 15 arginine residues were highly conserved. Arginine residues were individually substituted by alanine residues to produce R-->A-substituted V-PPases, which were then heterologously expressed in yeast. The characteristics of mutant variants were subsequently scrutinized. As a result, most R-->A-substituted V-PPases exhibited similar enzymatic activities to the wild-type with exception that R242A, R523A, and R609A mutants markedly lost their abilities of PPi hydrolysis and associated H+-translocation. Moreover, mutation on these three arginines altered the optimal pH and significantly reduced K+-stimulation for enzymatic activities, implying a conformational change or a modification in enzymatic reaction upon substitution. In particular, R242A performed striking resistance to specific arginine-modifiers, 2,3-butanedione and phenylglyoxal, revealing that Arg242 is most likely the primary target residue for these two reagents. The mutation at Arg242 also removed F- inhibition that is presumably derived from the interfering in the formation of substrate complex Mg2+-PPi. Our results suggest accordingly that active pocket of V-PPase probably contains the essential Arg242 which is embedded in a more hydrophobic environment.

Differential regulation of Ku gene expression in etiolated mung bean hypocotyls by auxins.

Plant Ku genes were identified very recently in Arabidopsis thaliana, and their roles in repair of double-stranded break DNA and maintenance of telomere integrity were scrutinized. In this study, the cDNAs encoding Ku70 (VrKu70) and Ku80 (VrKu80) were isolated from mung bean (Vigna radiata L.) hypocotyls. Both genes were expressed widely among different tissues of mung bean with the highest levels in hypocotyls and leaves. The VrKu gene expression was stimulated by exogenous auxins in a concentration- and time-dependent manner. The stimulation could be abolished by auxin transport inhibitors, N-(1-naphthyl) phthalamic acid and 2,3,5-triiodobenzoic acid implicating that exogenous auxins triggered the effects following their uptake by the cells. Further analysis using specific inhibitors of auxin signaling showed that the stimulation of VrKu expression by 2,4-dichlorophenoxyacetic acid (2,4-D) was suppressed by intracellular Ca(2+) chelators, calmodulin antagonists, and calcium/calmodulin dependent protein kinase inhibitors, suggesting the involvement of calmodulin in the signaling pathway. On the other hand, exogenous indole-3-acetic acid (IAA) and alpha-naphthalene acetic acid (NAA) stimulated VrKu expression through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Altogether, it is thus proposed that 2,4-D and IAA (or NAA) regulate the expression of VrKu through two distinct pathways.