Abstract
Ku is a heterodimer consisting of two related subunits, Ku70 and Ku80. Orthologues of both subunits have been found in many eukaryotes from yeast to man. This multifunctional protein is involved in many cellular metabolic processes, such as transcriptional regulation, telomeric maintenance, and cell cycle regulation. Moreover, Ku, being an origin binding-protein, acts at the initiation step of DNA replication. Plant Ku genes were recently identified in Arabidopsis thaliana (AtKu). AtKu has role in the repair of DNA double-strand breaks by non-homologous end joining in response to DNA damaging agents and lack of AtKu results in a deregulation of telomere length control. Involvement of AtKu80 in T-DNA integration was also verified. Moreover, an interaction between the Werner syndrome-like exonuclease AtWEX and the AtKu heterodimer was identified. However, studies about its function and regulation in plant are still limited until now. Firstly, the cDNAs encoding Ku70 (VrKu70) and Ku80 (VrKu80) were isolated from mung bean (Vigna radiata L.) hypocotyls. Computational analysis showed a lot of information about plant Ku, such as phylogenetic relations of Ku proteins among different organisms, predicted its structure. The Ku-interacting proteins and Ku expression under different environmental conditions were studied. Secondly, we investigated how plant Ku is regulated by plant hormone, auxin. Both VrKu 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. 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 Ca2+ 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 α-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. Thirdly, we continuously studied how AtKu is regulated during abscisic acid (ABA) induced slow growth in three-week-old seedlings. Bioinformatic analysis firstly predicted the existence of several ABA responsive elements and binding sites of transcription factor, AREB6 and ATHB5, on AtKu promoters. AtKu promoter-β-glucuronidase (GUS) analysis in transgenic Arabidopsis showed reduced activity of AtKu promoter upon ABA application. AtKu gene repression by ABA treatment is in a time- and concentration-dependent manner using GUS assay and real time quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. However, addition of ABA biosynthesis inhibitors, fluride and tungate, could not abolish the AtKu suppression. Moreover, AtKu repression in response to ABA was mediated through the pathway of extracellular Ca2+, phospholipase D alpha, p38-type mitogen-activated protein kinase (MAPK), MAPK6 and ABA transcription factors, ABI3 and ABI5 by analysis of inhibitor treatments and ABA responsive mutants. Finally, no cross-talk was found for modulating AtKu gene expression between ABA and antagonist hormones (auxins and gibberellic acid). Finally, we focused on how AtKu is regulated by environmental stress-heat shock. Bioinformatic analysis found several heat shock responsive elements and heat shock transcription factor binding sites on AtKu promoters. The expression of AtKu is down-regulated by heat stress in a time course with real time RT-PCR and AtKu promoter-GUS (β-Glucuronidase) analysis using 3-week-old young seedlings. On the other hand, the high-temperature repression of AtKu is mediated through ABA biosynthesis, as shown by reversed repression of the AtKu in ABA-biosynthesis mutant, aba3 and increased ABA level analyzed by reverse high performance liquid chromatography. The involvement of ethylene signaling, DNA repair pathway and fatty acid synthesis in AtKu regulation by heat were also shown. Furthermore, our results showed heat regulated tissue-specific AtKu repression at different developmental stages. Taken together, bioinformatics tools and online databases gave us extended knowledge about functions of plant Ku protein. In addition, the experimental results provided molecular evidence for plant Ku gene regulation by plant hormones and stress.