| Bidirectional gene pairs comprise two adjacent genes located on opposite DNA strands, with the distance between their transcription start sites (TSS) within 1,000 bp.The sequence between the two such TSSs has been suggested to represent a potential bidirectional promoter. Divergent organization of two protein-encoding genes has been known for many years, and which is common in different organisms, including bacteria, yeasts, plants and animals. The most likely proposed function of such bidirectional promoter is to co-regulate two oppositely directed genes. Given the joint transcription regulatory elements for both directions, the transcripts of the divergent gene pair may be coordinately regulated.Although genome-wide expression analyses revealed that the bidirectional genes are more correlated in expression than other random gene pairs and most of all the correlations are positive,it has been also observed that some bidirectional genes display opposite regulation depending on the experimental conditions. To our knowledge, relatively few studies have addressed the mechanisms involved in the anti-correlated regulation of bidirectional gene pairs. To investigate the molecular mechanism of bidirectional gene pairs will be helpful for better unstanding of their transcrition regulation as well as their physiological function.The A. thaliana genes Atlg71850 and At1g71860 are arranged in a head-to-head configuration and their TSSs are separated by a 347-bp intergenic sequence, which is assumed to act as a bidirectional promoter. Atlg71850,named as AtUCH4 by us,·encodes a putative ubiquitin carboxyl-terminal hydrolase family protein. The Atlg71860 gene encodes PTP1, a tyrosine-specific protein phosphatase and has been proved to be down-regulated by cold treatment. Analysis based on the AtGenExpress Visualization Tool (AVT) shows that both of the AtUCH4 and AtPTPl expression are localized to the germinated seeds, the shoot meristems, and the flowers. In silico analysis using AVT indicates that AtUCH4 is also a cold-responsive gene, but the transcripts of AtUCH4 is significantly induced by cold stress. Then,the bidirectional genes AtUCH4 and AtPTP1 are anti-regulated by abiotic stress.In the present study, we first investigated whether the intergenic region between AtUCH4 and AtPTP1 can be a true bidirectional promoter. The expression pattern of AtUCH4 and AtPTPl was also examined during the development and growth of Arabidopsis plants as well as during stress situations. In addition, we investigated the functions of the AtUCH4 and AtPTPl divergent genes under salt stress. The main results and conclusions of our study are indicated below:1 The intergenic sequence between the AtUCH4 and AtPTPl divergent genes is a functional bidirectional promoterTo test whether the putative bidirectional promoter can activate gene expression in both orientations, the intergenic sequence between the AtUCH4 and AtPTP1 divergent genes was fused to the GUS gene in either orientation and then introduced into wild type A. thaliana plants.Histochemical and fluorometric assays of GUS activity indicated that the intergenic sequence between the AtUCH4 and AtPTPl divergent genes is indeed a functional bidirectional promoter, able to drive expression, but in an asymmetrical manner according to its orientation. The strength of the forward promoter was significantly greater than that of the reverse promoter in all tissues and at all developmental stages tested. The bidirectional promoter activity of the 578 bp intergenic region was again confirmed by using two reporter genes in both orientations. Results suggest that this bidirectional promoter could direct GFP and GUS transient expressions in the heterologous tobacco system.2 The AtUCH4 and AtPTPl divergent genes were co-expressed in the same tissues Histochemical and fluorometric assays of GUS activity showed that the AtUCH4 and AtPTP1 promoters have similar expression patterns, with preferential expression in the germinated seeds, the pollen and the shoot meristems. The entire AtPTP1 promoter showed to be localized to the germinate seeds, the shoot and root meristems,the pollen, and the vascular system. The expression of the entire AtUCH4 promoter was largely limited to the germinated seeds and the pollen. However, the larger deleted AtUCH4 promoter directed GUS expression in the germinate seeds, the shoot and root meristems, the pollen, and the vascular system. Therefore, the AtUCH4 and AtPTP1 promoters have similar expression patterns, co-expressed in the germinated seeds, the pollen and the meristems.3 The AtUCH4 and AtPTP1 divergent genes were co-regulated under abiotic stressesFluorometric measurement of GUS activity revealed that cold treatment changed the expression level of the AtUCH4/AtPTPl bidirectional promoter in either orientation. After cold treatment, the transgenic plants containing the AtUCH4 promoter-GUS fusion showed a higher expression level of the reporter gene compared to the untreated transgenic plants. However, transgenic lines containing the AtPTP1 promoter fused to GUS showed lower expression level compared to the untreated controls. Therefore, the up-regulation of AtUCH4 expression during cold stress was accompanied by a simultaneous down-regulation of AtPTP1 expression. The expression of AtUCH4 and AtPTP1 under salt stress was also estimated using GUS histochemical assay and QRT-PCR analysis. The results showed that the AtUCH4 and AtPTP1 bidirectional genes were to be salt-reducible. Therefore, the AtUCH4 and AtPTP1 bidirectional genes were co-regulated under salt stress.4 The AtUCH4/AtPTPl bidirectional promoter harbors positive and negative regulatory cis-elementsDeletion analyses have showed that the AtUCH4/AtPTP1 bidirectional promoter could be divided into three sub-regions, region ?, region ? and region ?. The. basal expression level of promoter activity in reverse orientation was regulated by one weak positive regulatory region (region ?) and one weak negative regulatory region (region?), whereas the activity of the promoter in forward orientation was regulated by one strong positive regulatory region (region ?) and one weak negative regulatory region(region ?). Deletion analyses also demonstrated that the site ? elements and the telo-box acted in concert to maximize the strength of the forward promoter, whereas the effectiveness of the reverse promoter depended mainly on the presence of the site II elements and the ACGT motif.The cold-responsive expression level of promoter activity in AtPTPl orientation was regulated by a negative regulatory region (region ?), whereas the activity of the promoter in AtUCH4 orientation was regulated by one positive regulatory region(region ?) and one negative regulatory region (region ?) under the cold stress.Further analyses demonstrated that the as-1/ocs-like element was a cold-responsive element, and acted in an orientation-dependent manner.5 The AtUCH4 and AtPTPl divergent genes were involved in salt stress responseTo study AtUCH4 and AtPTP1 functions in plants, two T-DNA mutants, uch4 and ptpl were identified. The uch4 and ptpl mutants were indistinguishable from Col-0 in morphology under standard growth conditions. However, they showed a different phenotype to Col-0 plants under salt stress. Under salt stress (100 mM and 150 mM NaCl) conditions, the germination rates of the uch4 and ptpl. mutants were significantly lower than wild-type plants.To further investigate AtUCH4 and AtPTP1 functions, the AtUCH4 and AtPTP1 genes, under the control of an ubiquitin promoter, were transformed into the uch4 and ptpl mutants respectively and the homozygous lines with the similar AtUCH4 and AtPTP1 expression to Col-0 were selected for further analysis. The complemented lines expressing AtUCH4 in the uch4 mutant and AtPTP1 in ptpl were indistinguishable from Col-0 in morphology under salt stress and standard growth conditions. Under salt stress conditions, the germination rates of the complemented lines expressing AtUCH4 in the uch4 mutant and AtPTP1 in ptpl were similar to those of wild-type plants. These results indicate that both of the AtUCH4 and AtPTPl genes function in salt stress response.In conclusion, our findings will enhance the understanding of the regulatory mechanisms of plant bidirectional promoters and co-expression and co-functionality of the divergent genes. |
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