articleIdentification of a new 130 bp cis-acting element in the TsVP 1 promoter involved in the salt stress response from Thellungiella halophila

Background: Salt stress is one of the major abiotic stresses affecting plant growth and productivity. Vacuolar H+pyrophosphatase (H+-PPase) genes play an important role in salt stress tolerance in multiple species. Results: In this study, the promoter from the vacuolar H+-pyrophosphatase from Thellungiella halophila (TsVP1) was cloned and compared with the AVP1 promoter from Arabidopsis thaliana. Sequence analysis indicated that these two promoters had seven similar motifs at similar positions. To determine which tissues the two promoters are active in, transgenic plants were produced with expression of the GUS reporter gene under the control of one of the promoters. In transgenic Arabidopsis with the TsVP1 promoter, the GUS reporter gene had strong activity in almost all tissues except the seeds and the activity was induced in both shoots and roots, especially in the root tips, when treated with salt stress. Such induction was not found in transgenic Arabidopsis with the AVP1 promoter. By analyzing different 5' deletion mutants of the TsVP1 promoter, an 856 bp region (-2200 to -1344) was found to contain enhancer elements that increased gene expression levels. Two AAATGA motifs, which may be the key elements for the anther specific expression profile, in the deleted TsVP1 promoters (PT2 to PT6) were also identified. A 130 bp region (-667 to -538) was finally identified as the key sequence for the salt stress response by analyzing the different mutants both with and without salt stress. GUS transient assay in tobacco leaves suggested the 130 bp region was sufficient for the salt stress response. Bioinformatic analysis also revealed that there may be novel motifs in this region that are the key elements for the salt stress responsive activity of the TsVP1 promoter. Conclusions: The TsVP1 promoter had strong activity in almost all tissues except the seeds. In addition, its activity was induced by salt stress in leaves and roots, especially in root tips. A 130 bp region (-667 to -538) was identified as the key region for responding to salt stress. Background Salt stress is one of the major abiotic stresses for plants in the world. High concentrations of sodium in soils are deleterious to the growth and development of non-halophytes. Global crop production is affected by salinity stress and this problem is becoming more and more serious [1,2]. Most of the major crops are salt-sensitive and irrigation-induced soil salinization causes the lost of large tracts of agricultural land [3]. It is important to study the salt-tolerance mechanisms to improve crop plants. Arabidopsis (Arabidopsis thaliana) has been considered an excellent model system for determining molecular pathways in plants since its genome was sequenced in 2000. In the past ten years it has been used extensively to investigate salt tolerance in plants [4]. However, as a true glycophyte, it is difficult for Arabidopsis to survive at even moderate salinity (~100 mM NaCl). Studying only Arabidopsis could not provide enough information on salt-tolerant mechanisms. Thellungiella halophila is a salt-tolerant close relative of Arabidopsis that has been considered a model system for studying salt tolerance in plants [1,5]. Because of the high similarity between these two species at the cDNA level (>90% nucleotide identity in cDNA sequences), the resources of Arabidopsis such as gene and protein information can be used to study T. halophila. Furthermore, T. halophila shared several advantages with Arabidopsis. It has a small genome, a * Correspondence: jrzhang@sdu.edu. cn 1 School of Life Science, Shandong University, Jinan, China Full list of author information is available at the end of the article BioMed Central © 2010 Sun et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sun et al. BMC Plant Biology 2010, 10:90 http://www.biomedcentral. com/1471-2229/10/90 Page 2 of 12 short life cycle, enough seed production and can easily be transformed [1,5]. Interestingly, these two species have obvious differences in stress-tolerance while sharing high similarity in cDNA sequences. This may due to differences in the gene regulatory regions between these two species [4]. Therefore, it is important to study the promoter sequences of T. halophila to discover how they differ from those of Arabidopsis. Strong constitutive promoters, such as the CaMV 35S promoter and the maize ubiquitin gene promoter [6,7], have been widely used in transgenic plants to express foreign genes. However, it may be harmful to express a foreign gene constitutively in host plants. This may lead to sterility, delayed development, abnormal morphology, yield loss, changes in grain composition or transgene silencing [8-13]. To solve this problem, a strong tissuespecific or inducible promoter can be used to restrict gene expression to only the required tissue or at a specific time. Arabidopsis promoters rd29A and rd29B were found to response to multiple stresses including high salinity, drought, cold and ABA [14]. Then with the development of cDNA microarray technology and sequencing of the Arabidopsis genome, many stress inducible genes were identified in Arabidopsis [15-18]. In addition, a large number of tissue-specific or inducible genes and their promoters have been identified from other species [1923]. However, there has been no report of a promoter that responds to salt stress particularly in the root tips. Vacuolar H+-pyrophosphatase (H+-PPase) genes play an important role in abiotic-stress tolerance. Transgenic plants overexpressing the vacuolar H+-pyrophosphatase gene AVP1 from Arabidopsis are much more resistant to high concentrations of NaCl and to water deprivation than wild-type strains [24]. Gao et al. cloned a novel H+PPase gene named TsVP1 from Thellungiella halophila. The heterologous expression of TsVP1 or AVP1 in yeast mutant ena1 partly restored its salt tolerance [25]. Heterologous expression of TsVP1 in tobacco also improved its salt tolerance [25]. Although they have a similar function in salt tolerance, these two genes had different expression pattern under salinity stress. When treated with 200 mM NaCl solution, TsVP1 expression increased 3-fold in the aerial roots and 5-fold in the roots during the first 16 h. But AVP1 expression was not changed [25]. Thus, there must be some differences between the promoter regions of these two homologous genes. In this report we cloned and analyzed the promoter regions of the TsVP1 and AVP1 genes. In transgenic Arabidopsis, the GUS reporter gene driven by the TsVP1 promoter was obviously induced under salt stress, especially in the root tips. However, the GUS gene driven by the AVP1 promoter was not changed under salt stress. By analyzing the sequences and a series of deletion mutants, we identified a 130 bp region from the TsVP1 promoter which may be the key sequence for responding to salt stress.