Identification of a new 130 bp cis-acting element in the TsVP1 promoter involved in the salt stress response from Thellungiella halophila

Abstract

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.

Figure 1

The construction of the promoter-reporter plasmids. a. A set of 5' deleted promoters fused to the GUS reporter gene. A series of 5' deletions of the TsVP1 promoter region were transcriptional linked to the GUS reporter gene. The number stands for the nucleotide position from the translational initiate site, ATG (A as +1). b. The full length AVP1 promoter was linked to the GUS reporter gene.

Figure 2

Histochemical staining of transgenic Arabidopsis PT1 and PA1. A. PT1 whole plant; B. PT1 flower; C. PT1 silique; D. PT1 seeds; E. PT1 root tip without salt stress. F. PT1 root tip with salt stress. G. PA1 whole plant without salt stress; H. PA1 whole plant with salt stress. I. PA1 root tip without salt stress. J. PA1 root tip with salt stress.

Figure 3

GUS enzymatic activity quantification of different parts of transgenic Arabidopsis. A. Expression of the GUS gene was driven by promoters PT1, PA1 and P35S under normal and salt stress conditions. B. Expression of the GUS gene was driven by promoters PT1 to PT9 under normal conditions.

Figure 4

Histochemical staining of different transgenic Arabidopsis lines where GUS expression was driven by different fragments of the TsVP1 promoter. A. PT1 10-day old plant; B. PT2 10-day old plant; C. PT8 10-day old plant; D. a PT2 flower; E a PT7 flower; F. silique from a PT2 plant.

Figure 5

Histochemical staining of transgenic Arabidopsis lines PT2 and PT8 under normal and salt stress conditions. A. Whole PT2 plant under normal conditions; B. Whole PT2 plant under salt stress conditions; C. the roots from a PT2 plant under normal conditions; D. the roots from a PT2 plant under salt stress treatment; E. whole PT8 plant under normal conditions; F. whole PT8 plant under salt stress treatment.

Figure 6

GUS transient assay in tobacco leaves. A. Fusion constructs used in the transient assay: (a), the control construct P-mini35S in which the GUS gene was driven by the mini 35S (-46 to +10) promoter. (b), the test construct P-130-mini35S in which the 130 bp region (-667 to -538) identified in the TsVP1 promoter was fused to the mini 35S promoter to drive the GUS reporter gene. B. GUS activities resulting from the transient transformation with constructs P-mini35S and P-130-mini35S under both normal and salt stress conditions.

Figure 7

Comparing of the identified 130 bp region in TsVP1 promoter to the corresponding sequence from the AVP1 promoter using the EBI tool ClustalW2.