Populus euphratica HSF binds the promoter of WRKY1 to enhance salt tolerance

Highlights

• Salt-elicited WRKY1 expression in Populus euphratica contributes to salt tolerance.

• PeWRKY1 was down-regulated in salinized P. euphratica when PeHSF was silenced.

• PeWRKY1 promoter harbours four tandem repeats of cis-acting heat shock element (HSE).

• PeHSF directly binds the cis-acting HSE of PeWRKY1 promoter.

• Salt-enhanced expression of PeWRKY1 in P. euphratica was due to promoter activity.

Abstract

Poplar species increase expressions of transcription factors to deal with salt environments. We assessed the salt-induced transcriptional responses of heat-shock transcription factor (HSF) and WRKY1 in Populus euphratica, and their roles in salt tolerance. High NaCl (200 mM) induced PeHSF and PeWRKY1 expressions in P. euphratica, with a rapid rise in roots than in leaves. Moreover, the salt-elicited PeHSF reached its peak level 6 h earlier than PeWRKY1 in leaves. PeWRKY1 was down-regulated in salinized P. euphratica when PeHSF was silenced by tobacco rattle virus-based gene silencing. Subcellular assays in onion epidermal cells and Arabidopsis protoplasts revealed that PeHSF and PeWRKY1 were restricted to the nucleus. Transgenic tobacco plants overexpressing PeWRKY1 showed improved salt tolerance in terms of survival rate, root growth, photosynthesis, and ion fluxes. We further isolated an 1182-bp promoter fragment upstream of the translational start of PeWRKY1 from P. euphratica. Promoter sequence analysis revealed that PeWRKY1 harbours four tandem repeats of heat shock element (HSE) in the upstream regulatory region. Yeast one-hybrid assay showed that PeHSF directly binds the cis-acting HSE. To determine whether the HSE cluster was important for salt-induced PeWRKY1 expression, the promoter–reporter construct PeWRKY1–pro::GUS was transferred to tobacco plants. β-glucuronidase activities increased in root, leaf, and stem tissues under salt stress. Therefore, we conclude that salinity increased PeHSF transcription in P. euphratica, and that PeHSF binds the cis-acting HSE of the PeWRKY1 promoter, thus activating PeWRKY1 expression.

Introduction

Naturally existing saline and sodic soils present a global problem [1]. On land unsuitable for arable crop production, planting trees can both expand tree afforestation and promote silvicultural management of salt-affected soils [1]. Assessing the potential of tree species to cope with salt stress requires elucidation of the molecular mechanisms underlying salt acclimation and adaptation, particularly those employed in salt-resistant tree species. Populus euphratica is a model species for investigating salt tolerance in trees [1], [2], [4], [3]. Whole genome microarray analyses have revealed major transcriptome differences related to the capacity of P. euphratica to tolerate salinity compared to the salt-sensitive poplar species [3], [5], [6]. Under non-stressed conditions, P. euphratica shows constitutive upregulation of genes involved in carbohydrate metabolism, ATPase activity, ion transport, oxidoreductase, and cell wall organization compared with the Populus  × canescens transcriptome [5]. Microarray analysis shows that short-term exposure to salinity induces the expressions of genes related to Na⁺/K⁺ and ROS homeostasis in P. euphratica leaves [6].

Poplar species respond to salt stress with upregulated expressions of numerous transcription factors, such as HSF [7], CBL [8], bZIP [9] and WRKY [10]. In herbaceous species, transcription factors interact with cis-elements in the promoter regions of various stress-related genes [11], [12], [13]. The transcriptional response of HSF in P. euphratica to salinity and the relevance to NaCl-induced oxidative defence have been clarified in our previous study [7]. Binding of HSF to heat shock elements (HSEs) leads to transcriptional regulation of heat shock genes [14], [15], [16]. In higher plants, the importance of HSEs for heat-dependent transcriptional regulation has been demonstrated by promoter deletions and HSE sequence integration in a truncated CaMV35S promoter [17]. It is likely that, in P. euphratica, salt-inducible HSF bind with HSEs in the promoter of salinity-responsive genes, thus increasing their expressions. However, the activation pathways of stress-responsive genes have scarcely been investigated in tree species.

The WRKY gene family plays a crucial role in plant responses to abiotic and biotic stress [18]. WRKY proteins recognize and activate a variety of plant defence or defence-related genes that contain W-box sequences in their promoters, making these proteins important in pathogen responses [19]. Expression profiling and functional analysis have shown that Populus WRKY23 plays a regulatory role in the defence response to Melampsora rust [20]. Increasing evidence demonstrates that the stress-regulated WRKY gene is also important in abiotic stress responses. In Arabidopsis, WRKY genes are induced by drought, cold, or high-salinity stress [21], and a tobacco WRKY transcription factor is specifically induced by drought and heat shock [22]. In barley, the Hv-WRKY38 gene is continuously induced during cold and drought stress [23]. Transgenic overexpression of soybean WRKY13, WRKY21, and WRKY54 in Arabidopsis plants conferred differential tolerance to abiotic stresses [24]. Chen et al. reported that Arabidopsis WRKY18, WRKY40, and WRKY60 form a highly interacting regulatory network that modulates gene expression during the plant responses to abscisic acid and abiotic stress [13]. Arabidopsis WRKY8 is predominantly expressed in roots, is highly upregulated following salt treatment, and directly binds the RD29A promoter to modulate salinity stress tolerance [11]. Furthermore, WRKY8 affects plant Na⁺/K⁺ homeostasis, a critical determinant of salt adaptation in plants under salt stress [11]. Similarly, TaWRKY79 enhances a plant's tolerance to both salinity and ionic stress, while reducing sensitivity to ABA [25]. Using di-haploid Populus simonii × Populus nigra plants, Wang et al. examined spatio-temporal expression patterns of WRKY genes in response to salinity stress, and found that the thirteen tested WRKY genes were up-regulated in roots and leaves during NaCl stress [26]. Microarray analysis revealed that a variety of WRKY family genes were constitutively up-regulated in non-salinized P. euphratica, compared with the salt-sensitive poplar, P. popularis (our unpublished data). We noticed that P. euphratica increased PeWRKY1 expression after exposure to NaCl saline. However, the precise roles of Populus WRKY family genes in salt tolerance and its regulatory mechanisms are not yet fully understood.

In the present study, we assessed the salt-induced transcriptional responses of heat-shock transcription factor (HSF) and WRKY1 in the salt-resistant poplar species P. euphratica, and their roles in salt tolerance. We observed that salinized P. euphratica plants upregulated PeWRKY1 expression in roots and leaves, but PeWRKY1 was down-regulated when PeHSF was silenced under NaCl treatment. To evaluate the functional role of WRKY1 in salinity tolerance, PeWRKY1 was isolated from P. euphratica and transferred into tobacco under control of the CaMV35S promoter. We found that PeWRKY1 overexpression enhanced salinity tolerance in the transgenic plants, suggesting that the salt-induced WRKY1 up-regulation in P. euphratica helped plants adapt to highly saline environments. We further found that PeWRKY1 promoter regions harboured cis-acting heat shock elements (HSEs), implying that heat shock transcription factors (HSFs) may upregulate PeWRKY1 expression. We further investigated the subcellular localizations of PeHSF and PeWRKY1, the binding of PeHSF with cis-acting HSE in the PeWRKY1 promoter, and the salt-induced expression of PeWRKY1 promoter in root and shoot tissues. Overall, our findings confirm our hypothesis that the PeHSF domain can bind to the HSE in the PeWRKY1 promoter regions, thus exerting regulation of target gene transcription and promoting salt tolerance.