Heat shock transcription factorA2 (HsfA2) is a key regulator in response to heat stress in Arabidopsis (Arabidopsis thaliana), and its heat shock (HS)-induced transcription regulation has been extensively studied. Recently, alternative splicing, a critical posttranscriptional event, has been shown to regulate HS-inducible expression of HsfA2; however, the molecular mechanism remains largely unknown. Here, we demonstrate a new heat stress-induced splice variant, HsfA2-III, is involved in the self-regulation of HsfA2 transcription in Arabidopsis. HsfA2-III is generated through a cryptic 5′ splice site in the intron, which is activated by severe heat (42°C–45°C). We confirmed that HsfA2-III encodes a small truncated HsfA2 isoform (S-HsfA2) by an immunoblot assay with anti-S-HsfA2 antiserum. S-HsfA2 has an extra leucine-rich motif next to its carboxyl-terminal truncated DNA-binding domain. The biological significance of S-HsfA2 was further demonstrated by its nuclear localization and heat shock element (HSE)-binding ability. In yeast (Saccharomyces cerevisiae), the leucine-rich motif can inhibit the transcriptional activation activity of S-HsfA2, while it appears not to be required for the truncated DNA-binding domain-mediated binding ability of S-HsfA2-HSE. Further results reveal that S-HsfA2 could bind to the TATA box-proximal clusters of HSE in the HsfA2 promoter to activate its own transcription. This S-HsfA2-modulated HsfA2 transcription is not mediated through homodimer or heterodimer formation with HsfA1d or HsfA1e, which are known transcriptional activators of HsfA2. Altogether, our findings provide new insights into how HS posttranscriptionally regulates HsfA2 expression. Severe HS-induced alternative splicing also occurs in four other HS-inducible Arabidopsis Hsf genes, suggesting that it is a common feature among Arabidopsis Hsfs.

A cadmium-induced PvERF15 transcription factor binds to an AC-rich element to enhance the expression of transcription factor PvMTF-1 in bean, thus forming a PvE

The bean (Phaseolus vulgaris) stress-related gene number 2 (PvSR2) gene responds to heavy metals but not to other forms of environmental stresses. To elucidate its heavy metal-regulatory mechanism at the transcriptional level, we isolated and characterized the promoter region (−1623/+48) of PvSR2. Deletions from the 5′ end revealed that a sequence between −222 and −147 relative to the transcriptional start site was sufficient for heavy metal-specific induction of the promoter region of PvSR2. Detailed analysis of this 76-bp fragment indicated that heavy metal-responsive elements were localized in two regions (−222/−188 and −187/−147), each of which could separately confer heavy metal-responsive expression on the β-glucuronidase gene in the context of a minimal cauliflower mosaic virus 35S promoter. Region I (−222/−188) contains a motif (metal-regulatory element-like sequence) similar to the consensus metal-regulatory element of the animal metallothionein gene, and mutation of this motif eliminated the heavy metal-inducible function of region I. Region II (−187/−147) had no similarity to previously identified cis-acting elements involved in heavy metal induction, suggesting the presence of a novel heavy metal-responsive element. Transformed tobacco (Nicotiana tabacum) seedlings expressing β-glucuronidase under control of the PvSR2 promoter region (−687/+48) showed heavy metal-specific responsive activity that depended on the type and concentration of the heavy metal and the type of organ. These findings further our understanding of the regulation of PvSR2 expression and provide a new heavy-metal-inducible promoter system in transgenic plants.

Cadmium (Cd) is highly toxic to plants. Modulation of Cd-responsive transcription is an important way for Cd detoxification in plants. Metal-responsive element (MRE) is originally described in animal metallothionein genes. Although functional MREs also exist in Cd-regulated plant genes, specific transcription factors that bind MRE to regulate Cd tolerance have not been identified. Previously, we showed that Cd-inducible bean (Phaseolus vulgaris) stress-related gene2 (PvSR2) produces a short (S) PvSR2 transcript (S-PvSR2) driven by an intronic promoter. Here, we demonstrate that S-PvSR2 encodes a bean MRE-binding transcription factor1 (PvMTF-1) that confers Cd tolerance in tobacco (Nicotiana tabacum). PvMTF-1 expression was up-regulated by Cd at the levels of RNA and protein. Importantly, expression of PvMTF-1 in tobacco enhanced Cd tolerance, indicating its role in regulating Cd resistance in planta. This was achieved through direct regulation of a feedback-insensitive Anthranilate Synthase α-2 chain gene (ASA2), which catalyzes the first step for tryptophan biosynthesis. In vitro and in vivo DNA-protein interaction studies further revealed that PvMTF-1 directly binds to the MRE in the ASA2 promoter, and this binding depends on the zinc finger-like motif of PvMTF-1. Through modulating ASA2 up-regulation by Cd, PvMTF-1 increased free tryptophan level and subsequently reduced Cd accumulation, thereby enhancing Cd tolerance of transgenic tobacco plants. Consistent with this observation, tobacco transiently overexpressing ASA2 also exhibited increased tolerance to Cd. We conclude that PvMTF-1 is a zinc finger-like transcription factor that links MRE to Cd resistance in transgenic tobacco through activation of tryptophan biosynthesis.