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 59 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.

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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 (21623/148) of PvSR2. Deletions from the 5# end revealed that a sequence between 2222 and 2147 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 (2222/2188 and 2187/2147), each of which could separately confer heavy metal-responsive expression on the b-glucuronidase gene in the context of a minimal cauliflower mosaic virus 35S promoter. Region I (2222/2188) contains a motif (metalregulatory 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 (2187/2147) had no similarity to previously identified cis-acting elements involved in heavy metal induction, suggesting the presence of a novel heavy metalresponsive element. Transformed tobacco (Nicotiana tabacum) seedlings expressing b-glucuronidase under control of the PvSR2 promoter region (2687/148) 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.In plants, a number of heavy metals are essential for normal growth as cofactors and as structural and catalytic components of proteins and enzymes. These micronutrients, as well as nonessential heavy metals such as cadmium, are toxic at high levels. Plants have evolved a suite of mechanisms that control and respond to the uptake and accumulation of both essential and nonessential heavy metals. These mechanisms include the chelation and sequestration of heavy metals by particular ligands including small peptides, organic acids, and amino acids, which bind free metal ions. They contribute to metal detoxification by buffering cytosolic metal ions (Clemens, 2001). The two best-characterized heavy metal-binding ligands in plant cells are the phytochelatins (PCs) and metallothioneins (MTs; for review, see Cobbett and Goldsborough, 2002). MTs are Cys-rich polypeptides encoded by a family of genes. In contrast, PCs are a family of enzymatically synthesized Cys-rich peptides. The induction of PC synthesis is based on the posttranscriptional activation of preformed PC synthase and is not under transcriptional control.Several heavy metal-inducible genes have been reported in plants (Hagen et al., 1988;Lescure et al., 1991;Berna and Bernier, 1999), but surprisingly, little is known about the transcriptional regulation of gene expression in response to heavy metals. In particular, the cis-...

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