Galactinol synthase (GolS) is a key enzyme in the synthesis of raffinose family oligosaccharides that function as osmoprotectants in plant cells. In leaves of Arabidopsis (Arabidopsis thaliana) plants overexpressing heat shock transcription factor A2 (HsfA2), the transcription of GolS1, -2, and -4 and raffinose synthase 2 (RS2) was highly induced; thus, levels of galactinol and raffinose increased compared with those in wild-type plants under control growth conditions. In leaves of the wild-type plants, treatment with 50 μ m methylviologen (MV) increased the transcript levels of not only HsfA2, but also GolS1, -2, -3, -4, and -8 and RS2, -4, -5, and -6, the total activities of GolS isoenzymes, and the levels of galactinol and raffinose. GolS1- or GolS2-overexpressing Arabidopsis plants (Ox-GolS1-11, Ox-GolS2-8, and Ox-GolS2-29) had increased levels of galactinol and raffinose in the leaves compared with wild-type plants under control growth conditions. High intracellular levels of galactinol and raffinose in the transgenic plants were correlated with increased tolerance to MV treatment and salinity or chilling stress. Galactinol and raffinose effectively protected salicylate from attack by hydroxyl radicals in vitro. These findings suggest the possibility that galactinol and raffinose scavenge hydroxyl radicals as a novel function to protect plant cells from oxidative damage caused by MV treatment, salinity, or chilling.

Summary: We isolated 76 high‐light and heat‐shock (HL + HS) stress‐inducible genes, including a putative heat‐shock transcription factor (HsfA2), by suppression‐subtractive hybridization from Arabidopsis. The transcript level of HsfA2 was significantly increased under the several stress conditions or by the H₂O₂ treatment. Furthermore, the induction of HsfA2 expression was highest among those of other class A HSFs in response to HL + HS stress conditions. The promoter assay revealed that HsfA2 is induced mainly in rosette leaves under HL + HS stress conditions. In the HsfA2‐overexpressing Arabidopsis (Pro_35S:HsfA2) plants, 46 genes, including a large number of heat‐shock proteins, ascorbate peroxidase 2 and galactinol synthase 1 and 2, were highly expressed compared with those in the wild‐type plants. The transcript levels of the HsfA2 target genes are highly correlated with those of HsfA2 in the Pro_35S:HsfA2 plants. The transcript levels of the HsfA2 target genes, as well as HsfA2 transcripts, were induced by treating with exogenous H₂O₂. In the knockout HsfA2 Arabidopsis plants, the induction of 26 HsfA2 target genes was strongly reduced for up to 2 h under HL + HS stress conditions. Furthermore, the Pro_35S:HsfA2 plants showed increased tolerance to combined environmental stresses. Our present results indicate that HsfA2 is a key regulator in the induction of the defence system under several types of environmental stress.

We have isolated a combination of high-light and heat-shock (HL + HS) stress-inducible genes, including a NAC transcription factor designated ANAC078. Here we explored the physiological function of ANAC078 under HL stress. Yeast transcription activity assays showed that ANAC078 functions as a transcriptional activator. A fusion protein composed of green fluorescent protein (GFP) and full-length ANAC078 was detected in the nucleus and cytoplasm, while fusion proteins comprising GFP and ANAC078 deleted of a putative transmembrane motif were found only in the nucleus. In ANAC078-overexpressing Arabidopsis plants (Ox-ANAC078-43), the transcription of 166 genes was up-regulated compared with the levels in wild-type plants under HL (1200 μmol m⁻² s⁻¹, 30°C). These genes included some for transcription factors regulating the expression of genes related to the biosynthesis of flavonoids. Interestingly, the transcript levels of some genes related to flavonoid biosynthesis and the levels of anthocyanins were significantly increased in Ox-ANAC078 plants and reduced in knockout ANAC078 plants (KO-ANAC078) compared with wild-type plants under HL stress. The present findings suggest that ANAC078 protein is associated with the induction of genes related to flavonoid biosynthesis, leading to the accumulation of anthocyanins, in response to HL stress.

Heat shock transcription factor A2 (HsfA2) acts as a key component of the Hsf signaling network involved in cellular responses to various types of environmental stress. However, the mechanism governing the regulation of HsfA2 expression is still largely unknown. We demonstrated here that a heat shock element (HSE) cluster in the 5′-flanking region of the HsfA2 gene is involved in high light (HL)-inducible HsfA2 expression. Accordingly, to identify the Hsf regulating the expression of HsfA2, we analyzed the effect of loss-of-function mutations of class A Hsfs on the expression of HsfA2 in response to HL stress. Overexpression of an HsfA1d or HsfA1e chimeric repressor and double knockout of HsfA1d and HsfA1e Arabidopsis mutants (KO-HsfA1d/A1e) significantly suppressed the induction of HsfA2 expression in response to HL and heat shock (HS) stress. Transient reporter assays showed that HsfA1d and HsfA1e activate HsfA2 transcription through the HSEs in the 5′-flanking region of HsfA2. In the KO-HsfA1d/A1e mutants, 560 genes, including a number of stress-related genes and several Hsf genes, HsfA7a, HsfA7b, HsfB1 and HsfB2a, were down-regulated compared with those in the wild-type plants under HL stress. The PSII activity of KO-HsfA1d/A1e mutants decreased under HL stress, while the activity of wild-type plants remained high. Furthermore, double knockout of HsfA1d and HsfA1e impaired tolerance to HS stress. These findings indicated that HsfA1d and HsfA1e not only regulate HsfA2 expression but also function as key regulators of the Hsf signaling network in response to environmental stress.