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 mM 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.

SummaryWe isolated 76 high-light and heat-shock (HL þ 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 H 2 O 2 treatment. Furthermore, the induction of HsfA2 expression was highest among those of other class A HSFs in response to HL þ HS stress conditions. The promoter assay revealed that HsfA2 is induced mainly in rosette leaves under HL þ 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 H 2 O 2 . In the knockout HsfA2 Arabidopsis plants, the induction of 26 HsfA2 target genes was strongly reduced for up to 2 h under HL þ 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.

The active oxygen species (AOS) that arise from normal metabolic processes are kept under tight control by various antioxidant mechanisms. AOS are important signal molecules that regulate many physiological processes, including environmental stress responses. In this work, we have investigated the effect of lowered cytosolic ascorbate peroxidase (APX) activity in transgenic tobacco BY-2 cells, using two transformed BY-2 cell lines, cAPX-S2 and cAPX-S3, resulting from co-suppression by expression of Arabidopsis APX1 cDNA under the cauliflower mosaic virus (CaMV) 35S promoter. cAPX-S2 and cAPX-S3 possessed 50 and 75% lower cytosolic APX activity, respectively, compared with that in the untransformed cells. Chemical fluorescence analysis indicated that the AOS levels were markedly higher in the two APX-suppressed cell lines than in the wild-type cells. However, there were no substantial differences in the activity levels of the various other antioxidant enzymes. Interestingly, the APX-suppressed cells showed different responses and tolerances to environmental stresses, such as heat and salinity. Suppression subtractive hybridization revealed that several heat- and salt stress-inducible genes were up-regulated in cAPX-S3 cells. HSP70, DnaJ-like protein and purple acid phosphatase were among the constitutively induced genes. An in-gel kinase assay suggested that a mitogen-activated protein (MAP) kinase of approximately 46 kDa was predominantly active in the APX-suppressed cells, and transcript levels of both nicotiana protein kinase 1 (NPK1) and nucleoside diphosphate kinase 2 (NDPK2) were up-regulated. These data suggest the possibility that MAP kinase cascades are activated by subtle imbalances in the homeostasis of the cellular redox status caused by lowered cytosolic APX activity.

Preeclampsia is a pregnancy-induced disorder that is characterized by hypertension and is a leading cause of perinatal and maternal–fetal morbidity and mortality. HLA-G is thought to play important roles in maternal–fetal immune tolerance, and the associations between HLA-G gene polymorphisms and the onset of pregnancy-related diseases have been explored extensively. Because contiguous genomic sequencing is difficult, the association between the HLA-G genotype and preeclampsia onset is controversial. In this study, genomic sequences of the HLA-G region (5.2 kb) from 31 pairs of mother–offspring genomic DNA samples (18 pairs from normal pregnancies/births and 13 from preeclampsia births) were obtained by single-molecule real-time sequencing using the PacBio RS II platform. The HLA-G alleles identified in our cohort matched seven known HLA-G alleles, but we also identified two new HLA-G alleles at the fourth-field resolution and compared them with nucleotide sequences from a public database that consisted of coding sequences that cover the 3.1-kb HLA-G gene span. Intriguingly, a potential association between preeclampsia onset and the poly T stretch within the downstream region of the HLA-G*01:01:01:01 allele was found. Our study suggests that long-read sequencing of HLA-G will provide clues for characterizing HLA-G variants that are involved in the pathophysiology of preeclampsia.