SummaryIn order to assess specific functional roles of plant heat shock transcription factors (HSF) we conducted a transcriptome analysis of Arabidopsis thaliana hsfA1a/hsfA1b double knock out mutants and wild-type plants. We used Affymetrix ATH1 microarrays (representing more than 24 000 genes) and conducted hybridizations for heat-treated or non-heat-treated leaf material of the respective lines. Heat stress had a severe impact on the transcriptome of mutant and wild-type plants. Approximately 11% of all monitored genes of the wild type showed a significant effect upon heat stress treatment. The difference in heat stress-induced gene expression between mutant and wild type revealed a number of HsfA1a/1b-regulated genes. Besides several heat shock protein and other stress-related genes, we found HSFA-1a/1b-regulated genes for other functions including protein biosynthesis and processing, signalling, metabolism and transport. By screening the profiling data for genes in biochemical pathways in which known HSF targets were involved, we discovered that at each step in the pathway leading to osmolytes, the expression of genes is regulated by heat stress and in several cases by HSF. Our results document that in the immediate early phase of the heat shock response HSF-dependent gene expression is not limited to known stress genes, which are involved in protection from proteotoxic effects. HsfA1a and HsfA1b-regulated gene expression also affects other pathways and mechanisms dealing with a broader range of physiological adaptations to stress.

Heat shock factors (HSFs) are transcriptional regulators of the heat shock response. The major target of HSFs are the genes encoding heat shock proteins (HSPs), which are known to have a protective function that counteracts cytotoxic effects. To identify other HSF target genes, which may be important determinants for the generation of stress tolerance in Arabidopsis, we screened a library enriched for genes that are up-regulated in HSF3 (AtHsfA1b)-overexpressing transgenic plants (TPs). Galactinol synthase1 (GolS1) is one of the genes that is heat-inducible in wild type, but shows constitutive mRNA levels in HSF3 TPs. The generation and analysis of TPs containing GolS1-promoter::b-glucuronidase-reporter gene constructs showed that, upon heat stress, the expression is transcriptionally controlled and occurs in all vegetative tissues. Functional consequences of GolS1 expression were investigated by the quantification of raffinose, stachyose, and galactinol contents in wild type, HSF3 TPs, and two different GolS1 knockout mutants (gols1-1 and gols1-2). This analysis demonstrates that (1) raffinose content in leaves increases upon heat stress in wild-type but not in the GolS1 mutant plants; and (2) the level of raffinose is enhanced and stachyose is present at normal temperature in HSF3 TPs. These data provide evidence that GolS1 is a novel HSF target gene, which is responsible for heat stress-dependent synthesis of raffinose, a member of the raffinose family oligosaccharides. The biological function of this osmoprotective substance and the role of HSF-dependent genes in this biochemical pathway are discussed.

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In order to assess the specific functional roles of different plant heat shock transcription factors (HSFs) we have isolated T-DNA insertion mutants in the AtHsf1 and AtHsf3 genes of Arabidopsis thaliana. Complete and selective loss of the promoter binding activities of AtHSF1 or AtHSF3, verified by immunoprecipitation assays, had no obvious effects on the heat shock (HS) response in the individual mutant lines. Only hsf1(-) /hsf3(-)double mutants were significantly impaired in HS gene expression. In these plants the inability to form high-molecular-weight HSE-binding complexes correlates with a dramatic change in the kinetics of mRNA accumulation from all HSF target genes tested, including members of the Hsp100, Hsp90, Hsp70 and small Hsp families, and genes for two heat-inducible class B-HSFs. After prolonged HS, the amounts of most heat shock mRNAs expressed, except transcripts of Hsp18.2, reached approximately the same levels as in wild type plants. Our data indicate that AtHSF1 and AtHSF3 are key regulators of the immediate stress-induced activation of HS gene transcription, and consequently determine the kinetics of the negative feed back loop that is responsible for the transience of HS gene expression in wild type.

Heat stress transcription factors (HSFs) are central regulators of the heat stress response. Plant HSFs of subgroup B lack a conserved sequence motif present in the transcriptional activation domain of class A-HSFs. Arabidopsis members were found to be involved in non-heat shock functions. In the present analysis we investigated the expression, regulation and function of HSFB2a. HSFB2a expression was counteracted by a natural long non-coding antisense RNA, asHSFB2a. In leaves, the antisense RNA gene is only expressed after heat stress and dependent on the activity of HSFA1a/HSFA1b. HSFB2a and asHSFB2a RNAs were also present in the absence of heat stress in the female gametophyte. Transgenic overexpression of HSFB2a resulted in a complete knock down of the asHSFB2a expression. Conversely, asHSFB2a overexpression leads to the absence of HSFB2a RNA. The knockdown of HSFB2a by asHSFB2a correlated with an improved, knockdown of asHSFB2a by HSFB2a overexpression with an impaired biomass production early in vegetative development. In both cases the development of female gametophytes was impaired. A T-DNA knock-out line did not segregate homozygous mutant plants, only heterozygots hsfB2a-tt1/+ were viable. Approximately 50 % of the female gametophytes were arrested in early development, before mitosis 3, resulting in 45 % of sterile ovules. Our analysis indicates that the “Yin–Yang” regulation of gene expression at the HSFB2a locus influences vegetative and gametophytic development in Arabidopsis.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-014-0202-0) contains supplementary material, which is available to authorized users.

SummaryUpon heat stress, heat shock factors (HSFs) control the expression of heat shock protein (HSP) genes by transcriptional activation. The perplexing multiplicity of HSF genes in Arabidopsis ± 21 potential genes have been identi®ed ± renders it dif®cult to identify mutant phenotypes. In this study, we have attempted to generate a transdominant-negative mutant of HSF by transgenic expression of a protein fusion construct, EN-HSF1, consisting of the Drosophila engrailed repressor domain (EN) and the complete Arabidopsis AtHSF1. Transgenic lines were screened for impaired ability to induce high levels of low-molecular-weight heat shock proteins (sHSPs). Two lines, EH14-6 and EH16-3, which showed quantitative differences in the expression of EN-HSF1, were further analysed for induction of thermotolerance and heat-stress-dependent mRNAs of a number of different HSF target genes encoding different HSP and HSF. The mRNA levels of all genes tested were moderately downregulated in EH14-6 but strongly reduced in EH16-3 plants compared to wild-type (Wt) and HSF1-overexpressing control plants. The inhibition of the induction of heat shock response correlated with impaired basal and acquired thermotolerance of the EH16-3 line. The kinetics of HSP expression suggest that the negative effect of EN-HSF1 is stronger in the early phase of the heat shock response, and that the reduction in mRNA levels is partially compensated at the translational level.

Objectives Despite the superior patency of internal thoracic artery (ITA) grafting compared with saphenous veins, frequency of bilateral ITA (BITA) grafting in Europe is still approximately 10%. The aim of the present study was to compare the early outcome of patients receiving either BITA or single ITA (SITA) grafting. Methods A total of 11,496 patients with isolated coronary artery bypass grafting (CABG), operated between January 1996 and December 2012, were analyzed retrospectively; 0.6476 patients (mean age 65.2 years, 81.3% males) received BITA and 5,020 patients (mean age 66.6 years, 76.7% males) SITA grafting. Mean body mass index (BMI) was 27.2 versus 27.4, p ¼ 0.017. Incidence of diabetes was 28.9 versus 28.4%, p ¼ 0.08. Ejection fraction (EF) > 50 was 71.3% (BITA) versus 66.3% (SITA), p < 0.001. Elective operations were performed in 88.4% (BITA) versus 83.3% (SITA), and urgent/emergent surgery was necessary in 11.6% (BITA) versus 16.7% (SITA), p < 0.001. Results Number of grafts was 3.76 (BITA) versus 3.06, p < 0.001. Duration of surgery (194.4 vs. 180.4 minutes) as well as X-clamp time (60.4 vs. 51.7 minutes) was prolonged for BITA, p < 0.001. Perioperative infarction rate revealed 3.2% (BITA) versus 3.6%, p ¼ 0.54. Frequency of rethoracotomy due to bleeding was higher in the BITA group (3.8 vs. 2.1%), p < 0.001. Sternal instabilities occurred in 2.3% (BITA) versus 2.2%, p ¼ 0.749. Duration of mechanical ventilation < 12 hours was 74.6 versus 77.1%, p ¼ 0.09 and duration of in-hospital stay was 10.5 versus 10.4 days, p ¼ 0.68. Thirty-day mortality was 2.4% (BITA) versus 3.0%, p ¼ 0.09. Multivariate analysis identified prolonged duration of surgery, BMI > 30, emergent operations, advanced age, and BITA grafting as predictor for sternal instabilities. EF < 30%, advanced age plus emergency were associated with increased 30-day mortality. Conclusion CABG using BITA can be performed routinely with good clinical results and low mortality. Compared with SITA grafting, bleeding complications were enhanced.

ABSTRACTForward genetics is a powerful tool to establish phenotype-genotype correlations in virtually all areas of plant biology and has been particularly successful in the model plant Arabidopsis. This approach typically starts with a phenotype in an M2 mutant, followed by identifying a causal DNA change in F2 populations resulting from a cross between the mutant and a wildtype individual. Ultimately, two additional generations are needed to pinpoint causal DNA changes upon mutant identification. We postulated that genome-wide allele frequency distributions within the mutants of M2 families facilitate discrimination of causal versus non-causal mutations, essentially eliminating the need for F2 populations. In a proof-of-principle experiment, we aimed to identify signalling components employed by the executor-type resistance (R) protein, Bs4C, from pepper (Capsicum pubescens). In a native setting, Bs4C is transcriptionally activated by and mediates recognition of the transcription activator-like effector AvrBs4 from the bacterial pathogen Xanthomonas. Arabidopsis containing an estradiol-inducible Bs4C transgene was used in a conditionally lethal screen to identify second-site suppressor mutations. Whole genome sequencing was used for M2 mutant allele-frequency distribution (MAD) mapping in three independent M2 families. MAD-mapping uncovered that all three families harboured mutations in XRN4, a novel component of executor R protein pathways. Our work demonstrates that causal mutations observed in forward genetic screens can be identified immediately in M2 families instead of derived F2 families. Notably, the timesaving concept of MAD mapping should be applicable to most crop species and will advance the appeal of forward genetics beyond applications in fundamental research.SIGNIFICANCEForward genetics has uncovered numerous genes that govern plant immune reactions. This procedure relies on mutant plants with modified immune reactions followed by identification of causal DNA changes in derived F2 progeny. We developed a novel forward genetics concept where causal DNA changes are identified in the initial M2 mutants, making time consuming establishment of F2 populations obsolete. To confirm the feasibility of the concept, we mutagenized transgenic Arabidopsis seeds containing the cell death executing resistance gene Bs4C from pepper. Whole-genome sequencing of identified mutant families that lack a Bs4C-dependent cell death revealed the XRN4 gene as a novel component of Bs4C-dependent cell death. This confirms our hypothesis that causal mutations can be identified directly within phenotypically selected mutant families.