Friedrich Schöffl

Heat Stress- and Heat Shock Transcription Factor-Dependent Expression and Activity of Ascorbate Peroxidase in Arabidopsis

To find evidence for a connection between heat stress response, oxidative stress, and common stress tolerance, we studied the effects of elevated growth temperatures and heat stress on the activity and expression of ascorbate peroxidase (APX). We compared wild-type Arabidopsis with transgenic plants overexpressing heat shock transcription factor 3 (HSF3), which synthesize heat shock proteins and are improved in basal thermotolerance. Following heat stress, APX activity was positively affected in transgenic plants and correlated with a new thermostable isoform, APXS. This enzyme was present in addition to thermolabile cytosolic APX1, the prevalent isoform in unstressed cells. In HSF3-transgenic plants, APXS activity was detectable at normal temperature and persisted after severe heat stress at 44°C. In nontransgenic plants, APXS was undetectable at normal temperature, but could be induced by moderate heat stress. The mRNA expression profiles of known and three new Apx genes were determined using real-time PCR. Apx1 and Apx2 genes encoding cytosolic APX were heat stress and HSF dependently expressed, but only the representations of Apx2 mRNA met the criteria that suggest identity between APXS and APX2: not expressed at normal temperature in wild type, strong induction by heat stress, and HSF3-dependent expression in transgenic plants. Our data suggest thatApx2 is a novel heat shock gene and that the enzymatic activity of APX2/APXS is required to compensate heat stress-dependent decline of APX1 activity in the cytosol. The functional roles of modulations of APX expression and the interdependence of heat stress and oxidative stress response and signaling mechanisms are discussed.

Heat Shock Factors HsfB1 and HsfB2b Are Involved in the Regulation of Pdf1.2 Expression and Pathogen Resistance in Arabidopsis

In order to assess the functional roles of heat stress-induced class B-heat shock factors in Arabidopsis, we investigated T-DNA knockout mutants of AtHsfB1 and AtHsfB2b. Micorarray analysis of double knockout hsfB1/hsfB2b plants revealed as strong an up-regulation of the basal mRNA-levels of the defensin genes Pdf1.2a/b in mutant plants. The Pdf expression was further enhanced by jasmonic acid treatment or infection with the necrotrophic fungus Alternaria brassicicola. The single mutant hsfB2b and the double mutant hsfB1/B2b were significantly improved in disease resistance after A. brassicicola infection. There was no indication for a direct interaction of Hsf with the promoter of Pdf1.2, which is devoid of perfect HSE consensus Hsf-binding sequences. However, changes in the formation of late HsfA2-dependent HSE binding were detected in hsfB1/B2b plants. This suggests that HsfB1/B2b may interact with class A-Hsf in regulating the shut-off of the heat shock response. The identification of Pdf genes as targets of Hsf-dependent negative regulation is the first evidence for an interconnection of Hsf in the regulation of biotic and abiotic responses.

Arabidopsis HEAT SHOCK TRANSCRIPTION FACTORA1b overexpression enhances water productivity, resistance to drought, and infection

Heat-stressed crops suffer dehydration, depressed growth, and a consequent decline in water productivity, which is the yield of harvestable product as a function of lifetime water consumption and is a trait associated with plant growth and development. Heat shock transcription factor (HSF) genes have been implicated not only in thermotolerance but also in plant growth and development, and therefore could influence water productivity. Here it is demonstrated that Arabidopsis thaliana plants with increased HSFA1b expression showed increased water productivity and harvest index under water-replete and water-limiting conditions. In non-stressed HSFA1b-overexpressing (HSFA1bOx) plants, 509 genes showed altered expression, and these genes were not over-represented for development-associated genes but were for response to biotic stress. This confirmed an additional role for HSFA1b in maintaining basal disease resistance, which was stress hormone independent but involved H2O2 signalling. Fifty-five of the 509 genes harbour a variant of the heat shock element (HSE) in their promoters, here named HSE1b. Chromatin immunoprecipitation-PCR confirmed binding of HSFA1b to HSE1b in vivo, including in seven transcription factor genes. One of these is MULTIPROTEIN BRIDGING FACTOR1c (MBF1c). Plants overexpressing MBF1c showed enhanced basal resistance but not water productivity, thus partially phenocopying HSFA1bOx plants. A comparison of genes responsive to HSFA1b and MBF1c overexpression revealed a common group, none of which harbours a HSE1b motif. From this example, it is suggested that HSFA1b directly regulates 55 HSE1b-containing genes, which control the remaining 454 genes, collectively accounting for the stress defence and developmental phenotypes of HSFA1bOx.

Detection of in vivo interactions between Arabidopsis class A-HSFs, using a novel BiFC fragment, and identification of novel class B-HSF interacting proteins.

Class A heat shock factors (Hsfs) of Arabidopsis are known to function as transcriptional activators of stress genes. Genetic and functional analysis suggests that HsfA1a and HsfA1b are central regulators required in the early phase of the heat shock response, which have the capacity to functionally replace each other. In order to examine Hsf interaction in vivo, we conducted interaction assays using bimolecular fluorescence complementation (BiFC) on Arabidopsis protoplasts co-transformed with suitable Hsf-YFP fusion genes. BiFC assays were quantified with confocal laser scanning microscopy and flow cytometry, and confirmed with immunoprecipitation assays. For each Hsf we could not only demonstrate homomeric interactions but also detect heteromeric interaction between HsfA1a and HsfA1b. Truncated versions of these of Hsfs, containing deletions of the oligomerization domains (ODs), provided clear evidence that the ODs are required and sufficient for the HSF interaction in vivo. By contrast there was only homomeric but no heteromeric interaction detected between two different class B Hsf transcription factors (HsfB1 and HsfB2b) in a yeast two-hybrid assay. HsfB1/HsfB2b functions are not directly linked with the expression of conventional heat shock genes; class B Hsfs are devoid of the activation domain motif conserved in class A Hsfs. In order to identify other proteins interacting with HsfB1 and HsfB2b we performed yeast two-hybrid screenings of cDNA libraries. Three of the identified proteins were common to both screenings. This suggests that HsfB1 and HsfB2b may be involved in complex regulatory networks, which are linked to other stress responses and signaling processes.

Promoter specificity and interactions between early and late Arabidopsis heat shock factors.

The class A heat shock factors HsfA1a and HsfA1b are highly conserved, interacting regulators, responsible for the immediate-early transcription of a subset of heat shock genes in Arabidopsis. In order to determine functional cooperation between them, we used a reporter assay based on transient over-expression in Arabidopsis protoplasts. Reporter plasmids containing promoters of Hsf target genes fused with the GFP coding region were co-transformed with Hsf effector plasmids. The GFP reporter gene activity was quantified using flow cytometry. Three of the tested target gene promoters (Hsp25.3, Hsp18.1-CI, Hsp26.5) resulted in a strong reporter gene activity, with HsfA1a or HsfA1b alone, and significantly enhanced GFP fluorescence when both effectors were co-transformed. A second set of heat shock promoters (HsfA2, Hsp17.6CII, Hsp17.6C-CI) was activated to much lower levels. These data suggest that HsfA1a/1b cooperate synergistically at a number of target gene promoters. These targets are also regulated via the late HsfA2, which is the most strongly heat-induced class A-Hsf in Arabidopsis. HsfA2 has also the capacity to interact with HsfA1a and HsfA1b as determined by bimolecular fluorescence complementation (BiFC) in Arabidopsis protoplasts and yeast-two-hybrid assay. However, there was no synergistic effect on Hsp18.1-CI promoter-GFP reporter gene expression when HsfA2 was co-expressed with either HsfA1a or HsfA1b. These data provide evidence that interaction between early and late HSF is possible, but only interaction between the early Hsfs results in a synergistic enhancement of expression of certain target genes. The interaction of HsfA1a/A1b with the major-late HsfA2 may possibly support recruitment of HsfA2 and replacement of HsfA1a/A1b at the same target gene promoters.

Overexpression of AtHsfB4 induces specific effects on root development of Arabidopsis

The functions of plant class B-heat shock factors (Hsfs) are not well understood. Hsfs belonging to this group differ from class A-Hsfs in structural features of the oligomerization domain and by the absence of a typical AHA motif for transcriptional activation. AtHsfB4 is expressed in different parts of the plants with highest levels in root tissue. Transgenic Arabidopsis plants overexpressing (OE) HsfB4 by CaMV-35S-promoter showed massively enhanced levels of Hsf mRNAs. The root surface of OE-plants was rough and cells became detached. Crossings with cell type specific root marker lines and confocal laser scanning microscopy provided clear evidence for a duplication of cells in the ground tissue and ectopic layers of lateral root cap (LRC) cells in HsfB4-OE plants. A duplication of endodermis cells occurs already during embryonic development, while the ectopic LRC cells are only detected during postembryonic growth. The mutant phenotypes of Hsf-OE plants are without precedence and indicate that class B-Hsfs may play an important role in root development.