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, APX S . This enzyme was present in addition to thermolabile cytosolic APX1, the prevalent isoform in unstressed cells. In HSF3-transgenic plants, APX S activity was detectable at normal temperature and persisted after severe heat stress at 44°C. In nontransgenic plants, APX S 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 APX S 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 that Apx2 is a novel heat shock gene and that the enzymatic activity of APX2/APX S 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.There is increasing evidence for considerable interlinking between the responses to heat stress and oxidative stress. Both stresses induce pathways resulting in the expression/accumulation of heat shock proteins (HSP) in plants (Banzet et al., 1998; Dat et al., 1998; Schett et al., 1999; Lee et al., 2000) and, in fruit fly (Drosophila melanogaster), transient expression of small HSP (sHSP) decreases sensitivity of cells to heat and hydrogen peroxide stresses (Mehlen et al., 1993). On the other hand, there is also evidence that heat induces oxidative stress and/or expression of antioxidative enzymes in bacteria (Morgan et al., 1986), yeast (Davidson et al., 1996), and plants (Gong et al., 1998;Storozhenko et al., 1998; Lee et al., 1999). Thermotolerance can be generated by compounds that induce oxidative bursts, and very short heat pulses can induce bursts of superoxide and/or hydrogen peroxide (Vallelian-Bindschedler et al., 1998).Reactive oxygen species (ROS) such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals are continuously formed in aerobic organisms. Excess production of ROS causes oxidative damage of cellular components, and their involvement in a number of biotic and abiotic stresses is well documented (Bowler et al., 1992). Accumulation of hydrogen peroxide has not only negative consequences on living c...

The mechanisms of sensing and signalling of heat and oxidative stresses are not well understood. The central question of this paper is whether in plant cells oxidative stress, in particular H(2)O(2), is required for heat stress- and heat shock factor (HSF)-dependent expression of genes. Heat stress increases intracellular accumulation of H(2)O(2) in Arabidopsis cell culture. The accumulation was greatly diminished using ascorbate as a scavenger or respectively diphenyleneiodonium chloride (DPI) as an inhibitor of reactive oxygen species production. The mRNA of heat shock protein (HSP) genes, exemplified by Hsp17.6, Hsp18.2, and the two cytosolic ascorbate peroxidase genes Apx1, Apx2, reached similar levels by moderate heat stress (37 degrees C) or by treatment with H(2)O(2), butylperoxide and diamide at room temperature. The heat-induced expression levels were significantly reduced in the presence of ascorbate or DPI indicating that H(2)O(2) is an essential component in the heat stress signalling pathway. Rapid (15 min) formation of heat shock promoter element (HSE) protein-binding complex of high molecular weight in extracts of heat-stressed or H(2)O(2)-treated cells and the inability to form this complex after ascorbate treatment suggests that oxidative stress affects gene expression via HSF activation and conversely, that H(2)O(2) is involved in HSF activation during the early phase of heat stress. The heat stress induction of a high mobility HSE-binding complex, characteristic for later phase of heat shock response, was blocked by ascorbate and DPI. H(2)O(2 )was unable to induce this complex suggesting that H(2)O(2) is involved only in the early stages of HSF activation. Significant induction of the genes tested after diamid treatment and moderate expression of the sHSP genes in the presence of 50 mM ascorbate at 37 degrees C occurred without activation of HSF, indicating that other mechanisms may be involved in stress signalling.

Origin and rearrangement of ribosomal DNA repeats in natural allotetraploid Nicotiana tabacum are described. Comparative sequence analysis of the intergenic spacer (IGS) regions of Nicotiana tomentosiformis (the paternal diploid progenitor) and Nicotiana sylvestris (the maternal diploid progenitor) showed species-specific molecular features. These markers allowed us to trace the molecular evolution of parental rDNA in the allopolyploid genome of N. tabacum; at least the majority of tobacco rDNA repeats originated from N. tomentosiformis, which endured reconstruction of subrepeated regions in the IGS. We infer that after hybridization of the parental diploid species, rDNA with a longer IGS, donated by N. tomentosiformis, dominated over the rDNA with a shorter IGS from N. sylvestris; the latter was then eliminated from the allopolyploid genome. Thus, repeated sequences in allopolyploid genomes are targets for molecular rearrangement, demonstrating the dynamic nature of allopolyploid genomes.

The potential of different house-keeping genes for their use as internal standards of gene expression under changing environmental conditions and in different organs of plants was assessed. Using real-time PCR mRNA levels were precisely quantified for preselected actin and ribosomal protein genes in Arabidopsis thaliana (L.) Heinh. and Nicotiana tabacum L. grown at normal temperature and following heat stress. In tobacco leaves the mRNA levels of the constitutively expressed ribosomal protein gene Nt-L25 and the actin genes Nt-ACT9 and At-ACT66 were strongly reduced (to approximately 10%) during heat stress. Heat stress applied at the temperature optimum (37 degrees C) for elicitation of a heat stress response to Arabidopsis leaves resulted in a strong induction (several thousand-fold) of the mRNA heat shock protein genes, At-HSP17.6 and At-HSP18.2. Concomitantly, the mRNA levels of constitutively expressed actin 2 (At-ACT2) and ribosomal protein L23 (At-L23a) genes were reduced to approximately 50% of the levels in leaves incubated at room temperature. Conversely, under severe heat stress conditions (44 degrees C), the induction of At-HSP17.6 and At-HSP18.2 mRNAs was insignificant, the mRNA levels of At-ACT2 remained at approximately the same levels as in leaves incubated at room temperature, whereas the mRNA level of At-L23 declined. The mRNA levels of At-ACT2 and At-L23a examined in stem, flower and siliques of Arabidopsis plants grown under non-stress condition showed differential alterations; the mRNA level of ribosomal protein L23 correlates with the metabolic activity of tissues. The potential use of house-keeping gene expression as standards in expression profiling and the mechanisms modulating the mRNA levels are discussed.