Heat stress response is part of the isothiocyanate-induced response in Arabidopsis thaliana
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- Institutt for biologi 
Heat stress responses (HSR) represent a multi-pathway system of adaptive responses to environmental changes. Common downstream targets of HSR pathways are the heat shock proteins, amongst them HSP101 (At1g74310). In heat-stressed plants, the highly conserved HSP101 hexamerises and forms a disaggregase capable of untangling protein aggregates comprised of misfolded and denaturated proteins. HSP101 is essential to ameliorate proteotoxicity of these protein aggregates, as protein aggregates impair cel- lular functions and integrity. The importance of thermoresponsive HSP101 induction should not be understated, as the protein confers the organism a signi cant capacity to tolerate supraoptimal temperatures (thermotolerance). When exposed to a moderate temperature increase (37-38°C for Arabidopsis thaliana), plants are primed to tolerate near-future heat stresses, a phenomenon known as acquired thermotolerance. Certain low molecular weight compounds have been found to stimulate HSR akin to moder- ate temperature priming, such as allyl isothiocyanate. Allyl isothiocyanates (AITC) is a negative growth regulator likely due to perturbation of the cytoskeleton. However, the A. thaliana HSP101 null mutant line hot1-3 has only residual capacity to tolerate heat stresses but possesses elevated resistance to the herbicidal e ects of AITC. To in- vestigate the connection between HSP101 and AITC di erent strategies were pursued. First, the AITC resistance and thermotolerance pro les of other null and missense mu- tant lines of HSP101 and HSA32 (At4g21320) have been characterised. hot1-3 was the only mutant with abrogated HSP101 expression that displayed enhanced resistance to AITC. Second, the hot1-3 allele was backcrossed twice to Col-0. Backcrosses homozy- gous for the HSP101 allele of hot1-3 was more susceptible to the growth inhibitory ef- fects of AITC than both parental lines. Third, hot1-3 was complemented with wild type AtHSP101 allele. Complemented lines expressing a functional, transgenic HSP101 did not rescue the hot1-3 AITC resistance phenotype. Fourth, AtHSP101 was constitutively overexpressed in the Col-0 background. Overexpression lines were signi cantly more thermotolerant than wild type, but AITC resistance was unchanged. Taken together the results generated by these di erent approaches indicate that the AITC-resistance phenotype of hot1-3 is not due to knocking-out HSP101 but likely caused by one or more additional mutation(s). Luciferase reporter lines driven by the AtHSP101 promoters were produced in order to quantify temporal and spatial AITC-induced AtHSP101 ex- pression. Luciferase activity due to heat stress and AITC was detected primarily in rosette leaf laminae and petioles. A 1,329 bp full-length promoter region including the 5 -UTRs of the neighbouring gene AtCK2 (At1g74320) drives luciferase activity twice as strongly as the 810 bp AtHSP101-only promoter region. hot1-3 phenotype was likely caused by one or more secondary mutation. The role of AITC as a small molecule ther- motolerance inducer was also examined, but experimental methods must be optimised to give more consistent results.