Background

It is well-established that plants are able to acclimate to temperatures above or below the optimal temperature for their growth, and many studies have identified genes necessary for, or associated with temperature acclimation. Typically, acclimation requires a period of exposure to a non-damaging temperature treatment, either above optimal to induce heat tolerance, or below optimal to induce cold or freezing tolerance. In Arabidopsis and other plants, freezing tolerance can be achieved within a period of 24 h of cold treatment (cold hardening), with maximum freezing tolerance occurring after several days of hardening (Gilmour et al., 1988; Thomashow, 1999). Tolerance to normally lethal or damaging high temperatures can develop more rapidly, within a few hours. This type of rapid heat acclimation was extensively documented by Yarwood (1967) and others in the 1960s using a variety of plant species. Research on high temperature acclimation accelerated when it was recognized in the early 1980s that all organisms, including plants, responded to heat stress with a ‘heat shock response’ that involves transcription and translation of a conserved set of ‘heat shock proteins’ (Lindquist, 1986; Vierling, 1991). Work with soybean seedlings, similar to the earlier work of Yarwood, defined a simple assay for hypocotyl elongation that allowed investigation of the relationship of heat acclimation to the heat shock response (Lin et al., 1984). This assay was then extended to Arabidopsis and used to identify mutants altered in heat acclimation, in both forward and reverse genetic screens (Hong and Vierling, 2001; Larkindale et al., 2005; Kim et al., 2012). Other assays for heat acclimation were also developed and used for mutant screening in Arabidopsis, including heat acclimation of greening of dark grown seedlings (Burke et al., 2000) and seedling viability (Wu et al., 2013).

Because plants can experience rapid daily temperature cycles, it is perhaps not surprising that heat tolerance can be acquired on a time scale consistent with diurnal changes in temperature, and that acclimation treatments afford maximal protection if they are administered 24 h or less before the imposition of damaging heat stress. It is also important to recognize that plant responses to temperature vary significantly with the length and severity, as well as the developmental timing of the temperature treatment. A review by Yeh et al. (2012) provides an excellent description of how variations in temperature treatments can affect phenotypic outcomes. Here we describe in detail basic methods that have been used to assess the ability of plants to acclimate to severe high temperature using Arabidopsis thaliana. Done precisely, these assays can provide quantitative information on the relative heat tolerance of different plant genotypes or of plants grown under different conditions. They can also readily be developed to use with other plant species.