With the onset of post-genomic capabilities in Arabidopsis thaliana, and particularly the availability of large collections of genome-indexed insertion mutants, the plant biology community has been thoroughly employing reverse genetic strategies . The most critical step in this approach is the identification of a phenotype that is distinct from the wild-type, thus providing new insights on gene function [2, 3]. Searching for a noticeable phenotype can be a strenuous process, often hindered by gene redundancy, natural variability, or subtle imperceptible changes in growth or development . Previously, Boyes et al.  developed a sensitive and dynamic method to detect and interpret phenotypic differences over the entire span of Arabidopsis development, based on a time-dependent map of well-defined morphological traits. However, detection of differential phenotypes is often dependent on the application of particular experimental conditions, specific environmental challenges or a combination of several inputs . Thus, selection of the most suitable experimental conditions is critical for success in the identification of a phenotype.
In the specific case of heat tolerance, a standardized experimental protocol designed to detect temperature-related phenotypes has not been reported to the best of our knowledge. Distinct assays have been used by different laboratories to distinguish responses to temperature, including the analysis of seedling survival [6–14], adult plant survival/fresh weight [9, 15], seed germination and cotyledon greening [9, 10, 13], chlorophyll accumulation [16, 17], shoot elongation , hypocotyl elongation [6, 7, 9, 18–20], root growth [9, 13, 17], ion leakage , TBARS accumulation , and reporter enzyme activity . In addition to the way the phenotype is scored, other variables lead to differentiated thermotolerance assays, including the plant species, the plant’s developmental stage and the type of heat stress regime (reviewed by ). The importance of different time and temperature treatments was precisely depicted in pioneer studies in soybean, leading to important advances in the study of heat stress resistance (e.g. ). Unfortunately, the use of such different conditions promotes high variability in the results, with implications in the capacity to detect differential phenotypes.
Plants can rapidly acquire heat tolerance to otherwise lethal temperatures, if they are pre-exposed to a moderate high-temperature or exposed to a gradual increase in temperature. This phenomenon, known as acquired heat tolerance, is clearly distinct from basal heat tolerance, which refers to the innate ability of plants to survive exposure to temperatures above those optimal for growth . As differences have also been detected when using different recovery periods between the acclimation treatment and the high temperature challenge, the term ‘thermotolerance diversity’ was proposed to describe the multiple mechanisms that plants use in response to changes in the environment . The type of heat tolerance involved (basal or acquired) and the type of shifting temperature (instant or gradual) must then be considered for the experimental design, in order to enclose most of the potential conditions that could generate a visible phenotype. From the diverse methods used for evaluating thermotolerance phenotypes, the basal thermotolerance and short-term acquired thermotolerance (usually less than 2 h) are the most common forms of phenotyping strategies . Using in vitro assays, the acclimation treatment is usually performed at 37-38°C for 60–90 min [6–9, 13, 14, 17–20]. Temperature and duration of the stress imposition, application of a pre-conditioning treatment (which can differ in temperature and/or in time length) and duration of the recovery period are only some of the conditions that could be arranged in multiple ways to establish a phenotypic assay. An often underestimated aspect is the time point for determining plant traits. Even for similar morphological assessments, some researchers obtain their results a few days after heat treatment, while others wait for several days. Therefore, it is significantly important to perform a time-course analysis of the phenotypic traits in question, which can reveal a phenotype per se, to facilitate the discovery of masked alterations between mutant and wild-type plants. The growth stage of the treated plant also interferes with the recognition of phenotypic alterations. While the analysis performed in seeds is normally restricted to the germination stage, different assessments could be achieved both in early (2.5-10 day-old) or late (10–25 day-old) seedlings.
The diversity of existing protocols compels the creation of comprehensive surveys for heat-associated phenotypes; however these can be time-consuming and unproductive. In order to create a simplified framework for the phenotypic detection of heat-associated mutants, it is necessary to first assess the heat tolerance response of Arabidopsis wild-type plants. Motivated by the same purpose, Burke et al.  characterized the acquired heat tolerance of Col seedlings by chlorophyll accumulation assays. However, to our knowledge there is no study that characterizes the heat response of Arabidopsis plants, based on the assessment of important and highly practical germination and seedling survival traits. The present report provides a comprehensive primary approach to identify and interpret phenotypic differences of heat-associated mutants at early developmental stages. In seedlings, basal tolerance was characterized by temperature and exposure time variations, and acquired heat tolerance by characterizing survival after different acclimation conditions. We introduced a seed-based assay that was thoroughly surveyed in terms of temperature and exposure time variations, and subsequently validated by characterizing several natural occurring ecotypes and heat stress-associated functional mutants.