In the past hundred years, the average global temperature has increased by 1.1°C and is projected to continue increasing at a rate of 0.2°C per decade [1]. In addition to increasing average temperature, global climate change has been linked to more frequent and severe extreme temperature events such as heat waves which have increased in frequency, intensity, and duration [2, 3]. Heat stress is a major environmental factor that negatively affects plant growth and productivity, and ultimately threatens world food security. For instance, extreme heat events have reduced global cereal production by 9.1% from 1964 to 2007, mainly due to grain yield deficit [4]. Furthermore, crop models also predict that each degree Celsius increase in global average temperature would reduce global yields of wheat by 6%, rice by 3.2%, maize by 7.4%, and soybean by 3.1% [5, 6].
To cope with heat stress, plants are able to develop tolerance upon exposure to increased but non-damaging temperature, known as thermo-priming, which can allow them to survive subsequent heat stress [7]. The involvement of many genes including HSFs (heat shock transcription factors) and HSPs (heat shock proteins) in thermo-priming has been extensively studied [8]. Distinct from this acquired thermotolerance, plants have the innate ability to survive under heat stress (i.e., basal thermotolerance) which depends on plant species and genotype. Using thermotolerant germplasm and/or well-studied genes in heat response, multiple programs are currently underway to improve plant thermotolerance mainly through traditional breeding or bio-design engineering efforts [9, 10]. However, an emerging approach to enhance thermotolerance is through plant associations with beneficial microbes. Studies have investigated the bacterial association on plant growth, commonly termed plant growth-promoting bacteria (PGPB), under heat stress. Several examples of PGPB enhanced plant thermotolerance have been reported using wheat, sorghum, soybean, and tomato [11–14]. Recent studies show that PGPB alter host metabolism which can reduce heat-induced membrane injury, increase HSP levels, and alter chromatin modification via heat stress memory loci [12, 15, 16]. However, plant - PGPB interactions are highly species- and genotype-specific [17, 18], therefore the mechanism underlying the bacterial-provided thermal benefits is largely unknown.
Assays for plant thermotolerance have been developed to study plant heat acclimation and screen mutant lines [7, 19]. Critical assay components to consider when designing a high-throughput reproducible experimental protocol to assess plant thermotolerance are the heat stress regime and plant phenotyping. Heat stress regime can be determined based on the intensity and duration, of which the impact largely depends on plant species, culture system, physiological state, and developmental stage [20]. Various indicators have been used to distinguish plant phenotypes under heat stress, including hypocotyl elongation, survival rate, biomass, ion leakage, chlorophyll content and fluorescence, and seed germination [16, 19, 21–24]. However, no standard approach has been developed to explore bacterial effects on plant host thermotolerance. A reliable, high-throughput assay to quantify such beneficial effects could help to identify the molecular genetics governing thermotolerance from the perspective of both the bacteria and their plant hosts.
In the present study, we developed a high-throughput assay to assess bacterial effects on Arabidopsis thermotolerance. We introduced a hydroponic-based culture system to simplify the inoculation process and provide consistent heat stress. The basal thermotolerance of seedlings was quantified by measuring chlorophyll content under different heat durations. We utilized this assay to screen Variovorax strains for the ability to provide benefits to plants to heat stress. We subsequently performed additional analyses on a promising strain to confirm the bacterial provided thermotolerance. This assay is not only amenable to high-throughput screening of individual bacterial strains but can also scale to include multiple Arabidopsis genetic variants and microbial consortia.