What Is Stress? Dose-Response Effects in Commonly Used in Vitro Stress Assays

Abstract

In vitro stress assays are commonly used to study the responses of plants to abiotic stress and to assess stress tolerance. A literature review reveals that most studies use very high stress levels and measure criteria such as germination, plant survival, or the development of visual symptoms such as bleaching. However, we show that these parameters are indicators of very severe stress, and such studies thus only provide incomplete information about stress sensitivity in Arabidopsis (Arabidopsis thaliana). Similarly, transcript analysis revealed that typical stress markers are only induced at high stress levels in young seedlings. Therefore, tools are needed to study the effects of mild stress. We found that the commonly used stress-inducing agents mannitol, sorbitol, NaCl, and hydrogen peroxide impact shoot growth in a highly specific and dose-dependent way. Therefore, shoot growth is a sensitive, relevant, and easily measured phenotype to assess stress tolerance over a wide range of stress levels. Finally, our data suggest that care should be taken when using mannitol as an osmoticum.

Figure 1.

Literature study of in vitro stress assays. A, Concentrations of NaCl and mannitol/sorbitol used to impose salt and osmotic stress on Arabidopsis in PubMed Central open-access articles (n = 216). The median is indicated with the black lines. B, Phenotypes recorded to assess stress tolerance in PubMed Central open-access articles (n = 106). The different types of growth measurements are further broken down in the diagram on the right.

Figure 2.

Sensitivity of germination, overall plant health, root length, and rosette area to abiotic stress. Root length (n = 20–24) and rosette area (n = 30–36) are expressed relative to nontreated plants. For germination rate and plant health (as scored by the appearance of visual stress symptoms, such as bleaching and anthocyanin accumulation), data come from three independent experiments. Error bars indicate se.

Figure 3.

Effects of stress on rosette growth. A, C, E, and G, Projected rosette areas at 22 DAS (final time point), with representative images of plants for a range of mannitol, NaCl, and H₂O₂ concentrations. The top right insets show rosette compactness. For H₂O₂ treatment, the inset at the bottom left shows rosette areas (dots) and their averages (lines) grouped by the presence (red) or absence (black) of substantial bleaching. B, D, F, and H, RGRs as a function of the stress levels at 8 DAS (red), 15 DAS (blue), and 22 DAS (green). At NaCl concentrations greater than 150 mm (E and F), germination or seedling establishment was inhibited, so no growth data could be obtained for these concentrations, as represented by the dashed lines on the graphs. Error bars indicate se. The letters above the error bars denote significance groups (ANOVA; P < 0.05; n = 30–36).

Figure 4.

Effects of stress on the expression of selected marker genes. Levels of oxidative stress markers (NAC032 and AKR4C9), ABA markers (CYP707A3 and NCED3), dehydration markers (LEA5, RD29B, and DREB2A), and mild osmotic stress markers (ERF5, WRKY33, and MYB51) in complete seedlings are represented as log₂ (fold change) compared with nonstressed plants. Significant changes (ANOVA; P < 0.05; n = 3) are indicated with asterisks.