Here we describe some basic experimental designs (Figure 4) that are suited to the evaluation of mutants and transgenic plants: a number of lines can be tested in a fairly high-throughput manner and relatively little specialized equipment or apparatus are required. Given this starting point, Arabidopsis is used as the example plant. However, the principles illustrated and, to a large extent, the experimental techniques described, are applicable to other plants as well. Particular attention is paid to consideration of which of the aspects of low-ψw response discussed above is tested by each type of experiment.
Leaf water loss
Perhaps the easiest experiment to perform is to simply remove the aerial portion of the plant (or an individual leaf) from the roots and measure the decline in fresh weight over time (Figure 4a). The experiment should be set up under controlled temperature, light and humidity conditions that allow a gradual decline in leaf water content to be observed. A decline to 50% water content over the course of 6 to 8 h is typical in Arabidopsis (Figure 4a). The rate of water loss is largely determined by stomatal conductance; thus, experiments on leaf water loss measure avoidance of low ψw and are typically not applicable to investigation of tolerance mechanisms. In addition to leaf water loss experiments, measurements of leaf conductance and direct microscopic observation of stomatal apertures in leaf epidermal strips (see for example Leymarie et al., 1999) can be performed. Rates of leaf water loss can also be estimated based on leaf temperature. Thermal imaging has been used to isolate Arabidopsis mutants with altered stomatal regulation and stress avoidance (Merlot et al., 2002; Wang et al., 2004) and at the field level to estimate plant water status (Cohen et al., 2005).
Because stomatal conductance is controlled in large part by ABA, measurements of leaf water loss are often most useful as an indicator of altered accumulation of or sensitivity to ABA. Mutants deficient in ABA and many (although not all) mutants with altered ABA sensitivity exhibit altered leaf water loss. In our laboratory, leaf water loss experiments are followed by, or performed concurrently with, other tests of ABA accumulation and response. These include the effect of ABA on seed germination and seedling growth and ABA-dependent gene expression and stress-induced accumulation of ABA. In many cases, these parameters are measured using the polyethylene glycol (PEG)-infused agar plate system described below. Measurements of ABA-responsive seed germination have been described in numerous studies (see for example Finkelstein, 1994) and typically involve plating seed on media containing ABA at a range of concentrations and scoring either emergence of radicles or the formation of green cotyledons over a period from 1 to 10 days after the end of stratification.
Soil drying experiments using pot-grown plants are typically done by removing the water supply and measuring some aspect(s) of plant growth, survival and water status after a fixed period of soil drying. Such soil drying experiments can at first seem quite straightforward but often turn out to be one of the most difficult types of experiment to interpret. This is because the severity of stress experienced by the plant is not determined directly by the investigator but rather by the plant itself based on the rate at which it depletes the available soil water. This can lead to confusion if the severity of the stress is not quantified by measuring leaf or soil ψw or if steps are not taken to ensure that the genotype of interest is exposed to the same severity of stress as a wild-type control.
An example of one of the complexities of soil drying experiments is the evaluation of mutants or transgenic plants with decreased stomatal conductance or decreased growth and leaf area. When water is withheld and the condition of the plants assessed after a given time, plants that have reduced stomatal conductance or reduced leaf area can be expected to deplete soil water more slowly (avoidance of low ψw) and may exhibit delayed wilting compared with wild-type plants. Such delayed wilting has been used to label such plants as stress or drought tolerant when instead the transgenic plant has avoided low-ψw stress by using the available water more slowly. In general, to establish whether a particular genetic modification leads to tolerance of low ψw, it must be shown that the stress response under study differs in plants exposed to the same severity of stress (same ψw) and that this difference leads to a desirable change in phenotype. A better-defined use of the term ‘tolerance’, as well as other terms related to the low-ψw response, could do much to clarify the literature on this topic.
These difficulties can be overcome in two ways. The first is by quantification of leaf and/or soil ψw during the drying cycle. This can be combined with control of humidity levels or partial rewatering of some plants to ensure that the comparisons of stress response are made only between plants exposed to the same ψw (see for example: Sharp et al., 2000; Thompson et al., 2004). Partial rewatering can also be used to extend the time for which the plants are exposed to low ψw, thus allowing physiological and molecular responses to low ψw be examined in more detail. These experiments are particularly relevant to more detailed evaluation of crop species (Sharp et al., 2000; Thompson et al., 2004) and numerous other studies where parameters such as osmotic adjustment and leaf growth have been evaluated in a number of crop species (see for example Babu et al., 1999; Puliga et al., 1996).
In the case of Arabidopsis, however, repeated measurements of leaf or soil ψw during the drying cycle are laborious and require a quantity of material that may be difficult to obtain. For genetic studies, where a mutant or transgenic plant is being compared with a wild type, the easiest way to ensure a valid comparison while avoiding extensive measurements of ψw is to grow the wild-type plant in the same pot as the genotype under evaluation (Figure 4b). Thus the roots of both genotypes will grow into the same soil and be exposed to the same ψw even if one genotype uses water more quickly than the other. This approach can be combined with measurement of soil ψw at the end of the drying cycle to quantify the final severity of the stress.
The rate of soil drying is a key factor in these experiments. A very rapid rate of soil drying allows little time for slow responses such as solute accumulation or cell wall modification to occur and can cause many important aspects of the low-ψw response to be overlooked. Using a sufficiently large and deep pot will avoid this situation. The soil type [we typically use a well-aerated potting mix such as Metro-mix 350 (Sungrow Horticulture, Bellevue, WA, USA): similar potting mixtures are also available from other suppliers], humidity, temperature and light intensity will also affect the rate of drying and these factors must be adjusted empirically for any given set of conditions. As a rule of thumb, leaf water content should decline by no more than 30–40% over a 10–12-day period after the cessation of watering.
Several measurements of response to low ψw can be used in conjunction with soil drying experiments. A general indication of plant performance can be obtained through measurements of growth (shoot fresh and dry weights, leaf area and root mass after soil removal), efficiency of water use or photosynthetic performance. Measurement of leaf relative water content and solute content and calculation of osmotic adjustment have been performed for many crop species (Babu et al., 1999; Zhang et al., 1999) and allow the capacity for dehydration avoidance to be accessed. If dehydration tolerance is the main interest, then measurements of plant survival after severe stress and measurements that quantify cellular damage such as loss of chlorophyll content, electrolyte leakage and ROS-induced damage (see below) can be performed.
Low-ψw treatment using PEG-infused agar plates
Many studies of low-ψw stress have used osmotica to lower the ψw of plant growth media. This approach has many advantages: ψw can be controlled precisely and reproducibly and a large number of treatments can be performed quickly. Osmoticum treatment does, however, bring up its own set of potential problems that become apparent when osmoticum treatment is compared with soil drying. In most cases, when soil water content decreases water is withdrawn from both the cell wall and the protoplast resulting in cytorrhysis, a process where both the cell wall and protoplast shrink (Oertli, 1985). This contrasts with the response to low molecular weight solutes such as mannitol that are often used to lower ψw. In this case the solute freely penetrates the pores of the cell wall and causes plasmolysis; a loss of water from and decrease in volume of the protoplast while the volume of the cell wall remains unchanged. Because it is not a part of the typical soil drying response and may cause cellular damage that is perceived and responded to differently from water loss caused by soil drying, plasmolysis should be avoided in studies of low ψw or salinity (Munns, 2002).
Experimentally, a cytorrhytic rather than plasmolytic low-ψw treatment can be imposed using solutions containing a high-molecular-weight solute such as PEG of molecular weight 6000 or above. Polyethylene glycol of this molecular weight range cannot enter the pores of plant cells (Carpita et al., 1979; Oertli, 1985) and thus causes cytorrhysis rather than plasmolysis. Polyethylene glycol is also a better choice for imposing low ψw than the often used solute mannitol because mannitol has been shown to be taken up by plant cells and can cause specific toxic effects on growth (Hohl and Schopfer, 1991; Verslues et al., 1998). An example of the toxic effects of mannitol and a similar solute melibiose are shown in Figure 5. For maize primary roots, transfer to a −1.6 MPa solution of mannitol or melibiose had less initial effect (0–10 h) on root growth than transfer to a −1.6 MPa PEG solution (Verslues et al., 1998). This is consistent with mannitol and melibiose being taken up by the roots, thus leading to less initial loss of turgor and less initial growth inhibition. After 48 h, however, PEG-treated roots had recovered and resumed steady-state growth, albeit at a reduced rate [root growth of the unstressed control at this time was approximately 4 mm h−1 (Verslues et al., 1998)] while growth of the mannitol or melibiose roots had stopped. This clearly demonstrates that mannitol, and other low-molecular-weight solutes, have toxic effects that can obscure the low-ψw response. In experimental systems such as PEG-infused agar plates (Protocol S1 in the Supplementary Material accompanying this article) where there is low transpiration, root damage is avoided, and the roots are not subjected to hypoxic conditions by submergence in PEG solution. Polyethylene glycol is the best solute that we are aware of for imposing a low-ψw stress that is reflective of the type of stress imposed by a drying soil (Verslues and Bray, 2004; Verslues et al., 1998; van der Weele et al., 2000).
Figure 5. Rates of primary root elongation in maize seedlings transferred from wet vermiculite to −1.7 MPa solutions of either PEG, mannitol or melibiose. In all cases, solutions were oxygenated to prevent root hypoxia (see Verslues et al., 1998 for methods). Rates of root elongation in seedlings transferred to high-ψw (no added solute) solution increased to approximately 4 mm h−1 by 50 h (data not shown). Thus, PEG treatment caused a reduction of approximately 60% in the steady-state root elongation rate but mannitol or melibiose of the same ψw completely stopped root elongation by 50 h. Data are from Verslues et al. (1998) and Verslues (1997).
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In addition to the choice of solute used to impose the low ψw stress, our experience, and that of others (van der Weele et al., 2000), shows that for many types of measurements, it is better to use media without sugar, or with a low level of sugar (0.5% or less). This is because sugar is well known to affect ABA responses (Finkelstein et al., 2002). Also, the addition of high a high level of sucrose itself can induce an osmotic response (the ψs of a 3.0% sucrose solution is approximately −0.2 MPa). Thus, seedlings in ‘control media’ containing a high level of sucrose can already be experiencing a low level of osmotic stress. This causes a high baseline level for many low-ψw responses. For example, ABA levels of more than 300 ng g−1 fresh weight (FW) have been reported for Arabidopsis seedlings on MS media with 3% sucrose (Ruggiero et al., 2004) whereas we routinely observe ABA levels of 1 to 4 ng g−1 FW in a half-strength MS medium without sucrose (Verslues and Bray, 2004). This high baseline and the possibility that sugar from the medium can accumulate in the plant tissue and reduce the water loss caused by further decreases in ψw means that many low-ψw responses can be difficult to detect in high-sugar media.
A system of using PEG-infused agar plates to impose low ψw has been described by van der Weele et al. (2000) and a modified version of this procedure is in use in our laboratory. A detailed protocol for the preparation and use of PEG plates is included as Supplementary Material with this article (Protocol S1). This system has the advantage of being able to easily make plates of a range of ψw without the complications that arise from using low-molecular-weight solutes. Another advantage is that as long as steps are taken to prevent drying of the plates use of PEG-infused plates allows the imposition of a constant ψw over time. Because ψw is constant and transpiration minimal in the PEG-infused plate system, avoidance of stress is not an issue; the seedlings must equilibrate with the ψw of the agar over time. Thus, the PEG plate system is ideal for studies of dehydration avoidance and mechanisms of dehydration tolerance. Measurements of growth, water and solute content, hormone accumulation and stress-regulated gene expression are examples of specific traits that can be quantified.
Seeds can be plated directly onto PEG-infused plates and seed germination and growth measured. However, in many cases the more useful experiment is to plate seeds on unstressed media (typically half-strength MS without sugar) and transfer them to PEG-infused plates after 5–7 days of growth (Figure 4c). To facilitate transfer of seedlings between plates, seed can be plated on a mesh overlaid on the original agar plate and transferred by moving the mesh and seedlings to the PEG-infused plate (Verslues and Bray, 2004; van der Weele et al., 2000). For ψw of −0.7 MPa or below, this transfer leads to rapid dehydration of the seedlings (Verslues and Bray, 2004). This loss of water in turn causes a number of rapid stress responses including high levels of ABA accumulation (Figure 4c) and, similar, to other systems, rapid induction of a number of stress- and ABA-regulated genes (P. E. Verslues and J.-K. Zhu, unpublished). These events, which we refer to as the ‘acute’ phase of the low-ψw response (Figure 4c) have been the focus of most studies of low-ψw response at the molecular and genetic levels. This acute response is followed by longer-term responses, such as solute accumulation and osmotic adjustment (Verslues and Bray, 2004) and changes in root and shoot growth (van der Weele et al., 2000) indicative of an adjustment to and recovery from the effects of the reduced ψw. These recovery and longer-term responses are also important aspects of the low-ψw response to be investigated by molecular and genetic studies. The PEG-infused plate system is in many ways (imposition of a constant low ψw with minimal transpiration) similar to the dry vermiculite system that has been used to study low-ψw responses of seedlings of maize and other crop species (Sharp et al., 1988, 2004).