Mimicking genuine drought responses using a high throughput plate assay

Reviewer #1 (Public Review):

High-throughput genetic screening is a powerful approach to elucidate genes and gene networks involved in a variety of biological events. Such screens are well established in single-celled organisms (i.e. CRISPR-based K/O in tissue culture or unicellular organisms; screens of natural variants in response to drugs). It is desirable to extend such methodology, for example to Arabidopsis where more than 1000 ecotypes from around the Northern hemisphere are available for study. These ecotypes may be locally adapted and are fully sequenced, so the system is set up for powerful exploration of GxE. But to do so, establishing consistent "in vitro" conditions that mimic ecologically relevant conditions like drought is essential.

The authors note that previous attempts to mimic drought response have shortcomings, many of which are revealed by 'omics type analysis. For example, three treatments thought to induce osmotic stress; the addition of PEG, mannitol, or NaCl, fail to elicit a transcriptional response that is comparable to that of bonafide drought. As an alternative, the authors suggest using a low water-agar assay, which in the things they measure, does a better job of mimicking osmotic stress responses. The major issues with this assay are, however, that it introduces another set of issues, for example, changing agar concentration can lead to mechanical effects, as illustrated nicely in the work of Olivier Hamant's group (e.g., https://elifesciences.org/articles/34460).

Reviewer #2 (Public Review):

The authors aim to make a reliable plate-based system for imposing drought stress (which for experiments like this would be better referred to as low water potential stress). This is an admirable goal as a reliable experimental system is key to conducting successful low-water potential experiments and some of the experimental systems in use have problems. They compare several treatments but seem to be unaware that such comparisons need to be based on the measurement of water potential as the fundamental measure of how severe the level of water limitation is. Only by comparing things at the same water potential can one determine if the methods used to impose the low water potential are introducing confounding factors. In this manuscript, they compare several agar-plate-based treatments to what they view as a baseline experiment of plants subjected to soil drying. However, that baseline soil drying (vermiculite drying, to be precise) experiment illustrates many of the problems present in the molecular drought literature in that they give no information on plant or soil water potential or water content. Thus, there is no way to know how severe the drought stress was in that experiment and no way for any other lab to reproduce it. It is directly akin to doing a heat stress experiment and not reporting the actual temperature.

They compare transcriptome data from this soil drying experiment to transcriptome data from agar plates with PEG, mannitol or salt added. However, this comparison is problematic, because none of the treatments being compared are at the same water potential (as mentioned above). Also, the PEG-infused agar plates have limitations in that no buffer is added and it is not clear that anything is done to check or control the pH. Adding PEG to the solution will reduce the pH. Thus, in their unbuffered PEG plates, the plants are almost certainly exposed to low pH stress and this can explain the supposed difference they observe between PEG and other treatments, especially since the plants are left on such stressful pH conditions for a relatively long period. It is also problematic that the comparison between soil drying and plate-based treatments is at different times (5 vs 14 days). They also show an over-reliance on the GO annotations of drought-induced gene expression. This GO annotation is based on experiments using very severe stress for a short time period. It is notorious for not accurately reflecting what happens on longer-term exposure to more moderate levels of low water potential stress. Thus, for example, we would not expect many of the canonical drought regulation genes (RD29A and similar genes) to be upregulated in the longer-term treatments as its expression is induced rapidly but also rapidly declines back to near baseline at the plant acclimates to the low water potential stress.

The authors have not always considered literature that would be relevant to their topic. For example, there is a number of studies that have reported (and deposited in the public database) transcriptome analysis of plants on PEG-plates or plants exposed to well-controlled, moderate severity soil drying assays (for the latter, check the paper of Des Marais et al. and others, for the former, Verslues and colleagues have published a series of studies using PEG-agar plates). They also overlook studies that have recorded growth responses of wild type and a range of mutants on properly prepared PEG plates and found that those results agree well with results when plants are exposed to a controlled, partial soil drying to impose a similar low water potential stress. In short, the authors need to make such comparisons to other data and think more about what may be wrong with their own experimental designs before making any sweeping conclusions about what is suitable or not suitable for imposing low water potential stress.

To solve the problem of using these other systems to impose low water potential stress, the authors propose the seemingly logical (but overly simplistic) idea of adding less water to the same mix of nutrients and agar. Because the increased agar concentration does not substantially influence water potential (the agar polymerizes and thus is not osmotically active), what they are essentially doing is using a concentrated solution of macronutrients in the growth media to impose stress. This is a rediscovery of an old proposal that concentrated macronutrient solutions could be used to study the osmotic component of salt stress (see older papers of Rana Munns). There are also effects of using very hard agar that is of unclear relationship to actual drought stress and low water potential. Thus, I see no reason to think that this would be a better method to impose low water potential.

Reviewer #3 (Public Review):

This work compares transcriptional responses of shoots and roots harvested from four plate-based assays that simulate drought and from plants subjected to water deficit in pots using the model plant Arabidopsis thaliana with the aim to select a plate-based assay that best recapitulates transcriptional changes that are observed during water-deficit in pots. Polyethylene glycol (PEG), mannitol, and sodium chloride (salt) treatments that are commonly used by molecular biologists to simulate drought were used for the plate-based assays as well as a new assay that uses increased concentrations of agar and nutrients to elicit drought which was developed by the authors and termed a 'low-water agar' assay since the amount of water added to the media mix and plates was lowered. Plants in pots were grown on vermiculite with the same nutrient mix as used in the plates and drought was induced by withholding watering for five days. Additionally, treatment with abscisic acid was conducted to study whether growth on plates itself led to artifacts compared to water deficit in pots. Shoot and root samples were harvested from all treatments for RNA sequencing analysis and differentially expressed genes were called against control samples.

The authors observed that gene expression responses of roots in their 'low-water agar' assay resembled more closely the water deficit in pots compared to the PEG, mannitol, and salt treatments (all at the highest dose). In particular, 28 % of PEG led to the down-regulation of many genes that were up-regulated under drought in pots. Through GO term analysis, it was pointed out that this may be due to the negative effect of PEG on oxygen solubility since downregulated genes were over-represented in oxygen-related categories. The data also shows that the treatment with abscisic acid on plates was very good at simulating drought in roots. Gene expression changes in shoots showed generally a high concordance between all treatments at the highest dose and water deficit in pots, with mannitol being the closest match. This is surprising, since plants grow in plates under non-transpiring conditions, while a mismatch between water loss by transpiration on water supply via the roots leads to drought symptoms such as wilting in pot and field-grown plants. The authors concluded that their 'low-water agar' assay provides a better alternative to simulate drought on plates.

Strengths:

The development of a more robust assay to simulate drought on plates to allow for high-throughput screening is certainly an important goal since many phenotypes that are discovered on plates cannot be recapitulated on the soil. Adding less water to the media mix and thereby increasing agar strength and nutrient concentration appears to be a good approach since nutrients are also concentrated in soils during water deficit, as pointed out by the authors. To my knowledge, this approach has not specifically been used to simulate drought on plates previously. Comparing their new 'low-water agar' assay to popular treatments with PEG, mannitol, salt, and abscisic acid, as well as plants grown in pots on vermiculite led to a comprehensive overview of how these treatments affect gene expression changes that surpass previous studies. It is promising that the impact of 'low-water agar' on the shoot size of 20 diverse Arabidopsis accessions shows some association with plant fitness under drought in the field. Their methodology could be powerful in identifying a better substitute for plate-based high-throughput drought assays that have an emphasis on gene expression changes.

Weaknesses:

While the authors use a good methodological framework to compare the different drought treatments, gene expression changes were only compared between the highest dose of each stress assay (Fig. 2B, 3B). From Fig. 1F it appears that gene expression changes depend significantly on the level of stress that is imposed. Therefore, their conclusion that the 'low-water agar' assay is better at simulating drought is only valid when comparing the highest dose of each treatment and only for gene expression changes in roots. Considering how comparable different levels of stress were in this study leads to another weakness. The authors correctly point out that PEG, mannitol, and salt are used due to their ability to lower the water potential through an increase in osmotic strength (L. 45/46). In soils, water deficit leads to lower water potential, due to the concentration of nutrients (as pointed out in L. 171), as well as higher adhesion forces of water molecules to soil particles and a decline in soil hydraulic conductivity for water, which causes an imbalance between supply and demand (see Juenger and Verslues, The Plant Cell 2022 for a recent review). While the authors selected three different doses for each treatment that are commonly used in the literature, these are not necessarily comparable on a physiological level. For example, 200 mM mannitol has an approximate osmotic potential of around -5 bar (Michel et al. Plant Physiol. 1983) whereas 28 % PEG has an osmotic potential closer to -10 bar (Michel et al. Plant Physiol. 1973). It also remains unclear how the increase in agar concentration versus the increase in nutrient concentration in the 'low-water agar' affect water potentials. For these reasons it cannot be known whether a better match of the 'low-water agar' at the 28% dose to water deficit in pots for roots in comparison to the other treatments is due to a good match in stress levels with the 'low-water agar' or adverse side-effect of PEG, mannitol, or and salt on gene regulation. Lastly, since only two biological replicates for RNA sequencing were collected per treatment, it is not possible to know how much variance exists and if this variance is greater than the treatments themselves.

Author Response:

eLife assessment

This work is an attempt to establish conditions that accurately and efficiently mimic a drought response in Arabidopsis grown on defined agar-solidified media - an admirable goal as a reliable experimental system is key to conducting successful low water potential experiments and would enable high-throughput genetic screening (and GWAS) to assess the impacts of environmental perturbations on various genetic backgrounds. The authors compare transcriptome patterns of plant subjected to water limitation imposed using different experimental systems. The work is valuable in that it lays out the challenges of such an endeavor and points out shortcomings of previous attempts. However, a lack of water relations measurements, incomplete experimental design, and lack of critical evaluation of these methods in light of previous results render the proposed new methodology inadequate.

We thank eLife for the initial assessment and comments to our work. In our revised manuscript we plan to address the main concerns raised by reviewers. Specifically, we plan to perform water relations measurements for all our treatment assays, as well as explore the separate effects agar hardening and nutrient concentration have in our low-water agar assay. We will also provide a more in depth critical review of our results compared to previously published results.

Reviewer #1 (Public Review):

High-throughput genetic screening is a powerful approach to elucidate genes and gene networks involved in a variety of biological events. Such screens are well established in single-celled organisms (i.e. CRISPR-based K/O in tissue culture or unicellular organisms; screens of natural variants in response to drugs). It is desirable to extend such methodology, for example to Arabidopsis where more than 1000 ecotypes from around the Northern hemisphere are available for study. These ecotypes may be locally adapted and are fully sequenced, so the system is set up for powerful exploration of GxE. But to do so, establishing consistent "in vitro" conditions that mimic ecologically relevant conditions like drought is essential.

The authors note that previous attempts to mimic drought response have shortcomings, many of which are revealed by 'omics type analysis. For example, three treatments thought to induce osmotic stress; the addition of PEG, mannitol, or NaCl, fail to elicit a transcriptional response that is comparable to that of bonafide drought. As an alternative, the authors suggest using a low water-agar assay, which in the things they measure, does a better job of mimicking osmotic stress responses. The major issues with this assay are, however, that it introduces another set of issues, for example, changing agar concentration can lead to mechanical effects, as illustrated nicely in the work of Olivier Hamant's group.

We thank the reviewer for their comments. We hypothesize that our low-water agar assay is able to replicate drought gene expression patterns through a combination of hardened agar and higher nutrient concentration. However, we did not explore the separate effects each of these factors may play in eliciting such responses. Thus, in our revised manuscript, we will explore what role the mechanical effects of changing agar concentration has on root gene expression. However, we suspect that the mechanical effects introduced by hard agar does not introduce another issue per se, but in fact may help with replicating the transcriptional effects seen under drought.

Reviewer #2 (Public Review):

[…] The authors have not always considered literature that would be relevant to their topic. For example, there is a number of studies that have reported (and deposited in the public database) transcriptome analysis of plants on PEG-plates or plants exposed to well-controlled, moderate severity soil drying assays (for the latter, check the paper of Des Marais et al. and others, for the former, Verslues and colleagues have published a series of studies using PEG-agar plates). They also overlook studies that have recorded growth responses of wild type and a range of mutants on properly prepared PEG plates and found that those results agree well with results when plants are exposed to a controlled, partial soil drying to impose a similar low water potential stress. In short, the authors need to make such comparisons to other data and think more about what may be wrong with their own experimental designs before making any sweeping conclusions about what is suitable or not suitable for imposing low water potential stress.

To solve the problem of using these other systems to impose low water potential stress, the authors propose the seemingly logical (but overly simplistic) idea of adding less water to the same mix of nutrients and agar. Because the increased agar concentration does not substantially influence water potential (the agar polymerizes and thus is not osmotically active), what they are essentially doing is using a concentrated solution of macronutrients in the growth media to impose stress. This is a rediscovery of an old proposal that concentrated macronutrient solutions could be used to study the osmotic component of salt stress (see older papers of Rana Munns). There are also effects of using very hard agar that is of unclear relationship to actual drought stress and low water potential. Thus, I see no reason to think that this would be a better method to impose low water potential.

We thank the reviewer for their comments. In our revised manuscript, we will address points regarding plant and soil water potential; similar concerns were also raised by Reviewer 1 and 3. We note that we report vermiculite water content in Supplementary Table 4.

We would like to clarify that both the PEG media and overlay solution were buffered - we did not include this within the written description in the methods, but will do in our revised manuscript.

We agree with the reviewer’s concern that it may be problematic to compare the transcriptomic profiles of seedling and mature plants. In light of this, we plan to explore what effects our treatment media has on mature rosettes.

We note that we do not claim that PEG is unable to produce low-water potential responses similar to partial soil drying. Indeed, we indicate that it is a good technique for eliciting phenotypes comparable to drought at the physiological level (line 48). Rather, we claim that PEG is unable to produce gene expression responses that are sufficiently similar to partial vermiculite drying.

Reviewer #3 (Public Review):

[…] The authors observed that gene expression responses of roots in their 'low-water agar' assay resembled more closely the water deficit in pots compared to the PEG, mannitol, and salt treatments (all at the highest dose). In particular, 28 % of PEG led to the down-regulation of many genes that were up-regulated under drought in pots. Through GO term analysis, it was pointed out that this may be due to the negative effect of PEG on oxygen solubility since downregulated genes were over-represented in oxygen-related categories. The data also shows that the treatment with abscisic acid on plates was very good at simulating drought in roots. Gene expression changes in shoots showed generally a high concordance between all treatments at the highest dose and water deficit in pots, with mannitol being the closest match. This is surprising, since plants grow in plates under non-transpiring conditions, while a mismatch between water loss by transpiration on water supply via the roots leads to drought symptoms such as wilting in pot and field-grown plants. The authors concluded that their 'low-water agar' assay provides a better alternative to simulate drought on plates.

Strengths:

The development of a more robust assay to simulate drought on plates to allow for high-throughput screening is certainly an important goal since many phenotypes that are discovered on plates cannot be recapitulated on the soil. Adding less water to the media mix and thereby increasing agar strength and nutrient concentration appears to be a good approach since nutrients are also concentrated in soils during water deficit, as pointed out by the authors. To my knowledge, this approach has not specifically been used to simulate drought on plates previously. Comparing their new 'low-water agar' assay to popular treatments with PEG, mannitol, salt, and abscisic acid, as well as plants grown in pots on vermiculite led to a comprehensive overview of how these treatments affect gene expression changes that surpass previous studies. It is promising that the impact of 'low-water agar' on the shoot size of 20 diverse Arabidopsis accessions shows some association with plant fitness under drought in the field. Their methodology could be powerful in identifying a better substitute for plate-based high-throughput drought assays that have an emphasis on gene expression changes.

Weaknesses:

While the authors use a good methodological framework to compare the different drought treatments, gene expression changes were only compared between the highest dose of each stress assay (Fig. 2B, 3B). From Fig. 1F it appears that gene expression changes depend significantly on the level of stress that is imposed. Therefore, their conclusion that the 'low-water agar' assay is better at simulating drought is only valid when comparing the highest dose of each treatment and only for gene expression changes in roots. Considering how comparable different levels of stress were in this study leads to another weakness. The authors correctly point out that PEG, mannitol, and salt are used due to their ability to lower the water potential through an increase in osmotic strength (L. 45/46). In soils, water deficit leads to lower water potential, due to the concentration of nutrients (as pointed out in L. 171), as well as higher adhesion forces of water molecules to soil particles and a decline in soil hydraulic conductivity for water, which causes an imbalance between supply and demand (see Juenger and Verslues, The Plant Cell 2022 for a recent review). While the authors selected three different doses for each treatment that are commonly used in the literature, these are not necessarily comparable on a physiological level. For example, 200 mM mannitol has an approximate osmotic potential of around -5 bar (Michel et al. Plant Physiol. 1983) whereas 28 % PEG has an osmotic potential closer to -10 bar (Michel et al. Plant Physiol. 1973). It also remains unclear how the increase in agar concentration versus the increase in nutrient concentration in the 'low-water agar' affect water potentials. For these reasons it cannot be known whether a better match of the 'low-water agar' at the 28% dose to water deficit in pots for roots in comparison to the other treatments is due to a good match in stress levels with the 'low-water agar' or adverse side-effect of PEG, mannitol, or and salt on gene regulation. Lastly, since only two biological replicates for RNA sequencing were collected per treatment, it is not possible to know how much variance exists and if this variance is greater than the treatments themselves.

We thank the reviewer for their comments. In our statistical analyses, we found that dose-responsive genes (as fit by a linear model) were very similar to those genes found differentially expressed at the highest dose. Thus, for clarity, we decided to simply present the genes differentially expressed at the highest dose. We see now that this might have been an oversimplification. In our revised manuscript, we will present genes that are dose responsive across the range of treatment doses, thus providing more evidence that lower doses of low-water agar are also capable of simulating drought (as is suggested by overlap analysis of Figure 2A).

Additionally, we will also explore the osmotic potential of each of our different assays to provide a better benchmark of how comparable each of our treatments are (as similarly requested by Reviewer 1 and 2). Lastly, to address concerns regarding the size of variance in gene expression, we will sequence a 3rd replicate of RNA.