Plant performance under aeroponic simulated drought
To address the effect of disturbances on root exudation dynamics, new and non-destructive sampling methods that incorporate drought and recovery need to be developed (17). In this study, we developed and assessed the efficacy of using a high-pressure aeroponic system to simulate progressive drought stress and nondestructively collect root exudates. An aeroponic method was used to precisely control the severity and recovery of drought at a desired timescale by empirically specifying the irrigation delivered to cotton roots.
As roots are irrigated in the aeroponic system, runoff droplets are drained by gravity to the bottom of a collection chamber. In theory, this creates a non-destructive "rinseate” containing root exudates and other rhizodeposition products such as root border cells (Fig. S3). The chamber containing the rooting system is easily replaceable, so sampling can be performed at desired time points with a clean collection pot. We hypothesized that by using the infrastructure of aeroponics a sampling solution could be applied to the root system with the resultant runoff droplets containing detectable root exudates. This hypothesis was validated by identifying ABA in response to treatments in the collected samples. We also hypothesized that drought and subsequent recovery could be simulated in aeroponics, which was assessed through evaluation of morphological traits, biomass, PSII fluorescence parameters, and visual drought severity index, with numerous effects of drought stress observed in cotton.
The morphological results of applying water-stress to cotton using aeroponics were similar to observations made in other field or greenhouse studies using soil media. We observed that drought reduced canopy height, number of green leaves, and leaf dry weight, which is consistent with other studies (46). Pace et al. (47) evaluated the effect of drought on two cotton cultivars in the field and reported a 28% decrease in stem height, 19% decrease in stem dry weight, and 35% decrease in leaf dry weight at the end of drought treatment (water withheld for 13 days). They found no differences in root dry weight between the well-watered and drought-treated plants. After 10 days of recovery, they reported that drought-treated plants continued to have lower stem height, stem dry weight, leaf dry weight, and root dry weight than the control (47). The timing of drought stress can have varying impacts on harvest yield depending on the growth stages at which it is applied (48). Zonta et al. (48) reported that water deficiency at first flower and peak bloom had the largest effect on yield, with the first square stage following in yield reduction. In our study, the treatments covered the critical growth stage of cotton from first square to first flower.
Chlorophyll fluorescence is a common and non-destructive technique to analyze PSII activity. It has been widely utilized to evaluate plant response to abiotic and biotic stresses, including drought. However, we did not observe a consistent response in fluorescence parameters attributable to drought. Measurements are sensitive to multiple factors, such as the time of measurement, leaf age, and the position of leaves in the canopy, so it is important to keep those factors consistent throughout the experiment. There is no consensus on reporting parameters or standard protocols for measuring chlorophyll fluorescence in cotton, such as the sampling time and leaf position. Several field studies have indicated that Fv/Fm may not be a good indicator of drought in cotton (39, 40, 44). In a 4-year field trial, Pettigrew (2004) measured Fv/Fm from two leaves per plot in the morning and afternoon and found no differences in Fv/Fm between two soil moisture treatments (irrigated and dryland) across both timepoints and eight genotypes (44). Similar findings were also reported in Massacci et al. (39) and Luo et al. (40), although the drought treatments, leaf age, dark-adapted time, and sampling time varied in different studies. In a greenhouse study with four soil moisture treatments, the author reported that Fv/Fm decreased in the drought-treated plants compared with well-watered control (49), but Fv/Fm was measured on the first fully expanded leaf in the morning before the leaf was exposed to direct sunlight. Besides leaf age and sampling time point, the plant growth stage is likely another factor that affects the sensitivity of fluorescence parameters. It has been reported that midday photosynthesis rates of cotton during the reproductive stage was less sensitive than during the vegetative stage (50). As for other fluorescence parameters, Tro/ABS and ETo/ABS reported no differences between soil moisture treatments; however, PIabs decreased in suboptimal irrigation treatments (45% field capacity) as compared to control (75% field capacity) in Luo et al. (40). Our results, as well as others, suggest that chlorophyll fluorescence measurements may not be as good of an indicator of drought in cotton compared to other morphological traits, such as canopy height, number of green leaves, and drought severity index.
Capturing drought associated root exudates using aeroponics
ABA is considered a universal abiotic stress-response hormone (51) and is ubiquitous in all plant parts and throughout the environment due to its involvement in many developmental processes, such as shoot growth inhibition, stomatal closure, leaf senescence and primary root growth. The biosynthesis of ABA has been well studied (52, 53). In response to stress, ABA is generally synthesized in the root tips. The largest accumulation is often located in root tips and is transported to aboveground tissues via the xylem. Yet, ABA can also be synthesized in leaf tissue and transported to roots via the phloem. In addition to synthesis and catabolism, ABA levels in the root can be regulated through absorption of exogenous ABA or release of ABA to maintain cell equilibrium (52). For instance, to enhance drought adaptation, upland rice actively exports more root-source ABA into the rhizosphere to reduce ABA concentrations in the roots (54). ABA has been detected in root exudates using a wide range of collection methods over diverse species including rice (Oryza sativa, (18, 54, 55), maize (Zea mays, (13, 56), barley (Hordeum vulgare, (57), holm oak (Quercus ilex, (1), Scotch pine (Pinus sylvestris, (58), castor bean (Ricinus communis, (59), and rehmannia (Rehmannia glutinosa, (60). However, to the best of our knowledge, there are no studies that have measured ABA in cotton root exudates.
ABA concentration in root exudates generally increases in response to stress including drought (1, 18, 57), elevated CO2 (55, 57), and warm temperature (58). With no common reporting parameters, studies usually report ABA concentration in root exudates by weight per unit volume of sampling solution (pmol, pg, or ng mL− 1) or per root dry or fresh weight (pmol, pg, or ng g− 1) (18, 57, 58). When discretely sampled over a period of days to weeks, and without precise sampling methodology defined, many past studies represent an unknown temporal exudate accumulation in their samples. Assuming the sampling wash using the aeroponic system effectively retrieves 100% of rhizodeposition products, the samples from this study represent no more than 72 hours of exudation processes and likely much less as excess irrigation spray drains from the chamber without collection. While the ABA concentration in this study is lower than other research (18, 57, 58), our finding using aeroponics is likely due to the nature of our continuous samplings and prevention of major root disturbance that would otherwise confound actual exudate with damaged and leaking root tissues. Difficulty in comparing exuded metabolites across studies (13) highlights potential biases introduced by high disturbance sampling methods and a lack of consistent reporting regarding root exudation.
Sampling the exudates, or the roots? Destructive sampling confounds results
There is a paramount need to reform the scientific language of sampling root exudates, particularly in regard to the consideration of experimental design and data interpretation (2). Root handling and damage, along with mechanical impedance of root systems, creates artefacts that may bias the exudation profiles that underlie rhizosphere dynamics (61, 62). Because of this, methodology for root exudate sampling should pursue minimal damage to roots. Drought is additionally a complex environmental disturbance that alters cotton root morphology through accelerating the senescence process of older roots to create new fine roots (63). Thus, it is probable that the roots are sensitive during water-stress and should be sampled with care. An advantage of using aeroponics is that root disturbance is limited during sampling and growth, root hair development is potentially greater than in hydroponics (29, 64), and rinseate collection is a passive, nondestructive process. As realized in other studies analyzing root exudates (13, 16, 65) the metabolic comparison of exudates is difficult even between plants of the same species, owing to the complex chemical nature of exudates and various collection methods, making a synthesis of the literature bemusing. To support the postulation that a stress mitigating microbiome may be harnessed to enhance plant protection and performance (66, 67), we recommend sampling strategies such as aeroponics for studying the effect of drought in a consistent and reproducible manner that limits root damage. In this way, root exudates are collected nondestructively to reduce tissues from becoming highly permeable during growth and sampling, which would otherwise lead to capturing the metabolites present within the root rather than those exuded from it.
Technical considerations using aeroponics for root exudate collection
Technical considerations of growth chamber conditions include ensuring microenvironmental conditions are homogenous throughout the experiment. During sampling, it was noticed that growth chamber conditions may have disturbed some plants by unintentionally blowing warm air onto vegetative surfaces causing the plants to perceive differences in temperature or wind stress on shoot tissues. Air circulation was fixed when the issue was first noticed, but the most intensively affected plants, located in the control group, produced ABA outliers. After the issue was resolved, the disturbed pots continued to exude higher quantities of ABA than other control pots but levels declined as recovery continued. This result highlights the delicate nature of exudation and the potential sampling bias that can occur if sampling strategies do not incorporate great care to reduce unintentional disturbances to the plant. Because the quantity, quality, and patterns of root exudation are vastly unknown for agronomically relevant crops, the identification of potential nuances that could modify the exudate molecular profile should be given thorough attention in future studies.
Future applications for aeroponic root exudate sampling
Future applications for this aeroponic root exudate sampling methodology would be to isolate the specific metabolites involved in signaling functions during abiotic stresses (nutrient, salt, heat, fire, elevated greenhouse gases), biotic stresses (pathogens, pests, herbivory), and the stabilization of soil organic matter and other biogeochemical cycles. Presumably, most globally important food and fiber crops could be screened with this aeroponic method for their unique exudate signals. Aeroponics could be used to evaluate the regions of the root system (i.e., their location) responsible for differing interactions, and also community dynamics (allelopathy, competition, or facilitation) of multiple crops or weeds growing together. Stable isotope tracing with labeled 13CO2 could show allocation of photosynthetically manufactured carbon compounds to rhizodeposition alongside disturbances or other treatments (68, 69). The influence of a mineral or organic soil environment, or inoculation with specific biota, on rhizosphere dynamics could be investigated using split-root design, which has been applied to study the systemically induced root exudation of metabolites (SIREM) in a hydroponic system (13). Lastly, the net flux of rhizodeposition throughout 24-hour cycles could be explored to better understand the temporal response underlying exudation behavior.