The ecological implications of interplant drought cuing

Plants can perceive, integrate and respond to multiple signals and cues informative of imminent threats and opportunities. Here, we tested the hypothesis that unstressed plants are able to perceive and adaptively respond to stress cues emitted from their drought‐stressed neighbours, and to induce adaptive responses in additional unstressed plants. Triplets of split‐root Stenotaphrum secundatum plants were grown in rows. One root of the first plant was subjected to drought while its other root shared its pot with one of the roots of an unstressed target neighbour, which in turn shared its other pot with an additional unstressed target neighbour. Cuing from drought‐stressed plants increased survival in both proximate directly cued and in more distant relayed‐cued target plants under drought. Drought cuing lowered plant performance under benign conditions. Experiments with a Wilty mutant and fluridone treatments showed that drought cuing was greatly reduced in ABA‐deficient Pisum Sativum plants. Synthesis. Our findings demonstrate for the first time the possible adaptive implications of both direct and relayed stress cuing among neighbouring plants, the possible cost of plant responsiveness to drought cues under benign conditions and the involvement of ABA in interplant drought cuing. The results suggest that interplant root communication of drought cues could have novel implications for plant interactions, survival and performance under both natural and agricultural settings.

In response to stress cues emitted from the roots of their drought-inflicted neighbours, unstressed plants readily close their stomata, and via relayed cuing induce stomatal closure in additional unstressed plants (Falik et al., 2011(Falik et al., , 2012. Nevertheless, while drought-stressed plants show persistent stomatal closure, their unstressed neighbours fully reopen their stomata 3-24 h following the onset of drought cuing (Falik et al., 2012). These findings raise important questions regarding the possible ecological implications of interplant drought cuing. What are the adaptive implications of drought cuing? Are such adaptations only expressed in directly cued plants or also in their relayed neighbours? What is the mechanism of interplant drought cuing?
Here, we studied the ecological implications of interplant drought cuing by subjecting unstressed Stenotaphrum secundatum target plants to short periods of root cuing from neighbours that were either stressed or unstressed by drought. The survival and performance of the target plants were monitored during a subsequent period, during which they were subjected to either benign or drought conditions. We predicted that both direct and relayed interplant cuing would increase the survival and performance of unstressed plants under a subsequent drought. In addition, we expected that drought cuing would inflict performance costs on target plants that would not be subjected to a subsequent drought.
The mechanism of interplant drought cuing was studied in additional experiments with Pisum sativum, a widely studied model plant in the field of stress physiology (e.g. Sharma et al., 2019). Previous findings have demonstrated that interplant drought cuing was attained among neighbouring roots, suggesting that the involved vectors are emitted by the roots of drought-inflicted plants. Abscisic acid (ABA) is a promising candidate that satisfies these specific criteria. ABA is produced in most plant tissues and is involved in the induction of resistance and tolerance to drought and other stresses (Nakashima et al., 2014). Previous studies have demonstrated that in some legumes and grasses, drought may cause ABA leakage from the roots (Harris, 2015;Shi et al., 2014). Other studies have shown that exogenous ABA can be taken up by roots and elicit stress responses in receiving plants (Hartung et al., 2002;Rogiers et al., 2012). We studied the possible involvement of ABA in two experiments in which plant responses to interplant drought cuing was monitored in ABA-deficient and control plants. Specifically, we compared stomatal closure in unstressed target P. sativum plants subjected to drought root cuing from neighbouring plants deficient in their ability to produce ABA (ABA-deficient wilty mutant plants; Wang et al., 1984), or in plants treated with fluridone that interferes with ABA biosynthesis (Mialoundama et al., 2009).

| Experimental design and setup
The experiment was conducted on S. secundatum (buffalo grass), a perennial stoloniferous grass native to the Caribbean, South America and parts of North America and Africa, which has been introduced to many other geographical regions (CABI, 2022). S. secundatum is a strong competitor typical to anthropomorphically disturbed habitats and is commonly used as a lawn grass (Busey, 2003). Testing for the effects of drought cuing required that specific induced plants (IND) would experience a drought event or benign conditions while their neighbouring target plants (T1, T2) would only experience cuing from the IND plants ( Figure 1). This was achieved using triplets of split-root S. secundatum plants planted in rows of four pots ( Figure 1).
One of the roots of the IND plant was subjected to either drought or benign conditions, while its other root shared a pot with one of the roots of its nearest unstressed neighbour (T1). The other root of T1 shared its pot with one of the roots of an additional unstressed target plant (T2). This configuration permitted T1 to exchange stress cues with both IND and T2, while preventing direct root cuing between IND and T2 and thus allowing to separately study the effects of direct and relayed drought cuing on T1 and T2, respectively (Falik et al., 2011, Figure 1 Figure 1) was filled with tap water and the other pots were filled with a commercial soil mixture (Deshanit). In clonal plants such as S. secundatum, resource translocation is predominantly acropetal (Price & Hutchings, 1992) and in response to herbivory, endogenous warning signals are more efficiently transferred acropetally than basipetally (Gutbrodt et al., 2011). To avoid potential variability due to axis polarity, all plants were planted with their proximal ramets nearest to the IND pot ( Figure 1). To increase root intermingling and plant uniformity, plant cuttings were grown in the experimental systems for 30 days, after which their tillers were clipped and they were allowed to regenerate for additional 7 days before starting the cuing period.
The cuing period started by evacuating the water from pot 1 and filling it with either dry (drought treatment) or wet (benign control) 9:1 mixture of grade 2 vermiculite (Agrekal) and bentonite (Agat), (VB mixture) (Falik et al., 2012), for either 1 or 5 days. The IND plants were removed at the end of the cuing period to ensure that drought cuing was restricted to the designated periods and that the performance of the target plants during the survival period would not be affected by the presence of the IND plant ( Figure 1).

| Plant survival and performance
During the survival period, pots assigned to drought treatment were not irrigated and pots assigned to benign conditions were irrigated every 3-4 days. Plant aboveground survival (AGS) was estimated every 7 days from colour analyses of 3216 × 2136 pixels F I G U R E 1 Experiment 1: Testing for the adaptive implications of drought cuing in Stenotaphrum secundatum-experimental setup. Triplets of two-ramet plants were planted in rows of four pots. One of the roots of the IND plant (pot 1) was subjected to either drought (yellow) or benign (blue) conditions while its other root shared pot 2 with one of the roots of its nearest unstressed neighbour (T1). The other root of T1 shared pot 3 with one of the roots of an additional unstressed target plant (T2). This configuration permitted T1 to exchange root stress cues with both IND and T2, while preventing direct root cuing between IND and T2, thus allowing to separately study the effects of direct and relayed drought cuing on T1 and T2, respectively. Following an establishment period, one of the roots of the IND plant (pot 1) was subjected to either drought (a, b) or benign (c, d) conditions for 1 or 5 days after which the IND plant was removed (white) and the target plants were subjected to either benign (b, d; blue) or drought (a, c; yellow) conditions. Establishment (37 d) Cuing (1 or 5 d) Survival (60 d)   1  2  3  4  1  2  3  4   IND T1  T2  IND T1  T2  T1  T2 (a) white-balanced JPEG digital photographs. The area of all aboveground organs of each plant was digitally separated from its background using Adobe Photoshop, and red (R), green (G) and blue (B) reflectance data were used to calculate excess green index (2G-R-B) (Richardson et al., 2007) using ImageJ ( compared to WT (Wang et al., 1984). In experiment 3 (fluridone; Figure 4c), the T3 plant was either untreated (control) or treated with 10 μM fluridone, known to drastically interfere with ABA biosynthesis (Mialoundama et al., 2009

| Data analyses
The effects of drought cuing on the survival of S. secundatum under drought (experiment 1) were analysed using log-rank analyses (Kleinbaum & Klein, 2010). greater shoot biomass, 88% more branches and 73% greater branch length than their uncued controls (Figure 3c,g,j); however, no such effects were found in directly cued (T1) plants (significant cuing treatment × target interaction for shoot growth; Table S1), or when drought cuing was implemented for 5 days (Figure 3d,h,l). T2 plants that experienced 5 days of relayed drought cuing and grew under benign conditions showed 40%, 36% and 42% lower shoot biomass, branch number and branch length, compared to their uncued controls (Figure 3b,f,k), but no such effects were observed in directly cued T1 plants or when drought cuing was implemented for 1 day ( Figure 3a,e,i, Table S1).

| Soil water
Soil water content during the cuing period was non-significantly different in drought-cued treatments and in their uncued controls ( Figure S1). Incidental differences in soil water content were observed during the drought period, but in all these cases values were higher in the drought treatment than in the benign controls ( Figure S1).

| DISCUSS ION
Plants are able to perceive and integrate intricate signals and cues that enable them to plastically adapt to imminent risks and opportunities (Novoplansky, 2016). It has been previously demonstrated that unstressed plants rapidly close their stomata in response to stress cues emitted by the roots of their drought-stressed neighbours, and that 'relay cuing' can elicit stomatal closure in additional increasingly distant unstressed plants (Falik et al., 2011). In the following, we discuss the adaptive implications of interplant drought cuing and its underlying mechanism as reflected from the results.
In S. secundatum, both direct and relayed drought cuing can significantly increase plant survival time and performance under a subsequent drought (Figures 2 and 3). The criticality of water limitation to plant survival, growth and functioning cannot be overemphasized and predictive information regarding forthcoming droughts could be Values are means ± SEM of shoot dry biomass (a-d), Number of branches (e-h) and branch length (i-l) of directly cued (T1) or relayed-cued (T2) plants. Significance values are for Tukey's posthoc tests evaluating the differences between target plants that either received (red) or not received (blue) drought cues, + 0.1 < p < 0.05, *p < 0.05, (n = 20).  T1  T2  T1  T2  T1  T2  T1  T2   *  *   *   T1  T2  T1  T2  T1  T2  T1  T2

Drought Benign
Shoot dry mass (g)  crucial for plant survival and fitness. Plants are known to respond to mild soil drying before any changes in leaf water potential are detectable (Davies & Zhang, 1991;Passioura, 1988) and competitive depletion of soil water has been shown to elicit large plastic modifications in resource allocation that facilitate increased survival under drought (Novoplansky & Goldberg, 2001). Our results show that 'eavesdropping' on neighbours allows plants to exploit even earlier indications of an ensuing drought, before they experience any decreases in water availability ( Figure S1). In arid environments, such predictive cues may help plants increase their survival and performance during dry spells within and between growing seasons (Goldberg & Novoplansky, 1997;Huxman et al., 2004;Robertson et al., 2009;Swemmer et al., 2007) and prompt annual plants to more timely and less traumatically modulate their resource allocation and phenology in preparation for the end of the growing season (Kozłowski, 1992;Shemesh & Novoplansky, 2013).
The findings demonstrate that drought cuing can be relayed via a chain response of multiple neighbouring plants (Figures 2 and   3). As the perception of drought cues elicits further cuing, relayed drought cuing is expected to be rapidly amplified by a feedforward positive-feedback loop (see Wenig et al., 2019 for a similar phenomenon related to volatile defence cuing). Nevertheless, for such a self-enhancing system to be ecologically relevant and reliable, it is essential that plants are not engaged in an inappropriate and costly permanent overly escalated state of alert (Farmer, 2007). tally cued responses such as seed germination (Gutterman, 2012) and shade avoidance (Novoplansky et al., 1990), where inappropriate responses could be critically detrimental for plant survival and fitness (Hauser, 1996).
As expected, the drastic limitation in water availability only allowed limited effects of drought cuing on plant growth under a subsequent drought (Figure 3), but the results show that induced stress tolerance and increased survival may incur performance costs under both drought and benign conditions. While 1 day of drought cuing increased plant size under drought and inflicted no costs under benign conditions, 5 days of drought cuing did not improve plant growth under drought yet caused some cost under benign conditions (Figures 2 and 3). Indeed, functional trade-offs between stress tolerance and growth rates under benign conditions have been commonly demonstrated in comparative studies (Díaz et al., 2016;Grime, 1979;Tilman, 1982 (Winter & Holtum, 2014).
Interestingly and perhaps counterintuitively, both survival and growth effects of drought cuing tended to be consistently greater in relayed target plants (T2) than in directly cued plants (T1) (Figures 2   and 3). While we cannot provide a conclusive explanation for this trend, it can be speculated that it might have been caused by the slightly faster soil drying of pots 3 and 4 (T2) compared to pots 2 and 3 (T1), 10-20 days following the onset of drought ( Figure S1).
Although this trend was similar in all cuing treatments, it is thus conceivable that the match between the experienced water status and the perceived drought cues was tighter and thus potentially more reliable for the relayed T2 than for the directly cued T1 plants. An alternative though not mutually exclusive hypothesis could be that T2 was experiencing stronger drought cuing than T1 plants, which in turn increased hydraulic conductance into the plant (Parent et al., 2009) and accelerated soil dehydration. Such a scenario could have increased plant survival under drought and decreased plant growth under benign conditions (Figures 2 and 3).
While it is relatively easy to understand the adaptive benefits of plant responsiveness to sufficiently reliable drought cues, the selective advantage of the emission of stress cues is much less obvious. Selection is only expected to prefer cue 'leakiness' from stressed plants where the average fitness benefits of stress cuing outweigh their production and competitive costs (Dicke & Bruin, 2001;Falik et al., 2012). Accordingly, leakiness of honest drought cues is expected to benefit emitters (a) if the presence of drought cues in the rhizosphere could increase drought resistance (Jiang & Hartung, 2008), (b) in large plants or plants with strict anatomical sectoriality (Espino & Schenk, 2009), where exogenous root signalling could be more rapid and efficient than endogenous signalling (Rodriguez-Saona et al., 2009), (c) in clonal plants or in plants where kin or clone mates are spatially clumped (Cheplick, 1993;Herben & Novoplansky, 2008), and thus the probability of benefiting genetically alien competitors is low, or (d) where eavesdropping neighbours reduce water uptake and by so doing improve the survival of larger patches of neighbouring plants. The latter scenario can be expected under extreme arid conditions, where the importance of facilitation could be greater than that of competition (Bertness & Callaway, 1994;Ploughe et al., 2019).
Roots exchange with the rhizosphere myriad substances, some of which carry vital adaptive information (Mommer et al., 2016). For example, under nutrient deficiency, some plants increase production and root exudation of strigolactones that promote mycorrhizal development and establishment (Waters et al., 2017). ABA is a plant hormone involved in growth and responses to abiotic stresses such as drought, salinity and nutrient deficiencies (McAdam & Brodribb, 2016). In drying soils, long-distance endogenous ABA signalling regulates multiple pathways related to stomata aperture, root development and hydraulic conductivity (Kuromori et al., 2018). In some plants, drought not only increases ABA production and accumulation, but also elicits ABA exudation to the rhizosphere (Hartung et al., 2002;Shi et al., 2014;Wilkinson & Davies, 2002). As roots can absorb ABA from the soil solution and transfer it to the shoots (Hartung et al., 2002), the combined effects of ABA exudation and uptake may be involved in the interplant drought cuing observed here (Falik et al., 2011; Figures 2 and 3).
The involvement of ABA in interplant drought cuing is supported by the significant reduction in drought cuing observed in P.
sativum plants with diminished ABA synthesis (Figure 4) shown to metabolize soil ABA (Belimov et al., 2014), which could potentially alleviate performance costs in previously stressed or communicated plants under benign conditions (Falik et al., 2011), and increase the reliability of ABA cuing by rapidly reducing soil ABA concentrations following the cessation of its exudation by stressed plants. The ubiquity of ABA as a stress hormone (Vishwakarma et al., 2017) and the existence of similar drought cuing in a variety of plant species (Shi et al., 2014) (Gorzelak et al., 2015;Karban, 2021;Tedersoo et al., 2020)

ACK N OWLED G EM ENTS
We thank Ishay Hoffmann and Tamar Sinai for technical help, and Pedro Aphalo for insightful discussions and very useful comments on an early version of the manuscript. The study was partially supported by research grants from the Israel Science Foundation to A.N.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1111/1365-2745.13991.