Coordinated Systemic Stomatal Responses in Soybean.

Rapid and coordinated systemic stomatal responses occur in the crop plant soybean and could be involved in acclimation to changes in light conditions occurring in the field as a result of sunflecks.


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Abiotic stress conditions such as drought, salinity, heat, and their different combinations, can devastate agricultural production (Mittler and Blumwald, 2010;Bailey-Serres et al., 2019;Zandalinas et al., 2020b). A rapid change in stomatal aperture and/or stomatal conductance, to control water loss, photosynthesis, and respiration rates, is one of the earliest responses plants mount when challenged with an abiotic stress (Raven, 2014;Kollist et al., 2019;Lawson and Vialet-Chabrand, 2019;Papanatsiou et al., 2019;Kostaki et al., 2020;Lawson and Matthews, 2020;Medeiros et al., 2020). Recent studies revealed that stomatal responses induced by a local abiotic stress treatment applied to a single Arabidopsis (Arabidopsis thaliana) leaf (i.e. local tissue) can induce similar stomatal responses in the entire plant (i.e. systemic tissues). This type of response was termed "coordinated systemic stomatal response" and occurs in Arabidopsis in response to light or heat stresses, wounding, or a transition from dark to light (Devireddy et al., 2018(Devireddy et al., , 2020Yoshida and Fernie, 2018;Kollist et al., 2019;McLachlan, 2020). A similar systemic stomatal response was recently shown to occur in two tree species in response to a sudden increase in ambient CO 2 concentration (from 400 to 1,100 mL L 21 ) or a sudden transition from light to darkness (Ehonen et al., 2020). The findings that very different plant species, i.e. the annual plant Arabidopsis and the perennial trees aspen (Populus spp.) and birch (Betula spp.), display coordinated systemic stomatal responses to different stimuli, suggest that this type of response is common among different plants. However, the nature of coordinated systemic stomatal responses, the common mechanisms underlying them, and their prevalence in important crops, such as soybean (Glycine max), has not been demonstrated. To address these questions we studied the systemic stomatal response of greenhousegrown soybean plants ('Magellan', PI595362) to rapid changes in light intensity (Figs. 1 and 2; Supplemental Fig. S1). Taken together, our findings reveal that rapid and coordinated systemic stomatal responses occur in a crop plant such as soybean and could be involved in the acclimation of soybean to rapid changes in light conditions, humidity, and temperature occurring in the field as a result of sunflecks and/or other rapid changes in environmental conditions (Pearcy, 1990;Kollist et al., 2019;Papanatsiou et al., 2019;Wang et al., 2020).
In our experimental design, we used two portable photosynthesis system apparatuses (LI-6800; LI-COR), one attached to an upper leaf, and one attached to a lower leaf, of the same plant, and monitored changes in stomatal conductance (gs), transpiration (E), photosynthesis (A), and leaf temperature (Leaf Temp) in response to a sudden change in light intensity (from 40 to 1,500 mmol m 22 s 21 ) applied to the upper or the lower leaf using the portable photosynthesis system light source ( Fig. 1; Supplemental Fig. S1; plants were acclimated to a low light intensity of 40 mmol m 22 s 21 for 1 h before the change in light-intensity treatment, to mimic cloud or canopy coverage). The change in light intensity was limited to one leaf and conducted within the portable photosynthesis system chamber. Young, fully expanded source leaves, at the same relative position, of same-age and developmental-stage plants (4-week-old) were used, and light intensities used were based on measured intensities at different heights within the soybean canopy under field conditions (Wang et al., 2020).
In contrast to Arabidopsis, which displays a stomatal closure response to a sudden increase in light intensity (Devireddy et al., 2018(Devireddy et al., , 2020, local leaves of soybean plants subjected to a 40 to 1,500 mmol m 22 s 21 lightintensity change displayed a local stomatal opening response that was characterized by an increase in local stomatal conductance, transpiration, and photosynthesis ( Fig. 1). Changes in these parameters were significant as early as 3 min after the application of increase in light intensity treatment, demonstrating that the plant response to the sudden change in light intensity was rapid. Changes in local leaf temperature (an increase of 2°C to 3°C) were also rapid after the increase in light intensity (leaf temperature was not controlled within the LI-6800 chamber), but subsided within 10 to 15 min when leaf transpiration increased (Fig. 1).
Interestingly, when the change in light intensity was applied to an upper (local) leaf, increases in stomatal conductance and transpiration of the untreated lower (systemic) leaf were observed within 6 min (Fig. 1B). By contrast, when the change in light intensity was applied to the lower (local) leaf, similar changes were not observed in the untreated upper (systemic) leaf (Fig. 1C). These findings demonstrate that systemic stomatal responses occur in a crop plant such as soybean. In addition, they suggest that the systemic signal that 1 This work was supported by the National Science Foundation (grant nos. IOS-1353886, MCB-1936590, and IOS-1932639)   mediates the response to changes in light intensity and/ or temperature in the local leaf primarily functions from upper to lower leaves. Notably, the changes observed in stomatal conductance and transpiration of the lower leaf in response to increased light intensity in the upper leaf ( Fig. 1B) were not accompanied by enhanced photosynthesis, suggesting that light intensity is the primary limiting factor for photosynthesis in these leaves (as their photosynthetic rates increased rapidly when light was applied to the lower leaves directly; Fig. 1C). The direction of the systemic signal (i.e. from the upper to the lower leaf) could result from the hierarchal architecture of soybean plants and the fact that, compared to lower leaves, upper leaves closer to the top of the canopy are more likely to sense changes in light intensity. Upon sensing these changes, upper leaves could generate a systemic signal that is transmitted along the main stem to lower leaves to regulate overall plant transpiration and adjust the response of the entire plant to the changes in environmental conditions, sensed at the upper canopy. In contrast to young leaves positioned at adjacent nodes at the top of the canopy, older leaves positioned at the bottom of the canopy did not display a measurable systemic stomatal response (not shown, and in agreement with the relative decline in overall performance and leaf hydraulic responses of older leaves found at the bottom of the canopy; Locke and Ort, 2014).
The differences observed between the stomatal response of soybean (opening) as described in this study ( Figs. 1 and 2), and Arabidopsis (closing) as described in Devireddy et al. (2018Devireddy et al. ( , 2020 and Balfagón et al. (2019), to an increase in light intensity, could be a result of differences in the photosynthetic light response curves and saturating light intensities between these two species. While for Arabidopsis a light intensity of 1,500 mmol m 22 s 21 is an excess light stress that would require an immediate stomatal stress response (e.g. Raven, 2014;Devireddy et al., 2018;Balfagón et al., 2019), for soybean a similar light intensity is within its normal photosynthetic light range (e.g. Wang et al., 2020), and would require stomatal opening to enhance photosynthesis under well-watered conditions. Systemic stomatal responses in Arabidopsis depend on systemic reactive oxygen species (ROS) signals and include systemic stomatal closure responses to light or wounding, as well as systemic stomatal opening responses to heat (Devireddy et al., 2018(Devireddy et al., , 2020Kollist et al., 2019;Zandalinas et al., 2020a). The systemic stomatal response of soybean appeared to occur at a rate comparable to that of Arabidopsis (6 min to transverse ;25-cm distance; Fig. 1), suggesting that it too could be mediated by systemic ROS signals (Fichman and Mittler, 2020). To test this hypothesis, we applied the ROS/redox inhibitor diphenyleneiodonium (DPI; 50 mM), or water, in agarose solution (0.3%) to the stem-petiole junction of the lower leaf for 40 min before applying the 40 to 1,500 mmol m 22 s 21 light-change treatment to the upper (local) leaf ( Fig. 2; Supplemental    S1D; the application of a drop of agar with water or DPI, without any change in light conditions, did not alter stomatal conductance). In contrast to the application of water, the application of DPI inhibited the systemic stomatal conductance and transpiration responses of the lower leaf to the light-change treatment applied to the upper leaf (Fig. 2). By contrast, and similar to our findings with Arabidopsis (Devireddy et al., 2018), application of DPI in the absence of changes in light conditions did not alter systemic stomatal conductance responses ( Fig. 2; Supplemental Fig. S1). These findings suggest that changes in ROS/redox levels are required along the path of the systemic signal that controls systemic stomatal responses in soybean.

Supplemental Data
The following supplemental materials are available.
Supplemental Data S1. Supplementary materials and methods.
Supplemental Figure S1. Experimental design used in Figure 1, A-C, and in Figure 2D, showing the position of the upper and lower leaves and the change in light intensity applied to each leaf using the LI-6800 portable photosynthesis system apparatuses (LI-COR).