Systemic signaling in the regulation of stomatal conductance

Systemic signaling is involved in the regulation of stomatal conductance in response to rapid changes in ambient light and intercellular CO2 concentration.

Over shorter time periods, low light and c i also affect stomatal conductance. However, it is unclear 35 whether local changes in light and intercellular CO 2 levels also regulate stomatal conductance and 36 photsynthesis in distal leaves. To address this question, we developed a unique experimental setup 37 that enabled the application of treatments on local and distal leaves separately while simultaneously 38 monitoring rapid changes in leaf gas exchange. Here we show that systemic signaling is involved in 39 rapid stomatal closure in response to changes in ambient light conditions and CO 2 concentration in 40 two different tree species, hybrid aspen (Populus tremula x tremuloides) and silver birch (Betula 41 pendula), but not in Arabidopsis (Arabidopsis thaliana). 42 43 In our experimental setup, two leaf gas exchange measuring devices (GFS3000,Heinz Walz GmbH,44 Germany) were placed inside a FitoClima growth chamber (Aralab, Portugal). This allowed us to 45 manipulate ambient CO 2 concentrations and light levels on local and distal leaves separately while 46 simultaneously monitoring rapid changes in stomatal conductance and photosynthesis (Fig. 1A).

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The treatments were applied in three different combinations: sudden increase in ambient CO 2 48 concentration (from 400 to 1100 ppm) or darkness treatment on 1) the measured leaf (cuvette), 2) 49 the rest of the measured seedling (chamber) or 3) the whole seedling (cuvette and chamber). During 50 each treatment, stomatal conductance, assimilation (A), and c i of the cuvette leaves were 51 continuously measured. Stomatal conductance of the measured leaf remained unchanged for the 52 untreated P. tremula x tremuloides, B. pendula, and A. thaliana samples ( Fig. 1; see also 53 Supplemental Table S1, Supplemental Table S2

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When CO 2 and darkness treatments were applied in the growth chamber only, stomatal conductance 55 was significantly decreased in the non-treated (cuvette) leaves of P. tremula x tremuloides and B.  The effect of systemic signaling on the regulation of stomatal conductance in the two tree species 66 was also evident when comparing whole seedling and local treatments. Stomatal conductance of B. 67 pendula decreased more when CO 2 and dark treatments were subjected to the whole seedling 68 www.plantphysiol.org on May 5, 2020 -Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

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(cuvette and chamber) than when the treatments were applied only on the measured leaf (cuvette).   The rapid systemic signaling seems to affect stomatal conductance in the opposite direction and at a 88 different time scale than would be expected for water potential-or sugar concentration-mediated 89 stomatal regulation (see supporting information for detailed explanation). We propose that the  In our study, the systemic stomatal response resulted in a subtle, but significant, decrease in 100 stomatal conductance of the measured non-treated leaf within a few minutes of the treatment onset 101 (Fig. 1, Supplemental Table S3). As the adjacent leaves were approximately two to five centimeters 102 away from the measured leaf and the stomatal response could already be seen within 1.5 minutes 103 after the onset of treatment, we estimated the maximum speed of the signal to be approximately 2 104 cm/min. ROS, Ca 2+ , or electric signals have been shown to travel at a rate of 2.5 to several

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The kinetics of stomatal conductance and CO 2 assimilation were also monitored and compared in 116 the distal untreated leaf to examine how the systemic signal is perceived in the measured leaf. A 117 decrease in stomatal conductance (Fig. 1) preceded the decrease in CO 2 assimilation rate 118 (Supplemental Fig. S3), while there was no change in the c i levels (Supplemental Fig. S4) in the 119 untreated leaf a few minutes after the initiation of darkness or CO 2 treatment in the growth 120 chamber. Moreover, the results suggest that the signal is likely perceived directly by the guard cells, 121 leading to a reduction in stomatal conductance followed by a decrease in assimilation rate due to a 122 decrease in the supply of CO 2 through the stomata.      The color of the asterisk shows the subject of the pairwise comparison.