# Element content and expression of genes of interest in guard cells are connected to spatiotemporal variations in stomatal conductance

Maxime Durand, David Cohen, Nathalie Aubry, Cyril Bure, Ivana Tomášková, Irène Hummel, Oliver Brendel, Didier Le Thiec

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Maxime Durand, David Cohen, Nathalie Aubry, Cyril Bure, Ivana Tomášková, et al.. Element content and expression of genes of interest in guard cells are connected to spatiotemporal variations in stomatal conductance. Plant, Cell and Environment, 2019, 10.1111/pce.13644. [hal-02352487](https://hal.science/hal-02352487)

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# Element content and expression of genes of interest in guard cells are connected to spatiotemporal variations in stomatal conductance

Maxime Durand<fnref n="1" /> | David Cohen<fnref n="1" /> | Nathalie Aubry<fnref n="1" /> | Cyril Buré<fnref n="1" /> | Ivana Tomášková<fnref n="2" /> |
Irène Hummel<fnref n="1" /> | Oliver Brendel<fnref n="1" /> | Didier Le Thiec<fnref n="1" /> ID

### Correspondence

D. Le Thiec, Inra, Université de Lorraine, AgroParisTech, SILVA, F-54280 Champenoux, France.

Email: didier.lethiec@inra.fr

### Funding information

FP7 Food, Agriculture and Fisheries, Biotechnology, Grant/Award Number: Watbio / 311929; Institut National de la Recherche Agronomique, Grant/Award Number: Dpt EFPA; Lorraine region; French National Research Agency, Grant/Award Number: ANR-12-LABXARBRE-01; European Union's Seventh Programme, Grant/Award Number: 311929

## Abstract

Element content and expression of genes of interest on single cell types, such as stomata, provide valuable insights into their specific physiology, improving our understanding of leaf gas exchange regulation. We investigated how far differences in stomatal conductance ($g_s$) can be ascribed to changes in guard cells functioning in amphistomateous leaves. $g_s$ was measured during the day on both leaf sides, on well-watered and drought-stressed trees (two *Populus euramericana* Moench and two *Populus nigra* L. genotypes). In parallel, guard cells were dissected for element content and gene expressions analyses. Both were strongly arranged according to genotype, and drought had the lowest impact overall. Normalizing the data by genotype highlighted a structure on the basis of leaf sides and time of day both for element content and gene expression. Guard cells magnesium, phosphorus, and chlorine were the most abundant on the abaxial side in the morning, where $g_s$ was at the highest. In contrast, genes encoding $H^+$-ATPase and aquaporins were usually more abundant in the afternoon, whereas genes encoding $Ca^{2+}$-vacuolar antiporters, $K^+$ channels, and ABA-related genes were in general more abundant on the adaxial side. Our work highlights the unique physiology of each leaf side and their analogous rhythmicity through the day.

## KEYWORDS

abaxial and adaxial surfaces, droughts, elements, gene expression, plant stomata, Populus, stomatal conductance

## 1 | INTRODUCTION

Stomata are small pores on the surface of the leaf responsible for almost all gas exchange between the leaf internal environment and the surrounding air. They are composed of two guard cells that modify their volume, adjusting the central aperture and controlling the flow of gases in and out of the leaf. These adjustments are regulated by a variety of internal and external factors, including but not limited to water availability (Monclus et al., 2006), irradiance (Shimazaki, Doi, Assmann,

& Kinoshita, 2007), and circadian rhythms (de Dios, 2017). Regulation of stomatal aperture is considered a key phenomenon, maximizing CO₂ uptake for photosynthesis with regard to water loss by transpiration (Cowan & Farquhar, 1977), whose importance increases as internal and environmental conditions become more limiting.

Poplars are dicots, model trees for molecular studies (Jansson & Douglas, 2007; Tuskan et al., 2006), with amphistomateous leaves. As such, despite being in the same environment, each leaf side experiences a different magnitude of irradiance and temperature (Clum,

<footnote n="1">¹Inra, Université de Lorraine, AgroParisTech, SILVA, F-54280 Champenoux, France</footnote>

<footnote n="2">²Department of Genetics and Physiology of Forest Trees, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences Prague, Prague 165 00, Czech Republic</footnote>
1926; Sheriff, 1979). Literature evidence suggests that guard cell sensitivity to irradiance (Kassam, 1973), vapour-pressure deficit (Pallardy & Kozlowski, 1979), leaf water potential (Kanemasu & Tanner, 1969), and ozone (Dumont et al., 2014) differs between leaf sides. Moreover, some species showed independent stomatal regulation between surfaces (Mott, 2007; Richardson, Brodribb, & Jordan, 2017), consistent with an optimal stomatal behaviour of each leaf side. This suggests that, in poplars, stomatal movements under drought and along the day may depend on the side of the leaf.

As a consequence of global climate change, droughts are expected to be more intense and frequent in the future (Dai, 2012; Intergovernmental Panel on Climate Change, 2014). A prolonged reduction in water availability will induce a wide variety of severe and long-lasting humanitarian, economic, and environmental impacts on ecosystems and societies (Touma, Ashfaq, Nayak, Kao, & Diffenbaugh, 2015). Among them, the impact of drought on forests is a central concern in global climate change research given the interrelation between forests and climate (Bonan, 2008). Poplars are commonly distributed along riparian ecosystems due to their high water requirements (Tschaplinski & Blake, 1989). Their cultivation however usually extend to mesophyte habitats where they are more frequently subjected to soil water deficit. Plant response to drought is complex, but stomatal closure and a reduction in plant productivity is generally observed (Ciais et al., 2005; Coopman et al., 2008; Monclus et al., 2006).

For soil water deficit and high vapor pressure deficit (VPD) conditions, it remains unresolved whether hydraulic or hormonal mechanisms are responsible for stomatal closure (Merilo et al., 2018; Rodriguez-Dominguez et al., 2016). One hypothesis states that a decrease in stomatal conductance ($g_s$) during drought is mostly driven by hormonal signals such as abscisic acid (ABA), which are generated independently of leaf water status (Davies & Zhang, 1991; Dodd, 2005; Tardieu & Simonneau, 1998). However, the complex regulation of stomatal functioning can involve a number of actors, from phytohormones to strigolactones (Acharya & Assmann, 2009; Lv et al., 2018). It has alternatively been proposed that stomatal closure during edaphic droughts is caused by an actively mediated negative feedback response of stomata to reduced leaf turgor (Brodribb & Cochard, 2009; Buckley, 2005; Sperry, Hacke, Oren, & Comstock, 2002). ABA is a central plant hormone responsible for promoting stomatal closure and inhibiting opening under drought (Chaves, Maroco, & Pereira, 2003; Huang, Wu, Abrams, & Cutler, 2008). In recent years, evidence has accumulated showing that guard cells possess the full ABA biosynthesis pathway (Bauer et al., 2013) and the early ABA signal transduction (Zhang et al., 2015). ABA binds and inhibits PP2C-type phosphatases by binding to the PYR/PYLs family of proteins (Merilo et al., 2013), including ABI1 (Park et al., 2009; Yoshida et al., 2006). This in turns relieves the repression on OST1, which inactivates the KAT1 inward-rectifying potassium channel (Sato et al., 2009) and activates the S-type anion channel SLAC1 (Geiger et al., 2009). These channels, among others, induce a reduction of osmotic potential inside the guard cells leading to a flow of water outward, which results in stomatal closure (Schroeder & Hedrich, 1989; Schroeder, Raschke, & Neher, 1987).

In addition, circadian rhythms in transcript accumulation (Wilkins, Waldron, Nahal, Provart, & Campbell, 2009) and gas exchange (de Dios, 2017) are limiting our ability to fully understand the molecular signals involved in stomatal movements. A number of genes involved in the regulation of stomatal movements were found to be gated by the circadian clock, including ABA biosynthesis and signalling pathways (for a complete review, see Seung, Risopatron, Jones, & Marc, 2012) and TIPs and PIPs (aquaporins) mRNA levels (Chaumont & Tyerman, 2014). Circadian rhythms appear to be cell-specific as stomata were shown to have a distinct rhythmicity compared with surrounding mesophyll cells in *Arabidopsis thaliana* (Hassidim et al., 2017; Yakir et al., 2011). Given the importance of stomata in regulating gas exchange and thus the productivity of plantations, there is a need to better understand the regulation of $g_s$ during the day and under different water availabilities.

Specifically studying stomata independently from the surrounding mesophyll cells is not trivial (Leonhardt et al., 2004). Hence, not much is known about the physiological and molecular processes in guard cells driving stomatal conductance in trees species such as poplars. To increase our understanding of the differences in such cellular processes among genotypes, between leaf sides, during the day and plastic responses related to soil water deficit, there is a need for studies targeting a specific cell type.

To test well-established processes exerted by the guard cells, quantification of their element content and their expression of 27 genes of interest were monitored separately on both leaf sides from leaves harvested in the morning and the afternoon. We used two *Populus nigra* × *Populus deltoides* (*Populus euramericana*, Moench) and two *P. nigra* (L.) genotypes grown in parallel under control and soil water deficit conditions. Given this experimental set-up, we aimed to answer the following questions: (a) Is there a constitutive difference among genotypes of element content and gene expression in poplar guard cells? (b) Do factors such as water regime, time of the day, and leaf side induce a plastic response in terms of guard cells (i) element content and (ii) gene expression?

## 2 | MATERIAL AND METHOD

### 2.1 | Plant material and growth conditions

Woody cuttings of four poplar genotypes belonging to different species or commercial hybrids (Carpaccio and I214: *P. deltoides* × *P. nigra*, Moench, 6J29 and N38: *P. nigra*, L.) were grown in a fully automated glasshouse under natural light with a free range for air temperature between 15°C and 25°C at INRA-Grand-Est (48°45′09.3″N, 6°20′ 27.6″E; Champenoux, France), as described previously (Durand, Brendel, Buré, & Le Thiec, 2019). Genotypes were chosen based on their contrasting drought response: I214 and 6J29 are generally found to be sensitive to drought (Chen, Wang, Altman, & Huttermann, 1997; Giovannelli et al., 2007; Muller & Lambs, 2009; Viger et al., 2016). Sixteen cuttings per genotype were planted in 9.5 kg (±1.5%) of a sand/peat mixture (1/1, v/v), complemented with fertilizer (1 g L⁻¹ of
CaMg (CO₃)₂ and 20 g of Nutricote T100, 13:13:13 NPK and micronutrients, FERTIL S.A.S., Boulogne-Billancourt, France). Each 10-L pot was covered with 1.4 kg of white marble gravel (8–12 mm). Pots were weighed and watered to 27.9% volumetric soil water content (SWC; 85% of field capacity) at least three times a day with a robot (Durand et al., 2019). After 60 days of precultivation, watering was stopped for half the individuals (randomly chosen) until an SWC of 18.3% was reached. This controlled soil water deficit was maintained constant for 25 days using a pot-specific linear regression between SWC measured by time domain reflectometry (HD2, IMKO, Ettlingen, Germany) and pot mass (R² > .8). Reference masses were adjusted each week to take the plant growth into account. Growth, transpiration, and meteorological data are available in Durand et al. (2019).

## 2.2 | Leaf gas exchange and leaf sampling

Leaf gas exchange and leaf sampling were performed sequentially in order to accurately capture stomatal conductance at the time of sampling. The first fully expanded leaf (selected before the drought) and the one immediately below were sampled 21 days after the start of the drought experiment at 10:30 a.m. and 3:00 p.m. local time, respectively. At the time, the natural daytime lasted 16 hr (starting at 4:30), global radiation was 297.2 ± 47.8 and 145.6 ± 45.1 W m⁻², air temperature was 24.9 ± 0.2 and 24.8 ± 0.2°C, and vapour-pressure deficit was 1.31 ± 0.02 and 1.20 ± 0.07 kPa for the two sampling events, respectively. From previous g_s time-course measurements, stomata were likely fully open in the morning and in a closing phase in the afternoon (Durand et al., 2019). The last watering event was completed 2 hr before each sampling. A leaf porometer was used in order to measure g_s separately on the abaxial and adaxial surfaces of the leaves (SC-1 porometer; Decagon Devices, Inc., Pullman, WA, USA). Stomata being on the leaf surfaces, sampled leaves were flash frozen in liquid nitrogen immediately after measurements, to guarantee both RNA integrity and that diffusive elements remained in guard cells (Amsellem et al., 1983; Jaiprakash et al., 2003).

## 2.3 | X-ray microanalysis

Sample preparation and microanalyses were performed following Dumont et al. (2014), except for the use of a tungsten coating on the samples and a probe intensity of 1 nA, in order to allow analyses in stomata specifically. Although the analysed X-rays originate from a lower layer in the sample than electrons (Amsellem et al., 1983), the electron beam diameter and depth of penetration is lower than 2 µm and has been simulated in a Monte Carlo procedure (Pouchou & Pichoir, 1991); 20 guard cells, each belonging to different stomata of freeze-dried leaves, were specifically targeted and analysed for each of the two leaf sides, the two times of day, the two water treatments, and the four genotypes with six replicates in each condition. Microanalyses were corrected by measuring the content of beech leaves powder of known mass fraction (CRM 100, BCR® certified reference material, Commission of the European Communities, Brussels,

Belgium) for sodium (Na), magnesium (Mg), phosphorus (P), chlorine (Cl), potassium (K), and calcium (Ca). All forms were measured, both organic and inorganic. We also computed a ratio of anions over cations for each guard cell (i.e., P and Cl over Na, Mg, K, and Ca later referred to as An/Cat). Values were converted from percent to “per mille” (or mg g⁻¹) of dry mass.

## 2.4 | Guard cell microdissection, RNA extraction and cDNA amplification

Guard cells microdissection, RNA isolation, RNA reverse-transcription, and cDNA amplification were performed following Dumont et al. (2014). A brief description of the methods is given hereinafter. If not otherwise specified, protocols were performed following the manufacturer's instructions; 1,000 stomata per sample were microdissected using the PALM MicroBeam system (Carl Zeiss Microlmaging GmbH, Jena, Germany). Total RNA was extracted using Qiagen RNeasy Plus Micro Kit (Qiagen, Courtaboeuf, France). Previous tests in our lab and in the literature show that the process of freeze-drying and preparing microscopic slide does not impact the RNA integrity (Damsteegt, McHugh, & Lokman, 2016; Dumont et al., 2014; García-Baldenegro et al., 2015; Heinen et al., 2014; Jaiprakash et al., 2003). Transplex® Whole Transcriptome Amplification Kit (MERCK, Saint-Quentin Fallavier, France) was used for RNA reverse transcription and cDNA amplification (100 µl twice by sample). The latter was purified by Gene Elute PCR (SIGMA, Saint-Quentin Fallavier, France). cDNA purity and concentration were measured (Nanodrop 1000; Thermo Scientific, Illkirch, France). All cDNA samples exhibited a good purity level (1.6 < A₂₆₀/A₂₈₀ < 2.0 and 1.3 < A₂₆₀/A₂₃₀ < 1.9), and each sample was diluted to get 2.5 ng µl⁻¹. Absence of genomic DNA was confirmed by PCR with intron-flanking primers.

## 2.5 | Expression of genes of interest and real-time PCR

The genes of interest were retrieved on the Populus genome v3.0 (Phytozome, RRID:SCR_006507, https://phytozome.jgi.doe.gov/pz/portal.html; Tuskan et al., 2006). The genes of interest were selected for their putative role in encoding ionic pumps, aquaporins, transporters involved in stomatal movements, and phototropins, β-carbonic anhydrases, and genes linked to ABA biosynthesis and signalling. Genes were selected in regard to previous studies (Cohen et al., 2010; Dumont et al., 2014; Heinen et al., 2014) on the basis of a high expression level in poplar guard cells (when available, and in leaves otherwise), their differential expression among genotypes, between leaf sides, water availability, and time of day. The design of primers, with adequate specificity for all four genotypes, was also a limiting step for gene selection. Only primers with a reduced risk of primer dimer formation or nonspecific amplification were selected. Because the expression of gene of interest approach is not exhaustive by definition, we chose to investigate genes with different roles, instead of a whole family of genes. Thus, our interpretation is not limited to a
single family of transporters but benefits from a more exhaustive assessment of guard cell processes linked with stomatal regulation. The targeted genes and their description are provided in Table 1 (additional information is given in Table S1).

Real-time PCR was performed in a 96-well Mx3005P thermocycler (Agilent, Waghaeusel-Wiesental, Germany) using the recommended cycling programme: 5 min at 95°C, 40 cycles of 5 s at 95°C, and 20 s at 58°C or 60°C depending on primers, followed by melting cycle to ensure a single amplicon (Dumont et al., 2014). The mix contained 10 ng of cDNA, the reference dye (ROX, Agilent technologies), gene-specific primers, and the Brilliant III UltraFast SYBR GREEN qPCR Master Mix (Agilent, Santa Clara, CA). No-template control reactions were prepared for each gene. The plate set-up included all samples

from two genotypes and the negative controls. The Ct values were determined with the same threshold. For each targeted genotype group, efficiency was calculated from standard curves over seven dilutions. Efficiencies varied from 65% to 102% ($R^2 \ge .985$) and were taken into account in all subsequent calculations (Bizet et al., 2015). Among the studied genes, *UBQ11* (Brunner, Yakovlev, & Strauss, 2004), *QUAC1* (Dumont et al., 2014), and *BAM1* were the most stable and used as references, together with an external control (GENORM v3.5, RRID:SCR_006763, https://genorm.cmgg.be/; Mestdagh et al., 2009; Vandesompele et al., 2002). The fluorescent threshold being set constant, the nonnormalized expression level was computed as $RQ = 1/(Ta \cdot E^{Ct})$, $Ta$ being the amplicon length in bp and $E$ the efficiency (Bizet et al., 2015; Gutierrez et al., 2008).

<table><caption>TABLE 1 Description of the genes studied and their localization</caption><thead><tr><td>Genes</td><td>Description</td></tr></thead><tbody><tr><td>NCED3.1</td><td>Key enzyme in ABA biosynthesis (9-cis-epoxycarotenoid dioxygenase).</td></tr><tr><td>NCED3.2</td><td>Key enzyme in ABA biosynthesis (9-cis-epoxycarotenoid dioxygenase).</td></tr><tr><td>ABI1</td><td>Protein phosphatases type 2C (PP2Cs), negative regulator of ABA promotion of stomatal closure.</td></tr><tr><td>OST1</td><td>SNF1-related protein kinase, involved in ABA-induced stomatal closure by activating of S-type anion channels and inhibiting inward rectifying potassium channel.</td></tr><tr><td>PYL2</td><td>PYR/PYL/RCAR family of protein, inhibits the activity of group-A protein phosphatases type 2C (PP2Cs) when activated by ABA.</td></tr><tr><td>PYL4</td><td>PYR/PYL/RCAR family of protein, inhibits the activity of group-A protein phosphatases type 2C (PP2Cs) when activated by ABA.</td></tr><tr><td>PYL8</td><td>PYR/PYL/RCAR family of protein, inhibits the activity of group-A protein phosphatases type 2C (PP2Cs) when activated by ABA.</td></tr><tr><td>CA1</td><td>Beta-carbonic anhydrase, involved in CO<sub>2</sub>-mediated stomatal closure (chloroplast).</td></tr><tr><td>CA4</td><td>Beta-carbonic anhydrase, involved in CO<sub>2</sub>-mediated stomatal closure (PM).</td></tr><tr><td>PHOT1</td><td>Blue light photoreceptors, mediate blue light-dependent activation of the plasma membrane H<sup>+</sup>-ATPases</td></tr><tr><td>PHOT2</td><td>Blue light photoreceptors, mediate blue light-dependent activation of the plasma membrane H<sup>+</sup>-ATPases</td></tr><tr><td>AKT2</td><td>Inward rectifying potassium channel (PM), responsible for the Ca<sup>2+</sup> sensitivity of the K<sup>+</sup> uptake channel.</td></tr><tr><td>KAT1.2</td><td>Inward rectifying potassium channel (PM), responsible for the Ca<sup>2+</sup> sensitivity of the K<sup>+</sup> uptake channel.</td></tr><tr><td>KAT3</td><td>Inward rectifying potassium channel (PM), responsible for the Ca<sup>2+</sup> sensitivity of the K<sup>+</sup> uptake channel. Involved in down-regulating AKT1 and KAT1 channel activity by forming heteromers.</td></tr><tr><td>OST2</td><td>Proton ATPases (PM)</td></tr><tr><td>AHA11</td><td>Proton ATPases (PM)</td></tr><tr><td>SLAC1</td><td>Slow anion channel (PM), involved in stomatal closure in response to a variety of signals including elevated CO<sub>2</sub>, ozone, ABA, darkness, and humidity.</td></tr><tr><td>CAX1</td><td>Calcium antiporter (V)</td></tr><tr><td>CAX1.6</td><td>Calcium antiporter (V)</td></tr><tr><td>NHX1.13</td><td>Potassium, sodium/proton antiporter (V) involved in salt tolerance and ion homeostasis</td></tr><tr><td>PIP1.2</td><td>Aquaporin: plasma membrane intrinsic protein (PM)</td></tr><tr><td>PIP2.1</td><td>Aquaporin: plasma membrane intrinsic protein (PM)</td></tr><tr><td>PIP2.5</td><td>Aquaporin: plasma membrane intrinsic protein (PM)</td></tr><tr><td>TIP1.3</td><td>Aquaporin: tonoplast intrinsic protein (V)</td></tr><tr><td>TIP1.4</td><td>Aquaporin: tonoplast intrinsic protein (V)</td></tr><tr><td>TIP2.1</td><td>Aquaporin: tonoplast intrinsic protein (V)</td></tr><tr><td>TIP2.2</td><td>Aquaporin: tonoplast intrinsic protein (V)</td></tr></tbody></table>

<footnote>ªAbbreviations: PM, plasma membrane; V: vacuole.</footnote>
The normalized expression of targeted genes were computed by divid-
ing their RQ by the geometric average of the reference gene's RQ
(Bizet et al., 2015).

2.6 | Statistics

Statistics were performed using R 3.5.1 (R Project for Statistical Com-
puting, RRID:SCR_001905, https://cran.r-project.org/, all data used
are available in Table S2). We used a type 2 factorial analysis of vari-
ance design to study the variation of g<sub>s</sub> (n = 6), element content (n =
6), and normalized mRNA levels (n = 4) by genotype, water regime,
time of day, and leaf side and their first-order interaction. Given the
complexity of the design, we decided to focus our interpretation of
the dataset on the significant main factors and their first-order interac-
tions, instead of computing all pairwise comparisons between our 32
groups. This enabled us to explore the major trends with a larger
sample size and a higher statistical power than if we specifically
addressed each pairwise comparison. Normalized transcript levels
were transformed with a logarithmic function of base 2 in order to
achieve a normal distribution of the data. Post hoc contrast analyses
were performed to test differences between specific modalities of
the studied factors depending their significance, and P values were
adjusted to control for the false discovery rate. The partial R<sup>2</sup> was cal-
culated to compare the contribution with the explained variable of
each of the four dependent variables. We performed a principal com-
ponent analysis (PCA) on the six elements studied and the ratio An/Cat
on one hand and on the expression data of 27 genes of interest on the
other hand. We fitted to every variable a linear model that included
the highly significant genotype effect as a dependent factor. The
residuals of these models were used as the data for the two PCA in
order to emphasize the other main effects (water regime, time of
day, and leaf side). Stomatal conductance data were fitted with a sim-
ilar model, and the residuals were added as a supplementary variable.

3 | RESULTS

3.1 | Stomatal conductance

Testing for genotypic, water availability, time of day, and leaf side
effects on g<sub>s</sub> measured immediately before sampling the leaves, we
found all four to be significant (Table 2, P < .002 in all cases).
Drought-stressed poplars had an overall 20% lower g<sub>s</sub> than well-
watered trees. Because there was no interaction with the water treat-
ment, we showed the average g<sub>s</sub> of both treatments (Figure 1). The
significant interaction between genotype and time of day (P = .023)
showed that the afternoon decrease of g<sub>s</sub> tended to be stronger in
both hybrid poplars than in the black poplars (Figure 1). Black poplars
exhibited in general a higher g<sub>s</sub> than the hybrid poplars, and g<sub>s</sub> was
overall higher on the abaxial side. Essentially, the highest g<sub>s</sub> tended
to be observed on the abaxial side, in the morning and on well-
watered trees for all genotypes, whereas the adaxial side, in the
afternoon on drought-stressed trees, showed the lowest g<sub>s</sub>.

3.2 | Genotypic diversity of element content and gene expression within guard cells

In this study, element content was measured as the whole guard cell
average. We found significant genotypic diversity in all measured ele-
ments (Figure S1, Table 2, in all cases P < .001). The genotype effect
was always the factor explaining the total variance of element content
the most (Table S3). To highlight these genotypic differences, we
decided to show the genotype means for each element in Figure 2.
Partial R² for genotype ranged from.16 for the P content to.7 for the
CI content (Na: .57, Mg: .49, P: .16, CI: .70, K: 0.33, Ca: 0.21). The main
elements in the guard cells were K and Ca. Na content was the highest
in the guard cells of the hybrid poplars, and especially in Carpaccio
were it was 3.1, 5.3, and 3.8 times higher than I214, 6J29, and N38
respectively. Additionally, Mg content was 1.7 times higher in Carpac-
cio than in the other three genotypes, but K content was 1.6 times
higher in I214 than in the other three genotypes. On the other hand,
CI content was 4.6 times lower in both hybrid poplars than in the
two black poplar genotypes. This effect was passed on in our calcula-
tion of An/Cat, which was 2.2 times lower in the two hybrid poplars
than in the P. nigra genotypes. Moreover the guard cells of 6J29
showed a P and Ca content, respectively, 17% and 24% lower than
in the other three genotypes on average.

The expression of genes of interest in the guard cells was also
structured according to genotype. Figures 3 and S2 show the back-
transformed mean of normalized mRNA levels by genotype, that is,
by applying a base 2 exponential to the means of the log2-
transformed data, to be consistent with statistical analyses. For all
measured transcripts (in all cases *P* < .001) except for *CAX1* (*P* = .34),
the genotype effect was always the factor explaining the total vari-
ance of normalized mRNA levels the most (Table S3). Partial *R*² for
the genotype effect ranged from 0.13 to 0.98 for *AKT2* encoding a
*K*⁺ channel and *PYL2*, encoding an ABA receptor, respectively, with
an average of 0.57. Overall, the normalized expression of *NCEDs*,
encoding 9-cis-epoxycarotenoid dioxygenases involved in ABA bio-
synthesis, were the highest in black poplars. Specifically, N38 and
6J29 exhibited the highest accumulation of transcripts for *NCED3.1*
and *NCED3.2*, respectively (Figure S2a,b). Conversely, ABA signalling
pathway-related transcripts appeared more expressed in I214 guard
cells, and especially the genes encoding the ABA receptor *PYL2* and
*PYL8* (Figure S2e,g). Carpaccio was consistently among the genotypes
with the lowest accumulation of transcripts related to both ABA bio-
synthesis and signalling pathways. Regarding β-carbonic anhydrases,
N38 exhibited equivalent levels of *CA1* and *CA4* mRNA, whereas for
the other genotypes, *CA1* transcripts were accumulated up to 100
times more than *CA4*. Hence, *CA1* normalized transcript levels were
the highest in the guard cells of Carpaccio and 6J29, but *CA4* levels
were the highest in the guard cells of N38 (Figure S2h,i). The transcript
accumulation of genes encoding phototropins *PHOT1* and *PHOT2*
were significantly different across all genotypes. Concerning ion chan-
nels, I214 was often the genotype with the lowest level of transcripts
for inward-rectifying *K*⁺ channels (*AKT2* and *KAT3*) and proton H⁺-
ATPase (*OST2* and *AHA11*). Among black poplars guard cells, these
<table><caption>**TABLE 2** Summary of the genotype, water treatment, time of day, and leaf side effect on stomatal conductance (n = 6), guard cell element content (n = 6), and expression of genes of interest (n = 4)</caption><thead><tr><th></th><th>Genotype (G)</th><th>Water treatment (T)</th><th>Time of day (D)</th><th>Leaf side (S)</th><th>Interaction G:T</th><th>Interaction G:D</th><th>Interaction G:S</th><th>Interaction T:D</th><th>Interaction T:S</th><th>Interaction D:S</th></tr></thead><tbody><tr><td>g<sub>s</sub></td><td>&lt;.001</td><td>.002</td><td>&lt;.001</td><td>&lt;.001</td><td>ns</td><td>.023</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>Na content</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>Mg content</td><td>&lt;.001</td><td>ns</td><td>.043</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>P content</td><td>&lt;.001</td><td>ns</td><td>.003</td><td>&lt;.001</td><td>.032</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>Cl content</td><td>&lt;.001</td><td>.003</td><td>.009</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>K content</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>.001</td><td>ns</td><td>ns</td><td>.015</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>Ca content</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>&lt;.001</td><td>.008</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>An/Cat</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>.001</td><td>.003</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>ABI1</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>.002</td><td>.004</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>.03</td></tr><tr><td>AHA11</td><td>&lt;.001</td><td>ns</td><td>&lt;.001</td><td>ns</td><td>.027</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>AKT2</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>&lt;.001</td><td>.004</td><td>.018</td><td>.001</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>CA1</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>.002</td><td>.025</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>CA4</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>ns</td><td>.024</td><td>ns</td><td>ns</td></tr><tr><td>CAX1</td><td>ns</td><td>ns</td><td>ns</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>ns</td><td>.029</td><td>ns</td><td>ns</td></tr><tr><td>CAX1.6</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>&lt;.001</td><td>.042</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>KAT1.2</td><td>&lt;.001</td><td>ns</td><td>.031</td><td>&lt;.001</td><td>&lt;.001</td><td>ns</td><td>.007</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>KAT3</td><td>&lt;.001</td><td>.005</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>NCED3.1</td><td>&lt;.001</td><td>ns</td><td>.023</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>NCED3.2</td><td>&lt;.001</td><td>ns</td><td>.049</td><td>.013</td><td>.022</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>NHX1.13</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>.015</td><td>.004</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>OST1</td><td>&lt;.001</td><td>.034</td><td>.001</td><td>&lt;.001</td><td>.01</td><td>ns</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>OST2</td><td>&lt;.001</td><td>ns</td><td>.014</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>PHOT1</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>.016</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>PHOT2</td><td>&lt;.001</td><td>ns</td><td>.011</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>PIP1.2</td><td>&lt;.001</td><td>ns</td><td>&lt;.001</td><td>&lt;.001</td><td>.005</td><td>ns</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>PIP2.1</td><td>&lt;.001</td><td>&lt;.001</td><td>.022</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>PIP2.5</td><td>&lt;.001</td><td>ns</td><td>.009</td><td>ns</td><td>ns</td><td>ns</td><td>.003</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>PYL2</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>.048</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>PYL4</td><td>&lt;.001</td><td>0.008</td><td>ns</td><td>ns</td><td>.037</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>PYL8</td><td>&lt;.001</td><td>ns</td><td>.032</td><td>&lt;.001</td><td>.015</td><td>ns</td><td>.003</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>SLAC1</td><td>&lt;.001</td><td>0.018</td><td>ns</td><td>.016</td><td>.039</td><td>ns</td><td>.008</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>TIP1.3</td><td>&lt;.001</td><td>ns</td><td>.001</td><td>ns</td><td>.02</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>TIP1.4</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>ns</td><td>.004</td><td>ns</td><td>.009</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>TIP2.1</td><td>&lt;.001</td><td>ns</td><td>.005</td><td>.049</td><td>ns</td><td>.001</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>TIP2.2</td><td>&lt;.001</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr></tbody></table>

<sup>a</sup>Note. A type two factorial analysis of variance design with first-order interactions was used. Normalized transcript levels were transformed with a logarithm function of base 2 in order to achieve a normal distribution of the data.

<sup>b</sup>Abbreviations: g<sub>s</sub>, stomatal conductance to water vapour; An/Cat, ratio of anions over cations (i.e., P and Cl over Na, Mg, K, and Ca); ns, not significant.

*P* < .05. **P* < .01. ***P* < .001.

genes were either similarly or more expressed in 6J29 than in N38 (AKT2, OST2, and SLAC1). As to vacuolar Ca<sup>2+</sup> antiporters, although transcripts accumulation for CAX1 did not show any significant genotype effect, CAX1.6 was on average 161 times more accumulated in the guard cells of hybrid poplars than in the black poplars. The same

pattern was found for the vacuolar Na<sup>+</sup>/H<sup>+</sup> antiporter NHX1.13. Its mRNA levels were also high in Carpaccio, compared with I214. Interestingly, NHX1.13 expression correlated to the Na content in guard cells (Figure 4, *P* < .001, *R*<sup>2</sup> = .35). Targeted aquaporins (PIPs and TIPs) mRNA were consistently expressed at the lowest levels in I214, with
<figure><img src="image_2.png" /><figcaption>**FIGURE 1** Stomatal conductance of four poplar genotypes under contrasting water availability. Each leaf side has been measured separately once in the morning and once in the afternoon (10:30 a.m. and 3:00 p.m.). Carpaccio and I214 are *Populus deltoides* × *Populus nigra*, whereas 6J29 and N38 are *P. nigra* genotypes. White and grey coloured bars show the contrasts used for statistical design. Letters show significant differences among groups for the genotype-time of day interaction, the water treatment and the leaf side (*P* < .05)</figcaption></figure>

the notable exception of *TIP2.1*, which was expressed at the highest level in I214 guard cells (Figure S2u-aa).

## 3.3 | Impact of water availability on element content and transcript accumulation in guard cells

CI was the only element whose content in the guard cells was significantly 13% higher in the drought-stressed trees across all genotypes (Figure S1d, *P* = .003). Moreover, the guard cells CI content was positively correlated with *g*<sub>s</sub> when controlling for genotype and water treatment differences (Figure 5, partial *R*<sup>2</sup> = .15, *P* < .001). Other modifications of element content in response to water regime were

genotype specific. The guard cells of I214 had a P content 11% lower under drought (for well-watered and drought-stressed trees, respectively: 1.92 and 1.71 mg g<sup>-1</sup>, *P* = .011, Figure S1c). Among black poplars, 6J29 and N38 exhibited an increased guard cells Ca content, respectively, 15% and 13% higher in the drought-stressed than in the well-watered trees (for control and drought-stressed, respectively, 6J29: 4.92 and 5.78 mg g<sup>-1</sup>, *P* = .028; N38: 6.60 and 7.55 mg g<sup>-1</sup>, *P* = .015, Figure S1f). Neither K nor any other element in guard cells were under a significant effect of the water regime, either as a main effect or as part of an interaction (*P* > .075, Table 2).

With respect to the normalized transcript accumulation, the water regime was highly genotype specific as 14 of 27 genes showed a significant interaction between the two factors (Table 2, Figure S2). Only *KAT3* and *PIP2.1* mRNA levels were significantly lower and higher, respectively, in the drought-stressed trees than in the control, in all genotypes (*P* < .006, Figure S2n-v). Concerning the hybrid poplars, the expression of three genes was down-regulated in response to water stress in the guard cells of Carpaccio (*KAT1.2*, *SLAC1*, and *TIP1.4*) but only two in the guard cells of I214 (*AHA11* and *TIP1.3*). For 6J29, the expression of *PYL8*, *CA1*, and *PIP1.2* was up-regulated in response to water stress. N38 showed the widest responsiveness to water stress. The expression of *PYL4* and *TIP1.3* was significantly down-regulated, whereas the expression of *ABI1*, *OST1*, *AKT2*, *KAT1.2*, and *PIP1.2* was up-regulated under water stress.

## 3.4 | Diurnal and leaf side differences of element content in guard cells

The PCA highlighted four groups consisting of every paired modality of the time of day and leaf side (Figure 6). The first and second axis explained 36.1% and 24.7% of the total variance with the third

<figure><img src="image_3.png" /><figcaption>**FIGURE 2** Guard cell element content of four poplar genotypes. Carpaccio and I214 are *Populus deltoides* × *Populus nigra*, whereas 6J29 and N38 are *P. nigra* genotypes (in dark and light blue, yellow and orange, respectively). Values inside each box are the genotypic mean expressed as "per mille" of dry mass, or mg g<sup>-1</sup> (n = 42-48). Boxes and bars show the 10th, 25th, 75th, and 90th percentiles. Letters show significant differences by post hoc contrast among the four genotypes for each element [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**FIGURE 3** Guard cell transcript accumulation heatmap of four poplar genotypes. Carpaccio and I214 are *Populus deltoides* × *Populus nigra*, whereas 6J29 and N38 are *P. nigra* genotypes. Values inside each box are back-transformed means by genotype of normalized transcript accumulation (n = 2-4). Colors range from white to orange to dark red to show the lowest, middling, and highest overall normalized expression values. Letters show significant differences by post hoc contrast among the four genotypes for each gene [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**FIGURE 4** Correlation between NHX1.13 normalized transcript accumulation and sodium content (as “per mille” of dry mass) inside the guard cells of four poplar genotypes. Carpaccio and I214 are *Populus deltoides* × *Populus nigra*, whereas 6J29 and N38 are *P. nigra* genotypes (in dark and light blue, yellow and orange, respectively; n = 115) [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

accounting for less than 14% (not shown). P and Ca content in the guard cells contributed the most to the first axis, whereas the An/Cat ratio, CI, and K content contributed the most to the second axis.

<figure><img src="image_6.png" /><figcaption>**FIGURE 5** Correlation between the stomatal conductance and the chlorine content inside the guard cells once the genotype and water treatment effect is taken into account. Data used are residuals of a linear model with genotype and water treatment as main effect, chlorine content as independent variable, and stomatal conductance as dependent variable (n = 10-22) [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

Focusing on the first two axis, the four groups formed a diagonal line parallel to the CI and Mg content and to the supplementary variable $g_s$. The group associated with the abaxial side in the morning was positively related to these variables, whereas the group formed
<figure><img src="image_7.png" /><figcaption>**FIGURE 6** Principal component analysis of guard cell element content for each leaf side and time of day. Data used are residuals of linear models with genotype as main effect and each element content as dependent variable (n = 48). Stomatal conductance was added as a supplementary variable (in red). Mean points and confidence ellipse are shown. Circles and squares are for the abaxial and adaxial side respectively. Green and orange are for the morning and blue and pink are for the afternoon [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

by the data associated to the adaxial side in the afternoon was neg-
atively related to them. The leaf sampled in the morning showed
overall a higher Mg, P, and Cl content in the guard cells and a higher
An/Cat ratio than in the leaf sampled in the afternoon (Figure S1b,c,
d,g; P = .043, P = .003, P = .009, and P < .001, respectively). The
afternoon decrease ranged from 7% for P to 13% for Cl. Moreover,
a higher amount of element was found in the guard cells on the
abaxial side than on the adaxial side, with a noticeable exception
of the Mg content (P = .07). P, Ca, and the ratio An/Cat were,
respectively, 15%, 17%, and 10% more abundant on the abaxial side
(P < .003). Cl and K were also significantly higher on the abaxial side
of black poplars (Figure S1d,e). Similar trends, however not signifi-
cant, were observed in hybrid poplars, as well as a 98% higher Na

content in the abaxial guard cells of Carpaccio, compared with the
adaxial side (Figure S1a, P < .001).

3.5 | Diurnal and leaf side differences of guard cells transcript accumulation in guard cells

Because the PCA on the guard cells element content revealed a strong
structuration of sampling modalities (Figure 6), we performed a similar
analysis on the gene expression for the 27 studied genes (Figure 7).
The first three axis explained 21.3%, 13.1%, and 7.6% of the total var-
iance. Among the genes contributing the most to the first axis, we
found genes involved in the ABA biosynthesis and signalling pathways
(NCED3.1, NCED3.2, ABI1, OST1, and PYL8), K⁺ channels (AKT2 and
KAT1.2), and some of the aquaporins (PIP1.2, PIP2.5, TIP1.4, and
TIP2.1). The genes contributing highly to the second axis were those
encoding H⁺-ATPase (AHA11 and OST2), Ca²⁺ vacuolar antiporters
(CAX1 and CAX1.6), K⁺ channels (KAT1.2 and KAT3), and NHX1.13.
Similar to the PCA on guard cells element content (Figure 6), the four
modalities composed by the crossed factors leaf sides and time of day
were even more clearly separated by the first two axis, compared with
the element content. Morning and afternoon measures were sepa-
rated on the upward diagonal, whereas leaf sides were separated on
the downward diagonal. Overall, the differences between time of
day were related to the expression levels of gene encoding H⁺-ATPase
and aquaporins, whereas the differences of leaf sides could be
ascribed to genes associated mostly with the ABA response, Ca²⁺
vacuolar antiporters, and K⁺ channels.

Out of the 27 genes we tested, 20 showed some degree of differ-
ential expression in the guard cells between the morning and the
afternoon (Table 2, Figure S2). Only three genes were overall less
expressed in the afternoon (OST1, KAT1.2, and KAT3) than in the
morning by 16%, 17%, and 22%, respectively (Figure S2d,m,n). The
remaining 17 genes were expressed at the highest level in the after-
noon. This included both NCEDs (NCED3.1 and NCED3.2), both H⁺-

<figure><img src="image_8.png" /><figcaption>**FIGURE 7** Principal component analysis of guard cell normalized transcript accumulation for each leaf side and time of day. Data used are residuals of linear models with genotype as main effect and each transcript as dependent variable (n = 30-31). Stomatal conductance was added as a supplementary variable (in red). Mean points and confidence ellipse are shown. Circles and squares are for the abaxial and adaxial side, respectively. Green and orange are for the morning and blue and pink are for the afternoon [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>
ATPase (OST2 and AHA11), the three tested PIPs (PIP1.2, PIP2.1, and PIP2.5) as well as PYL8, PHOT2, and TIP1.3. ABI1 normalized mRNA levels were higher in the afternoon but only in the guard cells on the abaxial side (Figure S2c, P = .004). Few significant interactions occurred between genotype and times of day. Both NHX1.13 and TIP2.1 transcripts levels were higher in the afternoon, but in the guard cells of hybrid poplars (P < .05) or of black poplars (P < .002), respectively (Figure S2t,z). Both AKT2 and PYL2 genes were more expressed in the afternoon but only in the guard cells of I214 and N38 (P < .04), respectively (Figure S2e,l).

With respect to the transcripts levels between leaf sides, 18 of the 27 tested genes exhibited a differential expression. In general, we observed the highest expression in the guard cells on the adaxial side of the leaf. Solely, CAX1 and CAX1.6 genes were, respectively, 12.6 and 10.1 times more expressed in the guard cells on the abaxial side than on the adaxial side (Figure S2r,s, P < .001). The same pattern was found for *TIP1.3* and for *PIP2.5* but only in I214 (Figure S2w,x). The tested *NCEDs* transcripts levels were 13.5 and 6.8 times more abundant on the adaxial side (P < .01). Similarly, the two β-carbonic anhydrases (CA1 and CA4), *PHOT1* and *ABI1* were also overall more expressed on the adaxial side, the accumulation of ABI1 transcripts being only significant in the morning (Figure S2c, P = .002). A number of genes showed a genotype-specific differential expression between leaf sides. *OST1*, *PYL8*, *AKT2*, and *KAT1.2* genes were more expressed in the adaxial guard cells only in Carpaccio and N38. *TIP1.4* mRNA level was higher in the guard cells on the adaxial side in Carpaccio. *SLAC1*, *NHX1.13*, *PIP1.2*, and *PIP2.5* genes exhibited the same pattern in N38 only. *PYL8* was also 3.3 times more expressed in the guard cells on the adaxial side than on the abaxial side of 6J29 (P = .013).

4 | DISCUSSION

4.1 | Genotypic diversity of element content and gene expression in the guard cells

Our study focused on the relationships between guard cell element
content, expression of genes of interest, and stomatal conductance.
By modulating the water regime, leaf sides, and times of day on four
poplar genotypes, we intended to gain insight into the molecular
drivers of gas exchange regulation and the unique physiology of
guard cells.

We found a tremendous diversity for both element content and
gene expression in the guard cells of four poplar genotypes. This adds
to previous investigations highlighting the considerable diversity of
transcript accumulation and responses to environmental factors in dis-
tinct organs of poplars (Bizet et al., 2015; Cohen et al., 2013; Dumont
et al., 2014; Wilkins et al., 2009). Interestingly, Carpaccio exhibited the
lowest expression of genes related to ABA biosynthesis and signalling
pathways, especially compared with I214. This was not associated
with a lower plasticity of stomatal closure under drought, as previous
studies found a lack of stomatal response under various water deficits
in I214 but not in Carpaccio (Durand et al., 2019; Muller & Lambs,

2009). Instead, exogenous supply of ABA to the I214 clone induced
leaf abscission (Chen et al., 1997), which may be part of its drought
response (Giovannelli et al., 2007). Additionally, we found a lower
expression for the genes encoding K⁺ channels, H⁺-ATPase, and aqua-
porins in I214. These differences could in theory contribute to a
reduced stomatal control by limiting the exchange of solutes and
water across the plasma membrane. However, of the four genotypes
studied, I214 showed the highest stomatal density and the smallest
stomata (Durand et al., 2019). As such, one could expect that not only
less water is required to modify the guard cell turgescence but a
smaller change in aperture added over the whole leaf could result in
a massive change in stomatal conductance. Durand et al. (2019) also
showed that I214 had the fastest stomatal closing speed; here we
found that it may not be due to an increased expression of genes
encoding transporters on the guard cell plasma membrane.

Along with differences in transcript accumulation, we found evi-
dence of a genotypic diversity of elements contributing to stomatal
movements. Little is known on this topic in poplars as the majority
of studies focus only on three species (Assmann, 1993), namely, A.
*thaliana* (L.), *Commelina communis* (L.), and *Vicia faba* (L.). *NHX1.13*
encodes a vacuolar Na⁺ and K⁺ transporter that has been shown to
regulate cell turgor and stomatal function in A. *thaliana* (Barragan
et al., 2012). The higher amount of Na in the guard cells of hybrid
poplars, linked with higher *NHX1.13* mRNA levels, highlights the
importance of this element for stomatal control in these genotypes.
Because K⁺ channels and some aquaporins may be permeable to Na⁺
as well (Byrt et al., 2017; Jezek & Blatt, 2017), one could speculate
that these transporters have an increased permeability for Na⁺ com-
pared with black poplars. In contrast, the higher Cl content in black
poplars emphasize its role in stomatal function in these genotypes.
Both malate and Cl⁻ act as counter-ions to balance the uptake of K⁺
into the guard cells in various proportions depending on species, Cl
availability, and starch metabolism (Raschke & Schnabl, 1978; Santelia
& Lawson, 2016).

4.2 | Impact of water availability

A relatively weak effect of the water regime occurred on element con-
tent and normalized mRNA levels, especially as compared with the
other tested factors (Table S3). This might be related to the leaf sam-
pling, which occurred 21 days after the start of a mild drought, when
short-term gene expression responses were replaced by acclimation
of physiological processes. Similarly in *Populus tremula*, few changes
in the expression of genes of interest were found in leaves sampled
more than 20 days after the start of the drought (Possen et al.,
2011). The drought response at the transcript level was highly geno-
type specific, in agreement with transcriptomic studies on poplar
genotypes, showing divergent expression profiles under drought even
when the physiological response was similar (Cohen et al., 2010; Wil-
kins et al., 2009). *PIP2.1* was the only up-regulated gene in all four
genotypes under drought. Despite not always being the case, similar
results were found on various hybrid poplars (Almeida-Rodriguez,
Cooke, Yeh, & Zwiazek, 2010; Cohen et al., 2013). AtPIP2.1 and ZmPIP2.1 increased water permeability when expressed in Xenopus oocytes and protoplasts of Lilium longifolium pollen (Lopez et al., 2003; Sommer et al., 2008). Moreover, AtPIP2.1 was related to ABA-induced stomatal closure, mediated by OST1 by increasing guard cell permeability for water enabling its efflux (Grondin et al., 2015; Maurel, Verdoucq, & Rodrigues, 2016). Increasing PIP2.1 expression under drought may allow for a faster stomatal closure.

Under drought, there was an increased Ca content in black poplars together with a higher Cl content for all genotypes. During stomatal closure, free Ca²⁺ entering the cytosolic spaces of guard cells is known to be responsible for the inactivation of inward-rectifying K⁺ channels in V. faba (Grabov & Blatt, 1999; Schroeder & Hagiwara, 1989) and the activation of anion channels, such as SLAC1 when expressed in Xenopus laevis oocytes (Geiger et al., 2010). However, Ca²⁺ elevation in the cytosol has been found to be mostly due to influx from the vacuole and the endoplasmic reticulum as opposed to entry from the apoplast (Chen et al., 2012). Unfortunately, we are not able to localize specific subcellular compartments. Concurrently to the increased Ca content, the Cl content increased as well under drought. This suggests that an increased solute concentration in guard cells may be in part a response to a decline in leaf water content in poplars under drought (Guo, Zhang, & Huang, 2010; Marron et al., 2003).

## 4.3 | Leaf side and time of day shape guard cell element content and transcript accumulation

Gene expression and element content in the guard cells followed opposite patterns over the leaf and along the day. Although stomatal conductance and element content reached a maximum on the abaxial side in the morning, transcript levels was overall at the lowest. The same pattern occurred, but reversed on the adaxial side in the afternoon.

There was no significant change of K content in guard cells of various apertures despite the overall element content being higher in more opened stomata, particularly with the increased Cl content (Figure 6). These results were checked by X-ray microanalyses under cryogenic conditions (−145°C). Both techniques (cryo and freeze-dried samples) showed no difference of K content inside the guard cells between leaves with markedly different stomatal conductance. The lack of decrease in K concentration in stomatal guard cells with lower stomatal aperture was quite unexpected as K is known to be of primary importance for stomatal movements. A previous study on hybrid poplar genotypes found an increased K content in guard cells under ozone while having lower gs (Dumont et al., 2014). Langer et al. (2004) found only a 25% decrease in guard cell K content between P. tremula × Populus tremuloides leaves grown under CO₂-free environment and leaves in the dark subjected to 100-µM ABA, resulting in an 80% gs decrease.

In our study, stomatal conductance was impacted by the different water regimes, leaf sides, and times of day. Because the difference in gs between well-watered and drought-stressed poplars was relatively

small, differences of K content would most likely also be relatively small. Consequently, such small changes may not be identified using this technique, especially when considering the many roles K has in multiple processes, from homeostasis to enzymes activation (Clarkson & Hanson, 1980). Despite a significantly lower K content on the adaxial side of the leaf, differences in gs between leaf sides may be predominantly attributed to differences of stomatal density rather than apertures (Durand et al., 2019). Hence, differences of gs between times of day were our best candidate to find corresponding differences in guard cell K content. However, unlike stomatal opening, it has been proposed that stomatal closure in the afternoon may be more related to sucrose than K (Amodeo, Talbott, & Zeiger, 1996; Granot & Kelly, 2019; Kottapalli et al., 2018; Talbott & Zeiger, 1996). When the rate of sucrose production is higher than its loading into the phloem, excess sucrose accumulate in the apoplast and may enter guard cells (Ewert, Jr, Zhang, Aghoram, & Riddle, 2000; Kang, Outlaw, Andersen, & Fiore, 2007), inducing stomatal closure mediated by hexokinases (Kelly et al., 2013). Clearly, more research is needed to decipher changes of element content in poplar guard cells related to stomatal movements, which will necessitate the input of multiple techniques.

Diurnal rhythms in transcript accumulation were reported by previous studies for the expression of aquaporin-encoding genes and genes involved in ABA regulation. In contrast to our findings, whole leaves and roots generally have an increased accumulation of aquaporin transcripts in the morning, or in the few hours following an increase of light (Hachez, Heinen, Draye, & Chaumont, 2008; Henzler et al., 1999; Lopez et al., 2003; Lopez et al., 2013; Vandeleur et al., 2009). On the other hand, even if leaf ABA concentrations tend to increase at the end of the day (Novakova et al., 2005), the genes involved in the ABA-related pathways seem to present a diversity of diurnal rhythms in leaves (Seung et al., 2012). Despite their close proximity to mesophyll cells, stomata have specific circadian rhythms (Hassidim et al., 2017; Yakir et al., 2011). Given their relatively low proportion in the leaf compared with other cell types, the different expression profile along the day in guard cells compared with whole leaves could result from the cellular complexity of the organ that hides cell-type specific regulations (Nourbakhsh-Rey & Libault, 2016). In addition to the changes in element content, guard cells are also subjected to pH variations during stomatal movement. In general, stomatal closure induces cytosolic and vacuolar alkalinization (Irving, Gehring, & Parish, 1992; Zhang, Dong, Gao, & Song, 2001), whereas stomatal opening induces acidification (Huang, She, Zhang, & Zhao, 2013), linked with H₂O₂ and Ca²⁺ signalling during both ABA and dark-induced stomatal responses (Agurla & Raghavendra, 2016; Ma & Niu, 2017). Moreover, external pH changes strongly altered transcript levels, which was linked to ABA-responsive element motifs in the promoter region of pH-responsive genes (Lager et al., 2010). Little is known about the effect of intracellular pH on transcript accumulation, and whether or not guard cell movements affect its own expression pattern remains to be established.

Potential differences in the physiology of guard cells between leaf sides is seldom investigated, and even fewer studies compare the
patterns of transcript accumulation and/or element content. We
reported here an overall stronger expression of genes of interest on
the adaxial side for a number of genes, including *NCEDs* and β-
carbonic anhydrases. However, the higher expression of vacuolar
Ca²⁺ antiporters on the abaxial side was associated with a higher Ca
content on this side, in agreement with Dumont et al. (2014). It has
been hypothesized that guard cells on each side may have different
pathways for Ca²⁺-mediated signal transduction (Wang, Wu, &
Assmann, 1998). There is evidence of an independent control of gas
exchange between leaf sides (Mott, Cardon, & Berry, 1993; Richard-
son et al., 2017). Such control would allow amphistomatal leaves to
regulate more efficiently in response to the environmental conditions
specific to each side. Previously reported thermal and irradiance gradi-
ents between leaf surfaces of a few degrees (Buckley, John, Soffoni,
& Sack, 2015; Clum, 1926; Sheriff, 1979) could result in different per-
ceived environmental conditions between leaf sides (e.g., light, VPD,
and temperature), prompting different stomatal control, as was found
on poplar clones in response to a change in light and VPD (Ceulemans,
Hinckley, & Impens, 1989; Pallardy & Kozlowski, 1979). Differences in
the intensity and nature of perceived stresses, such as evaporative
demand, light, and heat (Urban, Ingwers, McGuire, & Teskey, 2017)
may induce a distinct conjugation of stress between sides, which
may affect the synergic or antagonistic response to environmental
cues (Bigot et al., 2018). This would result in a different perception
and signalization between sides; for example, stomata of *Gossypium
barbadense* (L.) have distinct sensitivity to light between sides linked
with different pigment contents (Lu, Quiñones, & Zeiger, 1993). A dif-
ferent sensitivity between sides would lead to a diversity of gene
expression (see Bigot et al., 2018), and element content in the guard
cells, which may be a part of what we observed. A stronger gene
expression on the adaxial side might be seen as the result of a more
extreme condition on the side facing the light source.

In conclusion, even though we found a strong genotypic diversity,
our data show how changes in stomatal conductance between leaf
sides and time of day are linked to guard cell element content and
transcript accumulation profiles in a similar way across the four
studied genotypes (Figures 6 and 7). Future technical developments
such as cryogenic electron microscopy will allow for an easier element
cellular compartmentation. Because stomata are responsible for opti-
mizing carbon gain with regard to water loss, their functions affect
not only the plant but the ecosystem as well (Berry, Beerling, & Franks,
2010). Understanding the specificities of guard cells functioning will
further expand our current knowledge of stomatal physiology and
their global consequences.

ACKNOWLEDGMENTS

This work was conducted in the frame of the WATBIO (Development
of improved perennial biomass crops for water-stressed environ-
ments), a collaborative research project funded from the European
Union's Seventh Programme for research, technological development
and demonstration under grant agreement 311929. The research
received funding from the French National Research Agency through

the Laboratory of Excellence ARBRE (ANR-12-LABXARBRE-01). M.
D. received a PhD scholarship from the Lorraine Région and EFPA
(INRA research department). We thank the nursery of Guéméné
Penfao for providing the Euramerican poplar cuttings. The authors
acknowledge Marc Villar and Catherine Bastien from the UR0588-
INRA Unit for access to the referenced *P. nigra* clones 6J29 and N38
as well as SILVATECH from UMR 1434 SILVA, 1136 IAM, 1138
BEF, and 4370 EA LERMAB EEF research centre INRA Grand-Est for
its contribution to X-ray microanalyses. SILVATECH facility is
supported by the French National Research Agency through the
Laboratory of Excellence ARBRE.

CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence
of any commercial or financial relationships that could be construed
as a potential conflict of interest.

AUTHOR CONTRIBUTION

D. L. T., O. B., C. B., M. D., and D. C. developed the experimental
design. C. B. carried out the experimental drought set-up. D. L. T. col-
lected leaf porometer data; D. L. T., D. C., I. H., and N. A. sampled the
leaves; M. D. performed X-ray microanalysis; M. D., D. C., and I. T.
microdissected the stomata and extracted the RNA. N. A. performed
cDNA amplification, RT-PCR, and data standardization. M. D., D. L.
T., D. C., and I. H. performed data analysis. All contributors were
involved in the writing and reviewing of the manuscript.

ORCID

Didier Le Thiec https://orcid.org/0000-0002-4204-551X

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## SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section at the end of the article.

Table S1 List of gene studied with their specific primers.

Table S2 Dataset used for statistical analysis (see separate Excel file).

Table S3 Summary of the genotype, water treatment, time of day and leaf side partial R² of stomatal conductance, guard cell element content and expression of genes of interest.

**Figure S1** Guard cell element content of four poplar genotypes under contrasting water availability with each leaf side measured separately once in the morning and once in the afternoon (10:30 and 15:00).

**Figure S2** Normalized guard cell transcript accumulation of four poplar genotypes under contrasting water availability with each leaf side measured separately once in the morning and once in the afternoon (10:30 and 15:00).

Data S2: Supplementary Information

**How to cite this article:** Durand M, Cohen D, Aubry N, et al. Element content and expression of genes of interest in guard cells are connected to spatiotemporal variations in stomatal conductance. *Plant Cell Environ.* 2019;1-16. https://doi.org/10.1111/pce.13644