# Nighttime transpiration represents a negligible part of water loss and does not increase the risk of water stress in grapevine

Silvina Dayer, Jose Carlos Herrera, Zhanwu Dai, Régis Burlett, Laurent J.

Lamarque, Sylvain Delzon, Giovanni Bortolami, Hervé H. Cochard, Grégory

A. Gambetta

► To cite this version:

Silvina Dayer, Jose Carlos Herrera, Zhanwu Dai, Régis Burlett, Laurent J. Lamarque, et al. Nighttime transpiration represents a negligible part of water loss and does not increase the risk of water stress in grapevine. Plant, Cell and Environment, 2021, 44 (2), pp.387-398. 10.1111/pce.13923. hal-03013821

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ORIGINAL ARTICLE

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WILEY

# Nighttime transpiration represents a negligible part of water loss and does not increase the risk of water stress in grapevine

Silvina Dayer<fnref n="1" /> ID

José Carlos Herrera<fnref n="2" /> ID

Zhanwu Dai<fnref n="3" /> ID

Régis Burlett<fnref n="4" /> |

Laurent J. Lamarque<fnref n="4" />,<fnref n="5" /> ID

Sylvain Delzon<fnref n="4" />

Giovanni Bortolami<fnref n="6" /> |

Hervé Cochard<fnref n="7" />

Gregory A. Gambetta<fnref n="1" />

**Correspondence**

Silvina Dayer, EGFV, Bordeaux Sciences Agro, INRAE, xUniv. Bordeaux, ISVV, 210 Chemin de Leysotte 33882 Villenave d'Ornon, France.

Email: silvinadayer@gmail.com

**Abstract**

Nighttime transpiration has been previously reported as a significant source of water loss in many species; however, there is a need to determine if this trait plays a key role in the response to drought. This study aimed to determine the magnitude, regulation and relative contribution to whole plant water-use, of nighttime stomatal conductance ($g_{night}$) and transpiration ($E_{night}$) in grapevine (Vitis vinifera L.). Our results showed that nighttime water loss was relatively low compared to daytime transpiration, and that decreases in soil and plant water potentials were mainly explained by daytime stomatal conductance ($g_{day}$) and transpiration ($E_{day}$). Contrary to $E_{day}$, $E_{night}$ did not respond to VPD and possible effects of an innate circadian regulation were observed. Plants with higher $g_{night}$ also exhibited higher daytime transpiration and carbon assimilation at midday, and total leaf area, suggesting that increased $g_{night}$ may be linked with daytime behaviors that promote productivity. Modeling simulations indicated that $g_{night}$ was not a significant factor in reaching critical hydraulic thresholds under scenarios of either extreme drought, or time to 20% of soil relative water content. Overall, this study suggests that $g_{night}$ is not significant in exacerbating the risk of water stress and hydraulic failure in grapevine.

**KEYWORDS**

circadian regulation, grapevine, nighttime transpiration, stomata, stomatal conductance, water loss

1 | INTRODUCTION

The opening of the stomata at night is observed in many species and challenges some assumptions regarding plant function, namely that plants constantly strive to maximize carbon gain while minimizing water loss (Cowan & Farquar, 1977; Farquhar, 1973). Stomata are theorized to be closed overnight when no net photosynthetic carbon fixation is possible, however, this contrasts with most observations

that nighttime stomatal opening is commonplace (Costa et al., 2015; Dawson et al., 2007; Fuentes, Mahadevan, Bonada, Skewes, & Cox, 2013; Schoppach, Claverie, & Sadok, 2014; Zeppel, Tissue, Taylor, Macinnis-Ng, & Eamus, 2010). Reports across different species indicate that water loss due to nighttime stomatal opening represents around 12% of daily transpiration (Forster, 2014) and in some cases it can be as high as 30% (Caird, Richards, & Donovan, 2007). However, the relative importance and impact of this nighttime water

<footnote n="1">¹EGFV, Bordeaux Sciences Agro, INRAE, Univ. Bordeaux, ISVV, Villenave d'Ornon, France</footnote>

<footnote n="2">²Institute of Viticulture and Pomology, University of Natural Resources and Life Sciences (BOKU), Tulln, Austria</footnote>

<footnote n="3">³Beijing Key Laboratory of Grape Science and Enology and Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Beijing, China</footnote>

<footnote n="4">⁴INRAe, BIOGECO, Université Bordeaux, Pessac, France</footnote>

<footnote n="5">⁵Département des Sciences de l'Environnement, Université du Québec à Trois-Rivières, Trois-Rivières, Quebec, Canada</footnote>

<footnote n="6">⁶SAVE, Bordeaux Sciences Agro, INRAE, Univ. Bordeaux, ISVV, Villenave d'Ornon, France</footnote>

<footnote n="7">⁷Université Clermont-Auvergne, INRAE, PIAF, Clermont-Ferrand, France</footnote>

<footnote>This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2020 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.</footnote>
loss to overall plant performance and water use is still not well
understood.

Several hypotheses have been raised to explain the significance or potential functions of nighttime stomatal conductance (g_night). The most common explanation is that plants simply lack complete stomatal control at night and/or nighttime transpiration (E_night) is due to water leakage through the cuticle (Barbour et al., 2005). Nighttime fluxes could, however, serve a purpose of enhanced nutrient uptake or nutrient distribution to distal parts of the plant (Scholz et al., 2007), the delivery of dissolved oxygen to woody tissues (Daley & Phillips, 2006), and the prevention of excessive leaf turgor (Donovan, Linton, & Richards, 2001). A recent meta-analysis including published datasets from 176 different species found that the variation of g_night across plants was not consistent with the hypotheses mentioned above (i.e., simple leakage, nutrient uptake enhancement, or delivery of dissolved oxygen) and that changes in g_night could be partially explained by the circadian clock (Resco de Dios, Chowdhury, Granda, Yao, & Tissue, 2019).

Quantifying and understanding water losses at night is still challenging because some methods are not sensitive enough to accurately measure the low nighttime fluxes of water (e.g., sap flows, lysimeters; Tolk, Howell, & Evett, 2006; Zeppel et al., 2010; Fuentes et al., 2013) and direct measurements of g_night through porometry or gas-analyzers do not always respond to environmental drivers as during the daytime (Ogle et al., 2012). Drawbacks for each method must be recognized, particularly when comparing species or environments. For instance, vapor pressure deficit (VPD) has been well documented as the main factor driving daytime transpiration and, after light, stomatal conductance (Monteith, 1995; Oren et al., 1999). However, the VPD at night is much lower than during the day and responses of g_night to VPD have been observed to be variable among species: it can be invariable (Barbour et al., 2005), or decrease with increasing VPD (Bucci et al., 2004; Cirelli, Equiza, Lieffers, & Tyree, 2016). Despite being intimately associated with each other, E_night and g_night are different processes and may respond differently to environmental conditions (Caird, Richards, & Donovan, 2007).

Although g_night and E_night are increasingly the subject of study
many gaps in our knowledge remain, in particular an understanding of
the costs and/or benefits of high g_night and E_night. Many studies have
emphasized that g_night represents a significant source of water loss for
the plant with reductions in water use efficiency (WUE) under
drought conditions (Caird, Richards, & Hsiao, 2007; Coupel-Ledru
et al., 2016; Kavanagh, Pangle, & Schotzko, 2007; Rogiers, Greer,
Hutton, & Landsberg, 2009). Furthermore, common quantitative trait
loci (QTLs) underlying genetic variability in both growth and E_night
have been reported (Coupel-Ledru et al., 2016). However, drought
studies focused on E_night and g_night need to be carefully interpreted
because they generally attribute water loss to the stomata only. When
stomata are mostly closed, water loss continues at a very low rate
through the cuticle (Duursma et al., 2019). This residual rate of water
loss is referred to as the minimum conductance (g_min) and is generally
not directly measured.

Different mechanisms have been postulated to explain the signifi-
cance of nighttime transpiration, and there is a need to better integrate
the physiological traits that determine its contribution to whole plant
water use. One limitation in understanding the significance of E_night
and g_night for whole plant water balance is that we still do not know if
this trait plays a key role in exacerbating water stress and the risk of
hydraulic failure. In a recent study in grapevine (Dayer et al., 2020), we
observed that the maximum water use given by maximum daytime
transpiration (E_max) was strongly correlated with other drought-related
traits such as the water potential at stomatal closure (Pgs90), the leaf
turgor loss point (πtLP) and the leaf water potential inducing 50% of
loss of hydraulic conductance (P50), highlighting the importance of
integrating multiple traits in characterizing drought tolerance. In the
current study, we used the same three cultivars to explore E_night and
g_night in natural conditions (i.e., outdoors, to avoid any artefactual VPD
effects brought about by a greenhouse environment) to address the
following question: Is nighttime water loss a key trait in exacerbating
water stress? Accordingly, we examined here (a) the relative impor-
tance of water loss at night relative to daytime transpiration (E_day),
(b) the drivers of transpiration at night and if they are the same as dur-
ing the day, (c) the potential association between E_night and g_night with
daytime variables related to productivity (g_max or Pn_max), and (d) the
modelling of the contribution of different conductance's (i.e., g_night, g_min
and g_day) to the time necessary to reach 20% of soil relative water con-
tent (representing impacts on productivity and yield in an agronomic
context) and the time to hydraulic failure (representing the risk of
drought induced mortality) under different experimental conditions.

2 | MATERIALS AND METHODS

2.1 | Plant material

The experiment was conducted in 2019 in one-year-old plants of own
rooted Vitis vinifera L. "Grenache," "Syrah" and "Semillon" from INRAE
nursery (Villenave d'Ornon, France) planted in 7 L pots containing
1 kg of gravel and 5.5 kg of commercial potting media (70% of horti-
cultural substrate and 30% sand). Plants were grown outside in a drip
irrigated platform, well-watered (without stress) with nutritive solu-
tion (NH4H2PO4 0.1 mmol L-1; NH4NO3 0.187 mmol L-1; KNO3
0.255 mmol L-1; MgSO4 0.025 mmol L-1; 0.002 mmol L-1 Fe, oligo-
element [B, Zn, Mn, Cu, Mo]) to avoid any deficiency during their
development. The surface of the pots was covered with a plastic bag
to prevent water loss by soil evaporation. The cultivars were distrib-
uted following a completely randomized design.

2.2 | Gas-exchange dynamics

Dynamics of leaf gas exchange measurements were conducted peri-
odically at different days of the season, from July fourth to August
23rd (DOY 185, 186, 193, 204, 205, 206, 215, 216, 221, 234 and
235) in four plants per cultivar (n = 4). Stomatal conductance (g) and
transpiration (E) were measured at different times during the day ($g_{day}$ and $E_{day}$) and night ($g_{night}$ and $E_{night}$). In addition, maximum stomatal conductance ($g_{max}$) and photosynthesis ($Pn_{max}$) were registered on 5 days (DOY 185, 193, 205, 216 and 235). At each DOY, different time points were registered every 2 hr, starting and finishing at different times. For example, on DOYs 185–186 we started at 06:00 (solar time) and finished at 04:00 the following day, and in DOY 206 we started at 02:00 and finished at 07:00 the same day. In this way, we could cover the range of most of all times during the day and night. Measurements were performed on mature, well-exposed leaves using a portable open-system including an infrared gas analyzer (GFS-3000, 180 Heinz Walz GmbH, Effeltrich, Germany) equipped with CO$_2$, humidity, temperature and light control modules. During the daytime measurements, conditions inside a 3 cm$^2$ cuvette were controlled and set to conditions easily reproducible all along the experimental period (i.e., PPFD = 1,500 µmol m$^{-2}$ s$^{-1}$, temperature = 20°C, vapor pressure deficit ~1.0–1.3 kPa, relative humidity ~50%, impeller speed = 7 and CO$_2$ = 400 ppm).

During the night, we allowed the cuvette to follow the ambient conditions, except for the CO₂ that was set at 400 ppm. Impeller speed was set to standard value, corresponding to a boundary layer conductance close to 4,200 mmol.m⁻².s⁻¹ for our cuvette (Burlett, personal communication). To maximize the differential of water vapour mole fraction (and therefore increase the resolution), the instrument flow rate was set to 650 µmol s⁻¹. Leaves were enclosed in the cuvette, and the instantaneous gas exchange was logged following stability in cuvette conditions (after approximately 1.5 min). All the plants were kept well-watered (no stress from soil moisture).

## 2.3 | Environmental conditions

Air temperature, relative humidity and radiation were obtained hourly from a meteorological station (Climatik meteo station 33550003) located in very close proximity to the experimental site (Figure S1). Leaf temperature was recorded every 20 min using TinyTag Talk 2 data loggers (Gemini Data Loggers, Ltd., UK) associated with temperature probes that were carefully attached to the abaxial side of the leaf by adhesive tape. This type of probe records temperature from -40 to 125°C with an accuracy of 0.4°C and a resolution of 0.05°C. The values of leaf temperature obtained with these probes were used to calculate the leaf-air vapor pressure deficit (VPD$_{leaf-air}$).

## 2.4 | Total leaf area

Total leaf area (TLA) was estimated through the relationship obtained between the leaf midrib length and the leaf area (measured with a leaf area meter Model LI-3000, LI-COR, Lincoln, NE) prior the experiment for each cultivar using approximately 100 leaves per cultivar. The leaf midrib length was measured once every 6–7 days on all the leaves of each plant.

## 2.5 | Minimum conductance ($g_{min}$)

In the same set of plants used to measure nighttime $g_{night}$, minimum conductance ($g_{min}$; minimum water loss after stomatal closure) was determined in eight leaves from each of the three cultivars by using the mass loss of detached leaves (MLD) technique (Billon, Ruiz, Sleiman, Hitmi, & Cochard, 2019; Duursma et al., 2019). The technique consists in measuring the leaf mass loss monitored over time as the leaf dries out. Leaves from well-watered plants were detached and suspended by their petiole (to allow the lamina to transpire from both sides) in a controlled chamber (Fitoclima 1200, Aralab, Portugal) set to a constant temperature of 25°C and relative humidity of 45%. The petioles were sealed with parafilm immediately after the cutting to avoid water losses. Photosynthetic Photon Flux Density (PPFD) at the position of the samples was around 400 +/−50 µmol m⁻² s⁻¹. The mass loss of the leaves was measured every 5–10 min for the first hour and then every 15–20 min as long as the leaves dehydrated with a 0.0001 g resolution balance (Sartorius LE5201 Expert, Goettingen, Germany). The evaporation (E) of each sample was computed from the relationship between leaf mass and time, once water loss rates reached a steady state. The minimum conductance ($g_{min}$) was calculated by using the measured vapor pressure deficit (VPD) according to the Equation E = $g_{min} \cdot D/P$ where D is the VPD in kPa and P is the atmospheric pressure expressed in mmol m⁻² s⁻¹ (Duursma et al., 2019).

## 2.6 | Scanning electronic microscopy (SEM) for stomatal observation

Stomata cryo-SEM observations were performed in three plants per cultivar that were kept in a dark chamber overnight. Leaf samples were collected in dark conditions using a red headlight lamp in a very low intensity (0–23 µmol m⁻² s⁻¹) and for no more than 3–5 s (the time needed to punch and drop the leaf sample in N2), avoiding any response of the stomata (Raven 2014). Three leaf discs (9 mm diameter) from different leaves were sampled with a punch holder ("Biopsy Punch") from the middle zone of the lamina between major veins. Leaf samples were immediately mounted in copper specific supports with cryo-glue, freshly made up as a 50/50 mixture of colloidal graphite and Tissue-Tek mounting medium, and frozen in slush nitrogen (mix of solid and liquid nitrogen). Frozen samples were transferred under nitrogen to the preparation chamber (maintained at −140°C) in which sublimation at −95°C for 5 min and metallization with Platinum at 10 µA for 15–20 s was performed. Finally, samples were transferred under vacuum to the cold stage of the microscope chamber in which stomata were observed and photographed in high vacuum mode at 3kV acceleration voltage. Cryo-SEM observations were carried out in a Gemini SEM 300 FESEM (ZEISS, Germany) microscope coupled with a CRYO-SEM PP3010T module (Quorum Technologies, England).
**2.7 | The impact of decreasing E<sub>night</sub> on leaf water potential**

Eight well-watered plants of approx. 1.0 m height from each cultivar were chosen for a bagging experiment. On DOY 182 after sunset (radiation <50 µmol m⁻² s⁻¹) four plants (n = 4) were randomly selected and entirely bagged with a transparent polyethylene film that was hermetically sealed with tape. The plants were left bagged over-night and the pre-dawn (Ψ<sub>PD</sub>) and midday (Ψ<sub>leaf</sub>) leaf water potentials were measured the next day prior any light exposure and at midday respectively. A set of four more plants were left un-bagged and kept as controls. The Ψ<sub>PD</sub> and Ψ<sub>leaf</sub> were measured in basal fully expanded leaves from all plants (bagged and unbagged) using a “Hammel-Scholander” pressure chamber (DG Meca, Gradignan, France).

**2.8 | Model simulations of hydraulic traits**

A soil-plant water transport model (SurEau; Cochard, Martin-StPaul, Pimont, & Ruffault, 2020) was used to determine the predicted time it would take under drought to reach a particular threshold (thresholds are described below). A detailed explanation of the model is given in Martin-StPaul, Delzon, and Cochard (2017). Briefly, the plant is described as a series of variable symplasmic and apoplasmic hydraulic conductances and capacitances that determine water flows and water potential along the soil-plant-atmosphere continuum. The model computes the leaf transpiration, driven by leaf-air VPD, its regulation by stomatal closure and thus the variation in soil water content. Beyond the point of stomatal closure, residual leaf transpiration is maintained, leading to plant dehydration and hydraulic failure under extreme water stress. Environmental conditions were either constant (T<sub>air</sub> = 25°C, RH<sub>air</sub> = 50%, PPFD = 400 µmol m<sup>-2</sup> s<sup>-1</sup>) or variable with day/night cycles (day as above; night: T<sub>air</sub> = 20°C, RH<sub>air</sub> = 90%, PPFD = 0 µmol m<sup>-2</sup> s<sup>-1</sup>; day as above). In each simulation, the stomatal conductance's, g<sub>night</sub>, g<sub>day</sub> and g<sub>min</sub> were varied one at a time and the total leaf area was maintained constant to avoid any effect of this variable on dehydration. The modeling takes boundary layer effects into consideration (Cochard et al., 2020). The simulations were performed at a time step of 0.01 s and stopped when two different thresholds of water stress were reached in the leaf apoplasm: (a) the time needed to reach hydraulic failure (THF) corresponding to a total loss of conductivity (100% PLC), and (b) the time needed to reach a soil relative water content (RWC) of 20%.

**2.9 | Statistical analyses**

Data were analyzed by a one-way ANOVA using the general linear model procedure for completely randomized design. Means were compared by Fisher's LSD test (p ≤ .05), and significant interactions between treatments are indicated by letters and described in the text. Time series data, such as gas exchange values, were fitted using linear

mixed-effect models and Hotelling's multiple test (p ≤ .05) for comparison of means between treatments. Statistical analyses and fit were performed using R software (http://www.R-project.org) and Infostat version 2017 for Windows (Grupo InfoStat, FCA, Universidad Nacional de Córdoba, Argentina. URL http://www.infostat.com.ar).

**3 | RESULTS**

**3.1 | Dynamics of stomatal conductance and transpiration**

Stomatal conductance (g<sub>night</sub>) and transpiration (E<sub>night</sub>) measured at night showed constant, minimal rates from 20:00 to 00:00 (solar time) with a significant increase at 02:00 for the three cultivars (Figure 1b, e). Mean g<sub>night</sub> ranged from 8 to 17 mmol m<sup>-2</sup> s<sup>-1</sup> in Grenache and Syrah and from 15 to 25 mmol m<sup>-2</sup> s<sup>-1</sup> in Semillon (Figure 1 inset B') while E<sub>night</sub> mean values ranged from 0.1 to 0.22 mmol m<sup>-2</sup> s<sup>-1</sup> in Grenache and Syrah and 0.25 to 0.3 mmol m<sup>-2</sup> s<sup>-1</sup> in Semillon (Figure 1 inset E'). For both variables, Semillon had significantly higher nighttime rates that were about two times higher than Grenache and Syrah (Figure 1 inset B', E').

Since g<sub>day</sub> and E<sub>day</sub> values are constantly changing across each day, the choice of these values in calculating a night/day ratio is somewhat subjective. As an alternative approach, we calculated multiple night/day ratios using maximum daytime values across the whole day (Figure S2). When plotting all the data estimated from these ratios we observed that the overall variation of g<sub>night</sub>/g<sub>day</sub> ranged from 8 to 15% and that of E<sub>night</sub>/E<sub>day</sub> from 5 to 13% with no significant differences between cultivars (Figure 2).

**3.2 | Potential drivers of E<sub>night</sub>**

A significant positive relationship between transpiration and VPD<sub>leaf-air</sub> was observed for the three cultivars (Figure 3). This correlation was significant (p < .0001) when plotting all E data during the day and night at different dates during the season. When correlating only E<sub>night</sub> values to VPD<sub>leaf-air</sub> no significant relationship was obtained for any cultivar (space delimited by dotted lines in Figure 3).

To further explore possible drivers of E<sub>night</sub> we examined the variation of E<sub>night</sub> and g<sub>night</sub> along different times during the night (from 20:00 to 02:00) and we observed that not all the values were the same in magnitude despite radiation being 0 µmol m<sup>-2</sup> s<sup>-1</sup>. For example, when comparing “early night” (20:00) and “late night or predawn” (02:00) stomatal conductance, pre-dawn g<sub>night</sub> was significantly higher than early night g<sub>night</sub> for the three cultivars (Figure 4). Differences between pre-dawn and early night g<sub>night</sub> was significantly higher in Semillon followed by Syrah and finally Grenache (Figure 4). This increase at 02:00 was not associated with increases in VPD<sub>leaf-air</sub> which decreased progressively across the nighttime hours (Figure S3).
<figure><img src="image_3.png" /><figcaption>**FIGURE 1** Leaf stomatal conductance ($g_s$) and transpiration (E) before and during sunset (a, d) at night (b, e) and before and after sunrise (c, f) in potted grapevines of Grenache (Gre), Semillon (Sem) and Syrah (Syr). $n = 4$. Inset panels (B') and (E') correspond to the same data than in panels (b and e) respectively, but with a zoomed range on y axis (0 to 32 mmol m⁻² s⁻¹) for better visualization. Values are means of six different dates (DOYs 186, 193, 204, 206, 215 and 221). Different lower-case letters indicate statistically significant differences between cultivars at $p \le .05$ by Hotelling's test. Light grey area represents the light intensity (PPFD; µmol m⁻² s⁻¹) [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

## 3.3 | Relationship between $g_{\text{night}}$ and daytime productivity variables

Significant positive relationships between maximum stomatal conductance, photosynthesis and total leaf area (Max $g_s$, Max $P_n$ and TLA) were observed with $g_{\text{night}}$ at pre-dawn (Figure 5). However, no correlations were observed when correlating other values of $g_{\text{night}}$ (i.e., measured at 20:00, 22:00 or 00:00) with any of these daytime variables (data not shown). Semillon showed the highest values relative to Grenache and Syrah in all correlations.

We also investigated the reduction of nighttime transpiration on plant water status by enclosing the plants overnight and measuring the pre-dawn ($\Psi_{\text{PD}}$) and midday ($\Psi_{\text{leaf}}$) water potentials the following day. Two of the three cultivars evaluated, Semillon and Syrah, showed significantly more negative $\Psi_{\text{PD}}$ values than Grenache (Figure 6a, unbagged plants). When $E_{\text{night}}$ was stopped (via bagging the plants overnight) bagged plants all exhibited identical average $\Psi_{\text{PD}}$ (Figure 6a, bagged plants) suggesting that the unbagged differences in $\Psi_{\text{PD}}$ reflected differences in $E_{\text{night}}$. Bagging also reduced daytime leaf water potential for all cultivars but differences in midday $\Psi_{\text{leaf}}$ between bagged and unbagged plants were not significant (Figure 6b).

## 3.4 | Minimum conductance and overnight stomata observations

The leaf minimum conductance ($g_{\min}$) presented mean values of around 9.5 µmol m⁻² s⁻¹ with no differences among cultivars as previously reported (Dayer et al., 2020; Table S1).

When nighttime stomatal opening was assessed visually by cryo-SEM we observed variability in the extent to which the stomata were closed, and additionally, the extent to which the leaf cuticle covers the stomatal aperture (Figure 7). For some stomata, the guard cells appeared slightly open (Figure 7a), while for others they were clearly closed (Figure 7b). However, there was an additional state commonly observed where the cuticle completely covers the stomatal opening (Figure 7c).

## 3.5 | Modelling time to 20% of soil relative water content and to hydraulic failure

Using the “SurEau model” and the data collected in this study allowed us to compare the contribution of different conductance’s ($g_{\text{night}}$, $g_{\min}$,
<figure><img src="image_4.png" /><figcaption>**FIGURE 2** Variability of the night to day stomatal conductance ratio (a) and of the night to day transpiration ratio (b) expressed in percentage in potted Grenache (Gre), Semillon (Sem) and Syrah (Syr) grapevines. (n = 4). Night values are the mean of measurements taken at 22:00, 00:00 and 02:00 whereas day values correspond to measurements taken during from 07:00 to 16:00 with saturating levels of radiation (>800 µmol m⁻² s⁻¹). Data include values from all DOYs measured during the season (11 dates) [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

or g<sub>day</sub>) in controlling water loss and changing the time to reach partic-
ular thresholds during a simulated drought. We observed that when
considering a small rooting volume g<sub>min</sub> had a stronger influence on
the THF than g<sub>night</sub> or g<sub>day</sub> (Figure 8a). For example, a decrease in g<sub>min</sub>
by 60% increases the THF by nearly 100%, when reductions of similar
magnitude in g<sub>night</sub> or g<sub>day</sub> have almost no impact (Figure 8a). In terms
of days, when keeping g<sub>day</sub> and g<sub>night</sub> constant, decreasing g<sub>min</sub>
increased the simulated THF from 5 to 30 days (Figure S4A). In con-
trast, when keeping g<sub>day</sub> and g<sub>min</sub> constant, the impact of changing
g<sub>night</sub> was not significant, and THF remained at 7 days at any value of
g<sub>night</sub> (0–50 mmol m<sup>-2</sup> s<sup>-1</sup>). These results changed when using a larger
rooting volume, where g<sub>day</sub> became much more important than g<sub>min</sub> in
determining the THF (Figure 8b; Figure S4B). When looking at the
time to reach 20% of soil RWC we observed that g<sub>day</sub> was more
important in determining the time to reach this threshold, indepen-
dent of the rooting volume (Figure 8c,d; Figure S4C,D). In any

<figure><img src="image_5.png" /><figcaption>**FIGURE 3** Response of leaf transpiration (E) to increasing leaf-air vapour pressure deficit (VPD<sub>leaf-air</sub>) in three grapevine cultivars (Grenache, Semillon and Syrah). Each dot is a single value registered with an InfraRed Gas Analyzer at different dates and hours (day and night) during the season. The VPD<sub>leaf-air</sub> was calculated using the leaf temperature registered by TinyTag probes. Significant linear relationships were observed for each cultivar E(Gre) = 0.572 VPD<sub>leaf-air</sub> - 0.301; E(Sem) = 0.964 VPD<sub>leaf-air</sub> - 0.503; E(Syr) = 1.024 VPD<sub>leaf-air</sub> - 0.808. Nighttime transpiration (from 20:00 to 02:00 solar time) corresponds to the values delimited by the dotted black lines [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**FIGURE 4** Nighttime stomatal conductance measured at 20:00 (early night) and at 02:00 (pre-dawn) the previous night, in three grapevine cultivars (Grenache, Semillon and Syrah). The data include different nights of the season, that is, DOYs 185, 186, 193, 205, 206, 215, 216, 234, 235. Different lower-case letters indicate statistically significant differences between g<sub>s</sub> measured at 20:00 and 02:00 solar time for each cultivar at *p* ≤ .05 by Fisher's LSD [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

situation considered by the model, varying g<sub>night</sub> did not have a signifi-
cant impact on the time to reach the THF or the 20% of soil RWC
under water stress.
<figure><img src="image_7.png" /></figure>

**FIGURE 5** Correlations of maximal conductance (Max g<sub>s</sub>; a) maximal photosynthesis (Max P<sub>n</sub>; b) and Total Leaf Area (TLA; c) to the stomatal conductance measured before dawn at 02:00 solar time (Pre-dawn g) in three grapevine cultivars (Grenache, Semillon and Syrah). Values are means ± SE (n = 4). A single relationship was fitted in each panel for all the cultivars since no significant differences were obtained among them: In (a) [Max g<sub>s</sub> = -0.438 Pre-dawn g<sup>2</sup> + 20.993 Pre-dawn g - 32.765; r<sup>2</sup> = 0.73], in (b) [Max P<sub>n</sub> = -0.006 Pre-dawn g<sup>2</sup> + 0.511 Pre-dawn g + 3.391; r<sup>2</sup> = 0.40] in (c) [TLA = 0.005 Pre-dawn g + 0.068; r<sup>2</sup> = 0.80] [Colour figure can be viewed at wileyonlinelibrary.com]

<figure><img src="image_8.png" /></figure>

**FIGURE 6** Effect of nighttime bagging of potted Grenache (Gre), Semillon (Sem) and Syrah (Syr) vines on pre-dawn (Ψ<sub>PD</sub>; a) and midday (Ψ<sub>leaf</sub>; b) leaf water potentials. The plants were bagged (filled symbols) after sun-set and left overnight until the Ψ<sub>PD</sub> was measured the next day at pre-dawn. Unbagged plants (empty symbols) were used as controls. Values are means ± SE (n = 4). Different lower-case letters indicate statistically significant differences between bagged and unbagged plants for each cultivar at p ≤ .05 by Fisher's LSD [Colour figure can be viewed at wileyonlinelibrary.com]

suggested that nighttime water loss was relatively low compared to daytime transpiration and reductions in soil and plant water potentials were mainly explained by g<sub>day</sub> and E<sub>day</sub>. The drivers of E<sub>night</sub> were not the same climate variables as previously reported for E<sub>day</sub> (i.e., VPD) and possible effects of circadian regulation were observed. Pre-dawn g<sub>night</sub> was correlated with maximum gas exchange and total leaf area in the three cultivars suggesting a potential benefit of increased g<sub>night</sub> on growth. Overall, g<sub>night</sub> did not represent a key trait in exacerbating water stress and further hypotheses need to be explored to test its potential benefit for the plant.

4 | DISCUSSION

In this study, we investigated the regulation of nighttime transpiration, its contribution to whole plant water use, and its potential benefits and/or consequences. This is the first work to assess the relative importance of different conductances (g<sub>day</sub>, g<sub>night</sub>, and g<sub>min</sub>) in exacerbating water stress and the risk of hydraulic failure. Our results

4.1 | Quantification of E<sub>night</sub> and g<sub>night</sub>

Daily dynamics of transpiration and stomatal conductance revealed that the loss of water at night and the proportion of this loss relative to daytime values were rather low in grapevine for the three cultivars examined. The night/day ratios of E and g<sub>s</sub> were calculated considering different values of E<sub>day</sub> as the rate of transpiration is a dynamic
<figure><img src="image_9.png" /><figcaption>**FIGURE 7** Cryo-SEM microscopy images of stomata slightly open (a), closed with cuticles open (b) and completely closed (c) on the abaxial side of leaf vines after 12 hr spent under dark conditions. Bar scale = 10 µm</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**FIGURE 8** Simulated reduction of stomatal conductance's ($g_x$ = $g_{day}$, $g_{night}$ or $g_{min}$) and the corresponding increase of the time to hydraulic failure (THF; a, b) and to 20% soil relative water content (0.2 RWC; c, d) in 7 L and 70 L rooting volumes under the progression of a drought. Data correspond to a “mean” grapevine cultivar constructed by pooling the data of Grenache, Semillon and Syrah [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

variable that changes significantly across each day according to the environmental conditions (e.g., light, VPD, etc.). The vast majority of the time these ratios never exceeded 15% in any cultivar (Figure 2) which falls within the lower end of the range reported in the literature from 5 to 15%, and up to 30% in some species (Caird, Richards, & Donovan, 2007; Daley & Phillips, 2006). For grapevine specifically, a range, across different grapevine cultivars and studies, from 0.05 to 0.46 mmol m⁻² s⁻¹ for $E_{night}$, and 5 to 40 mmol m⁻² s⁻¹ for $g_{night}$, have been reported (Table S2). Only three of these studies measured $E_{day}$ and the reported $E_{night}/E_{day}$ ratios were no higher than 15% in any case (Coupel-Ledru et al., 2016; Rogiers et al., 2009; Rogiers & Clarke, 2013). In addition, in most of these studies, experiments were

performed in greenhouses where the VPD is difficult to control at night, generally remaining higher relative to natural conditions.

Regarding differences between cultivars, Semillon exhibited higher $E_{night}$ and $g_{night}$ values than Grenache and Syrah, similar to previous observations under field conditions (Rogiers et al., 2009). Despite these differences, the dynamics performed before/during sunset, at night and before/after sunrise in this study (Figure 1) showed that in addition to the absolute nighttime values it is also relevant to observe the timing and speed at which stomata open and close, which can vary significantly across species (Lawson & Vialet-Chabrand, 2019). In this study, we observed a higher rate of $g_s$ in Semillon relative to Grenache after sunrise (Figure 1c), although we
cannot make conclusions about the mechanisms leading to these dif-
ferences. It is important to note that the stomatal density (number per
mm of leaf and per leaf area) was not significantly different across the
three cultivars studied (data not shown).

## 4.2 | Drivers of E$_{night}$: VPD Or circadian regulation?

In a recent study, we reported that E$_{day}$ was mainly driven by VPD in the same three grapevine cultivars (Dayer et al., 2020). While daytime VPD$_{leaf-air}$ can be as high as 4–5 kPa or even more under extreme events such as heat waves, nighttime VPD$_{leaf-air}$ is typically much lower. In the current study, most of nighttime VPD$_{leaf-air}$ values ranged from 0.8 to 1.8 kPa, with the exception of the night of 24th July (DOY 205) where an episode of extreme heat led to VPD$_{leaf-air}$ values as high as 2.5 kPa registered at 20:00 solar time (leaf temperature was 32°C and air relative humidity 48%). It is important to highlight that July 2019 has been the hottest month in this region (Bordeaux) ever recorded, with a daytime temperature of 41.6°C on the 23rd, meaning that our nighttime VPD values were not underestimated. Despite the positive correlation between leaf E and VPD$_{leaf-air}$ for all data (including daytime and nighttime values) we did not observe any significant relationship between E$_{night}$ and VPD$_{leaf-air}$ (Figure 3). An absence of any relationship between temperature and VPD, and nighttime transpiration or stomatal conductance has been reported for other species (Barbour et al., 2005; Resco de Dios et al., 2015).

Examining the regulation of g-night as a dynamic across the night-
time hours we observed an increase during pre-dawn for most nights
and cultivars (Figure 4) and this increase was not associated with
increases in VPD<sub>leaf-air</sub>. In fact, VPD<sub>leaf-air</sub> decreased progressively until
pre-dawn. According to these observations, it seems unlikely that the
increase of g-night from early night to pre-dawn was driven by VPD<sub>leaf-
air</sub>. A possible explanation is that temporal changes in g-night appear to
be driven partly or perhaps largely by the circadian clock (Bucci
et al., 2004; Caird, Richards, & Donovan, 2007; Resco de Dios
et al., 2013, 2015). It is well known that the circadian clock regulates
other key traits for plant fitness, including seed germination, gas
exchange, growth, and flowering among others (Dodd et al., 2005).
However, given that there is no photosynthesis at night; water loss
would be detrimental, unless there is an unknown benefit underlying
this mechanism and/or if it is necessarily linked to other advantageous
traits.

## 4.3 | Benefits and/or consequences of g$_{night}$

To gain insight into the potential benefits and/or consequences of
nighttime water loss by open stomata we correlated early night and
pre-dawn g$_{night}$ against daytime variables related to productivity. In
the current study, Semillon exhibited the highest E$_{night}$ and g$_{night}$, con-
sistent with previous studies (Rogiers et al., 2009; current study), and

also showed the highest values in these correlations relative to
Grenache and Syrah. Thus, high E$_{night}$ and g$_{night}$ may be necessarily
linked to mechanisms by which intraspecific variation in plant carbon
gain occur. However, Semillon (and also Syrah) also showed a more
negative pre-dawn water potential than Grenache which could be
associated with a higher nighttime water loss, and these differences
were translated to some extent into more negative daytime Ψ$_{leaf}$ as
well (Figure 6). In addition, the correlations between g$_{night}$ at
pre-dawn and daytime max P$_{n}$ and g$_{s}$ appeared to level off. Thus, we
can hypothesize that higher pre-dawn g$_{night}$ values displayed by some
genotypes involve a benefit and a consequence; this is higher daytime
gas exchange (and productivity) at the expense of more negative
pre-dawn and midday water potentials (and soil–plant Ψ$_{PD}$
disequilibrium).

Potential effects of g$_{night}$ in enhancing growth could be mediated
indirectly by priming stomatal opening and photosynthesis. Genetic
variation in the capacity for anticipating sunrise translates into differ-
ences in carbon uptake and growth in some species (Resco de Dios,
Loik, Smith, Aspinwall, & Tissue, 2016). A faster stomatal opening
before dawn would accelerate the plant response to radiation, short-
ening the time needed to reach optimum conductance and carbon
gain by photosynthesis (Bucci et al., 2005; Caird, Richards, &
Donovan, 2007; Dawson et al., 2007). Consistent with this, we
observed an earlier stomatal opening in Semillon relative to Grenache
which may support its higher rates of maximum g$_{day}$ and Pn$_{day}$
(Figure 1c).

Alternative hypotheses regarding the benefits of g$_{night}$ have been
proposed in the literature such as enhanced nutrient uptake or nutri-
ent distribution to distal parts of the plant (Scholz et al., 2007), the
delivery of dissolved oxygen to woody tissues (Daley &
Phillips, 2006), and/or the prevention of an excess of turgor in species
with very negative leaf osmotic potentials (Donovan et al., 2001).
Nevertheless, a recent study across numerous species including
grapevine did not support any of these hypotheses, but instead
offered more support for the priming hypothesis discussed in the pre-
vious paragraph (Resco de Dios et al., 2019).

## 4.4 | The significance of minimum leaf conductance

The minimum leaf conductance includes two pathways; one across
the cuticle and the other through the incompletely closed stomata
(Duursma et al., 2019). While the cuticular component has received
much more attention in the literature, g_min is in general not directly
quantified. In this study, mean values of g_min measured by the MLD
technique were around 10 mmol m⁻² s⁻¹ for the three cultivars. This
value was very close to the values measured with the IRGA at night
for Grenache and Syrah (Figure 1b) indicating that g_min can be as high
as g_night. A similar finding has been reported for oaks where g_night and
g_min had comparable rates (Cavender-Bares, Sack, & Savage, 2007). In
that study, nighttime transpiration was reduced to a minimum under
drought through stomata closure, however, water loss through the leaf cuticle continued (Cavender-Bares et al., 2007).

In other studies, a wide range of variation in gmin across geno-
types has suggested that this trait cannot be easily explained by other
leaf traits or by the cuticle structure (Duursma et al., 2019). Genetic
variation in nighttime transpiration has been considered a relevant
drought tolerance trait in grapevine, but without considering the con-
tribution of gmin in explaining these differences (Coupel-Ledru
et al., 2016). However, the reported genetic differences in transpira-
tion in that study were assessed under well-watered and extremely
mild water deficit conditions (soil water potentials of -0.15 MPa;
Coupel-Ledru et al., 2016), rather than under the more negative water
potentials that grapevines regularly encounter in the field. Our study
suggests that during drought gmin is significantly more important.
Additional studies that include a larger range of stress levels are there-
fore needed to explore the contribution of gmin to drought tolerance
differences across genotypes.

4.5 | Time to 20% of soil RWC and THF: Contribution of different conductances

Simulations of the contribution of different conductances to the time to reach 20% of soil RWC or hydraulic failure (THF) varied according to the threshold and soil volume considered. The finding that gmin had a significantly higher contribution than gnight and gday in determining THF in small soil volumes (Figure 8a) indicated that gmin is a cornerstone trait that should be assessed in drought stress experiments and included in plant models (e.g., Zhu et al., 2018). Other studies have also emphasized the importance of gmin in controlling plant water potential decline after the stomata close and in determining thresholds of hydraulic failure (Brodribb, Powers, Cochard, & Choat, 2020; Duursma et al., 2019; Martin-StPaul et al., 2017). When considering the THF under higher soil volumes (e.g., in a vineyard), gday becomes more important than gmin, although both have a much more significant impact than gnight (Figure 8b). This result reinforces the observation that gnight has a very small impact in defining the time to reach mortality under extreme drought.

When looking at a more agronomic context, time to 20% soil RWC, gday is the most important variable and both gnight and gmin are insignificant in any soil volume (Figure 8c,d). This is a relevant finding since a reduction to 20% of the soil RWC is a frequent situation in most of vineyards suggesting that in a production setting it is gday that should be considered in estimating the overall plant water use. Our results challenge many of previous studies in grapevine that highly emphasize gnight as an important source of water loss affecting whole plant water balance and water use efficiency (Coupel-Ledru et al., 2016; Fuentes et al., 2013; Fuentes et al., 2014). In our study, simulations suggested that gnight was not as significant of a factor when compared to gday and gmin. Furthermore, eliminating Enight by bagging the plants did not significantly attenuate subsequent midday values of Ψleaf (Figure 6b). These observations suggested that gday and Eday were the predominant factors affecting daytime Ψleaf.

5 | CONCLUSIONS

This study evaluated the relative significance and potential benefits and/or consequences of nighttime water loss in different grapevine cultivars. To our knowledge, this is the first study that evaluates the relative importance between different conductances (gday, gnight and gmin) to whole plant water-use. Overall, the results of this study indicated that gnight cannot be considered a trait that exacerbates water stress within the cultivars examined here, and a much more important role was observed for gday and gmin. The potential benefits of higher nighttime stomatal conductance need to be explored further to test whether gnight increases growth and if so, whether this is driven by stomatal priming or other mechanisms. Finally, an important contribution of gmin was observed in determining the magnitude of hydraulic failure, stressing the importance of including this variable in drought experiments and water relations models.

ACKNOWLEDGMENTS

The authors thank the technical team of UMR Ecophysiology and Functional Genomic of Grapevine (INRAE, Bordeaux, France) for providing the plant material and logistics assistance at the "Serre Bordo" platform. This work was supported by the EU, ERA-NET, ARIMNET2 project, Opportunities for an Environmental-friendly Viticulture: EnViroS. This work was also supported by the "Investments for the Future" (ANR-10-EQPX-16, XYLOFOREST) program and the Cluster of Excellence COTE (ANR-10-LABX-45) of the French National Agency for Research, and the French Ministry of Agriculture, Agrifood, and Forestry (FranceAgriMer and CNIV) within the PHYSIOPATH project (program Plan National Dépérissement du Vignoble, 22001150-1506). Minimum leaf conductance measurements were performed at PHENOBOIS platform (Université de Bordeaux/INRAE, Pessac, France). This work is also conducted as part of the LIA INNOGRAPE International Associated Laboratory.

ORCID

Silvina Dayer ID https://orcid.org/0000-0001-5794-1600

José Carlos Herrera ID https://orcid.org/0000-0001-9532-5809

Zhanwu Dai ID https://orcid.org/0000-0002-7625-8337

Laurent J. Lamarque ID https://orcid.org/0000-0002-1430-5193

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## SUPPORTING INFORMATION

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

**How to cite this article:** Dayer S, Herrera JC, Dai Z, et al. Nighttime transpiration represents a negligible part of water loss and does not increase the risk of water stress in grapevine. *Plant Cell Environ.* 2020;1-12. https://doi.org/10.1111/pce.13923