Does night‐time transpiration provide any benefit to wheat (Triticum aestivum L.) plants which are exposed to salt stress?

Abstract

The study aimed to test whether night‐time transpiration provides any potential benefit to wheat plants which are subjected to salt stress. Hydroponically grown wheat plants were grown at four levels of salt stress (50, 100, 150, and 200 mM NaCl) for 5–8 days prior to harvest (day 14–18). Salt stress caused large decreases in transpiration and leaf elongation rates during day and night. The quantitative relation between the diurnal use of water for transpiration and leaf growth was comparatively little affected by salt. Night‐time transpirational water loss occurred predominantly through stomata in support of respiration. Diurnal gas exchange and leaf growth were functionally linked to each other through the provision of resources (carbon, energy) and an increase in leaf surface area. Diurnal rates of water use associated with leaf cell expansive growth were highly correlated with the water potential of the xylem, which was dominated by the tension component. The tissue‐specific expression level of nine candidate aquaporin genes in elongating and mature leaf tissue was little affected by salt stress or day/night changes. Growing plants under conditions of reduced night‐time transpirational water loss by increasing the relative humidity (RH) during the night to 95% had little effect on the growth response to salt stress, nor was the accumulation of Na⁺ and Cl⁻ in shoot tissue altered. We conclude that night‐time gas exchange supports the growth in leaf area over a 24 h day/night period. Night‐time transpirational water loss neither decreases nor increases the tolerance to salt stress in wheat.

1. INTRODUCTION

Plants take up water through the root system during the day and night. Most, if not all, the water taken up during the night is stored in the plant to replenish water resources, repair xylem embolism or minimize the chance of embolism during the subsequent day in some plant species (for review and discussion, see Caird et al., ²⁰⁰⁷; Dawson et al., ²⁰⁰⁷; Zeppel et al., ²⁰¹⁹). However, under most environmental conditions and in most plant species, particularly crops, the bulk of water taken up during the night is lost through transpiration (for reviews, see Caird et al., ²⁰⁰⁷; Fricke, ^2019a). This night‐time transpiration is <1%, yet in many cases, accounts for 5%–15% of daytime rates of transpirational water loss (Caird et al., 2007; Dayer et al., ²⁰²¹; Resco de Dios et al., ²⁰¹⁵; Yoo et al., ²⁰⁰⁹); it can also account for 50% and more in some genotypes (for review, see Caird et al., ²⁰⁰⁷; Coupel‐Ledru et al., ²⁰¹⁶; Rogiers & Clarke, ²⁰¹³; Schoppach et al., ²⁰¹⁴). Considering that night‐time transpiration causes water loss without carbon gain, one may argue that it should be a feature to breed against, not for (e.g., grapevine (Vitis vinifera L.), Coupel‐Ledru et al., ²⁰¹⁶; Dayer et al., ²⁰²¹). However, the mere existence of night‐time transpiration in today's plants could point to some evolutionary advantage unless it is an inevitable consequence of an inherent background permeability to water of the cuticle. Furthermore, as plants grow and leaf surface area increases, so do the capacity to lose water through transpiration and to assimilate and respire CO₂ from mature leaf tissue. There must be some functional link between the diurnal loss of water and CO₂ gas exchange and leaf area expansion (Figure 1).

FIGURE 1.

Linking diurnal gas exchange in mature to cell expansion in growing leaf tissue in a grass such as barley and wheat. Transpirational water loss during the day in the mature leaf zone (MZ) is a consequence of photosynthetic CO₂ assimilation; O₂ is also released. Some of the carbon is exported to the elongation zone (EZ), which is located near the root/shoot transition, and where cell expansion leads to an increase in leaf surface area. During the night, cells continue to expand and use up carbon and energy which have been provided through mature leaf tissue, while the latter respires residual resources to meet demands of base metabolism and possibly net biosynthesis of compounds. This respiration causes CO₂ to build up to potentially cytotoxic levels, and stomata need to open to let the CO₂ escape, and O₂ to enter, at sufficiently high rates. The result is night‐time transpirational water loss. Mature and elongation zone affect each other in positive and negative ways in their capacity to carry out gas exchange (MZ) and grow (EZ) during day and night. Day‐time transpiration also causes large tensions in the xylem, which may negatively affect cell expansion, as it lowers the water potential (ψ) of xylem and may make it harder for elongating cells to take up water. The aim of the present study was to make a detailed quantitative analysis of the processes shown, also in relation to salt stress, and to test to which extent night‐time transpirational water loss leads to significant tensions in the xylem. Does night‐time transpirational water loss provide any benefit(s) to plants when exposed to salt stress? The picture shows a 16 days‐old barley plant, and the shoot is about 20 cm tall; RGR, relative growth rate.

Plants produce new cells as they grow and develop, but the irreversible expansion of individual cells is the main means of achieving growth in size. Cell expansive growth is the prime mechanism through which water taken by the root system is stored within the plant. Leaves grow at night by almost as much, or more, than during the day (Poiré et al., 2010; Rozema et al., ¹⁹⁸⁷; Waldron et al., ¹⁹⁸⁵). Depending on the developmental stage of leaves and environment, cell expansive growth can be limited primarily metabolically (night) or hydraulically (day) (Tardieu et al., 2010). Night‐time cell expansive growth requires energy and carbon, which is acquired through day‐time processes (photosynthesis). This explains why there can be an effect of the provision of storage carbohydrates during the day on leaf growth rates during the subsequent night (Graf et al., 2010; Mora‐Garcia et al., ²⁰¹⁷; Scialdone & Howard, ²⁰¹⁵; Smith & Stitt, ²⁰⁰⁷).

As night‐time leaf growth provides an increase in leaf surface area, it will affect the capacity of plants to carry out photosynthesis during the day and provision of carbohydrates to growing tissue (Figure 1). In addition, as the leaf area increases, so should also the respiratory activity of the mature part of the shoot during the night. This respiratory activity has been proposed to be the main driver of night‐time transpirational water loss. The permeability of the cuticle to CO₂ is a factor 10⁴ lower than the permeability to water (vapor) (Boyer, 2015; Boyer et al., ¹⁹⁹⁷). Therefore, stomata need to open to allow respiratory CO₂ to escape at sufficiently high rates to avoid cellular acidosis (Marks & Lechowicz, 2007; Even et al., ²⁰¹⁸). It is evident from the above that mature and expanding leaf tissues must communicate their capacity to grow (expanding tissue) and perform gas exchange (mature blade) to each other during day and night (Figure 1). A thorough quantitative assessment of the processes involved is lacking.

Salinity reduces the rates of CO₂ assimilation, transpirational water loss and leaf expansion during the day in a range of crops (for review, see Munns & Tester, ²⁰⁰⁸). It is unclear whether salinity causes similar, combined reductions during the night in these sizes and whether water loss through stomata and cuticle are affected similarly. Growing leaf cells need to take up water from the xylem. Xylem ψ is less negative during the night compared with day, and this should make it easier for growing cells to take up water and expand. If so, a higher portion of shoot‐delivered water could be used in support of leaf growth during the night. How significant would this be in terms of the total use of water by plants? Furthermore, glycophytic plants such as barley and wheat, which are exposed to salt stress, need to take up water from a low water potential (ψ) root environment while minimizing the uptake of Na⁺ and Cl⁻ to potentially cytotoxic levels. The biophysical force that drives the uptake of water from the root environment into the xylem has a tension and osmotic component; if the latter acted alone, for example, during the night, it would require concentrations of salt in the xylem which are at least as high as in the root environment. Xylem tension associated with day‐time water loss is significant. Could it be that xylem tension also exists as a consequence of night‐time water loss through stomata and, through this, enables root water uptake during the night under salt stress? Xylem tensions drive the uptake of Na⁺ and Cl⁻ along apoplastic bypasses (Faiyue, Al‐Azzawi & Flowers, 2010) across the root cylinder. Having xylem tensions during the night would mean that such driving forces act throughout the 24‐day/night period.

We addressed the following questions on hydroponically grown wheat plants and in relation to salt stress:

1. Does night‐time water loss occur primarily through stomata?

2. Can night‐time water loss from the mature blade be related to CO₂ gas exchange?

3. How significant is the portion of water delivered to the shoot and stored through leaf growth during the day and night in relation to transpirational water loss?

4. Is night‐time transpiration associated with significant tension in the xylem?

5. Does night‐time transpiration provide any potential benefit to plants in a saline environment?

To test the above, we grew wheat plants hydroponically in a controlled environment under a 16/8 h day/night cycle. Plants were exposed to four different concentrations of NaCl (50, 100, 150, and 200 mM) when they were 10 days old and harvested when they were 14–18 days old. Plants were analyzed for transpirational water loss, leaf growth, gas exchange and the ψ and tension of leaf xylem during the day and night period. Night‐time transpirational water loss was altered experimentally by growing plants also under conditions of high (95%) relative humidity (RH) during the night. Shoot concentrations of Na⁺ and Cl⁻ were analyzed, as was the gene expression level of nine candidate aquaporins (AQP) genes. The latter analyses were carried out to obtain some information on potential molecular players which are involved in the regulation of diurnal water flow (Maurel et al., 2015) in growing and mature leaf tissue and in response to salt.

2. MATERIALS AND METHODS

2.1. Plant material and growth conditions

Wheat (Triticum aestivum L. Cv. Han6172) plants were grown on modified half‐strength Hoagland solution in a growth chamber (Microclima, MC1000HE, CEC Technology) as described previously (Knipfer & Fricke, 2011). The root medium was aerated. Plants grew at a day/night length of 16/8 h and temperature of 21/18°C. Relative humidity was 75%. The photosynthetically active‐radiation at plant level was 320–350 μmol m⁻² s⁻¹. The vapor pressure difference (VPD) was 0.516 kPa at night and 0.622 kPa during the day. Plants were germinated for 7 days in CaSO₄ solution (0.5 mM), before being transferred to the control nutrient solution (day 7), which contained 0.5 mM NaCl (to compare “high” with “low”, rather than “no” salt treatments). Salt stress was applied when plants were 10 days old by adding stock NaCl solutions to reach a final concentration of 50, 100, 150, and 200 mM. Plants were analyzed when they were 14–18 days old (control plants, 14–15 days; salt‐stressed plants, 15–18 days). Leaf two was the main photosynthesizing and transpiring leaf, while leaf three was the main growing leaf. We assumed that salt stress did not affect the length (ca 35 mm) of the elongation zone of leaf three (barley: Fricke & Peters, ²⁰⁰²; wheat: Hu & Schmidhalter, ²⁰⁰¹).

We also grew plants under conditions where the RH increased from the usual 75% to 95% during the night, and the VPD reduced from 0.516 to 0.103 kPa. The aim was to reduce plants' transpirational water loss during the night.

The osmotic pressure (π), equaling the negative of ψ, of growth media was determined with a VAPRO (Wescor Inc.) osmometer. The ψ averaged −0.040 MPa for the control nutrient solution and −0.271, −0.500, −0.733 and −0.964 MPa for the nutrient solution containing 50, 100, 150, and 200 mM NaCl, respectively.

2.2. Plant growth

At the start of the salt treatments, a set of 10 days‐old plants was analyzed to obtain a value for leaf and root surface area. Plants were analyzed 4–8 days later, with the control and lower salt treatments being analyzed at days 4–6 and the higher salt treatments being analyzed at days 7–8 to account for a slight delay in the development of plants (we wanted to have leaf three growing). Relative growth rate (RGR) was calculated as RGR = (Ln(Size‐2/Size‐1))/(t ₂ − t ₁), where Size‐2 and Size‐1 are the values obtained for the surface area at sampling point t2 and t1, the former representing the end, and the latter the start of the salt treatments. The relative growth rate presents the growth constant “k” (unit, d ⁻¹) of the general relation N _t  = N _o  × e ^{(k× t)}, with N _t , the size at time “t” and N ₀ the size at time zero.

2.3. Leaf growth

Leaf growth was assessed by measuring (ruler) the elongation rate of leaf three. Leaves were analyzed at the developmental stage when they elongated at (near‐) maximum and steady rates, which was the case when leaves had reached between 30–40% and 70–80% of their final length (also compare barley, Fricke et al., ¹⁹⁹⁷). The length of leaf three was measured on several successive days just before and after the end of the night period.

2.4. Plant transpiration and calculation of leaf conductance

Transpirational water loss was determined gravimetrically for plants in the growth chamber through continuous recording of weight loss using balances (Ohaus, SCOUT and SCOUT Pro models, Ohaus Corp.). Changes in weight were recorded every minute using computer software (sartoCollect 1.0; Sartorius; and SCOUT Pro USB interface kit with SPDC software, Ohaus Corp.). Experiments were carried out essentially as described in Even et al. (2018), except that plants were contained in 100 ml rather than 250 ml Erlenmeyer flasks to stay within the maximum weight limit of balances. Water loss by the plant setup (flask, nutrient solution, foam piece to support plant, and plant) was corrected for background water loss (everything except plant).

Leaf conductance was calculated by relating the transpirational water loss rate of a plant to the shoot surface area and then dividing the resulting figure by the difference in water vapor concentration between the outside and inside of leaf. Water vapor concentrations were calculated (Nobel, 1991) by assuming that the inside of the leaf was close to 100% relative humidity (RH); the RH of ambient air was taken as a measure of the outside water vapor concentration. The difference in water vapor concentration between leaf‐internal air spaces and outside air was 4.59 g m⁻³ during the day and 3.85 g m⁻³ during the night.

2.5. Residual transpiration

Residual transpiration measurements (Kerstiens, 1996) of detached leaves were used to determine cuticular water loss and calculate a residual (leaf) conductance (compare, Svenningsson, ¹⁹⁸⁸). Experiments were carried out essentially as described by Even et al. (2018) by recording the water loss over time from the combined, detached leaves of a plant, which had been sealed at their cut end by tape. The rate of water loss was followed for at least 6 h, and the final part of the time course (last 3 h) was used to calculate the rate of residual transpiration. The residual water loss rate was related to the leaf surface area and divided by the difference in water vapor concentration between the inside and outside of the leaf, using values for temperature and RH measured close to the detached leaves during experiments. This resulted in a residual (minimum) leaf conductance, approximating cuticle permeance (Schuster et al., 2016).

2.6. Surface area

Following transpiration analyses, the shoot of each plant was scanned (Canon 9900F model), and the scan was saved as a jpeg image file. Images were analyzed with the freely available software ImageJ (www.imagej.nih.gov/ij/). “Leaf surface area” refers here to the projected area of leaves, that is, half the combined area of the upper (adaxial) and lower (abaxial) surface of wheat leaves.

The surface area of the roots was also analyzed. Roots were stained in 0.25% Coomassie Brilliant Blue for 2–4 days prior to scanning to increase the contrast of root images.

2.7. Stomatal density

A double‐replica technique (first replica, dental impression material; second replica, clear, fast‐drying nail varnish) was used to determine the number of stomata per unit projected leaf area (stomatal density), following a procedure essentially as described in Even et al. (2018). Stomatal density was determined for the mature leaf two, halfway along the blade, on the abaxial surface.

2.8. Gas exchange analyses

A Li‐Cor 6400 portable photosynthesis analyzing system was used to determine net CO₂ assimilation rates during the day and night; the former represents the sum of photosynthetic activity and day‐time respiration and the latter the night‐time respiration. The dimension of the Li‐Cor measuring chamber was 30 × 20 mm. The CO₂ concentration in the chamber ambient air was set to 470 ppm. The photosynthetically active radiation at the plant level was around 260 μmol m⁻² s⁻¹ (growth chamber lights, no supplemental Li‐Cor light source). Between five to six recordings were obtained for one (leaf two) or two (leaves one and two) leaves combined. A picture of leaves clamped in the chamber was taken and used to determine the leaf surface area using ImageJ, and the chamber dimensions were used for calibration. Two types of measurements were carried out on mature leaf tissue. (1) The net rate of photosynthetic CO₂ assimilation was determined 14–15 h into the 16 h photoperiod by analyzing leaves halfway along the mature blade. (2) The net rate of respiratory CO₂ production was analyzed 2–3 h into the 8 h dark period, also by analyzing leaves halfway along the mature blade.

Respiratory CO₂ production was also analyzed in the dark for the isolated elongation zone of the growing leaf three. To this aim, leaf three was freed from the encircling sheaths of older leaves. The base 30 mm of the leaf was used as the elongation zone and placed onto a custom‐built piece of a photocopy transparency. This piece had dimensions made to be flush with the margins of the Li‐Cor chamber, and it had a 20 mm long and 10 mm wide slot cut into it, over which the leaf elongation zone section of two plants was placed next to each other, to increase the overall output (CO₂ consumption). The elongation zone was sealed at its cut end with tape onto the transparency. This setup made it possible to determine the gas exchange of the isolated elongation zone segment in the closed Li‐Cor chamber. It took less than a minute to remove two plants from the growth chamber to have the section of elongation zone mounted in the chamber.

2.9. Xylem tension and water potential

Xylem tension was determined with a PMS pressure chamber following the manufacturer's instructions (PMS Instrument Company). Plants were analyzed either 10–14 h into the 16‐h photoperiod or 3–6 h into the 8‐h dark period. Xylem tension was measured in the mature leaf two, within 20 s after leaf two had been severed from a plant in the growth chamber.

The pressure chamber provides a value of the hydrostatic pressure, rather than ψ, in the xylem (Boyer, 1995). The relationship between the mass flow rate through a root system and the π of collected xylem sap are hyperbolic in barley plants grown under control and salt stress conditions (Munns & Passioura, 1984). Applying this relationship to the closely related wheat and using flow rates obtained through transpiration measurements on the identical plants, which were used for pressure chamber analyses, it was possible to calculate xylem π and ψ for the plants analyzed during the light period. The π component comprised about 20% of xylem ψ in all treatments; about 80% of ψ was accounted for by the measured tension component (see File S1, sheet SH1). The transpirational water loss rates of plants analyzed during the dark period were too low to apply the same approach, as rates were not within the range of values used by Munns and Passioura (1984). Therefore, the pressure chamber was only used to determine xylem tension in detached leaves. The ψ of leaf xylem in the dark was determined through the combined use of the cell pressure probe technique and picolitre osmometry (Fricke & Peters, 2002; an osmolality of 40.75 mOsm kg⁻¹ corresponds to π of 0.1 MPa). Turgor and π were determined in epidermal cells, which were located above the veins on the abaxial surface, halfway along the expanded blade of the mature leaf two. The ψ of cells was calculated as the difference between turgor and π. Plants were analyzed in a laboratory environment under dim light (just illumination of cell pressure probe microcapillary through a cold light source).

2.10. Aquaporin gene expression

The entire elongation zone of leaf three (leaf elongation zone, LEZ) and a 4‐6‐cm long section halfway through the fully mature blade of leaf two (mature zone, MZ) of the identical plant was harvested; material from four plants was pooled to obtain one biological replicate of a LEZ and MZ sample. It was ensured that the LEZ of leaf three was freed from the younger 4th leaf developing inside leaf three. Three to four biological replicates were prepared for each plant treatment and time period. Plant material was immediately frozen in liquid nitrogen and stored at −80°C. Samples were ground (mortar with pestle), and the frozen powder was used to extract RNA with the RNeasy® Plant Mini kit (QIAGEN) following the manufacturer's instructions. The concentration and quality of RNA were determined using a NanoDrop®ND‐1000 Spectrophotometer, and integrity of RNA was verified through agarose gel electrophoresis. The RNA extract was treated with DNase I (Thermoscientific), and cDNA was synthesized using reverse transcriptase (Invitrogen/Thermo Fisher Scientific) and random hexamers. Gene expression analyses (qPCR) were carried out using a Fast SYBR® Green Master mix (Applied Biosystems/Thermo Fisher Scientific), by running samples on 96‐ well plates using an Applied Biosystems ViiA 7 Real‐Time PCR system (Life Technologies, Singapore). Gene expression data were calculated using the ΔCt method (Pfaffl, 2001). Four housekeeping genes were used as references of expression, glyceraldehyde‐3‐phosphate dehydrogenase (TaGAPDH), alpha‐tubulin (TaTUB), EST CJ705892 (TaEST) (Dudziak et al., 2020) and actin (TaACTIN). The sequence of primers and AQP genes studied is given in Supplemental File S3, as is the gene accession number. We identified candidate AQP genes of wheat, Triticum aestivum, by screening the gramene.org database and focusing on plasma membrane intrinsic proteins (PIPs), as these show generally water channel activity, particularly PIP2s (Maurel et al., 2015). We found one PIP2 AQP which was only listed for Triticum durum (TdPIP2‐2); the AQP detected in T. aestivum using primers for TdPIP2‐2 was referred to as “putative TaPIP2‐2” (PutTaPIP2‐2).

2.11. Ion analyses

The concentration of Na⁺ and Cl⁻, and their ratio were determined in shoot tissue using ion‐selective electrodes and following the manufacturer's instructions (sodium: ROSS sodium ion selective electrode, 8611BNWP, Thermo Scientific; chloride: Orion Chloride ion selective electrode, 9617BNWP, Thermo Scientific). The entire shoot of a plant was frozen, and sap was extracted through a centrifugation technique by spinning samples at 12,100g (Minispin Plus centrifuge, Eppendorf AG 22331 Hamburg, Germany) in tubes which contained a mesh insert close to the bottom to allow sap but not debris to pass.

2.12. Statistical analyses

Data were subjected to One‐way (factor: salt treatment or day/night) or Two‐way (salt treatment × day/night period) ANOVA analyses using the General Linear Model function in Minitab, followed by Tukey posthoc analyses. Values of p of Two‐Way ANOVA analyses (Figures 2, 3, 4, 5, 7, and 8) are provided in Supplemental File S1, Sheet SH5; values of three‐factor analyses (Figure 9) are provided in Supplemental File S1, Sheet SH6. Correlation and linear regression analyses were also performed using Minitab. Gauss' Law of error propagation was used to calculate the resulting standard deviation when individual means with their standard deviations were used to calculate another size (for details, see figure legends).

FIGURE 2.

Growth, transpiration, and stomatal density in wheat plants which were exposed to different concentrations of NaCl in the root medium. Plants were exposed to salt treatments when they were 10 days old and analyzed when they were 14–18 days old. (A) Relative growth rate between day 10 and harvest of root surface area and (projected) leaf/shoot surface area. (B–D) Transpiration rates per (B) plant and (C) unit (projected) leaf surface area (LSA) during day and night, and (D) the night‐rate expressed as a percentage of the day‐rate. (E) Stomatal density halfway along the blade of the mature leaf two. Results are averages and SE (error bars) of (n=) (A) 14, (B, D) 22–28, (C) 12–14, and (E) 12 plant analyses. Statistically significant (p < 0.05) differences in values between treatments for a particular time period (day or night) are indicated through different letters.

FIGURE 3.

Xylem water potential (ψ) and tension in salt‐stressed wheat. Plants were exposed to salt treatments when they were 10 days old and analyzed when they were 14–18 days old. (A) Xylem ψ and (B) tension in the mature leaf two during day and night; the ψ of the root medium is also shown. Results are averages and SE (error bars) of (n=) 4–8 plant analyses. The use of two different approaches to determine ψ in the dark (single‐cell analyses) and xylem tension in the dark (pressure chamber), and the use of different sets of plants may explain why the tension in (B) for control plants is slightly (0.04 MPa) more negative than ψ in (A). Statistically significant (p < 0.05) differences in values between treatments for a particular time period (day or night) are indicated through different letters.

FIGURE 4.

Conductance to water vapor of the shoot of wheat plants which were exposed to different concentrations of NaCl in the root medium. Plants were exposed to salt treatments when they were 10 days old and analyzed when they were 14–18 days old. (A) Total leaf conductance, which represents the sum of stomatal and cuticle conductance, was calculated from transpiration data of intact plants. (B) Residual leaf conductance, which represents cuticle conductance, was determined on detached shoots, and (C) expressed as percentage of total leaf conductance. The projected leaf surface area was used for the calculation of conductance. (D) Stomatal conductance was calculated as the difference between average values of total and residual leaf conductance. The resulting values for the day and night period were plotted against each other. The r ² and equation of the linear regression line are shown; the two sizes were correlated at p = 0.005. Results are averages and SE (error bars) of (n=) (A) 12–14 and (B) 8 plant analyses. Values in (C) were calculated by relating the average values shown in (A) and (B) to each other, and the error bars represent standard deviations based on Gauss' law of error propagation. Statistically significant (p < 0.05) differences in values between treatments for a particular time period (day or night) are indicated through different letters.

FIGURE 5.

Leaf elongation and water used of wheat plants which were exposed to different concentrations of NaCl in the root medium. Plants were exposed to salt treatments when they were 10 days old and analyzed when they were 14–18 days old. Growth was analyzed for leaf‐3, which was the main elongating leaf of plants at that developmental stage. (A) Leaf elongation rate during day and night, and (B) the night‐rate expressed as percentage of the day rate. (C) Water content of the leaf elongation zone (LEZ). (D) Water storage associated with leaf elongation growth, calculated as the numeric product of leaf elongation rate (A), water content (C) and using a total length of 35 mm for the LEZ (for details, see text) in all treatments; (A) and (C) were determined for the identical set of plants. (E) Water stored through growth expressed as a percentage of the total amount of water delivered to the shoot, the latter being the sum of water stored through growth and water lost through transpiration. Results are averages and SE (error bars) of (n=) (A–E) 12–15 plant analyses. Values in (E) were calculated by using transpiration data which had been obtained on the identical set of plants as in (A–D). Statistically significant (p < 0.05) differences in values between treatments for a particular time period (day or night) are indicated through different letters.

FIGURE 7.

The effect of growing plants at elevated relative humidity (RH during the night period on the (A) relative growth rate of shoot and (B–F) transpirational water loss. Plants were grown under the same conditions as usual (e.g., compare legend to Figure 2), except that the RH during the night was increased to 95%, causing plants to grow at 75/95% (day/night) rather than the usual 75/75% (day/night) RH. (A) Values of relative growth rates of 75/75% were taken from Figure 2A. Figures (E) and (F) show the percent changes in transpirational water loss per (E) plant and (F) leaf surface area (LSA) by growing plants at 75/95% RH rather than the usual 75/75% RH. Percent changes were calculated by comparing average values, whereas the statistical significance at p < 0.05 (*) was calculated by comparing the respective individual values. For example, in (E), the rate of plant transpirational water loss in plants treated with 50 mM NaCl was during the day significantly higher in plants grown at 75/95% RH compared with plants grown at 75/75% RH, and the rate increased on average by about 40%. Results are averages and SE (error bars), between (n=) 6–8 plants grown at 75/95% RH were analyzed. Statistically significant (p < 0.05) differences in values between treatments for a particular time period (day or night) and growth conditions are indicated through different letters and an asterisk, respectively.

FIGURE 8.

The effect of growing wheat plants at elevated relative humidity (RH during the night period on the (A–C) leaf elongation rate and (D–F) shoot ion concentrations. Plants were grown under the same conditions as usual (e.g., compare legend to Figure 2), except that the RH during the night was increased to 95%, causing plants to grow at 75%/95% (day/night) rather than the usual 75%/75% (day/night) RH. Figure (C) shows the percent changes in leaf elongation rate by growing plants at 75%/95% RH rather than the usual 75%/75% RH (compare legend of Figure 7). Percent changes which were statistically significant at p < 0.05 are indicated with an asterisk. Results are averages and SE (error bars) of between (A–C) 6–8 and (D–F) 16–48 plant analyses. Statistically significant (p < 0.05) differences in values between (A) treatments for a particular time period (day or night) and (D–F) growth conditions are indicated through different letters; the latter is also indicated through an asterisk in (C).

FIGURE 9.

Gene expression level of nine plasma membrane intrinsic (PIP) aquaporins (AQPs) in the leaf elongation zone (LEZ) of leaf three and the mature zone (MZ) of the blade of leaf two in wheat. Plants were grown throughout under control (non‐saline) conditions or were exposed to a 150 mM NaCl treatment when they were 10 days old. Plants were analyzed when they were 15 (control, CTRL) or 17 days (NaCl) old, at time when leaf three was elongating at maximum and steady rates. Results are averages and SE (error bars) of 3–4 biological replicates, each presenting the pooled sample of four plants. Statistically significant (p < 0.05) differences in values between treatments and leaf regions for a particular time period (day or night) are indicated through different letters.

3. RESULTS

3.1. Salt stress reduces the relative growth rate of shoot and root

Growth was assessed by analyzing the relative increase in the root and shoot surface area during the period between the addition of salt to the root medium and the harvest of plants. The relative growth rate of shoot and root surface area of plants grown under control conditions averaged 22–28% d⁻¹; it was little affected by the lower but was reduced increasingly and significantly by the higher salt concentrations tested (Figure 2A).

3.2. Salt stress reduces day‐ more than it reduces night‐time transpirational water loss

Plants grown under control conditions transpired water at a rate of 5.11 × 10⁻¹¹ m³ s⁻¹ during the day. This rate decreased significantly at the lowest salt concentration tested (50 mM) and reached 16% of the control level at 200 mM NaCl (Figure 2B). Night‐time transpirational water loss per plant also decreased significantly in response to NaCl, though not as much as daytime transpiration decreased (Figure 2B). The same applied to the rate of transpirational water loss per unit leaf surface area (Figure 2C). The rate of night‐time transpiration was 10.7% of the day‐rate in control plants. This percentage increased significantly and ranged from 15.4 and 16.5% at the three highest salt concentrations tested (Figure 2D).

3.3. Stomatal density increases in response to salt stress

The number of stomata per unit surface area in the fully mature and main transpiring leaf‐2 increased significantly in response to the higher salt concentrations tested (Figure 2E). This was unexpected, as salt stress reduced transpirational water loss, and can be explained with stomata had already been initiated in leaf‐2 when the salt stress was applied (Nunes et al., 2020). The subsequent reduction in leaf area expansion due to salt stress caused a smaller “dilution”, or spatial separation of stomata compared with the situation in control plants.

3.4. Xylem water potential (ψ) and tension become more negative in response to salt stress during day and night

Xylem ψ in the transpiring leaf‐2 of plants grown under control conditions averaged −0.79 MPa during the day and −0.14 MPa during the night. For comparison, the root medium ψ of control plants was −0.04 MPa (Figure 3A). As the ψ of the root medium decreased due to the addition of NaCl, so did the ψ and tension of the xylem during day and night (Figure 3A, B). In plants grown at 200 mM NaCl, xylem ψ averaged −1.97 MPa during the day and −0.99 MPa during the night; xylem tension averaged −1.75 MPa (day) and −0.70 MPa (night). The decrease in xylem ψ and tension in response to NaCl was generally significant. The difference in ψ between the root medium and the xylem was little affected by salt stress (Figure 3; compare bar height).

3.5. Day‐ and night‐time transpirational water loss occurs primarily through stomata

Total leaf conductance, which represents water loss through stomata and cuticle, was calculated from transpiration data for intact plants. Total leaf conductance averaged 6.86 × 10⁻³ m s⁻¹ in control plants during the day and decreased significantly in response to salt stress to 20.5% of the control value in plants treated with 200 mM NaCl (Figure 4A). Total leaf conductance during the night was 13% that during the day in control plants and decreased significantly too in response to salt stress (Figure 4A).

Residual transpiration experiments provided a measure of water loss through the cuticle. Residual leaf conductance changed little in response to salt stress compared with total leaf conductance. The exception was the 200 mM NaCl treatment, where residual leaf conductance decreased by almost 60% (Figure 4B). Residual leaf conductance accounted for between 1.8 and 5.0% of total leaf conductance during the day across treatments (Figure 4C). During the night, residual leaf conductance accounted for 13.8% of total leaf conductance in control plants (Figure 4C). This percentage figure increased to 19.7–26.7% for the three highest salt treatments tested (Figure 4C). As most of the leaf conductance was not accounted for by residual (cuticle) conductance, water exited leaves mainly through stomata, during day and night, and under salt stress.

Stomatal conductance was calculated as the difference between the total (Figure 4A) and residual (Figure 4B) leaf conductance. The calculated stomatal conductance during day and night was highly correlated with each other across treatments and in a positive and linear manner (Figure 4D).

3.6. Salt stress has minor effects on the relation between day‐ and night‐time leaf elongation rate

Leaf three was the main elongating leaf of plants at the developmental stage studied. The elongation rate of leaf three averaged 2.08 mm h⁻¹ during the day and 1.34 mm h⁻¹ during the night in control plants (Figure 5A). Leaf elongation rate decreased significantly in response to the higher salt concentrations tested during both day and night (Figure 5A). The rate of leaf elongation during the night accounted for between 54.5 and 75.6% of the elongation rate during the day, and this relation was not affected by salt stress, except for the 150 mM NaCl treatment which showed a significantly lower ratio compared with the other salt treatments (Figure 5B).

The water content per mm leaf elongation zone decreased significantly in response to salt stress (Figure 5C). Knowing this figure and the elongation rate of leaves, it was possible to calculate the net rate of water accumulation and storage associated with leaf growth (Figure 5D). This rate could then be related to the total rate of water delivery from root to shoot. The latter presented the sum of the transpiration rate of plants, which had been determined for the identical plants that had been used for the determination of the leaf elongation rate (not shown), and the rate of water accumulation in the leaf elongation zone. Between 0.97 and 1.27% of the water, which was delivered to the shoot was stored through leaf elongation growth during the day in plants grown under control conditions and exposed to the two lower levels of salt stress (Figure 5E). This figure doubled, significantly, in plants which were subjected to the 150  and 200 mM NaCl treatments (Figure 5E). Water accumulation in cells and storage associated with leaf growth accounted for 5.15 to 5.85% of the water delivered to shoots during the night and increased significantly to 8.31% in response to 200 mM NaCl (Figure 5E). When comparing night with day, the proportion of water, which was delivered to the shoot and stored through leaf elongation growth, was 3.1‐ to 5.6‐times higher during the night.

3.7. Salt stress affects daytime CO₂ assimilation more than it affects night‐time respiration in the mature leaf blade

Gas exchange measurements were carried out by clamping a 20  × 30 mm large chamber halfway along the mature blade. The net rate of photosynthetic CO₂ assimilation during the day averaged 10.7 μmol CO₂ m⁻² s⁻¹ in control plants. This rate decreased significantly in response to all salt concentrations tested and accounted for only 19% of the control value in plants treated with 200 mM NaCl (Figure 6A). Respiration rates in the mature leaf blade during the night, which caused the net release of CO₂ (negative figures) were comparatively little affected by salt stress and decreased significantly to 45% of the control value, at the highest salt concentration tested (Figure 6A).

FIGURE 6.

Carbon dioxide assimilation rates of wheat plants which were exposed to different concentrations of NaCl in the root medium. Plants were exposed to salt treatments when they were 10 days old and analyzed when they were 14–18 days old. Gas exchange was analyzed on (A) fully mature leaf blades of intact plants during either the day (photosynthetic net assimilation of CO₂) or night (respiratory net production of CO₂, negative figures); or on (B) the isolated elongation zone of leaf three during the night (respiratory net production of CO₂, negative figures). Results are averages and SE (error bars) of (n=) (A) 7–15 and (B) 3–5 plant analyses. Values were related to the (A) projected leaf surface area or (B) the entire leaf elongation zone (LEZ). Statistically significant (p < 0.05) differences in values between treatments are indicated through different letters. (C) Plot of respiratory (night) against photosynthetic (day) net assimilation rate of CO₂ in the mature leaf blade; a best fit quadratic equation is shown (y = 0.0295x ²–0.4622x + 0.1966; r ² = 0.9979). (D) Combined plot of the rates of net CO₂ exchange associated with photosynthesis (day) and respiration (night) in the mature blade against stomatal conductance as calculated for intact transpiring plants during the day and night period (compare, Figure 4D). Note that rates for respiration are also expressed as positive rates. Linear regression lines (y = 1477x + 0.6077, r ² = 0.9484; Pearson correlation coefficient 0.974, p < 0.001).

A plot of night‐time respiration against daytime photosynthetic net assimilation rate of CO₂ could be fitted almost perfectly through a quadratic equation (r ², 0.998), with respiration rates reaching a plateau in the control and lower salt stress levels and becoming increasingly smaller at the higher salt stress levels (Figure 6C). The daytime photosynthetic and night‐time respiratory net assimilation rate of CO₂ in mature leaves significantly correlated with the stomatal conductance during day and night and followed one overall trend (Figure 6D). Note that gas exchange was determined via Licor analyses, while stomatal conductance was determined through an independent approach for intact, undisturbed transpiring plants.

3.8. Night‐time respiration in the leaf elongation zone is not affected by salt stress

Respiration measurements were carried out on the isolated elongation zone of leaf three during the night. Data were expressed per unit elongation zone. The respiration rate per leaf elongation zone (LEZ) during the night was little affected by salt stress and ranged from 6.9 to 9.1 μmol CO₂ s⁻¹ LEZ⁻¹ (Figure 6B). If anything, moderate salt stress tended to increase the respiration rate. There was no consistent relationship between the rate of leaf elongation and the rate of respiration in the elongation zone during the night (compare Figures 5A and 6B).

3.9. How does the amount of carbon required for cell elongation during the night compare to the amount available through net photosynthesis during the day?

Most of the carbon required in support of cell elongation during the night was used to increase dry matter, a smaller portion was respired (File S1, sheet SH3; File S2). The combined carbon used for dry matter production and respiration during leaf elongation growth in the night (File S1, sheet SH3) approached 12% of the total extrapolated carbon assimilated by the shoot during the day in control plants (File S2). This figure increased in response to salt stress to 40% at the highest NaCl level tested (File S2). Night‐time leaf elongation became an increasingly significant sink for day‐time assimilated carbon as the NaCl concentration in the root medium increased.

3.10. Plants grown at elevated RH during the night transpire less water, increase leaf elongation rates, while the relative growth rate and ion concentrations of the shoot change comparatively little

Growing plants at 95% rather than 75% RH during the night hardly affected the relative growth rate of the leaf area (Figure 7A). Growth rates tended to be the same or 1–2% larger than the growth rates of plants grown at 75% night RH, except at 150 mM NaCl, where the relative growth rate increased significantly from 14 to 17% d⁻¹. High night RH did not affect the response of transpiration rate per plant or unit leaf surface area to salt stress and day/night changes, but it did alter absolute values of water loss and the ratio between night‐ and daytime water loss rates (Figure 7B–F). Night rates of water loss at 95% RH accounted for only 4.3 to 10.7% of the day rates, a reduction of 4–12% compared with plants grown at 75% night RH (Figure 7C; compare Figure 2D). High night RH not only reduced the rate of night‐time water loss but also increased significantly the rate of daytime water loss in some of the plant treatments (control, NaCl), both on a plant and leaf area basis (Figure 7E, F).

Leaf elongation rates responded similarly to salt stress in plants grown at 95% compared with plants grown at 75% night‐RH (Figure 7A, 8A). What changed, though, was that night rates almost matched day rates, which was due to large and significant increases in night leaf elongation rates at 95% RH (Figure 8B, C); the day rate also increased significantly in control plants and plants subjected to 50 mM NaCl (Figure 8C).

The average concentrations of Na⁺ and Cl⁻ in shoot tissue increased in response to salt stress, as did the Na⁺ to Cl⁻ ratio (Figure 8D‐F). The response to salt stress was not affected by night‐time RH, as none of the values differed significantly between salt‐stressed plants grown at 75% or 95% night RH (Figure 8D‐F).

3.11. Aquaporins are consistently differentially expressed in growing and mature leaf tissue and the expression patterns are little affected by salt stress and day/night

We studied nine candidate PIP AQPs, focusing on gene expression levels in the elongation zone of the growing leaf three and the blade of the mature leaf two, the main transpiring leaf at the developmental stage where plants were studied. None of the AQP isoforms was expressed at similar levels in the elongation zone and mature leaf tissue. Rather, isoforms were either expressed particularly in growing or, in six cases, in mature leaf tissue (Figure 9). Gene expression levels were generally little affected by day/night changes, except for two AQPs (TaPIP1;2; PutTaPIP2‐2), which showed large decreases in expression in control plants. Salt stress hardly affected the gene expression level of AQPs in growing leaf tissue (exception, TaPIP2‐26 increase during night) and caused no changes (TaPIP1‐1, TaPIP1‐4, TaPIP2‐2C3), an increase (TaPIP2‐4C1, day) or decreases (TaPIP1‐2, PutTaPIP2‐2, TaIP2‐3C1, TaAQP2) in gene expression in the mature blade; decreases in gene expression were particularly large during the day for TaPIP1‐2 and PutTaPIP2‐2 (Figure 9).

4. DISCUSSION

4.1. Night‐time transpiration in wheat occurs predominantly through stomata

We wanted to test whether night‐time transpirational water loss occurs primarily through stomata, also under salt stress. The data show that this is the case and that wheat plants must be able to regulate night‐time water loss in the short‐term. Water loss through stomata associated with night‐time transpiration has been reported for a range of species (for review, see Caird et al., ²⁰⁰⁷), including wheat (Rawson & Clarke, 1988), though previous data for salt stress are limited to barley (Even et al., 2018).

There exists no reason a priori why the rates of night‐ and day‐time water loss should be related to each other, even under constant environmental conditions (e.g., VPD). The present data show that there does exist a close relationship between the two (Figure 10A) and that this is the result of a regulation of stomatal conductance (Figure 4D). We did not test a possible contribution of water loss through leaf sheaths to night‐time transpiration (for discussion, see Sadok et al., ²⁰²⁰).

FIGURE 10.

Correlation analyses of a range of sizes which relate to the diurnal water use and growth in wheat plants under salinity. Plants were exposed to salt treatments when they were 10 days old and analyzed when they were 14–18 days old. (A) Relation between the rates of day‐ and night‐time transpirational water loss. (B) The percentage of water which was delivered to the shoot and used in support of leaf elongation growth as a function of leaf elongation rate. (C) The rate of water used for leaf elongation growth during day and night plotted against the water potential (ψ) of the xylem of the mature leaf two and of the root medium. (D) The ψ of leaf xylem in the dark plotted against the ψ of root medium. (E) Total amount of water accumulated through cell elongation in the leaf growth zone during an 8 h night period plotted against the total CO₂ assimilation during a 16 h day period in the mature blade; the latter was calculated as the numeric product of the CO₂ assimilation, rate (μmol m⁻² s⁻¹), leaf area (m²) and duration of day (s). Each data point in (A, B) represents data for one individual plant. Data points in (C–E) represent average values for a particular treatment. The Pearson correlation coefficient (PC) and p value of correlations was (PC/p value): (A) (0.787/<0.001), (B) (Night, 0.042/0.736; Day, −0.218/0.076), (C) (leaf xylem, day, 0.987/0.002; medium, day, 0.995/<0.001; leaf xylem, night, 0.983/0.003; medium, night, 0.986/0.002), (D) (0.981/0.003), and (E) (0.992/0.001). The linear regression lines had equations: (B) Night, y = 186.9x + 5.43, r ² = 0.0018; Day, y = −297x + 1.92, r ² = 0.0476.; (C) Leaf‐2, light, y = 0.0009x + 0.0023; r ² = 0.9743; Medium, light, y = 0.0012x + 0.0016, r ² = 0.9908; Leaf‐2, night, y = 0.0008x + 0.0012; r ² = 0.9661; medium, night, y = 0.0008x + 0.0011; r² = 0.9722), (D) (y = 0.99x − 0.13; r ² = 0.9616), and (E) (y = 7.32 × 10⁻⁶ + 1.40 × 10⁻³; r ² = 0.984).

Schoppach et al. (2014) observed significant variability in the quantitative relation between day‐ and night‐time transpirational water loss rates among wheat genotypes. Claverie et al. (2018) concluded for wheat growing under soil water deficit conditions that, night‐time transpirational water loss represents an increasing fraction of the daily water loss—and may be detrimental to the tolerance of drought—the opposite of what was observed here in plants grown on hydroponics. Tamang et al. (2019) concluded from a study of 77 genotypes of wheat exposed to changes in VPD that night‐time transpirational water loss is associated in some genotypes with the predawn opening of stomata. Such a mechanism could “prime” stomatal opening for the start of the day when CO₂ uptake is associated less with water loss than with the bulk of daylight. If so, why should stomata be open throughout the night, as observed here (see File S1, sheet SH4)? While we do not dismiss the idea of priming of stomata, we do not see any support for this idea through the present data.

4.2. Night‐time leaf expansion uses a higher portion of shoot‐delivered water compared with the day, but this portion is still minor

A consistently higher portion of shoot‐delivered water was stored through leaf expansive growth during the night (5%–8%) compared with day (1%–2%) (Figures 5E, 10B), as also proposed for barley using combined data from several studies (Fricke, ^2019b). It was not so much the salt treatment but the day period (day/night) which affected the portion of shoot‐delivered water that was retained through leaf expansive growth in the plant. This portion was still minor, at least under the conditions tested. It can be concluded that night‐time leaf growth does not offer any stress acclimation process under salinity, in a sense that it would be associated with the use of an exceptionally high portion of shoot delivered water at a time (night) when water uptake from the root environment is easier (less negative ψ compared with day).

4.3. CO₂ gas exchange is a driver of not only day but also night‐time water loss

The higher the rates of transpirational water loss were during the day, the higher the rates during the night (Figure 10A). In addition, the rates of net CO₂ assimilation during the day (photosynthesis) and loss during the night (respiration) from the mature leaf blade were highly correlated with stomatal conductance (Figure 6D). While this is not proof per se, it does support the idea that night‐time transpiration is a consequence of having to allow respiratory CO₂ to escape (and O₂ to enter) leaves at sufficiently high rates through stomata. This avoids the cellular formation of carbonic acid and acidosis (Even et al., 2018), yet need not apply to all species, nor may it apply to all growth conditions (for discussion, see Caird et al., ²⁰⁰⁷; Tamang et al., ²⁰¹⁹). The circumstance that the rate of net exchange of CO₂ through stomata linearly relates to stomatal conductance across the day and night period implies that the gradients in CO₂, which drove the uptake (day) and release (night) through stomatal pores, must have been similar during day and night. Any CO₂ sensing mechanism which controls stomatal apertures operated in a day‐period consistent manner.

4.4. Night‐time transpiration is associated with significant tensions in the xylem under salt stress

Xylem tension is a well‐studied feature typical of plants which transpire during the day. Far less is known about xylem tension during the night (Tang & Boyer, 2002), particularly under conditions where the ψ of root environment is very negative and where night‐time transpirational water loss occurs as in the present study through stomata (see Smith & Lüttge, ¹⁹⁸⁵, for a study on a CAM plant). The present data show that most of the xylem ψ during the day and night was due to the tension, as opposed to the osmotic component. Xylem tension during the dark increased to −0.7 MPa at 200 mM NaCl. These tensions aid radial water uptake across the root cylinder and avoid the need to establish high solute concentrations in the xylem to drive water uptake from a saline environment. This could not only save energy for xylem loading as well as for compartmentalizing Na⁺ and Cl⁻ in cells but facilitate water uptake during the night in the first place. Excised root systems of control plants which were suspended in a nutrient solution, exuded at a rate equivalent to 72% of the rate of night‐time transpirational water loss, yet root systems of salt‐stressed plants showed either very low (50 mM) or no exudation when placed in media containing 100–200 mM NaCl (Lu & Fricke, unpublished results). The above interpretation of xylem tension data relies on the accepted view that the pressure chamber measures the tension, not ψ in the xylem (Boyer, 1967, 1995). Our future aim is to independently verify these data using the cell pressure probe to measure xylem tension directly (Wei et al., 1999), particularly as we occasionally observed guttation, being indicative of positive root pressure, in control but not salinized plants (Lu & Fricke, personal observations).

4.5. Water uptake into expanding leaf cells relates linearly to the water potential in leaf xylem and root medium during day and night

The rate of net water uptake into growing leaf cells decreased in a linear fashion as the ψ in the leaf xylem and the root medium became more negative. This applied to the day and night period and across salt treatments (Figure 10C). The slopes of relations were almost identical when comparing leaf xylem with root medium ψ during either day or night. This means that any change in external ψ caused by the addition of salt to the root medium transmitted into a consistent change in leaf xylem ψ. A plot of leaf xylem ψ in the dark against root medium ψ gave a slope of 0.99 (Figure 10D). The slope is the root reflection coefficient for solutes (here, mainly Na⁺ and Cl⁻). A value of 1.0 stands for a perfectly semipermeable system, which means that the wheat roots of control and salinized plants studied here behaved like almost perfect osmometers, as previously also concluded for wheat grown under non‐stress conditions (Bramley et al., 2009) and salt‐stressed barley (Knipfer et al., 2021). Any transport of Na⁺ and Cl⁻ along an apoplastic bypass route driven by night‐time xylem tension must have been negligible (Knipfer et al., 2021).

The more negative xylem ψ is, the more difficult it is for growing cells to maintain a ψ difference between their protoplasm and xylem to drive the net uptake of water (for discussion and reviews, see Fricke, ²⁰⁰²; Tang & Boyer, ²⁰⁰²). A previous single‐cell study on hydroponically grown barley plants which were exposed to 75  and 120 mM NaCl showed that growing leaf cells lowered the ψ during the day by as much as the ψ was lowered in the root medium through the addition of NaCl (Fricke & Peters, 2002). If the same applied to growing leaf cells of the wheat plants analyzed here, and during the night, it would explain the close relationship between the rate of water uptake into growing leaf cells and xylem ψ; it would also imply that the hydraulic conductance of flow path between xylem and the protoplasm of cells was not affected by NaCl. The data on plants grown at high night‐time RH further support these conclusions. These plants showed significantly increased leaf elongation rates during the night, across treatments, a feature which can be easiest explained through diminished gradients in ψ between growing cells and xylem, thanks to the reduced VPD. Leaf growth seems to be limited hydraulically not just during the day (Tardieu et al., 2010), but also during the night period, at least on saline root media.

4.6. Night‐time water accumulation in growing leaf cells correlates with day‐time carbon assimilation

As the salt stress level increased, so did the portion of day‐assimilated carbon, which was required to support leaf growth during the night (Supplemental file S1, S2). This will have been the consequence of two processes, a reduced rate of CO₂ assimilation during the day, and an increased energy expenditure during the night, to osmotically drive water uptake into growing leaf cells and compartmentalize Na⁺ and Cl⁻. Furthermore, the rate of water use associated with night‐time leaf growth increased linearly with the calculated total day‐time photosynthetic output per plant (Figure 10E). These data not only support the idea, developed on the model plant Arabidopsis, that the day‐time provision of storage carbohydrates supports night‐time leaf growth (Graf et al., 2010) but show that the relation holds across a range of salt stress levels.

4.7. Does night‐time transpiration benefit wheat plants under salt stress?

The question central to this study was whether night‐time transpirational water loss confers any benefit to wheat plants, particularly under salt stress.

We can conclude from the present data that night‐time transpirational water loss neither significantly promotes nor decreases the tolerance of wheat plants to salt stress, as judged based on the relative growth rates (Figure 7A) and physical appearance of plants (Supplemental File S4) across all treatments. However, there was a tendency towards plants at the highest salt concentrations to appear healthier when grown at 95% night‐time RH, and plants subjected to 150 mM NaCl had a significantly increased RGR (17% d⁻¹ compared with 14% d⁻¹). When interpreting these data, it is important to distinguish between night‐time transpirational water loss and night‐time gas exchange in general, which also includes CO₂ and O₂. Growing plants at 95% RH during the night meant that the VPD, which drives stomatal water loss, was reduced by up to 80% compared with the VPD of plants grown at 75% night‐time RH – and high night‐time RH caused night‐time transpiration rates to decrease by almost as much in the highest three salt treatments. This implies that stomatal conductance and CO₂/O₂ gas exchange remained largely unaffected by the high RH during the night. At the same time, reduced water loss per unit transpiring leaf area will have facilitated more water uptake into growing leaf cells from the xylem with a less negative xylem Ψ, which would explain the much higher leaf elongation rates at night. Therefore, high night‐time RH benefited plants not so much through a reduction in night‐time water loss but through enabling higher leaf elongation rates while affecting stomatal gas exchange little. The accumulation of Na⁺ and Cl⁻ in the shoot was not affected by the decrease in night‐time transpiration (Figure 8D‐F). What intrigued us was that plants grown at high night‐time RH showed increased transpirational water loss rates during the day, also per unit leaf area, which was for some treatments significant. We can calculate the total amount of water being lost per unit leaf area by plants over a 24 h day/night period (Supplemental File S1, Sheet SH7). Experimentally reducing water loss during the night increased the total water loss per unit leaf area by 0% to 31%. We do not know whether this reflects some compensatory or over‐compensatory mechanism, in the sense that night‐time water loss can be sensed by plants and feeds back on daytime water loss.

Based on the present data, we propose a model in which night‐time transpirational water loss is as much as day‐time water loss a consequence of having to enable CO₂ exchange between the leaf's internal air space and the ambient atmosphere (Figure 11). This requires regulation of leaf conductance through stomata. Stomatal conductance, in turn, affects xylem ψ in leaves, which also responds to changes in ψ in the root environment and is affected by changes in root hydraulic conductivity, Lp, as well (e.g., Armand et al., ²⁰¹⁹). Assimilation rates of CO₂ during the day impact on day and night‐time leaf growth rates through provision of storage carbohydrates, yet also, the CO₂ respiratory activity in the mature blade during the night affects these growth rates—because any leaf area increase at a given relative growth rate (% d⁻¹) needs to be sustained not just through net CO₂ assimilation (day) but also through respiration (night). Solute and water transport processes, the latter involving AQPs, at the plasma membrane and tonoplast of growing cells, as well as biochemical processes which define wall properties, provide possible means of regulation of the rate of cell expansion and leaf growth (Fricke, 2002; Fricke & Peters, ²⁰⁰²; Cosgrove, ²⁰¹⁸). These processes require not only energy and carbon but can only exert their full potential when the ψ of the leaf xylem is in a range that allows the sufficiently fast uptake of water into growing cells (Fricke, 2002, ^2019b; Munns et al., ²⁰²⁰). Diurnal growth and transpiration rates should not be viewed in isolation, and the key players which this study identifies are stomatal conductance and the ψ and tension of the xylem. The quantitative link between stomatal conductance, xylem ψ and leaf cell expansion is the result of the (1) biochemical/metabolic capacity of tissues, as it defines relative growth rate, and (2) water relations of plant, as it affects the capacity to perform gas exchange in a changing VPD and ψ (e.g., salinity) environment.

FIGURE 11.

The current data show that night‐time transpirational water loss neither decreases nor increases the tolerance of wheat plants to salt stress. However, the data and suggested model emphasize the importance of night‐time transpiration as being an inevitable consequence of gas exchange through stomata and therefore forming an intergral part of diurnal plant growth. The relative growth rate of a plant at given environmental condition and developmental stage reflects the biochemical/metabolic capacity to carry out biosyntheses and gas exchange; it is also linked to the water relations of plant. Key to the growth of plant is the ability to net assimilate CO₂ during the day (photosynthesis) and respire CO₂ (night) in mature leaf tissue, to increase plant dry matter and the amount of fixed chemcial energy, store carbohydrates and fuel biochemical reactions. Stomatal conductance is the main facilitator of CO₂ gas exchange during day and night, and diurnal transpirational water loss, as well as xylem tension, is a consequence of this gas exchange. Day‐time photosynthesis in the mature blade provides resources not only for respiration in this tissue during the night but also for cell expansion in the leaf elongation zone during day and night. This in turn provides leaf area, which increases the capacity of mature leaf tissue to photosynthesise (day) and respire (night). Together, these processes enable a certain relative growth rate over a 24 h day/night period. Leaf elongation growth and gas exchange in the mature blade involve water flow between tissues and cells, which are likley to be facilitated by aquaporins (AQPs). Root hydraulics, which were not studied here, affect the relationship between the rate of transpirational water loss and the magnitude of xylem tension and may impact through this on cell and leaf area expansion, use of resources and, ultimately, the relative growth rate of plant. Environmental factors such as temperature (air, soil), vapor pressure deficit (VPD) and partial pressure of CO₂ (pCO₂) in the air, and water potential (ψ) and salt levels in the soil also affect the above process and can becoming limiting to growth.

4.8. The dominant factor which affects the gene expression level of PIP AQPs in wheat leaves is the tissue zone

We wanted to obtain information on the molecular control of water flow in growing and mature leaf tissue between xylem and peripheral cells. To this aim, we studied the gene expression level of nine candidate PIP AQPs (Figure 9). We do not know for sure whether these PIPs transport water or maybe also ions or CO₂ (Groszmann, Osborn & Evans, 2017; Tyerman et al., ²⁰²¹), as the functionality of wheat PIPs is poorly characterized, and it was beyond the scope of this study to carry out functionality tests. The major factor which determined the gene expression level of PIP AQP isoforms was the leaf tissue, as all of the isoforms tested were expressed either significantly higher in growing or in mature leaf tissue (also compare barley; Besse, Knipfer, Miller, Verdeil, Jahn & Fricke, ²⁰¹¹), irrespective of salt stress and day/night changes. The gene expression level does not have to reflect the protein level or water channel activity of AQPs (Yepes‐Molina, Bárzana, & Carvajal, 2020).

Nonetheless, we plotted the gene expression level against the respective net water flow rate through the leaf tissue, combining data from control and salt‐stressed plants, day and night, and growing and mature leaf tissue. Some AQPs (TaPIP1‐2, TdPIP2‐2 and TaAQP2) showed a positive correlation between the gene expression level and water flow rate, while others (TaPIP1‐1, TaPIP1‐4) showed a negative correlation, and again others showed no clear trend (Supplemental File S1, sheet SH8). It remains to be tested whether those PIPs, which display a positive correlation, have water channel activity and are involved in the diurnal regulation of growth and transpiration in wheat leaves.

AUTHOR CONTRIBUTIONS

Yingying Lu carried out analyses, except qPCR analyses which were also carried out by Ruth Jeffers, Anakha Raju, Tamara Kenny, and Evangeline Ratchanniyasamu as part of taught‐master projects. Yingying Lu and Wieland Fricke designed the project, analyzed the data and wrote the manuscript. Wieland Fricke submitted the manuscript.

Supporting information

Fig. S1: Plotting the gene expression level of aquaporins (y‐axis, 2^‐delta‐Ct‐value) against the net water flow rate (x‐axis, m3 s‐1 plant‐1) through the respective tissue.

Fig. S2: Dry weight and carbon use in the elongation zone of leaf three of wheat.

Fig. S3: data in relation to the wheat aquaporins (AQPs) studied.

Fig. S4: pictures of wheat plants grown at 75% / 75% RH day/night (‘RH 75%’) and 75% / 95 % day/night (‘RH 95%’).

Lu, Y. , Jeffers, R. , Raju, A. , Kenny, T. , Ratchanniyasamu, E. & Fricke, W. (2023) Does night‐time transpiration provide any benefit to wheat (Triticum aestivum L.) plants which are exposed to salt stress? Physiologia Plantarum, 175(1), e13839. Available from: 10.1111/ppl.13839

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.