Effect of Drought and Salinity on Water Relations and Photosynthetic Responses of Tamarix elongata and Haloxylon ammodendron in Wutonggou Desert Tourist Area, Northwest China

Abstract

It is still controversial whether photosynthesis is mainly restricted by diffusional or biochemical limitation under drought and salinity stress, and new adaptive mechanisms may appear when the two stresses interact. We measured water relations and photosynthetic responses under different water and NaCl treatments in two halophyte seedlings that coexist in northwestern China, a C₃ plant Tamarix elongata Ledeb and a C₄ plant Haloxylon ammodendron (C. A. Mey.) Bunge. Water potential values decreased with increasing salinity, and lower water potential values were measured in H. ammodendron and T. elongata under drought treatments. Leaf Na⁺ concentration increased and K⁺ concentration decreased with the intensification of the salinity treatment in H. ammodendron and T. elongata. The observed enhancement in Na⁺ concentration may be an adapting mechanism associated with osmotic regulation. The stomatal (L_s) and mesophyll (L_m) limitations co-controlled the photosynthetic rate of the two species. However, there were no significant differences in L_s but an increase in L_m under salinity stress with the well-watered condition, indicating mesophyll regulation of photosynthesis played a more important role than stomatal closure for the two studied species. Our results indicate that the reduction in photosynthesis for the two species is co-limited by both stomata and mesophyll under the combined drought and salinity conditions, but that L_m is the main limiting factor under salinity stress. It is also suggested that the C₄ plant H. ammodendron is more drought- and salt-tolerant than the C₃ plant T. elongata, so it is more widely distributed in arid saline environments.

1. Introduction

Salinity is a major environmental constraint with severe negative impacts not only on agricultural productivity but also on natural vegetation [1], particularly in arid and semi-arid regions of the world. Nearly 1.4 billion hectares of land throughout the world are salt affected, with an additional 1 billion hectares at risk due to the climate crisis and human mismanagement [2]. Halophytes represent an important potential as they can be used for fodder, fuel, fiber production and dune stabilization. Thus, it is of great significance to explore the salt tolerance mechanisms of halophytes for making full use of plant resources in arid areas [3,4].

Plants have many similar metabolic processes under salt and drought stress, such as a decline of photosynthesis and growth [5]. Moreover, most studies have addressed salt and drought stress individually. However, drought and salinity stress usually occur together under field conditions [6,7]. Some reports have shown combined drought and salt stress are often regarded as more severe stress than any single stress for plants. Others suggest salinity actually has a protective effect on plant yield under water-limited conditions. For example, an enhancement of growth by moderate NaCl under water stress has been observed for Populus tomentosa [8], Reaumuria soongorica [9] and Atriplex canescens [10]. In contrast, waterlogging under saline conditions has adverse effects on plant growth and survival [11]. Therefore, salinity constrains plant growth, but this effect depends on saline intensity, moisture levels and species differences. Based on all the above, although plant responses to drought and salinity stress have much in common, some aspects of plant physiology may differ when the plant suffers solely from salt or drought, or both combined stresses.

Photosynthesis is among the primary processes to be affected by water or salt stress. In a similar manner to drought, salinity stress may have an adverse effect on photosynthesis mainly through a reduction of stomatal conductance. Many studies suggest stomatal closure is a primary limitation on photosynthesis under salinity and drought stress [12,13,14]. However, the diffusional and non-diffusional limitations of photosynthesis should replace stomatal and non-stomatal limitations [15]. These diffusional limitations consist of stomatal limitation (L_s) and mesophyll limitation (L_m) [16]. The non-diffusional limitations depend on maximum capacity for Rubisco carboxylation [17]. It is still controversial whether photosynthesis is mainly restricted by diffusional or biochemical limitation under drought and salinity stress [18]. Recently, mesophyll conductance of CO₂ (g_m) has also been found to play a key role in photosynthesis rate limitation under several abiotic stresses [19]. Some works suggest that drought and salinity reduce mesophyll CO₂ conductance [20,21]. However, there are different tradeoffs between mesophyll and stomatal conductance under drought, salinity and temperature [22]. In addition, a study shows low salinity does not decrease but improves photosynthetic performance in Panicum antidotale under drought stress [23], which makes the adaptation strategies of plant photosynthesis to drought and salt stress more complex. Therefore, further study regarding the photosynthesis of halophytes is needed for a better understanding of their adaptation mechanisms.

C₄ plants are highly light-efficient species that evolved from C₃ plants, and the combined effects of climate warming, drought, increased salinity and low humidity are external drivers of C₄ evolution [24,25,26]. C₄ plants can increase their photosynthetic rates through the CO₂ concentrating mechanism due to reducing photorespiration. In C₃ plants, O₂ competes with CO₂ as a substrate for Rubisco when photorespiration occurs. Also, C₄ species can maintain high water use efficiencies under drought conditions. Thus, C₄ plants generally outperform C₃ plants under CO₂-limited conditions, especially under high temperature, drought and salinity. Although C₄ species constitute only about 3% of the global flora, they contribute about 23% of terrestrial gross primary production (GPP) [27]. The seedlings of C₃ and C₄ plants are more likely to be subjected to water deficit and salinity due to their shallow root systems. Drought and salinity are important for seedling establishment, which subsequently determines recruitment and distribution. Thus, understanding photosynthetic type differences in the physiological response and adaptation to drought and salinity stresses is valuable for the prediction of the relative abundance of C₃ and C₄ plants.

This study tested the interaction between salinity (four levels) and water stress (two levels) in two species (C₃, Tamarix elongata and C₄, Haloxylon ammodendron) differing in photosynthetic pathway and capacity to accumulate sodium. T. elongata and H. ammodendron are both dominant species that grow on the southern edge of the Gurbantunggut Desert. T. elongata is a Northwest Chinese C₃ phreatophytic shrub. It tolerances drought and salinity, and plays an important role in windbreak and sand-fixation [28]. The Tamarix species is also a salt-secreting halophyte which is able to grow and develop in saline soils [29]. H. ammodendron is a chenopodiaceae family, C₄ shrub. It is also tolerant to both salt and drought, can be used as firewood and livestock feed and plays a vital role in stabilizing sand dunes. Tamarix species only grow on the fringes of deserts, while H. ammodendron can coexist with Tamarix species on the fringes of deserts and also survive in more xeric conditions in desert hinterland [30]. The previous studies stimulated our interest to determine its adaptation mechanisms in response to a combination of salinity and drought stress on the water relations and photosynthetic capacity, but the adaptive strategies of halophytes and their tolerance towards drought and salinity stresses remain less understood. The objectives of the study were as follows: (1) to correlate water relations and photosynthetic responses with growth, (2) to verify whether a moderate salinity level with drought stress would have a positive effect on plant growth, as observed for other halophytes, and (3) to distinguish the stomatal (L_s) and mesophyll (L_m) limitations as well as the stomatal and non-stomatal limitations of photosynthesis.

2. Materials and Methods

2.1. Plant Material and Greenhouse Conditions

One-year seedlings Tamarix elongata and Haloxylon ammodendron with uniform sizes were selected from Fukang Halophytes Botanic Garden, Xingjiang. The seedlings were transplanted into pots with a diameter of 20 cm, and a height of 29 cm containing sandy soils in the Gurbantunggut Desert and watered with 500 mL 5 days⁻¹ and added Hoagland nutrient solution every week. Before the treatment, the sandy soil nutrient content is low, with nitrogen, phosphorus and potassium about 0.17, 0.37 and 0.19 g·Kg⁻¹, respectively. The sand was dried and sieved before use, and the pots were watered to field capacity after being filled with 12 Kg sand to ensure the same initial water conditions in every pot. During the experiment, plants in the greenhouse were supplied with an ambient photoperiod of 14 h day⁻¹. Daily temperature was set in the range of 18~30 °C and relative humidity in the range 20~30%.

After 30 days of cultivation, the salt stress treatment began. Four sets of plants were used and each set included 24 plants. The control set was watered with deionized water, while the other three sets were treated with 100, 300 or 500 mM NaCl. The target NaCl concentration was achieved within 20 days, and 100 mmol L⁻¹ was added every 5 days. Drought treatment began after NaCl treatment, the irrigation water of half plants was reduced to 200 mL 5 days⁻¹ with each NaCl treatment, and the control set (well-watered treatment) was also 500 mL 5 days⁻¹. One month later, the gas exchange measurements began.

2.2. Measurements of Soil Water Content, pH and Electrical Conductivity

Volumetric soil water content (VSWC, n = 4) at 0–10 cm soil depth was measured in in each pot using a portable soil moisture probe (Campbell Scientific Hydrosense, Logan, UT, USA). Air-dried soil was soaked with deionized water at a 5:1 water/solid ratio for one hour with agitation. Following a half-hour standing, the slurry was then measured for pH using a pH electrode and for electrical conductivity (EC) using a conductivity meter, four separate replicates for each treatment.

2.3. Water Potentials

Leaf water potential was determined before predawn (Ψ_pd) and at midday (Ψ_md) using a Model 3005 Pressure Chamber (PMS Instrument Company, Albany, OR, USA). Twigs were cut at the base and used for measurements, four separate replicates for each treatment.

2.4. Sodium and Potassium Concentration

For each leaf sample analyzed, 100 mg of dry material powder was ashed by a muffle furnace for 550 °C/5 h. The ashed sample was dissolved in 50 mL of distilled water and filtered. Then, 5 mL of filtrate and 1 mL of Al₂(SO₄)₃ solution (0.1 mol L⁻¹) were added into a 50 mL volumetric flask and metered volume with distilled water. Leaf Na⁺ and K⁺ concentrations (n = 4) were measured by a flamephotometer (FP640, Shanghai precision science instrument Co., Ltd., Shanghai, China).

2.5. Leaf Gas Exchange

The photosynthetic light-response curves and CO₂ response curves (n = 3) of the two studied plants in all treatments were measured at 8:00–12:00 local time on sunny days using a Li-6400 portable photosynthesis system (LI-COR, Lincoln, NE, USA). Photosynthetic photon flux density (PPFDi) was supplied by a 2 × 3 cm² leaf chamber with a red/blue light source (6400-02B). A CO₂ injecting device was attached to the system to control [CO2R]. Fully unfolded young leaves from near the top of the canopy in fully illuminated locations were measured for gas exchange. The gas flow rate was set at 500 μmol s⁻¹ to maintain reference relative humidity at 20–40%, which is close to ambient humidity. The chamber temperature was controlled at 30 °C and the reference CO₂ concentration was set at 400 mmol mol⁻¹, which was similar to ambient CO₂ concentration. For the light curve measurement, PPFDi gradients were set at 2000, 1600, 1200, 800, 400, 200, 150, 100, 50, 20 and 0 mmol m⁻²s⁻¹. CO₂ response curves were fitted to the exponential MnMolecular function:

$$y=A\times (1-e^{-k\times (x-CCP)})$$

(1)

where A represents the assimilation rate at the CO₂ saturation point, CCP represents [CO₂] at the CO₂ compensation point, and k × A represents the apparent CO₂ use efficiency of photosynthesis which indicates the activity of Rubisco [31]. Carboxylation efficiency (CE) was calculated from the initial slope of the curve. The relative stomatal limitation of the photosynthetic rate was calculated using the following equation:

$$L_s=(A_0-A)/A_0\times 100$$

(2)

where A₀ is the photosynthetic rate at C_i = C_a [32]. The relative mesophyll limitation was calculated using the following equation:

$$L_m=(A_C-A_S)/A_C\times 100$$

(3)

where A_C is the assimilation rate of control leaves at C_i =800 mmol mol⁻¹ and A_S is the rate of stressed leaves at the same C_i [33]. All the measurements of net assimilation rate (A) on the light-response curves and CO₂ response curves were corrected for leaf area, which was either calculated from leaf diameters (assimilating shoots of H. ammodendron seedlings approximate cylinders) determined by a caliper or from leaf pictures obtained from scanning (leaves of T. elongata seedlings are irregular). After photosynthetic measurement, all irregular leaves of T. elongata in the leaf chamber were removed and carefully spread and scanned by a scanner (Epson Perfection 2400 Photo, Seiko Epson, NA, Japan). The scanned images were processed and the pixels of the foliage recorded using Adobe Photoshop software (Adobe Systems Software Ireland Ltd., San Jose, CA, USA).

2.6. Chlorophyll Contents and Chlorophyll Fluorescence

Leaf chlorophyll contents (n = 4) were measured by an ultraviolet spectrophotometer. A 0.1 g leaf sample was cut into pieces and soaked in 95% alcohol and 80% acetone (1:1 volume ratio) mixed solutions for 24 h in dark conditions. Then, the extracted solutions’ absorbance was measured at 663 and 645 nm wavelengths, respectively. The chlorophyll a and b contents were calculated by Formulas (4) and (5):

$$c_a=12.7A_{663}-2.69A_{645}$$

(4)

$$c_b=22.9A_{645}-4.68A_{663}$$

(5)

In the two formulas, c_a and c_b indicate chlorophyll a and b content, and A₆₆₃ and A₆₄₅ indicate absorbance at 663 and 645 nm wavelengths, respectively.

Chlorophyll fluorescence was measured on attached dark-adapted leaves (n = 4) with a leaf fluorescence chamber (LI-6400-40). The maximum quantum yield of PSII (F_v/F_m) was measured in attached dark-adapted leaves or assimilating shoots.

2.7. Statistical Analysis

A two-way ANOVA was performed using the GLM procedure to calculate the effects of NaCl and water treatments and their interactions. When the ANOVA results were significant, the Tukey’s post hoc test was conducted to determine significant differences between the mean values at p < 0.05 in the analyzed groups. A bivariate correlation procedure was used to calculate the Pearson correlation coefficients. Data were analyzed using the SPSS 16.0 statistical package.

3. Results

3.1. Soil Water Content, pH and Electrical Conductivity

With the same water amount, the volumetric soil water content (VSWC) at 500 mM NaCl treatment was higher than other NaCl treatments (Figure 1a,b). VSWC in the well-watered pots was also higher than in the drought pots except for the 500 mM NaCl treatment. Soil pH values for growing H. ammodendron were affected by the interaction between water and NaCl treatments (Figure 1c). However, soil pH values for growing T. elongata were slightly smaller in the drought pots than in the well-watered pots (Figure 1d). The soil electrical conductivity (EC) for growing the two plants had a similar pattern, increased with increasing NaCl intensity (Figure 1e,f).

3.2. Plant Height

The plant height of H. ammodendron was significantly affected by the NaCl treatment but not the drought stress. With the increase in NaCl levels, plant height decreased gradually in both drought and well-watered conditions (Figure 2). Both NaCl and drought stress affected the plant height of T. elongata. The plant height of T. elongata was negatively correlated with the increase in NaCl levels in the well-watered condition, but the highest plant height was observed at the 100 mM NaCl with drought stress.

3.3. Water Potentials and Leaf Ion Contents

The predawn water potentials (Ψ_pd) and midday water potentials (Ψ_md) of the two species showed similar patterns in their responses to both NaCl and drought stress (Figure 3). Both Ψ_pd and Ψ_md were lower under drought and NaCl stress. Compared to the 0 mM NaCl treatment, Ψ_pd and Ψ_md declined 60% and 35% in H. ammodendron, while 234% and 76% in T. elongata at the 500 mM NaCl treatment, respectively. Under drought stress, Ψ_pd and Ψ_md declined 9% and 43% in H. ammodendron and 45% and 15% in T. elongata, regardless of the effects of NaCl treatments.

The K⁺ and Na⁺ content of the two species showed similar patterns in their responses to both saline and drought stress (Figure 4). NaCl stress led to a significant decrease in K⁺ content in both species (except for the 500 mM NaCl treatment under drought stress). At the 500 mM NaCl, drought significantly increased K⁺ content in T. elongata and led to an increasing trend in H. ammodendron (Figure 4a,b). In contrast, NaCl stress led to a significant increase in Na⁺ content in both species under drought and well-watered treatments (Figure 4c,d). At the 500 mM NaCl, drought significantly decreased Na⁺ content in H. ammodendron. However, at the 100, 300 and 500 mM NaCl treatments, drought significantly increased Na⁺ content in T. elongata, a significant increase of the Na⁺/K⁺ ratio under NaCl stress in both species (Figure 4e,f). At the 500 mM NaCl, the Na⁺/K⁺ ratio significantly decreased in T. elongata and had a decreasing trend in H. ammodendron under drought stress.

3.4. Leaf Photosynthetic Traits

The light-saturated point (LSP) and maximum assimilation rate (A_max) in H. ammodendron and T. elongata were affected by the interaction of drought and salinity stress (Figure 5). The light compensation point (LCP) had no significant difference in both drought and salinity stress. Salinity significantly reduced the apparent quantum yield (AQY) in T. elongata but not in H. ammodendron.

The net assimilation rate (A) and stomatal conductance (g_s) of the two species clearly indicated a positive correlation (Figure 6a). Under NaCl and water stresses, the variation of g_s could account for a 23% reduction of the A in H. ammodendron and a 59% reduction of the A in T. elongata, respectively. In addition, the A was also positively correlated with the transpiration rate (E) in H. ammodendron (r = 0.65, p < 0.001) and in T. elongata (r = 0.66, p < 0.001) (Figure 6b). The A was positively related to the ratio of intercellular CO₂ to atmospheric CO₂ (C_i/C_a) in T. elongata but not in H. ammodendron (Figure 6c).

Under drought stress, both the L_s and L_m of H. ammodendron increased with increasing NaCl levels except for the 100 mM NaCl treatment (L_s decreased but L_m increased). In well-watered conditions, the L_s of H. ammodendron remained nearly constant while the L_m decreased under 100 mM NaCl but increased under the 300 and 500 mM NaCl treatments (Figure 7a,c). Under drought stress, the L_s and L_m of T. elongata mutually compensated for the assimilation rate in the 100 mM NaCl treatment, but both increased in the 500 mM NaCl treatment. Under well-watered conditions, the L_s of T. elongata remained constant in low and moderate saline stress (100 mM and 300 mM NaCl), whereas the L_m increased with increasing salinity (Figure 7b,d).

3.5. Chlorophyll Contents

Treatments significantly affected leaf chlorophyll content (Chl). High NaCl concentration decreased Chl content in both species (Figure 8,

Table 1. Results of ANOVA on the effects of NaCl and drought on chlorophyll a and b content, leaf water content (LWC) and relative quantum yield of PSII (F_v/F_m).

Species	Water Treatment	NaCl Treatments (mM)	Chl a	Chl b	LWC (%)	Fv/Fm
H. ammodendron	Drought	0	5.67 ± 0.22 a	1.81 ± 0.10 a	69.78 ± 2.55 b	0.806 ± 0.004 a
	Drought	100	4.42 ± 0.11 bc	1.50 ± 0.05 b	80.70 ± 2.07 a	0.791 ± 0.010 a
	Drought	300	5.11 ± 0.21 ab	1.52 ± 0.05 b	77.66 ± 1.33 a	0.804 ± 0.007 a
	Drought	500	3.98 ± 0.41 c	1.35 ± 0.10 b	77.79 ± 0.33 a	0.807 ± 0.001 a
	Well-watered	0	6.02 ± 0.47 AB	2.40 ± 0.21 A	78.29 ± 0.70 A	0.800 ± 0.004 A
	Well-watered	100	6.60 ± 0.19 A	2.35 ± 0.12 A	78.59 ± 1.10 A	0.785 ± 0.005 A
	Well-watered	300	5.16 ± 0.18 B	1.88 ± 0.03 B	78.90 ± 1.21 A	0.797 ± 0.009 A
	Well-watered	500	5.11 ± 0.60 B	1.73 ± 0.17 B	79.58 ± 1.42 A	0.801 ± 0.003 A
T. elongata	Drought	0	9.05 ± 1.13 a	4.80 ± 0.53 a	73.69 ± 1.31 a	0.840 ± 0.003 a
	Drought	100	10.34 ± 0.37 a	4.74 ± 0.23 a	69.87 ± 1.18 ab	0.845 ± 0.002 a
	Drought	300	9.35 ± 0.74 a	4.72 ± 0.48 a	67.70 ± 2.02 b	0.844 ± 0.003 a
	Drought	500	10.07 ± 0.25 a	4.76 ± 0.28 a	60.17 ± 2.67 c	0.845 ± 0.002 a
	Well-watered	0	11.64 ± 0.67 A	5.16 ± 0.19 A	67.51 ± 0.69 A	0.838 ± 0.004 A
	Well-watered	100	11.63 ± 0.64 A	4.61 ± 0.24 A	67.20 ± 1.56 A	0.840 ± 0.005 A
	Well-watered	300	10.27 ± 0.20 AB	4.38 ± 0.14 A	65.25 ± 1.87 A	0.843 ± 0.005 A
	Well-watered	500	9.14 ± 1.02 B	4.22 ± 0.30 A	64.84 ± 1.42 A	0.834 ± 0.006 A

Note: Lowercase indicates NaCl treatment significant differences in drought conditions, and uppercase indicates NaCl-significant differences in well-watered conditions.

). The leaf Chl of T. elongata was far less affected compared to H. ammodendron, and it slightly increased in NaCl treatments compared with the 0 mM NaCl treatment under drought stress (Figure 8b). Drought stress led to a significant decrease of Chl content in H. ammodendron but not in T. elongata. However, drought significantly affected the ratio of Chl a to Chl b (Chl a/b) in the two species, with Chl a/b increased in H. ammodendron while decreased in T. elongata under drought stress (Figure 8c,d).

4. Discussion

4.1. Plant Growth Under Salinity and Drought Stress

Salinity or drought stress reduced the difference in water potential between soils and plants under salinity or water stress, which limits the water flux from soil to leaves. Therefore, soil salinity and drought can have similar effects on the physiology of plants [5]. For example, plants reduce growth and decrease photosynthesis under salt and water stress. In the present study, high salinity decreased the plant height of the two species in the well-watered conditions. The drought stress also decreased the plant height of T. elongata but not H. ammodendron, suggesting H. ammodendron is more drought-tolerant than T. elongata. In the Gurbantunggut desert, Tamarix species only grow on the fringes of the desert, while H. ammodendron can coexist with Tamarix species on the fringe of the desert and also survive in more xeric conditions in the desert hinterland. H. ammodendron is more resistant to drought than T. elongata, so it is more widespread in the Gurbantunggut desert.

In the present study, the highest plant height in T. elongata was detected at the 100 mM NaCl level under drought stress (Figure 3). This result is similar to those of other studies indicating that moderate salinity has a protective effect on growth and biomass production under water stress [8,9,10,34,35]. Moderate NaCl (100 mM) induced an obvious increase of Na⁺ accumulation in the leaves of T. elongata (Figure 4d), which may be beneficial for osmotic adjustment. It is likely that T. elongata growing under the 100 mM NaCl treatment had an ample amount of Na⁺ for osmotic adjustment, leading to high plant height and, consequently, high productivity. In contrast, H. ammodendron did not have a similar result. This would be due to inappropriate NaCl and/or water levels. The adverse effect of salinity on plant height in T. elongata was greater than that in H. ammodendron, which suggests H. ammodendron is more salt tolerant than T. elongata.

4.2. Water Relations Under Salinity and Drought Stress

Leaf or stem succulence (i.e., high water content) is an adaptative feature which contributes to the regulation of internal ion concentrations in many halophytes [33]. H. ammodendron is a succulent plant. In this study, the water content of assimilating shoots in H. ammodendron increased while the leaf water content of T. elongata decreased under NaCl stress (Table 1). Increasing the water content of leaves can dilute the salt absorbed by plants [36], or high concentrations of salt can enter into vacuoles to achieve the purpose of dilution. In contrast, no changes or even decreases in leaf water content were found in the halophyte T. ramosissima, suggesting that increased succulence is not a universal response to salinity.

Water potential (Ψ_w) is usually used as an indicator of water status for most plants [37]. Osmotic stress is the primary constraint on plant growth under salinity. Our results suggest H. ammodendron and T. elongata demonstrate significantly decreasing leaf water potential with increasing NaCl. This is in conformity with earlier observations of H. ammodendron [38], T. ramosissima [39] and other halophytes [6,40]. In this study, the two studied species also decreased the Ψ_w under drought stress. As the Ψ_w of the leaves or assimilating shoots was lower than the soil water potential, the plants seemed to be able to take up enough water and to maintain a positive water balance. The Ψ_pd of T. ramosissima decreased by 230% with an increase in NaCl concentration in the soil from 0 to 500 mM, while H. ammodendron only decreased by 60%, suggesting that T. ramosissima needed more energy to regulate osmotic pressure than H. ammodendron under high salinity. Plants with basically equal water potentials are isohydric plants, plants with large differences are non-isohydric plants. Isohydric plants have a strong stomatal regulation ability, which can reduce water loss through stomatal regulation and make the plant water potential relatively stable [41]. The stomatal regulation ability of non-isohydric plants is weak, so water potential is greatly reduced under water scarcity. In our study, H. ammodendron may be an isohydric plant, but T. elongata is a non-isohydric plant. Therefore, H. ammodendron showed little difference in water potential but T. elongata showed a large difference.

Halophytes can grow well in environments with a high concentration of salinity via accumulating Na⁺, but non-halophytes cannot. Therefore, halophytes can be used as resources in the fields of environmental and ecosystem protection. High salt tolerance was associated with a capacity for Na⁺ accumulation in H. ammodendron and T. elongata. In the present study, leaf Na⁺ contents increased with increasing NaCl levels in H. ammodendron and T. elongata. This is in conformity with earlier observations of the Atriplex species [6,10], Reaumuria soongorica [9], and Panicum antidotale [23]. Halophyte Suaeda salsa compartmentlizes inorganic ion (Na⁺ and Cl^-) in the vacuoles for osmotic adjustment [34]. In addition, more than 50% of halophytes store excess salt through epidermal bladder cells (EBCs), which can be up to 1000 times the volume of ordinary cellular vacuoles [42]. In general, plants keep an osmotic balance by accumulating organic osmolytes under drought stress. However, they mainly absorb inorganic ions (such as Na⁺ and Cl^-) to adjust osmotic balance under salinity stress. The contribution of Na⁺ to osmotic potential was over 50% for H. ammodendron in saline environments [38]. This data confirms that H. ammodendron is an includer, securing water and nutrient supply, turgor pressure and growth by NaCl accumulation [10,40,43]. However, Tamarix species are excluders, exuding salt through salt glands via an apoplastic xylem pathway. Excluders are very energy consuming and thus limit biomass production or energy-dependent adaptation mechanisms. In addition, osmotic adjustment through inorganic ion uptake is more efficient than adjustment through the production of organic solutes [35]. Compared to T. elongata, H. ammodendron can save energy because it does not need to synthesize high amounts of compatible solutes or to actively uptake mineral nutrients for osmotic adjustment. The rapid closure of stomata in C₄ plants in turn affects ion absorption. This is reflected in the Na⁺ content in the C₄ plant H. ammodendron tested in our study, which was lower than in the C₃ plant T. elongata. Similar results have been demonstrated in the behavior of H. aphyllum and Cynodon dactylon (C₄ plant), which showed a lower salt uptake in C₄ plants than in C₃ plants [44]. The increase of leaf K⁺ content in T. elongata under high salinity is consistent with t Kalidium foliatum [45]. The study suggests that the roots can specifically transport K⁺ from the roots to the leaves, thereby maintaining a low K⁺/Na⁺ ratio in the leaves to reduce ion toxicity caused by salt stress. In our study, the lower Na⁺/K⁺ values were indeed measured (Figure 4f).

4.3. Photosynthetic Traits Under Salinity and Drought Stress

An additional set of four-carbon fixation reactions enable C₄ plants to increase net photosynthesis in warm, high-light, arid and saline environments by reducing photorespiration [24]. C₄ plants are known to display a high affinity to phosphoenolpyruvate carboxylase for CO₂, enabling their high assimilation rate even at low concentrations. A C₄ species can absorb CO₂ with more tightly closed stomata than can a C₃ species. Therefore, C₄ plants generally have lower stomatal conductance (g_s) and transpiration rates than C₃ plants. In the present study, the C₄ plant H. ammodendron had a lower g_s and higher maximum net assimilation rate (A_max) than the C₃ plant T. elongata. Also, C₄ plants generally have higher water use efficiency than C₃ plants. In addition, the light saturation point (LSP) and light compensation point (LCP) were higher in H. ammodendron than in T. elongata, because the C₄ pathway is most advantageous in warm, high-light conditions. The innate properties of C₄ photosynthesis are thought to contribute to the persistence of C₄ chenopods in high temperatures and saline soils in the semi-arid deserts of Central Asia [13].

The decrease in g_s causes the decrease of A, indicating stomatal conductance as one factor that limits photosynthesis under drought and salt stress. This study found a strong positive correlation between A and g_s in H. ammodendron (r = 0.48, p < 0.05) and T. elongata (r = 0.77, p < 0.001). Therefore, we suggest that stomatal limitation was the main factor for the reduction in leaf photosynthetic activity in H. ammodendron and T. elongata. A previous study has reported that mesophyll limitation is also important for a decrease in the net assimilation rate [40]. In the present study, compared to the drought stress, the L_s of H. ammodendron remained nearly constant from the 0 mM NaCl to the 500 mM NaCl treatments under the well-watered conditions, while the L_s of T. elongata remained nearly constant from the 0 mM NaCl to the 300 mM NaCl treatments, whereas the L_m increased to 23% (500 mM NaCl) and 20% (300 mM NaCl), suggesting that mesophyll regulation of photosynthesis became more important than stomatal closure under single salinity stress. This is similar to Na₂CO₃ stress in Populus cathayana seedlings [46] and recovery from water stress in mediterranean plants [16], which show a dominant role of mesophyll conductance under severe stresses. The reduction of mesophyll conductance has been ascribed to a collapse of parts of the mesophyll under drought and alkaline stress, due to loss of turgor [47]. This is similar to T. elongata under salt stress with well-watered condition in the present study. However, other explanations would be required to justify reduced mesophyll conductance in H. ammodendron, since turgor loss was not likely in view of the almost increased tendency of LWC (see Table 1). The reduced mesophyll conductance resulted in a large decrease in CO₂ from C_i to CO₂ concentration in chloroplasts, which posed a large limitation on CO₂ fixation [46]. Thus, reduced mesophyll conductance in H. ammodendron under salinity might be due to a limited diffusion of CO₂ into chloroplasts or metabolic impairment. In any case, the precise mechanisms affected by drought and salinity stress remain to be further studied.

In H. ammodendron and T. elongata, both the light saturated point (LSP) and light saturated assimilation (A_max) decreased with increasing NaCl stress, but not in the drought stress. This suggests that either light harvesting or electron transport were affected by the NaCl stress but not by the drought stress. A_max in H. ammodendron was higher than in T. elongata under drought and salinity stress, which suggests H. ammodendron has a more efficient photosynthetic capacity than T. elongata. The apparent quantum yield (AQY) in T. elongata decreased while there was no significant change in H. ammodendron under high salinity, which suggests T. elongata had a lower ability to absorb and convert light energy than H. ammodendron under high salinity. In the present study, F_v/F_m in H. ammodendron and T. elongata was not affected by the drought and salinity stress (Table 1), which is in agreement with other studies [48,49], suggesting that PSII activity is very resistant to the imposed stresses.

In addition to ionic and osmotic components, salt stress, like other abiotic stresses, also leads to oxidative stress. The reduced gas exchange of H. ammodendron and T. elongata under salinity implies the danger of oxidative stress. Photo-inhibition can occur when the light harvesting array captures more light energy than can be released by electron transport. The reduction of the flow of electrons through the photosystems in T. elongata (the reduction of AQY) reduced the risk of photo-inhibition. However, the decrease in capturing light energy (the reduction of chlorophyll content) in H. ammodendron diminished the risk of photo-inhibition. An adaptation in light harvesting capacity could be used to optimize photosynthetic efficiency. Although a reduction of the chlorophyll content was observed under severe salinity stress, metabolic limitation is not the reason for the decline of photosynthesis because a general failure of metabolism occurs only when daily maximum stomatal conductance drops below 0.1 mol H₂O m^–2s^–1 [50]. In the present study, the stomatal conductance in the two species was far greater than the value. Therefore, in all treatments, the diffusional limitations set the constraints to photosynthetic rates. This can, in turn, induce an indirect secondary effect on photosynthetic metabolism.

The effects of drought on the leaf gas exchanges (A, g_s, E) of H. ammodendron and T. elongata were milder than the effects of salinity, which was opposite to Lycium nodosum [33]. In general, the decrease in A with declining Ψ_w and the consequent decrease in g_s may indicate that stomata were imposing a larger limitation on A under drought and salinity stress. In the present study, H. ammodendron and T. elongata had a rising trend or no significant change in A and g_s under the moderate salinity. Similar results with salinity stress have been reported in a halophyte, Suaeda salsa; its leaf gas exchange was unaffected by high salinity (400 mM NaCl) [48]. The two halophytes had a low net photosynthesis, minimum transpiration and low stomatal conductance at their threshold salinity. This strategy tends to reduce the salt loading into the leaves and helps to increase the longevity by maintaining salts at subtoxic levels longer than would occur if transpiration rates were not diminished [51]. Plants growing under long-term drought or salinity often face a dilemma. On the one hand, plants must minimize water loss, and this leads to a reduction of stomatal conductance, resulting in lower photosynthetic efficiency. Photosynthesis, on the other hand, is needed to sustain vital biochemical processes. In this study, the two studied plants reduce the photosynthetic rate and transpiration rate, reduce the growth and reach a balance between water loss and carbohydrate acquisition [52]. The trade-off of minimizing water loss and maximizing carbon gain is via hydraulic and stomatal regulation [53].

5. Conclusions

We found that the decrease of water potential in T. elongata under high salinity stress (500 mM) was much higher than that in H. ammodendron. In addition, the increase of Na⁺ concentration in T. elongata with increasing salinity was also much higher than that in H. ammodendron, which suggests H. ammodendron can maintain a better water status than T. elongata in drought and high salinity environments. The addition of moderate NaCl (100 mM) can improve plant height in T. elongata under drought conditions, since more Na⁺ participates in the osmotic adjustment to maintain higher water relations. However, the adverse effect of salinity on plant height in T. elongata was greater than that in H. ammodendron. The diffusion limitations (stomata and mesophyll) were both responsible for a decrease in the photosynthesis of H. ammodendron and T. elongata under salinity and drought stress. Under single salinity stress, the mesophyll limitation of photosynthesis became more important than the stomatal limitation. Stomatal and mesophyll limitation both increased in severe combined drought and salinity stress, but the photosynthetic rate in H. ammodendron and T. elongata could remain constant or rise slightly through the trade-off between the L_s and L_m in moderate salinity. In addition, H. ammodendron had a higher photosynthetic rate than T. elongata under salinity and drought stress. In summary, these results suggest that the C₄ plant H. ammodendron adapted more quickly to drought and salt stress than the C₃ plant T. elongata, which makes H. ammodendron more widespread in arid and saline ecosystems.