Shengrong Xu•Ruili Ma•Enhe Zhang•Yuan Chen,2•Qi Wang•Qinglin Liu

Abstract In order to further understand the effects of drought stress on hydraulic characteristics and the relationship between hydraulic conductivity and aquaporins or water channels of root systems of three wolfberry cultivars(Lycium chinense Mill.),hydraulic conductivity of 2-yearold pot-grown seedlings was measured under drought stress,rewatering,and treatment with exogenous mercuric chloride andβ-mercaptoethanol.Under moderate and severe drought stress levels,the most significant decrease of hydraulic conductivity was 37.3% and 24.0%,respectively,in the‘Ningqi 5’cultivar compared with the nonstressed control.After rewatering,the rate of recovery in specific conductivity was most rapid in the‘Mengqi 1’cultivar,at 0.058 and 0.072 kg MPa-1 m-2 s-1 h-1 under moderate and severe drought stress levels,respectively.The‘Mengqi 1’cultivar had the highest recovery degree of hydraulic conductivity under two concentrations ofβ-ME(500 or 1000μmol L-1),reaching 82.4% and 88.5%,respectively,of the initial conductivity.The adaptive capacity of hydraulic conductivity in the‘Ningqi 5’cultivar was weaker than in the‘Ningqi 1’and‘Mengqi 1’cultivars under drought stress.The recovery capacity of hydraulic conductivity in‘Mengqi 1’cultivar was stronger than the‘Ningqi 1’and‘Ningqi 5’cultivars after rewatering.Aquaporins of the‘Ningqi 1’cultivar root systems had the highest binding affinity with mercuric chloride,which was the most likely cause in the decrease in hydraulic conductivity,whereas aquaporins of‘Mengqi 1’root systems had the weakest binding affinity.The inhibitory effect of mercuric chloride was readily eliminated by β-mercaptoethanol in the‘Mengqi 1’cultivar.The hydraulic characteristics of this cultivar were more sensitive to drought,mercuric chloride andβ-mercaptoethanol than the other cultivars.

Keywordsβ-Mercaptoethanol·Aquaporins·Hydraulic conductivity·Mercuric chloride·Wolfberry(Lycium chinense)

Introduction

Drought stress is a major limiting factor for plant survival and growth in arid and semi-arid areas(Meijie et al.2013).Numerous physiological and biochemical mechanisms are involved in plant adaptation to drought such as dehydration,water potential reduction,cell membrane disruption,and xylem embolism or obstruction(Centritto 2005).Plants have mechanisms to protect the integrity of cell macromolecules and membranes to sustain tissue desiccation without metabolic inactivation(Ennajeh et al.2008).Understanding the water use characteristics of droughttolerant plants is important for sustainable agricultural production and management in arid and semi-arid regions.

Woody plants have developed xylem transport systems which efficiently transport water from the soil to aboveground organs to ensure regular water supply(Meinzer and McCulloh 2013).Root-mediated absorption is the main process by which soil moisture enters the plant,and hydraulic conductivity of the roots can indicate changes in plant water relations under adverse conditions(Mu et al.2006).Abiotic environmental stresses such as drought or heat usually result in water deficits and affect growth and productivity(Holmberg and Bu¨low 1998).Such stresses induce increased resistance to water uptake by roots(Steudle and Peterson 1998;Lee et al.2004).

High-efficiency transmembrane water-transport channels or aquaporins have recently been identified in plasma and vacuole membranes.Aquaporins belong to the ubiquitous major intrinsic protein superfamily(Noronha et al.2014),a highly conserved family of proteins ranging in size from 23 to 31 kDa(Gomes et al.2009).Increasing evidence implicates a crucial role for aquaporins in plant water relations(Tyerman et al.2002).They are moisture absorption and transportation channels which,in the internal membranes of root cells,can affect the cell-to-cell component of radial water flow(Seong et al.2005),regulate transmembrane water flow under stress conditions,and are associated with the water cycle,transpiration rate,and photosynthetic intensity(Tofimova and Zhestkova 2001).Rapid responses ofleaf hydraulic conductivity to changes in temperature and irradiance are attributable to the participation of aquaporins in water transport(Nardini et al.2005;Tyree et al.2005).

Mercuric chloride can specifically bind with aquaporins to reduce xylem hydraulic conductivity and thus ultimately influence leaf water transport capacity(Lovisolo and Schubert 2006),and root system(Carvasal et al.1996).Negative effects are usually observed in plants treated with mercury which is often used as an inhibitor of aquaporins(Aroca et al.2006;Verdoucq et al.2008).Mercuric chloride blocks the majority of aquaporins and is frequently used to estimate aquaporin contribution to total root water transport(Javot and Maurel 2002;Beaudette et al.2007).Inhibition ofleaf hydraulic conductivity by mercuric chloride shows that foliar protoplasts and apoplasts equally influence seasonal hydraulic conductivity dynamics(Aasamaa and So˜ber 2005).The inhibition by mercuric chloride on water diffusion in roots of maize seedlings has been investigated using the pulsed nuclear magnetic resonance method.Blocking water channels decreased water permeability of cell membranes by 1.5-2 times(Ionenko et al.2006).Mercuric chloride at three concentrations decreased water use efficiency and water transport of oriental hybrid lilies to varying degrees,compared with those of controls(Yang et al.2016).

β-Mercaptoethanol(β-ME)may reverse the specific binding between mercuric chloride and aquaporins and restore water transport capacity.The addition of mercuric chloride to a root-bathing solution resulted in a large and rapid reduction in pressure-induced root water flux;the inhibition was largely reversible upon the addition ofβ-ME(Albino and Robert 1995).Leaves that transpiredβ-ME following mercury application showed similar hydraulic conductivity to those of the control leaves transpiring distilled water,whereas in leaves pressure-infiltrated with mercuric chloride,hydraulic conductivity significantly declined by 25%(Voicu and Zwiazek 2010).

In this study,the objectives were to:(1)evaluate the water relations based on the hydraulic characteristics of three cultivars of wolfberry(Lycium chinense);(2)evaluate the relationship between aquaporins,mercuric chloride and β-ME;and,(3)investigate the effects of drought stress and mercuric chloride on hydraulic characteristics of roots.The results should contribute to an improved understanding of drought-response strategies in wolfberry,a better understanding of the inhibitory effects of drought and mercury chloride on water transport by aquaporins.

Materials and methods

Site description and growth conditions

The field study was conducted from 2016 to 2017 at the Gulang wolfberry model garden(37.30°N,103.29°E,1760 m altitude),located on the Loess Plateau of northwest China.The field site has a semi-arid monsoon climate,with mean annual air temperature of 5.6 °C.The mean annual rainfall is 300 mm and the frost-free period ranges from 130 to 150 days.Seedlings were transplanted into growth barrels(height:45 cm;diameter:30 cm)filled with a 20-cm mixture of topsoil and vermiculite,containing 14.5 g kg-1organic matter,rapidly-available phosphorus(Olsen-P)28.5 mg kg-1,rapidly-available potassium 396.9 mg kg-1,and inorganic nitrogen(NO3-N plus NH4-N)39.44 mg kg-1.The field capacity was 26.3%.

Plant material and water treatment

The plant material were three cultivars of Lycium chinense,‘Ningqi 1’,‘Ningqi 5’,and‘Mengqi 1’.The relative drought tolerance of these cultivars,on the basis of hydraulic characteristics,is Ningqi 1＞Mengqi 1＞Ningqi 5(Xu et al.2017).Two-year-old self-rooted cuttings from seedlings with comparable leaf areas were transplanted into growth barrels 100-120 cm in height.Seedlings were grown in a greenhouse covered with shade netting and watered weekly.Forty seedlings of each cultivar were deprived of water after 60 days.There were five treatments,including controls:normal water(controls,75-80% field capacity),moderate drought(TM,50-60% field capacity),severe drought(TS,45-50% field capacity),moderate drought and rewatering(TM+R,50-60% field capacity),and severe drought and rewatering(TS+R,45-50%field capacity).Rewatering was carried out after seven days of physiological stability under drought stress.Seedlings were weighed for drought stress.During the drought and rewatering treatments,water relations and hydraulic conductivity were measured with three plants of each cultivar.

Mercuric chloride andβ-mercaptoethanol treatment

When the physiological state of water potential was stable,the roots were connected to containers filled with a solution of mercuric chloride(50μmol L-1)and after 20 min of immersion,water relations and hydraulic conductivity were measured for each cultivar at 30 min intervals over a 2-h period.The roots were removed from the mercuric chloride solution and an identical procedure was carried out for treatment with aβ-ME solution,either 500μmol L-1(TME1)or 1000μmol L-1(TME2).The concentrations of mercuric chloride andβ-ME were based on Wang et al.(2003)and Voicu et al.(2010).

Absolute(Kroot)and specific(Ks,root)hydraulic conductivities of roots

The hydraulic characteristics of five seedlings per cultivar were used for measurements of absolute hydraulic conductivity using a high-pressure flow meter(HPFM;Dynamax Inc.,Houston,TX,USA).A detailed description of this instrument is provided by Tyree et al.(1995)and Cochard et al.(2007).Tyree et al.(1994)reported that during HPFM measurements of root systems,the direction of flow was opposite to the physiological direction of transpiration and thus solutes were pushed towards the root tips where they would concentrate,driving osmotic water counter-flows that would lead to an underestimation of the true root hydraulic resistance(Rroot).To minimize this effect,rapid transient measurements are preferred to the quasi-steady-state mode in the case of root systems(Bogeat-Triboulot et al.2002;Tyree et al.2005).Roots were cut 5 cm above ground from the basal stem,and were then connected to the HPFM via the basal stem using compression fittings.Pressure(P)was increased at the rate of 5-8 kPa s-1up to 400 KPa while measuring instant flow rates every 3 s.Root absolute hydraulic conductivity(Kroot)was determined as the slope of the linear regression of the measured flow to the increasing applied pressure.At the end of each experiment,stem diameter at the root base(Droot)was measured using a digital caliper.The specific conductivity of the root(Ks,root)was determined using Ks,root=Kroot/Sroot.Root hydraulic resistance(Rroot)was calculated as the inverse of absolute hydraulic conductivity of the root(Kroot).

Statistical analyses

Statistical analyses were performed using the SPSS version 17.0(SPSS Inc.,Chicago,IL,USA).Hydraulic conductivity was compared among treatments using an analysis of variance followed by Tukey’s honestly significant difference test to determine the significantly different individual means.Means and corresponding standard deviations(SD)are presented.All statistically significant differences were tested at the 0.05 significance level.

Results

Effect of water stress on hydraulic conductivity

Plant water potentials were reduced by drought stress,causing xylem embolism.Root hydraulic conductivity declined with increased severity of drought stress(Fig.1).It was highest in the‘Ningqi 5’cultivar in the controls,reaching 2.1×10-5kg MPa-1 s-1.Hydraulic conductivities of the roots did not differ significantly among cultivars under moderate drought stress.Hydraulic conductivity was highest in the‘Ningqi 1’cultivar under severe drought stress.For ‘Ningqi 1’, ‘Ningqi 5’, and ‘Mengqi 1’ cultivars,hydraulic conductivity under moderate drought stress decreased to 41.7%, 37.3%, and 40.8%, respectively, and under severe drought stress to 27.5%, 24.0%, and 27.7%,respectively,compared with the controls.

Fig.1 Hydraulic conductivity of seedling roots of three wolfberry(Lycium chinense)cultivars grown with adequate water or exposed to moderate or severe water stress.Hydraulic conductivity was measured at the end of the water stress period and under the physiology condition keep relative stabilization.Letters indicate significant differences among water-stress treatments(Duncan’s test;P＜0.05).Data are the mean of five replicates;bars indicate standard error.

Response of hydraulic conductivity under drought stress and rewatering

When plants under drought stress were rehydrated to restore normal water levels,physiological activities were gradually restored and the harmful effects of drought reduced.In this study after severe drought stress and during rewatering,specific conductivities of the roots gradually increased in a parabolic curve pattern and the rate of recovery was more rapid than after moderate stress (Fig. 2a, b). Specific conductivity of the roots was stable before rewatering,and after rewatering,the ‘Mengqi 1’cultivar recovery speed was the fastest (P ＜0.05), reaching 0.058 and 0.072 (kg MPa-1-m-2s-1h-1) under moderate and severe drought stress,respectively. The specific conductivity of the roots of the‘Ningqi 1’cultivar was greater than the ‘Ningqi 5’cultivar,although the difference was insignificant, and under moderate and severe drought stress reached 0.054 and 0.068 for the ‘Ningqi 1’ cultivar, and 0.055 and 0.066 (kg MPa-1-m-2s-1h-1)for the ‘Ningqi 5’cultivar,respectively.

Fig. 2 Hydraulic conductivity of roots of three Lycium chinense cultivars exposed to moderate stress and rewatering (a), or severe stress and rewatering (b). Root specific conductivity was estimated from the hydraulic conductivity and stem diameter during the period of water stress (1.5 h) and after rewatering (2-5.5 h). The rate of recovery was estimated from the specific conductivity and rehydration time after rewatering (2-5.5 h). Data are the means of five replicates.

Effects of mercuric chloride and β-ME on hydraulic conductivity

There were negative effects on aquaporins in seedlings treated with mercuric chloride (Fig. 3a, b). β-mercaptoethanol can cancel the specific binding between mercuric chloride and aquaporins and restore aquaporin water transport capacity. The TMCtreatment (mercuric chloride 50 μmol L-1) reduced root specific conductivities of the‘Ningqi 1’,‘Ningqi 5’,and ‘Mengqi 1’cultivars by 50.3%,52.4%,and 52.8%,respectively.The TMEtreatment(β-ME 500 μmol L-1or 1000 μmol L-1) stimulated the recovery of root conductivity of the three cultivars to different degrees. The treatment concentration of β-ME was significantly correlated with the degree of hydraulic conductivity recovery. The degree of recovery of the ‘Mengqi 1’cultivar was the most with TME1and reached 82.4%compared with the controls, the degree of recovery was almost identical in the ‘Ningqi 1’and ‘Ningqi 5’cultivars,73.7% and 73.5%, respectively. The degree of recovery of the ‘Mengqi 1’cultivar was the most significant with TME2and reached 88.5%compared to the controls,the degree of recovery did not differ between the ‘Ningqi 1’and ‘Ningqi 5’ cultivars, 80.4% and 82.4%, respectively.

Effect of water stress, mercuric chloride and β-ME on hydraulic conductivity

In this study,the dual effects of drought stress and different concentrations of β-ME had a significant effect on root hydraulic conductivity(Fig.4a,b).When treated with MC(50 μmol L-1) under different drought stress conditions,the decline in hydraulic conductivity under moderate drought stress was greater than under severe drought stress.Among the three cultivars,the ‘Mengqi 1’cultivar showed the greatest decline in root hydraulic conductivity.After β-ME treatment to stimulate recovery, root specific conductivity increased rapidly, and reaching a certain level,increased slowly. The degree of recovery in specific conductivity of roots under moderate stress was greater than under severe drought stress, but the specific conductivity under either drought stress treatment did not recover to the level before drought stress.The specific conductivity of the‘Mengqi 1’ cultivar roots showed a maximum degree of recovery (85.3%) under moderate drought stress; this was significantly higher than observed in the other treatments.

Fig. 3 Hydraulic conductivity of seedling roots of three Lycium chinense cultivars grown with exposed to TMC+ME1 (MC 50 μmol L-1 and β-ME 500 μmol L-1) (a), and TMC+ME2 (MC 50 μmol L-1 and β-ME 1000 μmol L-1) (b). Specific conductivity was estimated from hydraulic conductivity and stem diameter before treatment (1.5 h) and after mercuric chloride (2-3.5 h) and β-ME treatments (4-5.5 h). Data are the mean of five replicates.

Fig. 4 Hydraulic conductivity of roots of three Lycium chinense cultivars grown under moderate drought (a) or severe drought(b) conditions, and exposed to TMC+ME1 (MC 50 μmol L-1 and β-ME 500 μmol L-1). Specific conductivity of the roots was estimated from hydraulic conductivity and stem diameter before and after the moderate drought (TM) or severe drought (TS) treatments with TMC+ME1. Data are the mean of five replicates.

Discussion

Influence of water stress on hydraulic conductivity

The structure and degree of xylem differentiation and differences in the activity of water channel proteins aquaporins in cytoplasmic membranes affect xylem hydraulic conductivity (An et al. 2005; Yao et al. 2011). Drought stress can change internal root structures;air may infiltrate through pit membranes into the water-filled xylem conduits and lead to xylem embolism. This accelerates the rate of phellem formation, the degree oflignification of endothelial and exodermic cells, and casparian strip deposition in the endodermis cell walls, all of which can affect xylem water conductivity (An and Zhang 2005). Drought results in xylem embolism and root hydraulic conductivity declines with increasing drought stress (Xu et al. 2017). The hydraulic conductivity of the root system was highest in the‘Ningqi 5’ cultivar controls, but decreased rapidly in response to drought stress, indicating that the ‘Ningqi 5’cultivar was more sensitive to drought stress compared with the other cultivars. Previous research has shown that variations in root hydraulic conductivity may be used to measure the adaptive capacity of plants to respond to drought stress (Cochard 1992; Tyree et al. 1999). The adaptive capacity of the ‘Ningqi 5’ cultivars was the weakest under drought stress, whereas the adaptive capacity of the ‘Ningqi 1’ and ‘Mengqi 1’ cultivars to drought stress was much stronger.

Response of hydraulic conductivity under drought stress and rehydration

Under drought stress and subsequent rewatering to restore normal water conditions, the specific conductivity of the roots gradually increased in a parabolic curve pattern over the rewatering period in all seedlings. The recovery rate after rehydration was higher under severe than under moderate drought stress, and the recovery rate of root specific conductivity was most rapid in the ‘Mengqi 1’cultivar, and significantly higher than the other cultivars(P ＜ 0.05).The specific conductivity of‘Ningqi 1’cultivar roots was greater than that of the ‘Ningqi 5’cultivar under both moderate and severe drought conditions but the difference was insignificant. The recovery of hydraulic conductivity capacity of‘Mengqi 1’cultivars was greater than‘Ningqi 1’ and ‘Ningqi 5’ cultivars under drought stress,but there was no significant difference between the recovery of ‘Ningqi 1’ and ‘Ningqi 5’ cultivars. There are several possible reasons for variations in hydraulic conductivity of roots under drought stress; for example, exodermic and endodermal cell walls may be suberized and water channel root proteins may change (North and Nobel 1995), either of which may account for the sensitivity of root hydraulics to drought stress. During the evolutionary adaption process to different environments, the root xylem has differentiated in different directions and to different degrees (Hacke and Sperry 2001; Yin and Hao 2018).Sustained drought affects the rate of cell division (Kirdyanov et al. 2003), and tracheid diameter (Eilmann et al.2011); temperature affects the formation of xylem (Cobb et al.2007)and day length can affect the number and traits of vessels and tracheids (Grebner and Chaloner 1990).Root system architecture and activity of water channel proteins in the roots of the ‘Mengqi 1’ cultivar were the most sensitive to drought, but the rate of recovery of the hydraulic conductivity after rehydration was rapid.

Influence of mercuric chloride and β-ME on hydraulic conductivity

Aquaporins are intrinsic proteins that selectively transport water molecules across cell membranes. Several internal and external factors can control the opening and closing of aquaporins (Wan et al. 2004; Zhao et al. 2007). Negative effects of mercuric chloride on aquaporins have been observed in a variety of plants (Sellin et al. 2008; Voicu et al. 2008). β-Mercaptoethanol may reverse the specific binding between mercuric chloride and aquaporins and restore water transport capacity (Maggio and Joly 1995;Wang et al.2003).In this study,mercuric chloride reduced root specific conductivity to different degrees, with that of the ‘Ningqi 1’ cultivar changed the most significantly. β-Mercaptoethanol stimulated the recovery of the specific root conductivity to different degrees.The concentration of β-ME was significantly correlated with the degree of root conductivity recovery.The recovery degree of the‘Mengqi 1’ cultivar was the most significant under TME1and TME2.Mercuric chloride inhibits transmembrane water transport,owing to a mercury(Hg2+)reaction with cysteine residues of aquaporins, leading to the blockage of water channels,the suppression of the root water transmission system, and eventually the decrease in hydraulic conductivity(Belimov et al. 2015). β-Mercaptoethanol can reverse the decline in conductivity caused by mercuric chloride but recovery to prior to treatment was not observed.Therefore,β-ME only partially overcame the inhibition of Hg2+on aquaporins,and in some plants, the degeneration in conductivity is irreversible (Albino and Robert 1995). The aquaporins of the ‘Ningqi 1’ cultivar showed the highest binding affinity with mercuric chloride, the most likely cause of the decrease in hydraulic conductivity. The aquaporins of the‘Mengqi 1’ cultivar showed weaker binding with mercuric chloride,and thus the inhibitory effect of mercuric chloride was easily removed by β-ME in the ‘Mengqi 1’ cultivar.

The effect of water stress, mercuric chloride and β-ME on hydraulic conductivity

Drought and mercuric chloride can inhibit root hydraulic conductivity,whereas rehydration and β-ME can restore it.In the present study, the dual effects of drought stress and β-ME at different concentrations had significant effects on root hydraulic conductivity. Under double-stress conditions, the effect on the aquaporins of the ‘Mengqi 1’ cultivar was most significant. Under drought stress, some aquaporins were inactivated and the number that bonded with mercuric chloride were reduced.Severe drought stress increased the number of inactivated aquaporins compared with the number under moderate drought stress, but the degree of change in hydraulic conductivity was more significant than under severe drought conditions. Drought stress not only causes embolisms to form in the xylem but also affects the activity of aquaporins(Tyerman et al.2002;Maurel et al. 2008; Vandeleur et al. 2009). Under severe stress conditions, aquaporins combine with mercuric chloride with greater difficulty than under moderate stress,whereas after treatment with β-ME, the number of specifically bound aquaporins declined and reactivation was relatively high. Ionenko et al. (2006) observed that aquaporins in the root system of maize seedlings were insensitive to mercuric chloride, the effect of water stress on hydraulic conductivity was similar to that of mercuric chloride, and that mercuric chloride had no effect on hydraulic conductivity under water stress. In this study,root hydraulic conductivity was significantly different after treatment with mercuric chloride and β-ME, and the sensitivity of the ‘Mengqi 1’ cultivar was greater than that of the other two cultivars.

Conclusion

The adaptive capacity of hydraulic conductivity in‘Ningqi 5’ cultivar plants was weaker than in the ‘Ningqi 1’ and‘Mengqi 1’ cultivars under drought stress. However, the recovery capacity of hydraulic conductivity by the ‘Menqi 1’ cultivar was stronger than that by the ‘Ningqi 1’ and‘Ningqi 5’ cultivars after rewatering. The concentration of β-ME was significantly correlated with the degree of recovery in the specific conductivity of the roots. The aquaporins of the ‘Ningqi 1’ cultivar had the highest binding affinity with mercuric chloride, which is the most likely cause of decreased hydraulic conductivity, and the inhibitory effect of mercuric chloride was most readily eliminated by β-ME in the ‘Mengqi 1’ cultivar. The hydraulic characteristics of the ‘Mengqi 1’ cultivar were more sensitive to drought stress, mercuric chloride, and β-ME than the ‘Ningqi 1’ and ‘Ningqi 5’ cultivars. The results provide for further understanding of water transmembrane transport in wolfberry.