Jia Sun·Jiangbao Xia·Ximei Zhao·Li Su·Chuanrong Li·Ping Liu

Abstract Vegetation restoration is a main ecological remediation technology for greening saline and alkaline soils.The objectives of this study were to determine the effect of 1-aminobenzotriazole (ABT-1) on the growth and physiology of Tamarix chinensis under salt stress and to determine a suitable ABT-1 concentration and soil salinity (S c) for propagating T.chinensis-cuttings.Cuttings were soaked in water and ABT-1 solutions at three concentrations(50,100,and 200 mg L−1) and propagated in pots containing four soil salinity levels,mild (0.3%),moderate (0.6%),and severe(0.9% and 1.2%),and compared with a control.The cuttings were measured to determine growth indices and physiological and biochemical indices (e.g.,chlorophyll content,superoxide dismutase activity,peroxidase activity,and malondialdehyde content).ABT-1 was effective in improving survival,growth,and physiological processes of cuttings under salt stress.However,there was a threshold effect when using ABT-1 to facilitate propagation under salt stress.ABT-1 effects were insignificant when applied at low concentrations(＜ 100 mg L−1).At a high concentration (＞ 100 mg L−1),ABT-1 limited growth and physiological activities.Under a salt stress level (S c ≤ 0.9%),ABT applied at a 100 mg L−1 concentration increased chlorophyll content and superoxide dismutase and peroxidase activities in the leaves and reduced malondialdehyde accumulation and membrane lipid peroxidation effects.As a result,ABT-1 enhanced the resistance of T.chinensis to salt stress.However,under high salt stress (＞ 0.9%) and ABT-1 concentration (＞ 100 mg L−1),the physiological regulatory ability of T.chinensis seedlings weakened.T.chinensis grew well at a salt stress ≤ 0.9% and ABT ≤ 100 mg L−1 and exhibited relatively high physiological regulatory ability and high salt adaptability

Keywords Salt stress·Rooting powder·Growth·Physiological and biochemical indices·Tamarix chinensis

Abbreviations

ABT-1 1-Aminobenzotriazole

Sc Soil salinity

Chl Chlorophyll

SOD Superoxide dismutase

POD Peroxidase

MDA Malondialdehyde

ROS Reactive oxygen species

MLP Membrane lipid peroxidation

CAT Catalase

Chla Chlorophyll a

Chlb Chlorophyll b

ChlT Total chlorophyll

Introduction

Soil salinization has a significant impact on attempts to develop sustainable agriculture and on environmental quality in general.It can hinder normal plant growth and severely degrade vegetation (Glenn et al.2012).Site restoration measures focusing on planting local halophytic or salt-tolerant plants have become an important approach for ameliorating saline and alkaline soils (Qadir et al.2000).Some halophytic plants remove salts from soil by absorbing and accumulating the salts (Hasanuzzaman et al.2014;Bhuiyan et al.2017).These plants improve the physical and chemical properties of a soil by reducing its bulk density and improving porosity,which can increase permeability and facilitate salt leaching (Yang et al.2019).However,a main issue in implementing vegetation measures to improve saline and alkaline soils is the difficulty associated with adaptation of the planted seedlings to salt-alkali stress during the initial growth stage,resulting in slow root system growth and relatively low survival rates.Hence,there is an urgent need to develop methods for improving the survival of halophyte seedlings during the initial planting stage and to study their physiological adaptability.

Research indicates a significant increase in superoxide dismutase (SOD) and peroxidase (POD) activities and malondialdehyde (MDA) levels in leaves of bean seedlings (Farhangiabriz and Torabian 2017) and Jerusalem artichoke (Xue and Liu 2008) with increasing soil salinity.Hu et al.(2018) reported an increase in MDA content and SOD and POD activities ofPanicum virgatumL.with an increase in salt-alkali stress.Li et al.(2017a,b) found that salt stress inhibited the growth of branches and roots ofSalix matsudana Koidz.Additionally,they found that lowlevel salt stress induced increases in SOD,POD,and catalase (CAT) activities,whereas high-level salt stress inhibited the activities of antioxidant enzymes.Moreover,the authors reported that low-level salt stress led to an increase in the MDA content and that this increased rapidly as salt stress increased.Hong et al.(2017) found that as salt or sodium chloride (NaCl) concentration increased,there was a decrease in relative water content,chlorophyll a content,total chlorophyll,and chlorophyll a/ chlorophyll b ratios in the leaves ofSalixspp.but an increase in chlorophyll b,proline,and MDA levels.Additionally,they detected increases in the SOD activity and soluble protein content in leaves under mild salt stress and a significant decrease in these two parameters under moderate and severe salt stress.However,Zhu et al.(2015) found that,as salt stress levels increased,SOD and POD activities and MDA in the leaves ofTamarix chinensiscuttings first increased and then decreased.Evidently the physiological and biochemical indices of plants vary significantly among species and salt stress levels.

T.chinensisare the main protective shrubs in muddy coastal zones in saline and alkaline soils in the Yellow River Delta and play a vital role in maintaining ecosystem stability and improving these soils.In recent years,as a result of global warming,there has been a decrease in precipitation and an increase in evaporation in the Yellow River Delta.Additionally,seawater intrusion caused by natural factors and human activities has intensified soil salinization and degraded theT.chinensisshrub ecosystem.Therefore,there is an urgent need to develop amelioration measures that focus onT.chinensisshrub restoration.Determining the physiological and biochemical processes by whichT.chinensistolerates salts is necessary for site restoration.China’s research on the species has mainly focused on the spatial distribution characteristics of populations (Xia et al.2016;Zhu et al.2016),the effects of water and salt conditions on photosynthetic efficiency water consumption characteristics(Ren et al.2019;Zhao et al.2019),the soil characteristics of theT.chinensiscommunity (Sun et al.2016),and the physiological and ecological characteristics of the species under salt (Zhu et al.2012),salt-alkali (Li et al.2017a,b),and combined salt-drought stress conditions (Zhu et al.2015).In contrast,other researchers treatingT.chinensisas an invasive species,have investigated its role in ecosystems (Gaskin and Schaal 2002;Gaskin and Kazmer 2009) as well as biological and physical removal methods for controlling its growth and propagation (González et al.2017).The physiological adaptability ofT.chinensisto salt stress and its role in ameliorating saline and alkaline soils have also been examined (Newete et al.2020).

1-aminobenzotriazole (ABT-1) has been used extensively for afforestation projects,in seedling transplant programs,and in the propagation of cuttings.Parađiković et al.(2013) found that a commercial rooting powder of ABT played an active role in the rooting and development ofSalvia officinalisL.andRosmarinus officinalisL.cuttings and significantly improved their morphological properties (height,number of leaves,root length,fresh weight,and dry weight).Research has shown that,prior to the propagation of cuttings,treating current year semilignified branches with a rooting powder of ABT can significantly improve survival.Tan (2014) found that treatingT.chinensisshoot cuttings with ABT at 200 mg L−1for one hour increased biomass and rooting percentage to 84.4% on average.A combination of ABT and fertilizer significantly improved root activity ofT.ramosissimain different soil layers (Zhang et al.2009).However,these studies focused on propagation under salt or salt-free conditions and the results cannot be extended to field conditions.Relatively few studies have been carried out to examine the vegetative propagation ofT.chinensisunder salt stress conditions.In addition,the effects of ABT-1 at various concentrations on the growth,physiological and biochemical characteristics ofT.chinensiscuttings under salt stress have yet to be investigated.As a result,it is difficult to determine the optimal concentrations of ABT-1 forT.chinensiscuttings.

In this study,ABT-1 was applied at four concentrations toT.chinensiscuttings at fourSc levels.T.chinensiscuttings without salt stress were the control (CK).T.chinensiscuttings were analysed to determine their growth indices(survival rate,height,root length,root biomass and total biomass),and their physiological and biochemical indices(chlorophyll content,SOD and POD activities,and MDA levels).Growth conditions and physiological and biochemical characteristics ofT.chinensiscuttings treated with ABT at various concentrations under salt stress were investigated.The results may provide information for vegetative propagation techniques forT.chinensis.

Materials and methods

Experimental materials and design

Branch cuttings were collected fromT.chinensisshrubs in the Shandong Changyi National Marine Ecology Special Reserve.In mid-February 2018,before they began to sprout,branches 1-cm thick were cut and harvested,and sectioned into 15-cm-long cuttings.Each cutting was sectioned obliquely at the base and flattened at the top.Four soil salinity (Sc) stress levels were studied;mild (0.3%),moderate (0.6%),and severe (0.9% and 1.2%).ASc of ≤ 0.1% was the control (CK).EachSc level was based on soil dry weight,monitored every seven days,and additional salt added to maintain the previously establishedSc.An 8-cm-deep tray was placed beneath each pot and water that leaked from each pot into the tray was poured back into the pot.Additionally,the tray was washed,and the washing water was poured into the pot.This procedure prevented salt loss.The base of the cuttings were soaked to a 2 −3 cm depth in ABT-1 solutions of 50,100,and 200 mg L−1.Ten cuttings were planted in pots filled with soil at eachSc level.Three replicates were used for each treatment.A total of 600 cuttings were planted in 60 pots.During the initial propagation stage,the pots were given freshwater twice daily to maintain the soil water content at 60%–70% of field capacity.The simulation experiment was conducted in a smart greenhouse in the Shandong Key Laboratory of Eco-Environmental Science for the Yellow River Delta (37°22′56″N,117°58′57″E).The sunlight intensity in the greenhouse was approximately 85%of the natural light intensity,temperatures were 18–35 °C,and relative humidity 41–65%.After 90 days,growth (e.g.,biomass),physiological,and biochemical indices of the cuttings were measured and analysed.

Measurement of indices

Growth indices:three pots were selected for each treatment,five cuttings were selected from each pot for measurements.Heights were measured with a metre ruler,and aboveground and belowground biomass were measured by harvesting the whole plant.Branches,trunk,and root system of each seedling were labelled,subsequently dried at 105 °C for 30 min and then dried at 80 °C to a constant weight.

Physiological and biochemical indices:three pots were selected for each treatment and three cuttings randomly selected from each pot.Normally grown,mature leaves (controls) ofT.chinensiscuttings were collected to determine their physiological and biochemical indices.The leaves were stored in liquid nitrogen and maintained in an ultra-freezer.Chlorophyll contents were measured per unit fresh weight using the ethanol-acetone soaking procedure (Li 2000).SOD activity was determined by nitroblue tetrazolium photoreduction (Luo and Wang 1999).POD activity was calculated by guaiacol colorimetry (Mao et al.2003).The MDA content was tested by thiobarbituric acid colorimetry (Zhao and Li 1999).Three measurements of each index (survival rate,height,root length,root biomass and total biomass,chlorophyll content,SOD and POD activities,and MDA levels)were obtained and subsequently averaged.

Data processing

The data were analysed in Microsoft Excel 2010.An ANOVA was performed using SPSS 17.0 statistical software to determine the significance of differences between the data.Two-way ANOVA was used to examine the effects of salinity and ABT-1 concentrations on the growth,physiological and biochemical indices.Multiple comparisons were performed using the least significant difference method.

Results

Survival rates of Tamarix chinensis cuttings

With increasing salt stress,there was a significant decrease in the survival rate of cuttings treated with various concentrations of ABT-1 (Table 1,Fig.1).Survival of the controls(CK) was 100%.Survival of cuttings soaked in water at aSc of 0.3% was significantly lower than in the control group(P＜ 0.05).There was no significant differences in rates of survival between cuttings treated with each concentration of ABT-1 at aSc of 0.3% and the controls (P＞ 0.05).These findings demonstrate that ABT-1 concentrations had no effect on the survival of cuttings in the absence of salt stress,and a relatively minor impact under mild salt stress.At a soil salinity of 0.6%,however,survival of cuttingstreated with ABT-1 at CK,50,100,and 200 mg L−1were 30.0%,20.0%,23.4%,and 36.7% lower than those in the CK group,respectively.At aSc of 0.6%,the survival of cuttings treated with ABT-1 at 50 and 100 mg L−1were significantly higher than those ofT.chinensiscuttings treated with water(P＜ 0.05).This result suggests that salt stress significantly decreases and ABT-1 significantly increases the survival ofT.chinensiscuttings.At aSc of 0.9%,survival was highest for cuttings treated with ABT-1 at 200 mg L−1(53.3%).In comparison,the survival of cuttings soaked in water was only 30.0%,significantly lower thanT.chinensiscuttings treated with ABT-1 at 50 and 200 mg L−1(P＜ 0.05).At a soil salinity of 1.2%,noT.chinensiscuttings soaked in water survived and only 6.7% of cuttings treated with various concentrations of ABT-1.

Fig.1 Effects of salt stress on the survival rate of T.chinensis cuttings treated with various concentrations of ABT-1;in this and all subsequent figures,different letters signify a significant difference between treatments (P ＜ 0.05)

Table 1 Two-way ANOVA results of effects of salt stress and ABT concentration on survival rate and growth indexes of T.chinensis cuttings

Growth characteristics of Tamarix chinensis cuttings

Salinity had a significant effect on the growth of cuttings,whereas ABT-1 concentrations did not,but the interaction of the two was significant (Table 1).As salinity increased,biomass varied as a function of the concentration of ABT-1(Fig.2 a).At aSc of 0.9%,the biomass of individual cuttings treated with ABT-1 at 100 mg L−1peaked at 16.7 g.The biomass of cuttings treated with ABT-1 at 0,50,and 200 mg L−1was 48.6%,48.6%,and 54.7% lower than that of cuttings treated with ABT-1 at 100 mg L−1,respectively.At the sameSc,there was a significant difference in biomass among cuttings treated with various concentrations of ABT-1 (P＜ 0.05) and under different salt stress levels.At aSc of 1.2%,biomass of cuttings treated with each concentration of ABT-1 significantly decreased (P＜ 0.05).As salinity increased,root biomass cuttings showed a decreasing trend(Fig.2 b).AtSc levels of 0.6% and 0.9%,50 mg L−1and 100 mg L−1ABT-1 significantly increased root biomass.

The differences in height betweenT.chinensiscuttings became significant asSc increased (P＜ 0.05).Overall,height decreased as salinity increased (Fig.2 c).Under mild(0.3%) and moderate (0.6%) salt stress levels,there was no significant difference in heights of cuttings treated with ABT-1 at 100 and 200 mg L−1.At aSc of 0.9%,heights of cuttings treated with ABT-1 at 0,50,100,and 200 mg L−1was only 60.45,62.6%,52.7%,and 52.4% of that of the controls.Root lengths of cuttings treated with ABT-1 at 50 and 100 mg L−1decreased initially and then increased as salinity increased,reaching their respective minimum values atSc of 0.6%.AtSc levels of 0.3% and 0.6%,root lengths of cuttings treated with ABT-1 at CK mg L−1was significantly greater than those treated with any concentration of ABT-1(Fig.2 d).

Fig.2 Effects of salt stress on a total biomass,b root biomass,c height and d root length of T.chinensis cuttings treated with various concentrations of ABT-1

Chlorophyll content in Tamarix chinensis cuttings

Salinity and ABT-1 concentrations had significant,interactive effects on chlorophyll content (Table 2).Differences in Chl a (Fig.3 a),Chl b (Fig.3 b),and Chl T (Fig.3 c) levels between cuttings treated with ABT-1 at different concentrations became significant (P＜ 0.05) as salt stress increased.At aSc of 0.3%,Chl a,Chl b,and Chl T in leaves of cuttings soaked in water were lower than in control leaves,although the differences were insignificant (P＞ 0.05).At aSc of 0.9%,Chl a and Chl T levels in leaves treated with ABT-1 at 50 and 100 mg·L−1reached their maximum.As the salt stress increased,the chlorophyll levels began to decrease,suggesting that photosynthesis is enhanced by increases in chlorophyll levels to adapt to salt stress,but only within a certain range.

Fig.3 Effects of salt stress on a Chl a content,b Chl b content,and c total Chl content in leaves of T.chinensis cuttings treated with various concentrations of ABT-1

Table 2 Two-way ANOVA results of effects of salt stress and ABT-1 concentration on chlorophyll content of T.chinensis cuttings

At aSc ≤ 0.3%,there was no difference in chlorophyll contents among the cuttings.At a salinity of 1.2%,Chl a,Chl b,and Chl T contents first decreased and then increased.At aSc of 0.9%,all three initially increased and then decreased as ABT-1 concentrations increased.The total chlorophyll levels in cuttings treated with ABT-1 at 50,100,and 200 mg·L−1were 26.0%,23.0%,and 11.8%higher,respectively,than in cuttings soaked in water.This suggests that under severe salt stress,ABT-1 at 50 and 100 mg·L−1improves chlorophyll synthesis and reduces the inhibitory effects of salt stress.

SOD activity in Tamarix chinensis cuttings

Salinity and ABT-1 concentration had significant effects and interactive effects on superoxide dismutase (SOD) activity(Table 3).As shown in Fig.4 a,SOD activity in cuttings treated with ABT-1 at various concentrations increased and then decreased with increasing salt stress.Under various salt stress conditions,SOD activity was higher in the leaves of ABT-1-treated cuttings than in controls.At aSc ≤ 0.6%,SOD activity decreased with increasing ABT-1 concentration.AtSc ≥ 0.9%,SOD activity increased and then decreased as ABT-1 concentrations increased.This suggests that,under mild to moderate salt stress,ABT-1 can inhibit SOD activity.Under severe salt stress,ABT-1 at of 100 mg·L−1increased SOD activity.However,under severe salt stress and a high ABT-1 concentration,there was a significant decline in SOD activity.TheSc corresponding to peak SOD activity varied with ABT-1 concentrations.Maximum SOD activity with zero ABT-1 occurred at aSc of 0.6%,and was 6.0 times greater than the controls.Maximum SOD activity with ABT-1 at 50,100,and 200 mg·L−1occurred at a salinity of 0.9%,and increased 2.6,4.2,and 4.3 times,respectively,compared with controls.AtSc of 0.9%,SOD activity was higher with ABT-1 at 100 mg·L−1than at 50 mg·L−1(P＜ 0.05).SOD activity was 20.9% lower and 14.7% higher in cuttings treated with ABT-1 at 50 and 100 mg·L−1,respectively,than in cuttings soaked in water.

POD activity in Tamarix chinensis cuttings

Salinity and ABT-1 concentrations had significant,interactive effects on peroxidase (POD) activity (Table 3).In cuttings treated with 50 and 200 mg·L−1ABT-1 concentrations POD activity increased initially and then decreased with increasing salt stress,and reached a maximum at a salinity of 0.9% (Fig.4 b).POD activity was relatively low in cuttings treated with ABT-1 at 50 and 200 mg·L−1compared with the controls.At aSc ≤ 0.9%,POD activity remained relatively high in cuttings soaked in water.However,at a salinity ≤ 0.6%,there were no significant differences between cuttings in water (P＞ 0.05).AtSc levels of 0.3%,0.6%,0.9%,and 1.2%,POD activity was 37.2%,77.5%,125.3%,and 56.2% higher,respectively,in cuttings treated with ABT-1 at 200 mg·L−1than in the controls.This suggests that salt stress significantly increased POD activity in cuttings treated with ABT-1 but was not significantly different from cuttings soaked in water.

Fig.4 Effects of salt stress on a SOD activity,b POD activity,and c MDA content in leaves of T.chinensis cuttings treated with various concentrations of ABT-1

Table 3 Two-way ANOVA results of effects of salt stress and ABT-1 concentration on the antioxidant enzymes activity and MDA content of T.chinensis cuttings

POD activity decreased and then increased with increasing ABT-1 concentrations at each salinity level except for the most severe level of 1.2%,at which there was a gradual increase in POD activity.At the same salt stress level,there was a decrease in POD activity in cuttings treated with ABT-1 at 50 and 100 mg·L−1concentrations.In contrast,there was an increase in POD activity in cuttings soaked inwater or treated with a high concentration of ABT-1.These findings suggest that a suitable concentration of ABT-1 can significantly reduce POD activity,whereas ABT-1 at too low or too high a concentration can easily lead to an increase.At salinity levels of 0.6%–0.9%,there was no difference in POD activity between cuttings soaked in water and treated with ABT-1 at 200 mg·L−1(P＞ 0.05),whereas there was a significant difference in cuttings treated with other concentrations of ABT-1 (P＜ 0.05).At aSc of 0.6%,POD activity was 60.9% and 40.0% higher in cuttings treated with ABT-1 at 200 mg·L−1than in those treated with ABT-1 at 50 and 100 mg·L−1,respectively.At a salinity level of 0.9%,POD activity was 63.1% and 53.1% higher in cuttings treated with ABT-1 at 200 mg·L−1than in those treated with ABT-1 at 50 and 100 mg·L−1,respectively.

MDA content in Tamarix chinensis cuttings

Salinity and ABT-1 concentration had significant interative effects on the malondialdehyde (MDA) content (Table 3).As salt stress levels increased,MDA levels,a marker for oxidative stress,increased and then decreased in cuttings treated with ABT-1 at 50,and 200 mg·L−1.In comparison,as salt stress levels increased,there was a gradual increase in the MDA in cuttings treated with ABT-1 at 100 mg·L−1(Fig.4 c).This may be a consequence of enzyme system regulation.Owing to this regulation,there was an increase in the activities of relevant antioxidant enzymes which,in turn,reduced cell membrane damage caused by salt stress.The MDA content in leaves soaked in water reached a maximum of 30.21 nmol mg−1at aSc of 0.6%.At this level,the leaves sustained significant damage caused by membrane lipid peroxidation (MLP).

At the same salinity level,the MDA content in ABT-1 treated cuttings varied with the concentration.AtSc levels of 0.6% and 0.9%,MDA levels decreased and then increased.Additionally,at these two levels,malondialdehyde levels were significantly higher inT.chinensiscuttings soaked in water than inT.chinensiscuttings treated with ABT-1.At aSc of 1.2%,MDA contents increased and then decreased,suggesting that at concentrations of 50 and 200 mg·L−1ABT-1 prevented the cuttings from being damaged by severe salt stress,and led to a significant decrease in MDA content.

Discussion

Effects of salt stress on the growth of Tamarix chinensis

The survival rate ofT.chinensiscuttings under mild salt stress (Sc ≤ 0.3%) was high and differed insignificantly from the controls.This suggests thatT.chinensisis relatively tolerant to salt.As salt stress increased,the survival of cuttings decreased to zero atSc ＞ 1.2%.Increasing salt stress also caused greater damage to cuttings and significantly affected the normal growth of their root systems.The effects of growth regulator concentrations on the growth of cuttings varied significantly.An increase in concentration will prevent gymnosperms from rooting (Wang et al.2006).Research has also revealed that increasing the concentration will not increase rooting percentage but can improve the quality of the root system.This is related to differences in the type and concentration of hormones and hormone sensitivities among tree species (Jin et al.2013).A rooting powder,such as ABT-1,can facilitate growth of the root system ofT.chinensiscuttings.By increasing and regulating the amounts of endogenous hormones and the activities of important enzymes inT.chinensiscuttings,ABT-1 stimulates cell division and growth in the inner root sheath,strengthens root system development,facilitates vigorous growth,significantly reduces cell membrane damage caused by salt stress,and increases salt stress adaptability.In this study,under mild salt stress (Sc ≤ 0.3%),ABT-1 concentrations had no impacts on the survival ofT.chinensiscuttings.As salinity increased,ABT-1 at a specific concentration increased survival rate.However,as the level of salt stress increased,ABT-1 became ineffective.A relatively high soil salinity generally causes osmotic stress in plants and disrupts their nutrient ion balance,thereby affecting physiological and biochemical processes such as growth,photosynthesis,osmotic adjustment synthesis,and lipid metabolism (Zhang et al.2017),and ultimately limits their growth and biomass accumulation.In this study,there was a significant decrease in the biomass,root lengths,and heights ofT.chinensisunder salt stress.The species may reduce biomass accumulation and utilize more resources and energy to respond to high salinity-induced damage (Zhang et al.2017),which suggests that plants under stress respond to adverse external conditions by altering their biomass allocation patterns.By reducing the proportion of biomass allocated to root systems,some species reduce salt absorption and transport to their aboveground portions (Osone and Tateno 2005).Some species acquire more water and nutrients by increasing the proportion of biomass allocated to their root systems.This mechanism increases plant growth and salt dilution in cells(Guo et al.2018).In this study,T.chinensiscuttings treated with ABT-1 at 100 mg·L−1had a relatively high root biomass and the highest whole-plant biomass at a salinity level of 0.9%,and their aboveground and belowground portions showed consistent growth.

Effects of salt stress on chlorophyll contents in Tamarix chinensis cuttings

Chlorophyll is a critical substance in photosynthesis and its content reflects the ability of plants to assimilate substances.Under salt stress,chlorophyll levels gradually decreased as salinity levels increased.Research has shown that,as salinity increases,chlorophyll levels either increase and then decrease or gradually increase,and plants exhibit relatively high salt tolerance (Yang et al.2012).The stimulation of chlorophyll types by low-level stress preconditions plants to future,larger environmental threats by permitting the maintenance of homeostatic levels.For example,increases in chlorophyll due to low-level stress counteract predicted decreases due to high-level stress (Agathokleous et al.2020).In this study,as salinity increased,chlorophyll a,chlorophyll b and total chlorophyll in cuttings treated with various concentrations of ABT all decreased,then increased and then decreased again.This pattern may have resulted from lowSc levels which were conducive toT.chinensisgrowth and did not activate its mechanism of salt stress resistance.As salt stress increased,chlorophyll levels increased.Because chlorophyll synthesis requires proline,under low salt stress,a large amount of proline accumulated in the leaf cells,facilitating chlorophyll synthesis.Under high salt stress,a decrease in chlorophyll was observed,suggesting an increase in the inhibitory effects of salt stress onT.chinensis.Within a certain salinity range,salt stress facilitates the decomposition of chlorophyll by the chlorophyllase enzyme (Zhao 1993),resulting in a decrease in chlorophyll content.These alterations were similar to the changes in chlorophyll content observed inT.chinensiscuttings (Zhu et al.2015) and inRhaphiolepis umbellataMakino,Rosa chinensisJacq.,andMorus albaLinn (Qiu et al.2006) under salt stress.Salinity levels corresponding to the peak chlorophyll levels differed in leaves ofT.chinensistreated with ABT at various concentrations.The higher the ABT concentration,the higher the salinity level corresponding to the peak chlorophyll,suggesting that applying ABT at a specific concentration could reduce the effects of salt stress on chlorophyll synthesis,potentially because ABT can increase the rooting rate and activity and thereby allowing early adaptation to salt stress.

Effects of salt stress on antioxidant enzymes activities

Salt stress leads to an increase in reactive oxygen species(ROS) levels in plants.ROS affects many cell functions by damaging nucleic acids,oxidizing proteins,and causing lipid peroxidation (Foyer and Noctor 2005).Plants have an ROS removal system consisting of antioxidant enzymes as key components.SOD and POD are the primary antioxidant enzymes in plants under salt stress and play a vital role in removing superoxide ions,preventing membrane lipid peroxidation (MLP) and reducing plasma membrane damage (Farhangiabriz and Torabian 2017).When not under stress,plant cells exhibit a dynamic balance between the production and the removal of ROS.Under stress,this balance is disrupted and ROS levels accumulate.Plants remove excess ROS by increasing SOD and POD activities.SOD converts the ROS formed during the oxidation process into oxygen and H2O2through disproportionation reactions.Subsequently,POD decomposes and removes the formed H2O2(Farhangiabriz and Torabian 2017).Under salt stress,there is a threshold for the ROS level tolerable to plant cells.Below this threshold,plants are able to remove ROS by increasing the activities of antioxidant enzymes.Above this threshold,the activities of antioxidant enzymes are inhibited,

resulting in excess accumulation of reactive oxygen speciesleading to tissue damage (Jamal et al.2016).In this study,as the salt stress level increased,SOD activity increased and then decreased in the leaves of the cuttings treated with various ABT concentrations.This suggests that as the salt stress level increased,ROS began to accumulate in the leaf cells andT.chinensisremoved excess ROS by increasing SOD activity to adapt to salt stress.However,under relatively high salt stress,the amount of ROS that formed exceeded the regulatory ability of SOD.Consequently,SOD activity was inhibited and therefore decreased.This SOD pattern is consistent with previously described inTamarix austromongolicaandT.chinensis(Li et al.2017a,b) in saline and alkaline habitats and in the leaves ofPennisetum alopecuroides(L.) Spreng (Miao et al.2019) under NaCl stress.Plants reduce salt stress-induced damage by increasing POD activity.The causes of the increase include not only ROS production but also cell membrane damage and changes in calcium ion concentration.At various salinity levels,POD activity in leaves of cuttings soaked in water was relatively high,suggesting that the large amounts of H2O2produced in leaf cells of cuttings not treated with ABT resulted in increased POD activity to remove H2O2.Evidently,the membrane lipid peroxidation damage induced by salt stress was three times greater in cuttings that were not treated with ABT than in cuttings in the three ABT treatment groups.Li et al.(2017a,b) found that as the salt stress increased,POD activity inT.austromongolicaandT.chinensisincreased initially and then decreased.They also found that POD activity in leaves of both species reached a maximum at aSc of 1.2%,which was 3.5 and 3.6 times greater than in the controls,respectively.A similar pattern was also found in this study.POD activity in leaves ofT.chinensiscuttings treated with ABT at 200 mg·L−1first increased and then decreased with increasing salinity.AtSc levels of 0.3%–0.9%,there was a significant increase in POD activity.The antioxidant enzyme system decomposed the H2O2produced by SOD through disproportionation reactions by increasing POD activity.Under high salt stress (Sc ≥ 1.2%),excess ROS were produced in leaf cells,exceeding the removal threshold of POD.As a result,the accumulating ROS damaged the enzyme system,resulting in a decline in POD activity.Compared with that observed under treatment with ABT at 50 and 100 mg·L−1,the sensitivity of POD activity was relatively low in the leaves of cuttings treated with ABT at 200 mg·L−1,and the ability of POD to regulate salt tolerance in these cuttings was also relatively low.

Effects of salt stress on MDA content in Tamarix chinensis cuttings

Plants produce MDA (malondialdehyde) as a result of MLP under stress.MDA is the main indicator of cell membrane damage and free radical formation in plants (Karim et al.2012).As MDA accumulates,membrane damage increases,and the resistance of the plant declines.Thus,MDA can be used as an important index for evaluating the extent of membrane system damage under stress (Shi et al.2014).The MDA content in leaves varies significantly with their salt tolerance.As salt stress increases,there is a continuous increase in MDA in leaves of plants with relatively low salt tolerance,such asT.chinensistissue culture seedlings(Guo et al.2015).In comparison,with increasing salt stress,the MDA content in leaves of plants with relatively high salt tolerance first decreases and then increases (Luo and Wang 1999).In this study,the MDA content in the leaves of cuttings increased initially and then decreased as salt stress increased.This finding is consistent with those reported by Li et al.(2005) showing the effects of MLP in the seedling leaves of six gramineous forages but differs from the results of Qiu et al.(2018) regarding the MDA content inLagerstroemia indica‘Pink Velour’ under salt stress.This distinction may be related to the significant differences in salt stress tolerance regulatory mechanisms among plants.Here,the MDA content in the leaves peaked at aSc of 0.6% and decreased at 0.9%;SOD and POD activities were relatively high at 0.9%.These findings suggest that,through inter-regulation,SOD and POD can effectively remove excess ROS produced under salt stress and maintain a low level in the organism,thereby essentially preventing ROS-induced MLP and other damaging processes.However,under severe salt stress (Sc=1.2%),SOD and POD activities were relatively low and there was a significant decrease in MDA content,which might have been related to the adaptive regulation of a certain dominant factor under the combined action of salt stress and ABT.The relevant internal mechanism requires further analysis.

Conclusions

ABT-1,1-aminobenzotriazole,the rooting powder used in this study,enhanced the resistance ofT.chinensisto severe salt stress (Sc ＞ 0.6%).ABT treatment had a relatively high compensatory effect on cuttings under salt stress.ABT at 100 mg·L−1exhibited a positive effect on growth.The effectiveness of ABT in improving survival and growth of cuttings became increasingly pronounced as the salt stress increased.ABT was most effective in improving survival when applied at 50 and 100 mg·L−1.T.chinensiscuttings adapt to salt stress at moderate levels by increasing the chlorophyll content.Increasing the ABT concentration strengthens the tolerance ofT.chinensiscuttings to salt stress during chlorophyll synthesis.

ABT helped enhance the regulatory ability of the enzyme systems and significantly reduced cell membrane damage caused by low salt stress.The enzyme activities were highest in cuttings treated with ABT at 100 mg L−1,and cell membranes in these cuttings sustained the least significant oxidative damage.The following conditions were found to be suitable for vegetative propagation ofT.chinensis:Sc ≤ 0.9%and ABT ≤ 100 mg·L−1.T.chinensisgrew well in this range and exhibited a relatively high physiological regulatory ability and relatively high salt adaptability.