Environmental and Experimental Botany 138 (2017) 46–56

Rapid response of the carbon balance strategy in Robinia pseudoacaciaand Amorpha fruticosa to recurrent drought

Weiming Yana, Yangquanwei Zhongb, Zhouping Shangguana,*a State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Northwest A&F University, Yangling, Shaanxi 712100, ChinabCenter for Ecological and Environmental Sciences, Key Laboratory for Space Bioscience & Biotechnology, Northwestern Polytechnical University, Xi’an,710072, China

A R T I C L E I N F O

Article history:Received 11 August 2016Received in revised form 14 March 2017Accepted 15 March 2017Available online 18 March 2017

Keywords:Drought cyclesPhysiological responsesRewateredSoil respirationNon-structural carbohydrates

A B S T R A C T

Drought is becoming more severe and frequent in some regions due to climate change, which leads tocarbon imbalance in plants and has gained significant attention. However, it remains unclear how thecarbon balance responds to recurrent drought and recovery. To understand the carbon balance responseto recurrent drought, we monitored dynamic changes in the physiological traits of two species duringcycles of drought and recovery, Amorpha fruticosa and Robinia pseudoacacia, which are planted widely onthe Loess Plateau. We found that the two species performed similarly in response to drought; bothshowed growth cessation and a reduction of carbon assimilation and respiration under cycles of droughtand recovery. The soil water content (SWC) at the stress point of the fluorescence parameters was lowerthan those of gas exchange and water potential, which were higher in the second drought than in the firstdrought, except aboveground respiration, which may enhance the risk of drought-related mortality. Afterrewatered, leaf photosynthesis recovered fully and root respiration increased; however, the abovegroundcarbon flux of the plants did not fully recover due to leaf shedding. In addition, drought caused a decreasein the stem diameter, which impeded phloem function and carbon translocation and redistribution,resulting in a decrease in non-structural carbohydrates in local plant tissues under drought in bothspecies. Furthermore, A. fruticosa showed higher total non-structural carbohydrates. Our results suggestthat plants that had experienced drought were more sensitive when faced with subsequent drought, andrecurrent drought enhanced the risk of mortality in plants.

© 2017 Published by Elsevier B.V.

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Environmental and Experimental Botany

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1. Introduction

Drought is becoming more severe and frequent in some regions,which is reportedly due to climate change (Hoerling and Kumar,2003; Myhre et al., 2013) and is the main reason for the carbon (C)imbalance in terrestrial ecosystems (Zhao and Running, 2010;Reichstein et al., 2013). Severe drought could result in a significantdecline in net primary productivity in many different forest typesand large-scale tree mortality events, which has received extensiveattention in recent years (Breshears et al., 2009; Allen et al., 2010;Hicke and Zeppel, 2013); thus, it is necessary to study the responseof plants’ C balance strategies to recurrent drought.

The underlying mechanisms of plant C imbalance caused bydrought are the subject of ongoing research and have generated

* Corresponding author at: Xinong Rd. 26, Northwest A&F University, Yangling,Shaanxi, 712100, China.

E-mail address: shangguan@ms.iswc.ac.cn (Z. Shangguan).

http://dx.doi.org/10.1016/j.envexpbot.2017.03.0090098-8472/© 2017 Published by Elsevier B.V.

some debate (Leuzinger et al., 2009; McDowell, 2011). Plants ceasegrowth and close the stomata to prevent water loss under droughtconditions, which results in a simultaneous decrease in photosyn-thesis (McDowell et al., 2008; Zhao et al., 2013). However,maintenance respiration responds more slowly to drought thanphotosynthesis, resulting in a C deficit and forcing the plant toutilize stored carbohydrates (McDowell, 2011). If the C deficitpersists for a long time, carbohydrates will be depleted and plantswill experience C starvation (Sayer and Haywood, 2006; McDowell,2011), resulting in plant mortality. In addition, the refilling of bothembolized xylem conduits and woody growth after rewatered arecarbon-costly processes (Bucci et al., 2003; Salleo et al., 2009) thatrequire the utilization of stored carbohydrates and result in Cstarvation. Thus, it is necessary to study the dynamics of plantC balance strategies in response to recurrent drought and recovery.

Extreme meteorological events are predicted to appear morefrequently due to climate change, such as severe summer droughts(Field et al., 2012; Myhre et al., 2013), which cause plants toexperience recurrent drought. After a single drought, precipitation

Table 1The physical and chemical properties of the soil used in this study.

Property Value

Taxonomy Udic HaplustalfTexture

2000–50 mm (g kg�1) 6450–2 mm (g kg�1) 694<2 mm (g kg�1) 342

Bulk density (g cm�3) 1.27pH (H2O) 8.30Water-holding capacity (%) 22.8Soil organic carbon (g kg�1) 7.45Soil total nitrogen (g kg�1) 0.84Soil total phosphorus (g kg�1) 0.69

W. Yan et al. / Environmental and Experimental Botany 138 (2017) 46–56 47

is usually abundant and rapid, leading to soil moisture fluctuations.Previous studies have indicated that many plants can recoverduring this rehydration process, including the full recovery of leafgas exchange (Gallé et al., 2007; Brodribb et al., 2010). However, theC balance strategies of plants during recovery and after a seconddrought following a short recovery are unclear. Some previousstudies have reported that plants can adapt to an abiotic stressenvironment when experiencing stress (Bruce et al., 2007; Walteret al., 2011) by changing the plant phenotype (Aubin-Horth andRenn, 2009), and changes in pigment content can preventphotodamage under drought conditions (Munné-Bosch andAlegre, 2000). The change in phenotype can result in a “stressmemory”, which involves signaling proteins and transcriptionfactors and protects plants when they are faced with recurrentdrought (Bruce et al., 2007). Previous studies have also suggestedthat a single abiotic stress event can reduce the resilience of anecosystem and cause the deterioration of the ecosystem whenfaced with recurrent stress (Scheffer et al., 2001). For example,Plaut et al. (2013) conducted a rainfall experiment in a forest andfound that drought resulted in a downward spiral of the plantsbecause trees were unable to utilize the intermittent soil water.Therefore, more frequent droughts may enhance the risk ofdrought-related mortality. Moreover, some studies have reportedthat plants showed greater vulnerability in plant communitiesfaced with recurrent stress (Lloret et al., 2004; Mueller et al., 2005).The intensity and duration of drought cause changes in plantfunction by limiting photosynthesis in rainless periods, andchanges in physiology and structure could alter the plants’ abilityto utilize soil water after precipitation (Resco et al., 2009; Brodribbet al., 2010). Some physiological changes in response to droughtcould prevent plants’ immediate death; for example, leaf sheddingcould reduce the C demand and result in an improvement in thewater status of the remaining foliage and the subsequent survivalof the individual (McDowell et al., 2008; Sala et al., 2010). However,the mortality of the fine roots caused by drought could decreasethe ability of a tree to utilize soil water when it becomes available.Xylem cavitation is expected to occur when water uptake by rootsis insufficient to offset water loss from transpiration underdrought, which would reduce the xylem hydraulic conductanceand cause a lower leaf potential for gas exchange, eventuallyreducing the potential for C assimilation and leading to negativeeffects on plant growth (McDowell et al., 2008). Recent studieshave investigated the effects of a single drought event on the Cbalance of a single species (Gallé et al., 2007; Jentsch et al., 2009)and of plant communities (Van Peer et al., 2004; Kreyling et al.,2008) and ecosystems (Noormets et al., 2008). The frequency andmagnitude of droughts are expected to increase in the future; thus,it is necessary to better understand the effects of recurrentdroughts on plant functions, such as the physiological mecha-nisms, C balance strategies and responses of different plants duringand while recovering from drought (Blackman et al., 2009).

Two C3 woody legume seedlings, Amorpha fruticosa L. andRobinia pseudoacacia L., were widely planted on the Loess Plateauas pioneer afforestation species due to their high droughtresistance. The large-scale afforestation of R. pseudoacacia hascaused substantial problems, such as small old trees, which may becaused by the C imbalance caused by low soil water availability;however, this has not been observed in A. fruticosa. To determinehow recurrent drought affects the ability of these two plants toutilize water and changes their C balance, we monitored dynamicchanges in plant growth and the whole plant C balance duringcycles of drought, recovery and a second drought. The objective ofthis study was to identify differences in plant growth and thewhole-plant C balance as well as changes in non-structuralcarbohydrate (NSC) content in response to recurrent drought inthese two species. We hypothesized that (1) plants that experience

drought would exhibit an advanced stress point in the nextdrought cycle and that (2) the C distribution pattern of A. fruticosawould perform differently from that of R. pseudoacacia duringrecurrent drought.

2. Materials and methods

2.1. Study site and experimental design

This study was conducted at the Institute of Soil and WaterConservation in Yangling, Shaanxi Province (34�170N, 108�040E)from June to September 2015. The study site experiences atemperate and semi-humid climate; the mean annual temperatureis 13 �C, and the mean annual precipitation is 632 mm, of whichapproximately 60% occurs in July–September.

Two-year-old seedlings of two deciduous woody legumespecies were studied, Amorpha fruticosa L. (shrub) and Robiniapseudoacacia L. (tree), which have been widely planted on the LoessPlateau as pioneer afforestation species. Seeds of both plants weresown in a nursery at the same time during the previous year. Threemonths before the start of the experiment, A. fruticosa (30–50 cmtall and 3–5 mm in diameter) and R. pseudoacacia (40–60 cm talland 3–5 mm in diameter) were transplanted from the field to 400 Lpots (980 � 760 � 680 mm, length � width � height) because smallplots may affect the experimental results and undermine thepurpose of an experiment (Poorter et al., 2012). The soil used in thestudy was collected from the 0 to 20 cm soil layer, and the physicaland chemical properties of the soil are presented in Table 1

The plants were assessed during drought cycles and dividedinto two treatments: well-watered (80% field capacity) anddrought-rewatered-drought treatments. A completely randomizeddesign was used, and six replicates with four plants each wereplanted. All of the plants were well watered before the experimentonset. The control plants were watered every other day, anddrought was induced by ceasing to water plants until the netphotosynthetic rate was decreased to close to zero or until thepredawn water potential (Cp) decreased to between �3.0 and�3.5 MPa before rewatering, which took 35 and 28 days in A.fruticosa and R. pseudoacacia, respectively. The turgor loss points ofthe two species were �1.25 and 1.22 MPa, respectively. Then, thedrought plants were rewatered until net photosynthesis hadalmost completely recovered; recovery took seven days. Addition-ally, 40 L, 20 L and 20 L of water was added on the first, fourth andseventh days of the recovery stage, respectively. The soil watercontent (SWC) after rewatered can be seen in Fig. 1b. Subsequently,the plants were kept without water for approximately 42 days,which constituted the second drought cycle. Three replicates wereused to measure physiological parameters, such as water potential,gas exchange, chlorophyll fluorescence, soil respiration, and totalplant C exchange parameters. The drought and control plants wereidentical during the entire study. The other three replicates were

Fig. 1. Time courses of (a) the mean daily temperature and relatively air humidity and (b) the soil water content (SWC, %) in the drought pot of R. pseudoacacia and A. fruticosaduring the experimental period.

48 W. Yan et al. / Environmental and Experimental Botany 138 (2017) 46–56

sampled for NSC content analysis. One plant was sampled in eachreplicate at each time point, for a total of three plants per sampling.The sampled plants were divided into leaves, branches, stems, barkand roots, and then oven-dried at 105 �C for 30 min and at 75 �C to aconstant mass. All of the samples were ground into a uniformlyfine powder and sieved through a 1-mm mesh screen.

2.2. Measurements

The SWC was measured with an SWC reflectometer probe(CS650-L, Campbell Scientific, Australia). The soil moisture at 10and 40 cm was recorded every 30 min, and the average SWC wascalculated. The mean daily temperature, relative air humidity andSWC are shown in Fig. 1.

Throughout the experiment, the leaf Cp and gas exchange weremeasured on sun-exposed leaves on the upper crowns of the plantson a sunny day, for which at least three plants per replicate wererandomly selected. Spot Cp measurements were performedbetween 05:00 and 06:00 h using a PMS 600 pressure chamber(PMS Instruments Company, Albany, USA). Gas exchange traitswere measured in at least two leaves per plant selected during09:00–11:00 h using the Li-Cor model 6400 system (Lincoln, NE,USA). Fully expanded, mature leaves at the upper crowns wereselected and marked for the gas exchange measurements on eachsunny day or the second day after each rainy day during theexperimental period, and adjacent leaves were selected the leaf Cp

measurements. The saturating photosynthetic photon flux densitywas between 1000 and 1500 mmol m�2 s�1 in the leaf chamberduring the measurement periods. The temperature, CO2 concen-tration and relative humidity inside the leaf cuvette were alwaysclose to ambient air values.

Measurements of leaf chlorophyll fluorescence were performedon the same leaf with gas exchange by a portable pulse amplitude-modulated fluorometer on at least two leaves per plant from09:00–11:00 h (FMS-2.02 system, Hansatech Instruments, Norfolk,

UK). The Fm', Fo' and Fs were recorded after 20 min of darkness,and the following parameters were determined and calculated: Fv/Fm (the maximum quantum efficiency of photosystem II); non-photochemical quenching (NPQ, heat dissipation), which wascalculated as (Fm-Fm')/Fm'; and photochemical quenching (qP),which was calculated with the following formula: qP = (Fm'–Fs)/(Fm'–Fo') (Baker and Rosenqvist, 2004).

Repeated chamber measurements were performed using an LI-8100 automated soil CO2 flux measurement system and an LI-8150multiplexer with 8100-104 long-term chambers (Li-Cor Inc.,Lincoln, NE, USA), and a polyvinyl chloride collar (20 cm innerdiameter, 11 cm height) was installed in the center of each plot tomeasure the temporal variation in the CO2 flux. The collar wasinserted 6–8 cm into the soil to allow the roots to grow into it, andit was kept free of falling leaves. Soil respiration (SR) was recordedcontinuously every 0.5 h during the measuring day. The chamberwas closed for 120 s, and the linear increase in the CO2

concentration in the chamber was used to estimate the SR. Themean SR of one day was calculated during the experiment. The soilused in the study was air-dried soil with the plant litter removed.The 5 cm soil temperature and water content were measured usinga thermocouple probe and a moisture meter (EC5, Decagon, USA),respectively.

The aboveground C exchange of the plants was measuredusing an LI-8100 attached to a soil chamber in which the standardsoil chamber was replaced by a 250 L transparent Perspexchamber from 09:00–11:00 h. The air in the chamber was mixedusing small fans. To measure the aboveground C exchange, thetransparent chamber was placed on smooth plywood atapproximately 10 cm above the soil surface, and the bottomrim of the transparent chamber was sealed with a rubber sealstrip to prevent air leakage. The aboveground fixed CO2 (APn) wasmeasured for 130 s after allowing the chamber to equilibrate forapproximately 20 s after the chamber was placed on the plywood.The first 70 s of the measurement was used as an estimate of the

W. Yan et al. / Environmental and Experimental Botany 138 (2017) 46–56 49

APn rate. After the APn rate measurement, the chamber wasvented and repositioned, which was followed by the measure-ment of the aboveground respiration (AR) rate with the chambercovered by a thick layer of opaque tarpaulin (Street et al., 2007;Williams et al., 2014).

The total NSC content was calculated as the sum of the starchand soluble sugar contents, which were determined using theanthrone method with minor modifications (Yemm and Willis,1954; Luo et al., 2015). Sugar and starch determinations wereperformed spectrophotometrically at 625 nm. The sugar and starchcontents were presented as g g�1 of dry matter. The C concen-trations was assayed by dichromate oxidation (Bao, 2000).

Changes in the stem radius were monitored with an automatedDD dendrometer (Ecomatik Gmbh, Dachau, Germany) with aresolution of 0.1 mm. The dendrometer was mounted 5 cm abovethe ground on the stems of plants of both species. Stem radiuschanges were continually recorded at 1 min intervals. Diametervariations in stems can be used as a reliable predictor of phloemhydration (Zwieniecki and Holbrook, 2009; Hartmann et al.,2013b). In addition, the bark tissue was very thin in these youngsaplings; thus, the thickness of the bark was neglected (Hartmannet al., 2013b).

2.3. Statistical analyses

The first day that the values of the physiological traitssignificantly declined relative to the average values of the previousdays was defined as the stress point (SP) (t-test, P < 0.05). It wasassumed that no drought stress occurred before the SP and that anydifference in physiological traits reflected the influence of droughtalone. An independent samples t-test was used to test for statisticalsignificance of the SWC at the SP, and one-way ANOVAs were used

Fig. 2. Time courses of the stem diameter variation of R. pseudoacacia and A. fruticosa in tmean daily stem diameter of the drought plants. The red arrow indicates the point of rewais referred to the web version of this article.)

to test for statistical significance at the 95% confidence level usingSPSS software (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Treatment effects on stem diameter variation

The stem diameter increased before the SWC decreased to9.05% in R. pseudoacacia and 12.13% in A. fruticosa. Then, ashrinkage of the mean daily stem diameter was observed beforethe plants were rewatered in the first drought, which showed adecrease of 208.37 mm in R. pseudoacacia and 132.18 mm in A.fruticosa, indicating a decline in the hydration of the phloem andthe xylem (Fig. 2). After rewatered, the stem diameter increasedsignificantly and then began to shrink when the SWC decreased to11.98% in R. pseudoacacia and 10.12% in A. fruticosa. The daily stemdiameter variation was higher when the SWC was higher duringboth drought cycles. The two species had different growthpatterns when subjected to recurrent drought, the rate of stemdiameter increase in R. pseudoacacia in the rewatered stage wassimilar to that of the control plants; however, in A. fruticosa, therate of stem diameter increase was lower than in the controlplants.

3.2. Leaf gas exchange, water potential and chlorophyll fluorescenceparameters

Leaf gas exchange (Pn and gs) decreased rapidly when wateringwas stopped in both recurrent drought cycles (Fig. 3). Rewateredcaused instantaneous increases in Pn, which showed no differencefrom the control (P > 0.05) in either species; however, the gs of R.

he recurrent drought cycles (a and b). The inset figures show the time courses of thetered. (For interpretation of the references to colour in this figure legend, the reader

Fig. 3. Time courses of the photosynthetic rate (Pn) (a and d), stomatal conductance (gs) (b and e), and predawn water potential (Cp) (c and f) of the control and drought R.pseudoacacia and A. fruticosa plants during recurrent drought cycles. The black arrows indicate the stress point (SP). Before the SP, it is assumed that no water stress wasimposed because there were no differences in the drought (P > 0.05).

Table 2The soil water content (SWC%) at the stress point (SP) for the predawn waterpotential (Cp), photosynthetic rate (Pn), stomatal conductance (gs), abovegroundfixed CO2 rate (APn), released CO2 rate (AR), soil respiration (SR), the maximumquantum efficiency of photosystem II (Fv/Fm), photochemical quenching (qP) andnon-photochemical quenching (NPQ) in both species and drought cycles. *Indicatesa difference between the two drought cycles, and � indicates the SP was notdetectable in the drought.

R. pseudoacacia A. fruticosa

SP1 (%) SD SP2 (%) SD SP1 (%) SD SP2 (%) SD

Cp 10.82 0.21 13.21* 0.52 11.17 0.48 15.15* 0.31Pn 15.01 0.14 14.47 0.86 14.79 0.98 16.37 0.38gs 15.01 0.14 17.72* 0.54 14.79 0.98 16.37 0.38APn 11.64 0.28 13.21* 0.17 10.72 0.42 12.92* 0.18AR 14.16 0.25 13.21 0.52 12.91 0.69 10.20* 0.17SR 15.01 0.14 16.45* 0.27 16.41 0.42 18.14* 0.06Fv/Fm 8.38 0.30 9.45 0.90 9.33 0.53 9.72 0.22qP 9.79 0.09 12.69* 0.90 10.52 0.48 – –

NPQ 8.38 0.33 9.45 0.96 9.33 0.53 – –

50 W. Yan et al. / Environmental and Experimental Botany 138 (2017) 46–56

pseudoacacia did not fully recover to the control treatment value (P< 0.05) (Fig. 3b). Pn and gs during the two drought cycles droppedto close to zero (i.e., the physiological death point) when the SWCwas 7.58% in the first cycle and 6.72% in the second cycle for R.pseudoacacia and 8.05% in the first cycle and 7.67% in the secondcycle for A. fruticose. The Cp showed no significant difference whenthe SWC was higher than 10.82% in the first drought cycle andrapidly recovered in R. pseudoacacia, with no difference comparedto the control (P > 0.05); the Cp showed a significant decreasewhen the SWC was lower than 13.21% during the second drought.The Cp of A. fruticosa showed a similar trend to that of R.pseudoacacia. Specifically, it showed a significant decrease whenthe SWC decreased to 11.17% and 15.15% in the first and seconddrought cycles, respectively, and fully recovered after three days ofrecovery. The SWC parameters of Pn, gs and Cp at the SP werehigher in the second drought cycle than those during the firstdrought cycle (Table 2).

The fluorescence parameters did not change significantly untilthe SWC decreased below 10.41% and 10.52% in the first droughtcycle in R. pseudoacacia and A. fruticose, respectively (Table 2), andthen the Fv/Fm and qP significantly decreased, and the NPQincreased as the SWC declined further (Fig. 4). In addition, the SWCparameters of Fv/Fm and qP at the SP were higher in the seconddrought cycle than those during the first cycle in R. pseudoacacia.The change in the Fv/Fm, qP and NPQ was greater in R. pseudoacaciain the later stage of the second drought than that observed during

the first drought; however, there was no significant change in thesecond drought in A. fruticosa.

3.3. Aboveground net C exchange of the plants and soil respiration

In the first drought cycle, the APn and AR of R. pseudoacaciadecreased significantly when the SWC was lower than 11.64% and14.16% (Fig. 5a), respectively, and the SWC at the SP of the APn and

Fig. 4. Time courses of the maximum quantum efficiency of photosystem II (Fv/Fm) (a and d), photochemical quenching (qP) (b and e), and non-photochemical quenching(NPQ) (c and f) in the control and drought R. pseudoacacia and A. fruticosa plants during recurrent drought cycles.

W. Yan et al. / Environmental and Experimental Botany 138 (2017) 46–56 51

AR in A. fruticosa were 10.72% and 12.91%, respectively, indicatingthat the AR was more sensitive than the APn in the first drought. Inthe second drought, the SWC at the SP of the APn in R. pseudoacaciaand A. fruticosa were 13.21% and 12.92%, respectively, which washigher than that in the first drought; however, the SWC at the SP ofthe AR in the second drought was lower than that in the firstdrought in both species.

Drought also strongly inhibited root development and micro-bial activity. The control plants had well-developed root systems,in which the SR increased with the plant growth (Fig. 6). The SRdecreased during both drought cycles, and was more sensitive thanthe other parameters to decreases in the SWC. The SWC at the SPwas 15.01% and 16.41% of the SR in R. pseudoacacia and A. fruticosain the first drought, respectively, and then it decreased slowly asthe SWC decreased further. Interestingly, the SR values of bothspecies increased sharply after rewatered, and were higher thanthe SR when the experiment began. Similar to the other traits, theSWC at the SP of the SR was lower in the first drought than in thesecond drought (Table 2), with values of 16.45% and 18.14% for R.pseudoacacia and A. fruticosa in the second drought, respectively.

3.4. Recurrent drought effects on the NSC and organic C content

The C pools in the plants were different in various tissues andwere also affected by drought. The soluble sugar content washigher in the bark and root tissues in both species, and the starchcontent was higher in the roots, as was the total NSC content. We

also found that the starch and total NSC contents were higher in A.fruticosa than in R. pseudoacacia overall (Fig. 7). In addition, thesoluble sugar and starch and the total NSC concentrationdecreased in the leaves, branches and bark of R. pseudoacaciaafter the first drought, but showed a slight increase in the stems.Furthermore, the organic C content was increased in the varioustissues after the first drought, except in the roots. However, in A.fruticosa, the leaves and bark showed a decrease in the solublesugar and starch and the total NSC concentration after the firstdrought. In addition, the organic C content was increased in thevarious tissues, except in the leaves. The soluble sugar in the stems,bark and roots decreased after rewatered in both species. Incontrast, the starch content was increased, and the total NSCcontent showed a different response after rewatered. In addition,the organic C content varied in both species after rewatered; inparticular, the organic C content was increased in the leaves, stemsand roots of R. pseudoacacia, whereas it was decreased in thesetissues in A. fruticosa. The soluble sugar in the branches, stems,bark and roots showed an increase in both species after the seconddrought cycle, and the starch in the leaves, branches and rootsshowed a decrease. Furthermore, the total NSC showed a similarresponse in both drought cycles.

4. Discussion

Soil water availability is one important factor that limitsterrestrial biological activity in an ecosystem (Huxman et al.,

Fig. 5. Time courses of the aboveground fixed CO2 and released CO2 rates of the drought R. pseudoacacia and A. fruticosa plants during recurrent drought cycles.

52 W. Yan et al. / Environmental and Experimental Botany 138 (2017) 46–56

2004), and the importance of soil water availability is increasing asvariations in precipitation patterns due to climate change (East-erling et al., 2000; Hoerling and Kumar, 2003) may cause droughts

Fig. 6. Time courses of the soil respiration in the control and drought R

to become more frequent and severe in some regions (Myhre et al.,2013). Thus, it is necessary to better understand the effects ofdrought on plant functions, especially the underlying mechanisms

. pseudoacacia and A. fruticosa pots during recurrent drought cycles.

Fig. 7. Non-structural carbohydrate (NSC) concentrations (in g g�1 of dry biomass) for soluble sugar and starch, total NSC and organic carbon (C) concentration (in g kg�1)during the experimental period in the different plant tissues. “Before” indicates the beginning of the experiment, “drought 1” indicates the end of the first drought cycle,“rewatered” indicates 7 days after recovery, and “drought 2” indicates the end of the second drought cycle. The different lowercase letters indicate a significant difference atP < 0.05. The lowercase letters above and below symbols represent A. fruticosa and R. pseudoacacia, respectively.

W. Yan et al. / Environmental and Experimental Botany 138 (2017) 46–56 53

of the responses of plants to recurrent drought (Blackman et al.,2009).

4.1. Advanced stress point in recurrent drought

All the studied physiological traits showed significant changesas the drought progressed during recurrent drought; however, thesensitivity of the various physiological traits differed among thetraits and between the two species and the drought cycles. Theresults showed that the stem growth response was more sensitivethan the other physiological traits, especially in R. pseudoacacia, asthe stem increase rate began to decrease during the third day of theexperiment compared to that in the control plants. This resultsupports previous findings that plant growth is often the firstprocess to be affected due to the acute sensitivity of cell turgor andthe effects of cell division, enlargement and differentiation (Galvezet al., 2011; Mitchell et al., 2014). In addition, the SWC at the SP ofthe fluorescence parameters was lower than the other traits(Table 2), which indicated that the PSII reaction centers were morestrongly affected by the lower SWC (Maxwell and Johnson, 2000;Woo et al., 2008; Bresson et al., 2015).

During drought stress, C assimilation should be mainlyresponsible for the C balance because it shows larger decreasesthan respiration under drought conditions (Zhao et al., 2013).Plants close the stomata to prevent water loss under drought,which can reduce the CO2 diffusion in and out of the leaves,causing declines in Pn and assimilation (Flexas et al., 2006; Yanet al., 2016). Consistent with previous studies (Adams et al., 2009,2013; Hartmann et al., 2013a), we observed an earlier decline inthe AR compared to the APn under drought. The APn in thedrought-exposed plants significantly decreased until 10 and20 days and declined to the minimum level after 28 and 35 days inR. pseudoacacia and A. fruticosa (Fig. 5), respectively. The earlierdecrease in the AR during drought may be mainly caused by aturgor-mediated decrease in plant growth (Muller et al., 2011),which occurred in R. pseudoacacia but not in A. fruticosa (Fig. 2). Inaddition, a direct reduction in mitochondrial respiratory capacitycould also decrease the AR (Atkin and Macherel, 2009). We foundthat the SWC at the SP of the APn was lower than that for the ARand SR (Table 2) in the first drought, which indicated that thedrought might have caused an NSC surplus during early droughtbecause the drought caused greater reductions in growth and

54 W. Yan et al. / Environmental and Experimental Botany 138 (2017) 46–56

maintenance respiration than the APn (Hummel et al., 2010;McDowell, 2011; Pinheiro and Chaves, 2011). The lower APn in thelater stage of drought was also affected by the total number ofleaves because the drought caused leaf shedding in R. pseudoacaciaand A. fruticosa of 4.2 g and 0.99 g per plant in the first drought and4.6 g and 1.8 g per plant in the second drought, respectively. In therecovery stage, although the Pn at the leaf level almost recovered,the APn could not fully recover due to leaf shedding.

The SR is inhibited during both drought cycles (Bryla et al.,1997;Huang and Fu, 2000; Thorne and Frank, 2009). Roots are the firstplant organ affected by soil drying; both the root growth andnutrient uptake of plants decline (Espeleta and Eissenstat, 1998;Eissenstat et al., 1999), as well as the root cell integrity (Huanget al., 2005). Moreover, lower microbial activity, higher rootmortality (Harper et al., 2005) and a reduction in the photosyn-thetic C supply (Huang et al., 2005; Flexas et al., 2006; Atkin andMacherel, 2009) contribute to a further reduction in the SR duringdrought. However, the SR had a stimulating effect at the recoverystage, which was consistent with previous studies that showedthat after a rewetting event, the SR increased rapidly (Sponseller,2007; Aanderud et al., 2011) due to an increase in soil microbialand root activity. The later increase in the SR during the recoverystage was mainly due to increased root activity and biomass.However, lower SR rates were observed at a later stage of droughtin the second drought cycle than in the first, primarily due to leafshedding, which caused lower maintenance respiration (Scageland Andersen, 1997). In addition, we found that the SWC at the SPof the SR was earlier than the leaves’ physiological traits, indicatingthat the SR was the most sensitive to the drought because the rootswere the first plant tissue affected by soil drought (Eissenstat et al.,1999).

Although the two species examined here are morphologicallydifferent, a few general patterns occurred regarding the plants’responses during recurrent drought stress. The physiologicaltraits did not significantly decrease at the onset of treatment butshowed a sharp decrease as the SWC decreased further, whichindicated that the decline of the SWC in early stage of theexperiment did not affect the physiological traits in the firstdrought (Figs. 3 and 4). However, the results showed differentphysiological responses between the two drought cycles in bothspecies. In particular, the SWC at the SP in the first drought waslower than that in the second drought cycle, except the AR(Table 2). These findings indicated that plants respond to droughtmore rapidly to subsequent drought because the accumulation ofproteins and transcription factors involved the drought process,which can enhance the response rate to subsequent droughtstress (Bruce et al., 2007; Boyko and Kovalchuk, 2011; Backhauset al., 2014) and also optimize C partitioning for osmoticadjustment to adapt to subsequent drought (Hartmann et al.,2013b). The lower SWC at the SP of the AR in the second droughtobserved in this study may be because the plants’ response todrought, which involves signaling proteins and transcriptionfactors, was stronger during the second drought to protectthemselves (Bruce et al., 2007); however, all these metabolicprocesses require metabolite and energy from respiration. Inaddition, this less sensitive response in recurrent drought of theAR to the SWC caused the plants to utilize the restored C and mayhave enhanced the risk of drought-related mortality (Plaut et al.,2013).

4.2. Different C distribution patterns in recurrent drought

When the free mobile NSC cannot meet the requirements ofnormal plant growth, C starvation may occur (McDowell et al.,2011). Although the threshold at which NSC availability fails tomeet a plant’s growth demand is not defined, drought results in a

larger decline in C assimilation without a concomitant reduction inC consumption (McDowell et al., 2011). In this study, C assimilationand respiration decreased with drought as expected (Figs. 5 and 6),and C assimilation showed a greater decline than respiration. Inaddition, the decrease in stem diameter (Fig. 2) suggested that thestem water potential was very low as the SWC decreased further(Offenthaler et al., 2001), although a phloem water deficit couldexplain 90% of the diurnal variation in stem diameter (minus radialgrowth) due to the link between xylem water potential and phloemfunctioning (Zweifel et al., 2005). The reduction in stem diameterin both species indicated a severe impact of phloem function and astrong C limitation (no C left for stem growth) or a shift in Callocation from plant structural growth to maintenance respirationduring this period in drought-exposed plants (Offenthaler et al.,2001; Hartmann et al., 2013b). A lack of phloem transport wouldimpede C translocation because the translocation of stored C fromaboveground to belowground requires phloem loading andtransport (Hölttä et al., 2009); thus, the impairment of phloemfunction would affect the C translocation and redistribution,resulting in C depletion in local tissues rather than whole-plant Cstarvation. Stem growth increased suddenly and substantially afterrewatered, indicating relief from the limitation of phloem functioncaused by drought (Hartmann et al., 2013b) and suggesting arecovery of the C translocation and distribution pattern of thephloem.

In the present study, we found that the C concentrations indifferent issues showed no significant difference (Fig. 7);however, the soluble sugar content was higher in the bark androots, and the total NSC content was significantly higher in theroots in both species. Furthermore, the results also showed thatthe soluble sugar, starch and total NSC content were higher in A.fruticosa, which suggests a greater chance of survival underdrought (O’Brien et al., 2014). The different C distributions in thedifferent tissues suggest a different C allocation and balance underdrought, which also indicates a switch in carbohydrates fromgrowth to maintenance induced by drought to balance carbohy-drate accumulation and plant growth (Galvez et al., 2011). Wefound an indication of soluble sugar depletion in the leaves,branches and bark after the first drought because soluble sugar ispreferentially used by plants (Klein et al., 2014), whereas the rootsshowed an increase in soluble sugar but a decrease in starch,indicating that the roots began to utilize the starch anddemonstrating a C deficit in the roots (Galvez et al., 2011). Adecline in total NSC was observed in leaves due to maintenancerespiration and a larger decrease in photosynthesis. In addition,the impeded phloem transport during drought was likely alsoresponsible for the decreased NSC in the branches, bark and rootsbecause impeded phloem affected the C translocation from sourceto sink and forced the sink tissues to progressively rely more onlocal C reserves for respiration and even on other respirationsubstrates, such as lipids (Tcherkez et al., 2003; Hartmann et al.,2013b). The depletion in the leaves, branches and bark indicatedthat the NSC could not meet the normal growth requirements ofthese tissues, which is consistent with previous results (Mulleret al., 2011; Hartmann et al., 2013b; Mitchell et al., 2014). Afterrewatered, we observed an increase in NSC in the leaves due to therecovery of photosynthesis and an increase in roots and bark,which suggested recovery of C translocation and phloem function.However, a decrease in soluble sugar was observed in thebranches, stems, bark and root tissues, mainly caused by therecovery of tissue growth because the soluble sugar could be usedquickly for plant growth (Klein et al., 2014). The increase in starchindicated the synthesis of carbohydrates after rewatered wasstored in the form of starch, which could be decomposed to solublesugars for osmoprotection and osmoregulation when plantssuffered drought (Galvez et al., 2011).

W. Yan et al. / Environmental and Experimental Botany 138 (2017) 46–56 55

5. Conclusions

In conclusion, the two species studied here showed reduced leafgas exchange and limited growth as the SWC decreased. However,the responses of both species differed between the first and seconddrought. The SWC at the SP for the fluorescence parameters waslower than that of the other physiological traits, and all of the SWCfluorescence parameters at the SP were higher in the seconddrought cycle than in the first, except the AR. As expected, both theAPn and AR were reduced under drought. Although the leaf Pnrecovered fully, the APn did not fully recover due to leaf shedding.Moreover, the SR had a stimulatory effect at the recovery stage. Inaddition, drought strongly reduced the stem diameter, whichimpeded phloem function and caused a decrease in the NSCconcentration in the plant tissues in both drought cycles,specifically the leaves, branches and bark showed NSC depletionafter the drought. Finally, a decrease in soluble sugar was observedin the branch, stem, bark and root tissues after rewatered due torecovery of tissue growth.

Data accessibility

All the data used in this manuscript are presented in themanuscript.

Acknowledgments

This study was financially supported by the National NaturalScience Foundation of China (41390463) and the National KeyResearch and Development Program of China (2016YFC0501605).

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