Morphological, anatomical and physiological responses of Campylotropis polyantha (Franch.) Schindl. seedlings to progressive water stress

Morphological, anatomical and physiological responses of Campylotropis polyantha (Franch.) Schindl. seedlings to progressive water stress

Scientia Horticulturae 127 (2011) 436–443 Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: www.elsevier.com/locate...

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Scientia Horticulturae 127 (2011) 436–443

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

Morphological, anatomical and physiological responses of Campylotropis polyantha (Franch.) Schindl. seedlings to progressive water stress Fang-Lan Li, Wei-Kai Bao ∗ , Ning Wu Key Laboratory of Ecological Restoration, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, PR China

a r t i c l e

i n f o

Article history: Received 9 October 2009 Received in revised form 23 July 2010 Accepted 29 October 2010 Keywords: Water deficit Drought adaptation Leaf traits Morphological plasticity Chlorophyll a fluorescence Water threshold

a b s t r a c t In this study we implemented a potted water supply experiment for 100 days by a completely random sole-factored design with five treatments: 100% (W100 ), 80% (W80 ), 60% (W60 ), 40% (W40 ) and 20% (W20 ) of water holding capacity (WHC), corresponding to the soil volumetric water content (SVWC) maintained at 38.8 ± 0.3%, 31.6 ± 1.7%, 25.6 ± 1.3%, 16.5 ± 0.7%, and 8.1 ± 1.1%, respectively. The objective was to evaluate the ability of the 2-month-old Campylotropis polyantha (Franch.) Schindl. seedlings to tolerate drought and to explore the mechanism resisting drought. We monitored the growth process of seedling height and leaf number monthly and further investigated those changes in plant growth, dry mass accumulation and allocation, water-use efficiency (WUE), leaf functional traits, chlorophyll a fluorescence and pigment contents across the water deficit gradient. We found that the seedlings presented optimal growth, dry mass production, and physiological activity only at the W100 and W80 treatments and afterwards significantly decreased with progressive water deficit; the WUE was improved under moderate water stress (W60 and W40 ) but reduced under severe stress (W20 ). The serious leaf shedding, growth stopping and seedling death under the W20 condition revealed that the current-year shrub seedlings could not withstand severe drought. Water stress-induced decrease in total plant leaf area due to a combination of limited expansion of younger leaves and shedding of old leaves caused the leaf area ratio reduction under drought. The reduced mesophyll cell was a major anatomical response of leaves along the water stress gradient. The progressive water stress significantly damaged light harvesting complex and reduced photochemical processes and PSII activity. Our results clearly showed that the current-year shrub seedlings took the avoidance and tolerance mechanisms to withstand progressive drought stress and around 25.6% SVWC and around 12.3% SVWC separately are thresholds to limit the optimal growth and dry mass production and to last growing and surviving for the current-year shrub seedlings. © 2010 Elsevier B.V. All rights reserved.

1. Introduction An adverse effect of increased frequency and intensity of drought on plant growth and productivity had been predicted in the semi-arid and arid areas due to climate warming change (Gallé et al., 2007; IPCC, 2007). Planting activities will be potentially exposed to a further risk of drought stress endangering plant survival in these sites (Siam et al., 2009). The current-year seedlings especially have high moisture requirements and consequently are highly sensitive to water stress (Engelbrecht et al., 2005; Markesteijn and Poorter, 2009). Early dieback of the seedling for many plantation practices remains to be a problem in the semi-arid and arid areas. If we hope to promote the successful current-year seedling settlement, it is a key to understand how they

∗ Corresponding author. Tel.: +86 28 85231656. E-mail addresses: [email protected], [email protected] (W.-K. Bao). 0304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2010.10.017

are adapted to increasing drought, which is critical for cultivation program. Campylotropis polyantha (Franch.) Schindl. is a leguminous shrub native to distribute in arid and semi-arid sites of southeastern Asia, mainly in southwestern China, and was cultivated in European gardens (Chen, 1988). This species possesses considerable horticultural promise owing to its flowers repeatedly throughout the season, the numerous racemes of showy flowers (Barham, 1997). In addition, it is an important shrub for restoration of degraded arid and semi-arid lands due to the ability to fix nitrogen (Chen, 1988; Xu et al., 2008). We found more pronounced mortality of the current-year seedling in dry areas than in wet areas of the dry valleys of the Hengduan Mountains, SW China (Li et al., 2009), implying that soil water supply condition mostly determines the plant settlement process and consequently the spatial distribution in the dry valley area. Actually some works had observed that soil water content varies dramatically with season and space across dry valleys of the Hengduan Mountains where

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the climate with low precipitation and high evaporation leads to soil desiccation. For example, He (2002) reported that average water content at 0–20 cm soil depth was 3.8% in dry and 9.6% in rainy seasons in a slopeland of the Minjiang dry valley. Ma et al. (2004) and Xu et al. (2008) observed that mean soil water content was 24.5% in wet area and 7.0% in dry area in summer because the mountainous topography results in large spatial variations in microclimate and soil moisture. Furthermore, Ma et al. (2004) also found that plant composition, growth and community structure in dry valleys mostly depend on soil water availability. Therefore, understanding plant ability to cope with drought will necessarily contribute to exploring the possible mechanism behind its spatial distribution pattern. Nevertheless, how currentyear shrub seedlings respond to different water deficits keep to be known and the morphological, anatomical and physiological responses of the current-year shrub seedlings to progressive soil water stress have been poorly documented (Li et al., 2008; Wu et al., 2008a,b). Leaf relative water content (RWC) is an integrative indicator of plant water status which is used to assess the tolerance to drought (Ogbonnaya et al., 1998; Jeon et al., 2006; Gallé et al., 2007). Decline in RWC under drought condition leads to stomatal closure (Chartzoulakis et al., 1999; Gindaba et al., 2004), further resulting in decreased CO2 assimilation. As drought deepens, plant can change the physiological state of the photosynthesis apparatus which can be reflected by lower chlorophyll a fluorescence (i.e., Fv /Fm and ˚PSII ) and increased dissipation of excess excitation ´ ˙ energy (i.e., NPQ) of the leaves (Pukacki and Kaminska-Ro zek, 2005; Gallé et al., 2007). The pigment contents generally also decrease due to their low synthesis rate and rapid degradation with water stress (Mihailovié et al., 1997; Lei et al., 2006). Previous studies have suggested that leaf structure changes can alter tissue turgor maintenance (Kramer and Boyer, 1995) and the diffusion of CO2 from the substomatal cavities in the photosynthetic apparatus (Chartzoulakis et al., 1999). Hence, changes in leaf traits characteristics can be used to evaluate the effect of water stress on plant physiological processes. Under dry conditions the changes of plant dry mass allocation patterns among its components are often reflected by lower leaf area ratios and higher root biomass ratios. It closely relates to the water-use efficiency (WUE) and acclimation mechanism to water stress intensity (Brouwer, 1963; Arndt et al., 2001; Navas and Garnier, 2002). It has been found that the WUE is lower in water-stressed plants than in well-watered plants (Rodiyati et al., 2005; Bacelar et al., 2007) and believed that stomatal closure leads to a lower level of water loss per carbon assimilation, thereby improving the water-use efficiency (WUE) in water-stressed plants (Kramer and Boyer, 1995; Akhter et al., 2005; Gonzáles et al., 2008). However, the results keep to be controversial, because improving WUE may conflict with the high growth rate for many species (Hubick et al., 1986; Akhter et al., 2005; Wu et al., 2008a). The controversy focus is how plant WUE changes with water stress intensity. The present general response patterns of plant growth and WUE have primarily been tested in many crops and tree seedlings but most case studies are experimentally set only under two or three water stress regimes. Few data are available regarding leguminous shrubs that have been exposed to a series of water stress levels. Therefore, we subjected current-year C. polyantha seedlings under five water supply treatments to explore the adaptive changes in seedling growth, dry mass production and allocation, leaf water status (RWC), primary photosynthetic processes and leaf anatomical traits. The specific objectives were (1) to assess ability of the current-year C. polyantha seedlings to tolerate drought and to know the possible threshold value in soil water content to limit growth or to stop growth; and (2) to understand the mechanism of toler-

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ance to drought for the species in its critical settlement period-the current year seedling stage. 2. Materials and methods 2.1. Plant material and experimental design This experiment was conducted using current-year C. polyantha seedlings. Seeds of C. polyantha were collected in October 2005 in Maoxian County in the dry Minjiang River valley, Sichuan, China (103◦ 54 , 103◦ 56 E, 31◦ 37 –31◦ 44 N, and 1700–1900 m a.s.l.), which is its native habitat. The soil collected was a clay loam (He, 2002; Ma et al., 2004), with pH 7.82, bulk density 0.97 g/cm3 , organic matter 5.26 g kg−1 , total N 1.61 g kg−1 , total P 1.14 g kg−1 , and total K 11.65 g kg−1 . Fifty 1400 cm3 white plastic pots (25 cm in diameter × 28 cm in height) were each filled with 3.5 kg of soil. Slow release fertilizer (BAIELUOSI, 4–5 months containing micronutrients at 25 ◦ C air temperature, Wodi Chemical Co., Ltd., Nanjing, China) was mixed into the potting soil at a rate of 1 g kg−1 . Prior to sowing, the seeds were treated with 2.5% sodium hypochlorite (NaOCl) for 1 h. Four seeds of similar size were then sown in each of the 25 pots on April 5, 2006. Another 25 pots without seeds were arranged as controls. After sowing, all pots were moved into an open sided, plastic covered greenhouse at the Maoxian Mountain Ecosystem Research Station, Chinese Academy of Science (103◦ 53 58 E, 31◦ 41 07 , 1816 m above sea level). During the experiment, the average day and night temperatures in the greenhouse were 30 ◦ C and 11 ◦ C, respectively, and the relative humidity ranged from 45 to 85%. The pots were well-watered to ensure seed germination. Shortly after emergence, the seedlings were thinned to one plant per pot, and the watering treatment was initiated at 47 days after germination. At this time, the thinned seedlings were sampled and dried in an oven for 48 h at 70 ◦ C for dry mass determination (mean ± SE = 0.57 ± 0.02 g, n = 28 individuals). The experimental design was a completely random design with five water conditions, 100% (W100 ), 80% (W80 ), 60% (W60 ), 40% (W40 ) and 20% (W20 ) of water holding capacity (WHC). There were five replications per treatment as well as 5 pots that contained a dead C. polyantha twig at the centre of each treatment that served as a control to monitor water loss from the soil surface. Water loss due to transpiration was measured gravimetrically by weighing all of the pots and re-watering with tap water (pH 8.24, EC 0.69 mS/cm) every other day at 18:00. The watering amount for each pot was determined according to the difference between the weight of a re-watered pot and the weight of the pot 48 h later. The soil volumetric water content was maintained at 38.8 ± 0.3%, 31.6 ± 1.7%, 25.6 ± 1.3%, 16.5 ± 0.7%, and 8.1 ± 1.1% for the W100 , W80 , W60 , W40 and W20 treatments, respectively. Increases in seedling weight were estimated based on regression of the relationship between seedling fresh weight (y, g), plant height (x1 , cm) and seedling basal diameter (x2 , mm): y = −3.79 + 0.12x1 + 0.15x2 , r2 = 0.91, P < 0.001 (F.L. Li, unpublished data). Following periods of rapid growth, the pot weight changes resulted from plant growth and the relevant amount of water was adjusted every 15 days. After 100 days of the treatment beginning the experiment was terminated. 2.2. Measurements of growth and WUE Plant height and leaf number were recorded monthly from June to September. Dropped leaves were counted weekly during the experimental period, at which time the leaf biomass was recorded. The seedlings were then harvested on September 28, 2006, at which time the basal diameter and branch number were measured. Images of compound leaves were recorded with a scanner (Model F6580, Founder Electronics Co., Ltd., Beijing), and images were

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digitized by the Arcview 3.2a (Environmental Systems Research Institute (ESRI), Inc., New York) software in order to determine both total and leaf blade area. The roots were washed with water in a sieve (0.5 mm mesh size) to remove soil. Each seedling was then divided into roots, stems and leaves and dried in an oven for 48 h at 70 ◦ C. The total plant dry mass production was calculated by sum of the root, stem and leaf mass. R:S ratio and leaf area ratio (LAR) for each seedling and specific leaf area (SLA) were calculated as:

(F0 ), maximal fluorescence (Fm ) and maximal PS II photochemical efficiency (Fv /Fm ) were measured in dark-adapted leaves that had been enclosed in a leaf-clip holder (2030B, Heinz Walz) for 30 min. The effective quantum yield of PS II (˚PSII ), photochemical (qP) and non-photochemical (NPQ) fluorescence quenching coefficients were measured in light-adapted leaves under light conditions, with the pulse being delivered at 600 mmol m−2 s−1 for 0.8 s. The experimental protocol was as described previously ´ ˙ (Pukacki and Kaminska-Ro zek, 2005; Wu et al., 2008b).

(1) R:S ratio = root dry matter/shoot dry matter. (2) LAR = total leaf area/plant dry matter. (3) SLA = leaf area/leaf dry matter.

2.6. Analysis of pigment contents

The water-use efficiency (WUE) of each seedling was then evaluated by determining the ratio of dry mass production to water transpired during the experiment. Average dry matter of thinned seedlings at the beginning was subtracted from dry matter of final seedlings for WUE calculation. While calculating the amount of water transpired during the experiment, evaporative loss from the pots was taken into account by subtracting the average amount of water loss from the control pots without plants from each watering treatment.

Following measurement of the Chl a fluorescence, the concentration of pigments in 0.1 g of fresh leaflets per seedling was evaluated. The chlorophyll and carotenoids were extracted in chilled 80% acetone (v/v) in the dark for 24 h. The absorbance of the extracts at 664, 648 and 470 nm was then measured using a UV/visible spectrophotometer (UV-2450, Shimadzu Corporation, Tokyo, Japan), after which the contents of Chl a, Chl b and carotenoids, respectively, were determined using the method described by Lichtenthaler (1987). 2.7. Statistical analysis

RWC was determined gravimetrically at predawn. Three compound leaves per seedling were sampled from the mid-canopy position. The fresh weight (FW) of the sample was then determined immediately after harvesting, after which the samples were placed in a water-saturated vial for 24 h to achieve turgid weight (TW). Next, the samples were dried in an oven for 48 h at 70 ◦ C and the dry weight (DW) was determined. The RWC was calculated using the following formula: RWC (%) = [(FW − DW)/(TW − DW)] × 100 ´ ˙ (Pukacki and Kaminska-Ro zek, 2005).

Significant differences in the means of different treatments were determined using one-way analysis of variance (ANOVA). The LSD multiple comparison test was then used to compare significant differences in the means. The age effect was considered as a repeated effect, and repeated measurement analysis was conducted to determine the effects of watering on leaf number and plant height during the seedling growth cycle. Log-transformed data were used when the data did not meet the ANOVA assumption of homogeneity of variance. All of the statistical analyses were performed using SPSS (Standard released version 11.5 for Windows, SPSS Inc., IL, USA). All values were considered significant when P < 0.05.

2.4. Leaf anatomical measurements

3. Results

Three fully expanded leaflets were taken from the mid-canopy position of each seedling at the end of the experiment. Each leaflet was then cut across the mid-rib and a 5–7 mm portion that included the mid-rib was fixed in FAA (formalin–acetic acid alcohol; 10% (v/v) 37% formaldehyde, 5% acetic acid, 50% alcohol, 35% water). The fixed samples were dehydrated through an alcohol series (50, 60, 70, 85, 95 and 100%) and xylene (1 h per solution), infiltrated and embedded in paraffin. Next, 8–10 ␮m thick serial transverse sections were cut using a microtome (Leica-RM2135, Co., Ltd., Leica Microsystems CMS GmbH, Wetzlar). The sections were then stained with saturated safranin in 50% alcohol for 4 h and fast-green in 95% alcohol for 1 min. After successive steps in the aforementioned alcohol series to xylene (5–10 min per solution), each section was mounted on Canada balsam. Finally, three typical sections per leaflet were selected and used to determine the thickness of the entire lamina, epidermis, palisade mesophyll and spongy mesophyll. All specimens were examined using a light microscope (Leica-DMLB) with an eye-piece micrometer at a magnification of 40×. Finally, the ratios of palisade mesophyll to spongy mesophyll (P:S ratio) were determined.

3.1. Responses of growth parameters with water stress

2.3. Measurements of leaf relative water content (RWC)

2.5. Measurements of chlorophyll a fluorescence parameters They were determined on five fully expanded exposed leaves (one leaflet per seedling) using a modulated fluorometer (PAM 2100, Walz, Effeltrich, Germany) on August 25, 2006. All measurements were taken in the morning. The initial fluorescence

Water stress caused significant changes in plant height and number of green leaves of the seedlings (Fig. 1A and 1B). The differences in these two parameters increased with stress duration among the W100 , W60 , and W20 treatments (P < 0.01); however, no significant differences were found between the W100 and W80 or between the W60 and W40 treatments (P > 0.05). Both plant height and number of leaves increased from June to September in the W40 , W60 , W80 and W100 treatments, but no significant growth increases were observed in the W20 treatment after 2 months of treatment (P > 0.05). In addition, leaf shedding occurred at the second month after treatment and the biggest number of the senescent leaves was presented under the W20 treatment (Fig. 1C). Two seedlings subjected to the W20 treatment were observed to have died at the end of experiment. Total leaf area (TLA), leaf blade area, branch number and basal diameter significantly and linearly decreased along the water stress gradient from W100 to W20 treatments (P < 0.01; Table 1). When compared with the W100 treatment, TLA for the W60 , W40 and W20 treatments was reduced by 56%, 71% and 90%, respectively (Table 1), and the basal diameter was reduced by 30%, 44% and 60% under W60 , W40 , and W20 conditions, respectively (Table 1). The response patterns in leaf blade area and branch number under the water stress gradient were also similar to the TLA (Table 1). Comparatively, TLA presents the most serious reduction along the water stress gradient among those four parameters investigated.

A

Plant height (cm)

60

a

40

W20 W40 W60 W80 W100

-1

80

Number of green leaves (No. Plant )

F.-L. Li et al. / Scientia Horticulturae 127 (2011) 436–443

a a b

b b

20

a

c

c

0 Jun -1

Number of senescent leaves (No. Plant )

12

Jul

C

Aug

W20 W40 W60 W80 W100

10 8 6

240

439

B

W20 W40 W60 W80 a W100

200 160

a

120

40

b a Jun

b

b

c

c

Jul

Aug

Sep

0

Sep

b

a

80

a

a

b

4

b

2

b

c

0

Jun

Jul

Aug

Sep

Fig. 1. Dynamics on plant height (A) and leaf number (B and C) of Campylotropis polyantha (Franch.) Schindl. seedlings under the five water conditions. W100 , W80 , W60 , W40 and W20 were watered to 100, 80, 60, 40 and 20% water holding capacity (WHC), respectively. Bars represent means ± SE, n = 5. Different letters indicate significant differences across five water conditions according to LSD test, P < 0.05. Table 1 Total leaf area, leaf blade area, branch number and basal diameter of Campylotropis polyantha (Franch.) Schindl. seedlings under five water conditions. Values represent the means ± SE, n = 5. Different letters within a column indicate significant differences across five water conditions according to LSD test, P < 0.05. Water conditions

Total leaf area (cm2 plant−1 )

W100 W80 W60 W40 W20

1107.05 960.53 483.44 322.92 121.16

± ± ± ± ±

Leaf blade area (cm2 )

103.25a 106.58a 35.97b 39.05c 11.49d

10.80 10.34 8.86 6.58 3.58

± ± ± ± ±

Branch number

0.38a 0.89ab 0.35b 0.25c 0.16d

3.2. Responses of dry mass production, allocation and water-use efficiency to water stress

13.00 12.50 8.25 6.75 5.25

± ± ± ± ±

Basal diameter (mm)

1.29a 1.04a 0.48b 0.48bc 0.63c

9.81 10.07 6.88 5.49 3.90

± ± ± ± ±

0.22a 0.20a 0.59b 0.49c 0.20d

of the W80 and W100 treatments (P < 0.05; Table 2). We noticed that the highest WUEs were presented under the two moderate water stress treatments (W60 and W40 ) but the lowest for the severe stress (W20 ). Compared to W40 treatment, the WUE for the W20 and W100 treatments was reduced by 56 or 26%, respectively (Table 2). Seedlings presented a greater SLA under the W40 and W20 treatments than other treatments (Table 3). In contrast, the LAR was lower under the W60, W40 and W20 treatments than under the W80 and W100 treatments, totally presenting a linearly decrease tendency with increased water stress (Table 3).

As shown in Table 2, the dry mass of seedling and its components (root, stem and leaf) all were significantly and linearly reduced with water stress increased, but no significant differences were observed between the W80 and W100 treatments. Total dry mass was reduced by 19%, 51%, 65% and 85% under W80 , W60 , W40 and W20 conditions, respectively, when compared to the W100 treatment. The R:S ratio of the W60 , W40 and W20 treatments did not significantly differ (P > 0.05), but were greater than those

Table 2 Dry mass accumulation, root:stem (R:S) ratio, water-use efficiency (WUE) of Campylotropics polyantha (Franch.) Schindl. seedlings under five water conditions. Values represent the means ± SE, n = 5. Different letters within a column indicate significant differences across five water conditions according to LSD test, P < 0.05. Water conditions

Dry mass accumulation (g/plant) Total

W100 W80 W60 W40 W20

15.24 12.47 7.50 5.33 2.25

R:S ratio

Leaf ± ± ± ± ±

0.67a 0.58a 0.49b 0.79c 0.28d

5.07 4.52 3.09 2.15 1.28

Stem ± ± ± ± ±

0.35a 0.44a 0.37b 0.20bc 0.16c

5.99 4.58 1.64 1.13 0.45

WUE (g kg−1 )

Root ± ± ± ± ±

0.51a 0.27a 0.21b 0.25c 0.10c

4.18 3.37 2.76 2.04 0.83

± ± ± ± ±

0.24a 0.0.7a 0.0.6b 0.42c 0.09d

0.38 0.37 0.58 0.62 0.48

± ± ± ± ±

0.03a 0.01a 0.05b 0.04b 0.06b

2.12 2.02 2.83 2.87 1.23

± ± ± ± ±

0.06a 0.10a 0.26b 0.12b 0.11c

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Table 3 Specific leaf area (SLA), leaf area ratio (LAR), leaf relative water content (RWC) and anatomical characteristics of Campylotropics polyantha (Franch.) Schindl. seedlings under five water conditions. Values represent the means ± SE, n = 5. Different letters within a column indicate significant differences across five water conditions according to LSD test, P < 0.05. Water conditions

SLA (cm2 g−1 )

W100 W80 W60 W40 W20

152.44 159.68 190.01 189.39 182.98

± ± ± ± ±

LAR (cm2 g−1 )

9.06a 6.27a 4.79b 5.01b 2.35b

72.27 76.56 64.49 62.06 47.44

± ± ± ± ±

RWC (%)

1.14a 4.65a 2.94b 4.63b 0.82b

86.71 85.40 70.29 59.34 41.48

± ± ± ± ±

Leaf thickness (␮m) 0.50a 0.62a 1.80b 3.47c 2.87d

143.87 136.25 135.76 120.00 107.98

± ± ± ± ±

5.04a 6.57a 4.10a 4.21b 1.89c

Palisade mesophyll thickness(␮m) 56.80 58.72 59.22 49.88 40.67

± ± ± ± ±

2.15a 1.55a 3.53a 1.16b 0.69c

Spongy mesophyll thickness(␮m) 61.56 56.50 53.22 46.25 39.06

± ± ± ± ±

2.40a 0.96b 2.00b 1.04c 0.49d

Palisade/spongy ratio 0.92 1.04 1.11 1.08 1.04

± ± ± ± ±

0.07a 0.04a 0.05a 0.09a 0.06a

Table 4 The photochemical efficiency of photosystem II(Fv /Fm ), effective quantum yield of PSII (ФPSII ), photochemical quenching (qP), and non-photochemical quenching (NPQ) of Campylotropics polyantha (Franch.) Schindl. seedlings under five water supply conditions. Values represent the means ± SE, n = 5. Different letters within a column indicate significant differences across five water conditions according to LSD test, P < 0.05. Water conditions

Fv /Fm

W100 W80 W60 W40 W20

0.86 0.85 0.82 0.80 0.74

˚PSII ± ± ± ± ±

0.02a 0.00a 0.01b 0.01c 0.02c

0.57 0.55 0.38 0.32 0.33

qP ± ± ± ± ±

0.03a 0.05a 0.02b 0.04b 0.07b

0.73 0.74 0.66 0.54 0.51

NPQ ± ± ± ± ±

0.01a 0.03a 0.03b 0.01c 0.04c

0.40 0.41 0.59 0.66 0.68

± ± ± ± ±

0.00a 0.03a 0.01b 0.02bc 0.02c

3.3. Responses of leaf anatomical traits and water relative content (RWC) to water stress

3.4. Responses of chlorophyll (Chl) a fluorescence parameters and pigment contents to water stress

The leaf uniseriate cells were found to be slightly smaller in the lower epidermis (8.63 ± 0.38–10.26 ± 0.75 ␮m) than in the upper (10.81 ± 0.34–11.05 ± 0.31 ␮m) for the current-year seedlings under each of the water treatment. The arch-shaped vascular system of the midrib showed 4–6 bundles disposed near the lower epidermis. The mesophyll parenchyma of C. polyantha leaves was composed primarily of two parts in all treatments: the palisade and spongy tissues. The former contained 2–3 layers of elongated cells, whereas the latter contained larger intercellular spaces and variously sized vascular bundles with polygonal and randomly oriented cells. However, the different water stress did not affect the thickness of both upper and lower epidermis (Data not shown). The thickness of leaflet and both palisade and spongy mesophyll was significantly reduced in response to increasing water stress from W100 to W20 (Table 3). The slight to moderate water stress (from W80 and W60 treatments) did not result in differences significantly in thickness of spongy mesophyll, palisade mesophyll and leaflets, but severe drought (from W40 to W20 ) did result in significant decreases in the entire leaflet and palisade mesophyll thickness (P < 0.05; Table 3). Specifically, the entire leaflet thickness under the W20 treatment was decreased by 29% when compared to that under the W100 treatment. No significant difference was observed in the ratio of palisade mesophyll and spongy mesophyll due to a parallel decreasing trend in palisade mesophyll thickness and spongy mesophyll thickness (P > 0.05; Table 3). The relative water content (RWC) of leaves under the W100 and W80 treatments did not differ significantly (P > 0.05); however, when compared to the W100 the RWC for the W60 , W40 and W20 treatments decreased by 19%, 32% and 52%, respectively (Table 3).

Increasing water stress significantly reduced the Chl a fluorescence parameters (Fv /Fm , ˚PSII and qP) but increased NPQ (P < 0.05) (Table 4). The increasing water stress significantly increased the pigment contents by fresh weight basis (Table 5). However, the Chl a:b exhibited a decreasing trend along the water stress gradient from W100 to W20 treatment and CHl:Car did not present significant differences among five treatments (Table 5). No significant differences in the values of both Chl a fluorescence parameters and the pigment contents were present between the relative severe deficits: the W40 and W20 treatments (Tables 4 and 5). 4. Discussion 4.1. The tolerant degree of the current-year seedlings to water stress The present experiment with five water supply treatments allowed us a detailed analysis on responses of the current-year C. polyantha seedlings to the varied water stress. Our results displayed that the current-year seedlings can tolerate the progressed water deficit to some extent by morphological, anatomical and physiological acclimations because only two seedlings under the extreme W20 treatment died during the experiment, implying that the leguminous nitrogen-fixed shrub with beautiful flowers has a great potential to grow in the semi-arid and arid areas. Furthermore, we found that significant growth decline in plant height, basal diameter, branch/leaf number, total Leaf area, dry mass accumulation for the current-year seedlings all started to occur under the W60 treatment (Fig. 1 and Tables 1 and 2), telling us that around the water

Table 5 Chlorophylls and carotenoids contents in leaves of Campylotropics polyantha (Franch.) Schindl. seedlings under five water conditions. Values represent the means ± SE, n = 5. Different letters within a column indicate significant differences across five water conditions according to LSD test, P < 0.05. Water conditions

Chl a (mg g−1 )

W100 W80 W60 W40 W20

1.50 1.49 1.76 1.95 2.34

± ± ± ± ±

0.07a 0.04a 0.12b 0.16c 0.18c

Chl b (mg g−1 ) 0.42 0.48 0.53 0.68 0.86

± ± ± ± ±

0.02a 0.01a 0.04a 0.05b 0.06b

Chl a + b (mg g−1 ) 1.92 1.97 2.29 2.63 3.20

± ± ± ± ±

0.09a 0.06a 0.16ab 0.20bc 0.23c

Car (mg g−1 )

Chl a:b 3.57 3.10 3.32 2.88 2.71

± ± ± ± ±

0.07a 0.04b 0.12b 0.14bc 0.10c

0.22 0.21 0.25 0.31 0.38

± ± ± ± ±

0.02a 0.01a 0.02a 0.03b 0.04b

Chl:Car 8.73 9.38 9.16 8.48 8.42

± ± ± ± ±

0.22a 0.28a 0.35a 0.18a 0.24a

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content (25.6 ± 1.3%) is an initial threshold harmful to growth and productivity of the current-year C. polyantha seedlings. Therefore, before introducing the shrub in plantation efforts, water supply above 25.6% of soil water content could be recommended for this species to raise vigorous seedlings in the first year of growth. This threshold for the C. polyantha seedlings was similar with that for other two legumes under a similar experiment condition (Li et al., 2008; Wu et al., 2008a): Bauhinia faberi var. microphylla and Sophora davidii. With deepening water deficit (from W60 to W20 treatment), the current-year seedlings of C. polyantha still significantly reduced growth (Fig. 1 and Tables 1 and 2), but it was slower than the other two leguminous shrubs (mentioned above) with same natural distribution, resulting in higher values of plant height, basal diameter, branch/leaf number, total leaf area, dry mass accumulation for C. polyantha than the other leguminous shrubs under the W20 treatment (Li et al., 2008; Wu et al., 2008a), implying that C. polyantha seedlings among the three leguminous shrubs have relatively stronger ability to tolerate drought. Phenotypic plasticity is considered the major means by which plants cope with environmental change and consequently to survive, grow and even evolve (Valladares et al., 2007), indicating that loss of plasticity results in plant failing to tolerate the adverse environmental change by morphological and physiological modification. Our results showed that plastic responses to progressive water stress were reduced. Most growth parameters including plant height, basal diameter, branch number, stem biomass, leaf number and biomass, SLA, and LAR did not present apparent plastic change from W40 treatment to W20 treatment (soil water content from 16.5% to 8.1%), when compared to other slightly or moderate water stress treatments (from W60 treatment to W40 ), because no significant differences were checked (Fig. 1 and Tables 2 and 3). Moreover, under the W20 treatment the seedling height and green leaf number had stopped to grow since July after one month treatment (Fig. 1). Only root still presents significant decrease in dry mass from W40 treatment to W20 treatment, implying its stronger plasticity than other organs (branch, stem and leave). Therefore, we concluded that the soil water content between W40 and W20 treatments (from 16.5% to 8.1%; average 12.3%) should be the second key threshold to keep the growth and survival of the current-year C. polyantha seedlings. A sharper decline in leaf water status (RWC < 50%) and the lowest WUE at W20 treatment relative to W100 treatment indicated that the C. polyantha became severely dehydrated under severe drought (here soil water content 8.1 ± 1.1%). Moreover, the apparent leaf shedding and seedling mortality, as well as the extremely low dry mass production and stopped height growth observed under the W20 condition all revealed that the current-year C. polyantha seedlings cannot tolerate severe water stress (soil water content ≤8.1%), let alone living in some area of its natural distribution with extremely lower soil water supply with seasonal and spatial change (He, 2002; Ma et al., 2004; Xu et al., 2008), which could explain their unsuccessful existence in the extremely dry areas. We suggested that the water content around at 12.3% (between 16.5% and 8.1% or between W40 and W20 treatments) was a pre-condition required for growth by C. polyantha seedling for horticulture and vegetation restoration practices. 4.2. The morphological and anatomical modifications in response to water stress Plant can adapt to environmental change usually by morphological and anatomical modifications. In the case of C. polyantha seedlings, the total leaf area was reduced sharply in response to progressive water stress due to a combination of reduced expansion of younger leaves and shedding of older leaves (Table 1 and Fig. 1C). Therefore, the lowering LAR under water stress was facilitated by

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the reduction of total leaf area and leaf thickness (Tables 1 and 3) because a decreased LAR commonly associated with a high tissue density and total non-structural carbohydrate content in leaves under drought conditions (Castro-Díez et al., 2000). Additionally, the water-stressed C. polyantha seedlings reallocated relatively more dry mass to the roots in order to maximize water uptake from soil, being in line with the theory of functional balance, which states that plants will react to water deficit with a relative increase in the flow of assimilates to the root, leading to an increased root mass ratio (Brouwer, 1963). Comparative results had been obtained for a number of plants (Navas and Garnier, 2002; Lei et al., 2006; Gonzáles et al., 2008; Markesteijn and Poorter, 2009). Plants growing in dry conditions usually have thicker leaves and cuticles than those growing in wet conditions (Ludlow, 1989; Teklehaimanot et al., 1998; Kofidis et al., 2004; Guerfel et al., 2009). Such alterations have been described as the xeromorphic leaf structure (Teklehaimanot et al., 1998; Kofidis et al., 2004; Burghardt et al., 2008). On the contrary, the C. polyantha seedlings had mesomorphic leaves across all treatments. Thereby the decreased mesophyll cell size was a major structural response to increasing water stress. The cell size reduction in leaves occurs as a result of the role of water playing in the maintenance of turgidity being necessary for cell expansion (Ogbonnaya et al., 1998). This was supported by the lower leaf water status (RWC) observed in the W40 and W20 treatments (Table 3) and in previous studies (Cutler et al., 1977; Chartzoulakis et al., 1999). In our study the leaf thickness decrease was also accompanied by increased SLA (Table 3). Small cells can withstand turgor pressure better than large cells, and can contribute to turgor maintenance more effectively under drought conditions (Steudle et al., 1977; Kramer and Boyer, 1995; Burghardt et al., 2008). Thus, the cell size reduction is reasonably interpreted as a tolerance mechanism of the leaves to maintain tissue turgidity for the C. polyantha seedlings. The water-use efficiency (WUE) change is considered as an adaptive response to water supply (Brouwer, 1963; Arndt et al., 2001; Navas and Garnier, 2002). Although many studies have found that the WUE of woody seedlings increased with increasing water stress (Kramer and Boyer, 1995; Yin et al., 2004; Akhter et al., 2005; Rodiyati et al., 2005; Bacelar et al., 2007; Gonzáles et al., 2008), it is still unclear about how plant WUE changes with water stress intensity. Our results along the water stress gradient with five levels provided good opportunity to explore it. The response pattern of WUE for the current-year C. polyantha seedlings was indeed related to the degree of water stress, displaying the parabola relationships between WUE and water deficit intensity (Table 2). The WUE was only enhanced under moderate drought conditions (W60 and W40 ), but reduced from moderate drought (W40 treatment) to severe drought (W20 treatment). The WUE response pattern reinforced our conclusion that C. polyantha seedlings were tolerant to water stress by promoting the WUE, but more vulnerable to severe drought over the water stress condition under the W40 treatment. 4.3. The physiological activities of the photosynthesis II (PSII) of the seedlings in response to water stress Several works reported that water stress leads to a decreased level of chlorophylls and carotenoids in leaves (Reddy et al., 2004; ´ ˙ Kaminska-Ro zek and Pukacki, 2004; Guerfel et al., 2009) since water deficit usually causes an increase in chlorophyllase activity resulting in a decrease in the amount of chlorophyll (Mihailovié et al., 1997; Lei et al., 2006; Nunes et al., 2008). But because the pigment contents were expressed by a fresh weight basis (Lichtenthaler, 1987), in contrast to these early findings, the contents of Chl a, b and Carotenoids in leaves of C. polyantha seedlings linearly increased with water stress intensity in our experiment (Table 5). As a result, increased pigments in water-stressed

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seedlings were partially related to the reduced tissue hydration of the leaves. But our results about Chl a:b ratio decreasing with progressive water deficits were in agreement with previous studies mentioned above. Our results also displayed that photochemical processes and PSII activity were significantly restrained when soil water content decreased to 25.6 ± 1.3% under the W60 treatment and the photosynthetic damage was more significant under two relatively severe water stresses (W40 and W20 treatment) (Table 4). It was agreed with significant decreasing Chl a:b ratio along the progressive drought (Table 5), implying that the damaged light harvesting complex was an important reason why photochemical processes and PSII activity were lowered under the water stress condition (Jeon et al., 2006; Guerfel et al., 2009). Consequently, these changes in photosynthetic activity and leaf pigment composition pattern can greatly explain the lower seedling growth and dry mass production under the W60 , W40 and W20 treatments. Increasing NPQ observed in water-stressed seedlings indicated that the removal of excess excitation energy through thermal dissipation, a common photoprotective mechanism of PSII under drought conditions for many plants (Gallé et al., 2007). An important finding in the present work was that the current-year seedlings have lost the photochemical mechanism when water deficit surpassed the soil water content 16.5 ± 0.7% (from W40 treatment to W20 treatment) because no significant differences in Chl a:b ratios, photochemical processes, and PSII activity (Fv /Fm , ˚PSII , qP and NPQ) were found between W40 and W20 treatments (Tables 4 and 5). This suggested again that around soil water content between 16.5% and 8.1% (average 12.3%) may be a key limit to the growth and survival of the current-year C. polyantha seedlings. In conclusion, the current-year C. polyantha seedlings presented decreased responses to progressive water stress in the morphological, anatomical and physiological traits, showing a strong plasticity adaptive to drought. It coped with water stress by avoidance mechanisms such as significantly decreased leaf development, RWC and branch number and also by tolerance mechanisms such as an increased R:S ratio and thermal dissipation in leaves and decreased mesophyll cell size. The soil water content around 25.6% may be an initial threshold restricting to the optimal growth and dry mass production and around 12.3% was the second threshold to keep the growth and survival of the current-year C. polyantha seedlings. Acknowledgements Supported by the PhD foundation in Western Light Talent Training Plan (no: 08C2041100) and the Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization, Chengdu Institute of Biology, CAS (no: 08B2011106). Authors extend thanks to the Maoxian Mountain Ecosystem Research Station, CAS for providing technical assistance. References Akhter, J., Mahmood, K., Tasnweem, M.A., Malik, K.A., Naqvi, M.H., Hussain, F., Serraj, R., 2005. Water-use efficiency and carbon isotope discrimination of Acacia ampliceps and Eucalyptus camaldulensis at different soil moisture regimes under semi-arid conditions. Biol. Plant. 49 (2), 269–272. Arndt, S.K., Clifford, S.C., Wanek, W., Jones, H.G., Popp, M., 2001. Physiological and morphological adaptations of the fruit tree Ziziphus rotundifolia in response to progressive drought stress. Tree Physiol. 21, 705–715. Bacelar, E.A., Moutinho-Pereira, J.M., GonC¸alves, B.C., Ferreira, H.F., Correia, C.M., 2007. Changes in growth, gas exchange, xylem hydraulic properties and water use efficiency of three olive cultivars under contrasting water availability regimes. Environ. Exp. Bot. 60, 183–192. Barham, J., 1997. Campylotropis polyantha: Leguminosae-Papilionoideae. Curtis’s Bot. Mag. 14 (4), 203–207. Brouwer, R., 1963. Some Aspects of the Equilibrium Between Overground and Underground Plant Parts. Jaarboek IBS, Wageningen, pp. 31–39.

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