Morphological and photosynthetic responses of riparian plant Distylium chinense seedlings to simulated Autumn and Winter flooding in Three Gorges Reservoir Region of the Yangtze River, China

Acta Ecologica Sinica 31 (2011) 31–39

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Morphological and photosynthetic responses of riparian plant Distylium chinense seedlings to simulated Autumn and Winter flooding in Three Gorges Reservoir Region of the Yangtze River, China Li Xiaoling ⇑, Li Ning, Yang Jin, Ye Fuzhou, Chen Faju, Chen Fangqing Engineering Research Center of Eco-Environment in Three Gorges Reservoir Region, Ministry of Education, College of Chemistry and Life Science, China Three Gorges University, Yichang 443002, China

a r t i c l e

i n f o

Article history: Accepted 8 November 2010

Keywords: Distylium chinense Three Gorges Reservoir Region Simulated flooding Morphological adaptation Photosynthesis response Survival Recovery growth

a b s t r a c t To evaluate the tolerance of riparian plant Distylium chinense in Three Gorges Reservoir Region to antiseason flooding, a simulation flooding experiment was conducted during Autumn and Winter, and morphology and photosynthesis of D. chinense seedlings and their recovery growth after soil drainage were analyzed in different duration of flooding and flooding depth. The seedlings were submitted to four treatments: (1) 40 seedlings unflooded and watered daily as control (Unflooded, CK); (2) 120 seedlings flooded at 1 cm above the ground level (F-1 cm); (3) 120 seedlings flooded at 12 cm above the ground level (F-12 cm) and (4) 120 seedlings completely submerged with 2 m water depth (F-2 m, top of plants at 2 m below water surface). The flooding survival, plant height, stem diameter, adventitious roots, stem lenticels, epicormic shoots, chlorophyll content and photosynthesis parameters were determined at 0, 15, 30, 90 days in flooding stress and 15, 60 days after soil drainage. The results showed that the survival of the seedlings subjected to flooding was 100% for all repeated measurements in all treatments. Adventitious roots, hypertrophied lenticels and stem hypertrophy were observed in the seedlings flooded for more than 15 d, and increased with the prolonged flooding duration, while disappeared after the soil was drained. Flooding duration and flooding depth showed significant individual and interactive effects on leaf chlorophyll a (Chl a), chlorophyll b (Chl b), and their ratio, chlorophyll (a + b), the net photosynthesis rate (Pn), transpiration rate (Tr), stomatal conductance (Cs), and inter-cellular CO2 concentration (Ci) of D. chinense seedlings (P < 0.01). After 15 days of flooding, there was no significant decrease in Pn of the flooded seedlings as compared with that of the control seedlings. Pn of the flooded seedlings was significantly lower than that of the control seedlings after 30 days of flooding (P < 0.05), whereas Pn showed no significant difference among seedlings from three flooding depths. After 90 days of flooding, Pn of the F-2 m flooded seedlings was significantly lower than that of the controls, F-1 cm and F-12 cm flooded seedlings (P < 0.05), but still maintained high photosynthetic capacity. Pn of the F-1 cm and F-12 cm flooded seedlings rose gradually after soil drainage, while, it was significantly lower than that of the control seedlings after 15 days of recovery (P < 0.05). After 60 days of recovery, Pn of all seedlings flooded with different depths showed no significant difference as compared with that of the control seedlings and new leaves grew out in the F-2 m flooded seedlings. The effect of all flooding treatments on Gs, Tr, Chl a, Chl b, Chl a/Chl b and chl (a + b) was basically the same as their effect on Pn, while the effect of all flooding treatments on Ci was quite the contrary. Correlation analysis showed that Pn was positively relative with Gs, Tr, Chl a, Chl b and chl (a + b) (P < 0.05) and significantly negative with Ci (P < 0.05). Therefore, the present study demonstrates that D. chinense has high survival and good recovery growth after long-term flooding in anti-season flooding and could be taken as an excellent candidate species in the re-vegetation of water-level-fluctuation areas in Three Gorges Reservoir Region. Ó 2010 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

1. Introduction After the Three Gorges Dam (TGD) is constructed at Sandouping, Yichang, Hubei Province, China, the Three Gorges Reservoir inun⇑ Corresponding author. E-mail address: [email protected] (L. Xiaoling).

dates an area of 1080 km2 and forms a new vast hydro-fluctuation belt of 300 km2. The water level of the Three Gorges Reservoir reached a maximum of 175 m in Winter of 2010, would fluctuate from 145 m in summer (flood season) to 175 m in Winter (non flood season) thereafter. This hydrologic regime is the opposite of the Yangtze River’s natural regime before TGD construction when the peak flows occurred in summer (July–September) and

1872-2032/$ - see front matter Ó 2010 Ecological Society of China. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.chnaes.2010.11.005

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low flows occurred in Winter (January–March). The reversal of flooding time, prolonged flooding duration, and new hydro—fluctuation belt (up to 30 m in elevation) will dramatically alter environmental conditions in the hydro—fluctuation belt [1]. The flooding duration in riparian zone is relatively longer than before, which can reach as long as 6 months. Prolonged flooding duration may hinder the growth of some native species and even cause some flood-intolerant species to die, which may brings about a series of environmental issues such as lower biodiversity, decreased types of ecosystems, environmental pollution, etc. [2]. Therefore, it is imperative to re-establish quasi-natural river dynamics and the associated typical riparian vegetation, which is one of important restoration measures, for restoration of the fragile ecosystems and construction of the eco-barrier in new riparian areas of the Three Gorges Reservoir Region. However, the most pressing need is to select some flood-tolerant species and provide important insights into the ‘‘flooding tolerance’’ mechanisms in the new riparian zones during the course of the establishment of artificial vegetation. Soil flooding is a common environmental stress in areas prone to the high rainfall, poor soil drainage, wetlands and riparian zones, which has an important effect on metabolism, physiology and morphology of plants [3]. Flooding would affect water movement and oxygen diffusion in soil. Eco-physiological responses of plants to soil flooding are various under hypoxia or low light intensity. Under flooding conditions, the soil environment becomes deficient in oxygen due to reduced gas exchange rates at the soil surface with the atmosphere. Oxygen deficiency in roots induces anaerobic respiration and enhances the consumption of carbohydrate reserves. Inundation thus induces specific above-ground and below-ground stresses in wetland and riparian vegetations, adversely affecting their establishment, survival and growth [4–6]. Flood-induced changes in soil physicochemical properties affect several aspects of plant physiology, morphology and anatomy [3–9]. The initial eco-physiological response of most plants to flooding is wilting and stomatal closure within a day or two following root exposure [10]. A strong reduction in net photosynthesis, stomatal conductance and chlorophyll content was observed in some flood-intolerant plants as a result of flooding [7,11–14]. However, soil flooding had little influence on photosynthetic character for some flood-tolerant plants [9,15–17]. The stronger the plant recovery and growth performance after soil drainage, the better its flooding tolerance is. Especially, the plant photosynthetic production after soil drainage would be beneficial to its recovery growth [16,17,3,18]. Soil flooding leads to these physiological responses and further morphological adaptations that help plant tolerate flooding stress. Rapid biochemical changes are easily induced through short-term soil flooding or hypoxia, but anatomical and morphological changes such as adventitious roots, hypertrophied lenticels and aerenchyma formation are more likely to be involved in the long-term acclimation to flooding stress [4,19–24]. The responses of the plants to flooding vary with different species, their genetic characters, ages, flooding durations and flooding depths [25]. Distylium chinense (Fr.) Diels, the genus Distylium of the Hamamelidaceae [26], is a evergreen perennial shrub between 0.8 and 1.2 m in height, native to the riparian areas and wetlands and a dominant species in the riparian areas of the Three Gorges Reservoir Area of the Yangtze River and its branches. D. chinense was found to have acclimated to the water-level fluctuation of the natural river flood period for hundreds of years. When the flood period comes every year, D. chinense will be submerged with the water level rising. D. chinense is sometimes flooded for several months. Of course, its flooding duration and depth varies with the altitude of the vertical distribution on the river bank. In addition, through a long-term field observation we found that

D. chinense can survive the natural flood period and grow well, which showed that D. chinense possess high flooding tolerance. However, there is little information on how flooding durations and depths affect growth, morphology and photosynthetic physiology of this species in the riparian areas and wetlands. Furthermore, we are not also clear whether D. chinense will survive the antiseason flooding after the impoundment of the Three Gorges Reservoir and recover quickly after soil drainage. So, our general goal was not only (1) to demonstrate new mechanisms of adaptation or the eco-physiological mechanisms behind them for the plants in general, but also (2) to show what adaptations this very important species manifests if D. chinense can survive long periods of flood and thrive well after soil drainage. This would be very important for D. chinense to determine whether it is chosen as a candidate species in the re-vegetation of the water-level fluctuation zone in the Three Gorges Reservoir Region. To address this problem, (1) a simulation flooding experiment was conducted under different flooding duration and depth treatments during Autumn and Winter, (2) morphology and photosynthesis characteristics of D. chinense seedlings were measured during the flooding period and the recovery period after floodwaters receded in the present study and (3) in particular we will answer the two question below: (i) Whether will D. chinense survive the flooding under different flooding duration and depth treatments? What is the survival rate? Whether can it recover after soil drainage and how is the recovery growth? (ii) What influence will soil flooding impose on its morphology and photosynthesis if D. chinense can survive the flooding and restore growth after soil drainage? Whether does D. chinense adapt itself to soil flooding through morphological changes and photosynthetic production recovery? Therefore, a comprehensive knowledge of morphological and photosynthetic characteristics under soil flooding and after soil drainage is essential in understanding flooding tolerance of D. chinense in riparian areas and wetlands of the Three Gorges Reservoir Region of the Yangtze River, China. This knowledge might assist in finding new control strategies for this species to soil flooding.

2. Material and methods 2.1. Experiment material D. chinense is perennial shrub, belonging to the genus Distylium of the Hamamelidaceae. It forms dense colonies by adventitious shoots from deep-seated rhizomes. It grows from 0.8 to 1.2 m tall and has an extensive root system. Its young branches stout, internodes 2–4 mm long, older growth glabrescent; petiole densely lepidote, 1.5–2 mm long; leaf blade elliptic to oblanceolate, 2–4 cm long, 1–1.2 cm wide, both surfaces glabrous, base broadly cuneate, margin entire or with two or three teeth on each side near apex, apex subacute; lateral veins five on each side, reticulate veins obscure on both surfaces. It begins growth in late Autumn, its shoots begin to flower in early spring, and then produces seeds in Autumn [26]. D. chinense is widely distributed in the riparian areas and wetlands of the Three Gorges Reservoir Region, China, which was supposed to be excellent protective tree specie along banks and embankments. Two-year-old seedlings (approximately 20 cm in height) of D. chinense were collected in March 2007 from the Gaojiayan tree seedling nursery (111°210 E, 30°150 N, 200 m. . .) in Yichang city, Hubei province, China and delivered in soft-walled containers, reducing root loss during excavation and transport. After arrival, all seedlings were transplanted to plastic pots (23 cm width 

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18 cm height) containing a soil–sand mixture of 30% yellow brown soil, 30% rough-grained sand (1–2.2 mm), 30% fine–grained sand (0.7–1.22 mm), 10% perlite and 3 g L1 long-term fertilizer (pH = 6.25, soil moisture 27.32%, soil bulk density 1.38 g cm3). Before initiation of the soil flooding treatments, the plants were allowed to grow in the open air with one seedling per pot for at least 5 months and maintained under well-aerated soil conditions. Irrigation was performed every other day with tap water. Only healthy trees with intense root growth were used. The experiment was carried out in ecological experimental station of China Three Gorges University, Yichang city, Hubei province, China (111°180 E, 30°430 N, 134 m. . .. All the plants were under the same growing conditions and routine nursing measures such as weeding were used.

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structural surfaces of the stems, providing a medium for the direct exchange of gasses between the internal tissues and atmosphere) were counted. The shoots (including primary shoots, secondary shoots, and epicormic shoots) of all the seedlings were also counted before and after the flooding events. Twenty seedlings in each flooding treatment were selected to recover growth, which were under the same conditions as the control seedlings. Plants with new leaves at the end of the recovery period were supposed to be alive and begin recovery growth, otherwise they were regarded dead. The survival rate (SR) can be calculated by the following equation: SR% ¼ nn12  100; where n1 and n2 are the number of the surviving plants and all the recovery ones, respectively. 2.4. Photosynthesis parameters

2.2. Experimental design Afterwards, on 1 August 2007, 400 small plants were selected for uniformity in size and development. Average seedling heights and stem diameters at 1 cm above the ground level were 25 ± 1.8 cm and 9 ± 1.2 mm, respectively. The seedlings were submitted to four treatments: (1) 40 seedlings unflooded and watered daily as control (Unflooded, CK); (2) 120 seedlings flooded at 1 cm above the ground level (F-1 cm); (3) 120 seedlings flooded at 12 cm above the ground level (F-12 cm) and (4) 120 seedlings completely submerged with 2 m water depth (F-2 m, top of plants at 2 m below water surface). In flooding treatments water was periodically added to keep each water level. Those flooding conditions were maintained until November 1 for 90 days and the flooded seedlings were restored to grow after soil drainage until February 1, 2008 for 90 d. The flooding durations included three durations of 15, 30 and 90 days with two recovery growth durations of 15 and 60 d. The flooding depths were the above F-1 cm, F-12 cm and F-1 cm flooding treatments. All the F-1 cm and F-12 cm flooding seedlings were placed into plastic tanks (70 cm length  50 cm width  40 cm height) with five pots per tank. There, for the F-1 cm and F-12 cm flooding treatments were three treatments for every flooding depth with three flooding durations and each treatment had eight replicates with five pots per replicate, respectively. Therefore, there were 120 seedlings in the F-1 cm and F-12 cm flooding treatments, respectively. At the same time, 120 seedlings of the F-2 m flooding treatment were placed into a cistern (10 m length  2 m width  2.5 m height). Of them, each flooding duration treatment had 40 seedlings with four replicates and 10 pots per replicate, three flooding durations 120 seedlings. The 40 unflooded seedlings as control were watered daily in same place. The water of all flooding treatments was replaced every other day during the flooding events. Within each plastic tank, seedlings in all treatments were rotated randomly every 2 weeks to minimize positional effects. The seedlings of all the flooding duration treatments were put into plastic tanks or cisterns in batches according to the different flooding durations and taken out together to investigate their morphological and photosynthetic indexes at the end of experiments. During the experimental period, all the seedlings were not fertilized.

Photosynthesis parameters were measured on day 1 before flooding and also on days 2, 15 and 60 after flooded plants were unflooded with a portable photosynthesis system (Model LI-6400, Li-Cor, Inc., Lincoln, Nebraska, USA). But the photosynthesis parameters of the F-2 m flooding treatment for 90 days on day 15 after soil drainage (recovery) were not measured because of its leaf senescence and abscission on day 7 after soil drainage. The measurements were conducted from 9:30 to 11:30 a.m. to avoid midday depression of photosynthesis and made on well expanded, matured and developed leaves at the 3rd to 6th node from shoot apices. The leaves were acclimated to the environmental conditions inside the leaf cuvette for about 30 min. Air temperature (AT), relative humidity (RH) and CO2 concentration during photosynthesis measurements were recorded as follows: AT = 25–30 °C, RH = 50–60%, CO2 concentration = 360–380 lmol CO2 mol1, respectively. The saturated light intensity was set at 1000 lmol m2 s1 and 2 cm  3 cm standard leaf chamber was chosen. The area of the tested leaf was traced on a piece of paper and then the area of the paper was determined with a leaf area meter (Model LI-3000, Li-Cor, Lincoln, Nebraska, USA). Photosynthesis parameters comprise the net photosynthesis rate (Pn), transpiration rate (Tr), stomatal conductance (Cs), inter-cellular CO2 concentration(Ci), leaf temperature (Tl), atmosphere temperature (Ta), atmosphere CO2 concentration (Ca), etc. Their average values were calculated. According to the CO2 concentration of the air (Ca) and Ci, stomatal limiting value (Ls) for photosynthesis could be modeled as Ls = 1  Ci/Ca [27]. Five individuals were detected in each treatment and each plant was detected three times to calculate mean values. 2.5. Chlorophyll content Chlorophyll content (Chl) was measured on day 1 before flooding and also on days 2, 15 and 60 after flooded plants were unflooded. Fresh healthy mature function leaves (0.2 g) were abstracted with 80% acetone. Four samples were extracted from each treatment and the samples were detected with UV-1200 ultraviolet– visible spectrophotometer. Each sample was detected three times and mean value was obtained to calculate the photosynthetic pigment contents per unit fresh weight of leaf [28].

2.3. Morphological characteristics

2.6. Data processing

Plant height, stem diameter, adventitious roots, stem lenticels and epicormic shoots of all seedlings were measured before and after the flooding events. Plant heights were measured from the main stem base to its top leaf with a ruler. Stem diameters were measured at the stem base with vernier calipers. Adventitious roots and lenticels of the stem above the soil surface per plant in the flooded treatments (a lenticel functions as a pore within the

Variation was partitioned into two main effects and their interactions: the flooding depth and duration. Data were subjected to analysis of variance by SPSS (10.0 for windows) statistical program. Mean and standard error (SE) values of four replicates were calculated. Two-way ANOVA with the flooding depth and duration as the factors were employed to test any difference among four flooding depths, six flooding durations, and their interactions. If a

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significant difference were found in the flooding depths factor, the flooding depth effect on the same flooding duration would be tested again using one-way ANOVA. Multiple comparisons among treatments were performed by the method of Duncan’s multiple range test. 3. Results and analysis 3.1. Effects of flooding stress on morphological characteristics The survival rate of all the seedlings under different flooding treatments reached 100%. Flooding had significant influence on plant height and stem diameter of D. chinense seedlings. On day 90 after initial flooding, the 1 cm-flooded and 12 cm-flooded seedlings grew slower than those of control seedlings. The plant height increments in the 1 cm-flooded, 12 cm-flooded and the unflooded seedlings were 1.97 ± 0.24, 1.13 ± 0.21, and 5.20 ± 0.23 cm, respectively, while the 2 m-flooded seedlings showed almost no plant height increment. In all the flooded seedlings diameter increments were enhanced around the water levels. Of them, the diameter increments in the12 cm-flooded seedlings were comparatively larger than those of other seedlings with 4.4 times those of the control seedlings (Table 1). Primary roots of all the flooded seedlings became black, some of which even rotted to death with the increasing flooding duration. Meanwhile, adventitious roots and stem lenticels on submerged parts of stems were observed and kept increasing in the flooded seedling with the prolonged flooding duration (Fig. 1). In the first

15 days of flooding, a few adventitious roots and stem lenticels were observed in the 1 cm-flooded, 12 cm-flooded seedlings and large numbers of adventitious roots and stem lenticels occurred during 30–90 days of flooding. After 90 days of flooding, the number of adventitious roots and stem lenticels in the 12 cm-flooded seedlings was greater than those of other seedling, which showed significant difference between the flooded and unflooded seedlings, while the number of the adventitious roots and stem lenticels in the 2 m-flooded seedlings was much less than those of the 1 cm-flooded and 12 cm-flooded seedlings and the leaf senescence and abscission were observed in the 2 m-flooded seedlings. Almost all the stem lenticels swelled gradually when flooding duration lengthened while no obvious change was observed in control seedlings. The stem lenticels of all the flooded seedlings disappeared soon after soil drainage (recovery) and leaves in the 1 cm-flooded and 12 cm-flooded seedlings recovered quickly while old leaves in the 2 m-flooded seedlings continued falling off and new leaves occurred 15 days later. Epicormic shoots sprouted in the 1 cm-flooded, 12 cm-flooded and the unflooded seedlings, whereas the number of epicormic shoots in the 1 cm-flooded and 12 cm-flooded seedlings was significantly more than those of the unflooded seedlings and no obvious epicormic shoots were observed in the 2 m-flooded seedlings (Fig. 1 and Table 1). 3.2. Effects of flooding stress on photosynthetic characteristics ANCOVA showed that there was a significant effect of the flooding duration, flooding depth and their interaction on the net

Table 1 Effects of different flooding depth on the morphological characteristics of D. chinense seedlings after 90 days of flooding (Mean ± SE). Treatments

Plant height increment (cm)

Stem diameter increment (cm)

Number of adventitious roots

Number of stem lenticels

Number of epicormic shoots

CK F-1 cm F-12 cm F-2 m

5.20 ± 0.23a 1.97 ± 0.24b 1.13 ± 0.21b 0.00 ± 0.00c

0.12 ± 0.01c 0.24 ± 0.00b 0.53 ± 0.01a 0.22 ± 0.00b

0.00 ± 0.00d 14.33 ± 1.45b 29.67 ± 1.21a 5.00 ± 0.58c

0.00 ± 0.00d 47.67 ± 1.45b 67.67 ± 1.45a 31.33 ± 2.03c

4.33 ± 0.33c 9.67 ± 0.33a 6.67 ± 0.33b 0.00 ± 0.00d

The seedlings were flooded at 1 cm (F-1 cm), 12 cm (F-12 cm) above the ground level and submerged with 2 m water depth (F-2 m). Values without common superscripts in each column are significantly different at P < 0.05 using Duncan’s multiple range test.

Fig. 1. Morphological characteristics of D. chinense seedlings after 90 days of flooding.

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Table 2 Analysis of ANOVA of net photosynthetic rate (Pn), stomatal conductance (Cs), transpiration rate (Tr) and intercellular CO2 concentration(Ci) in leaves of the seedlings of D. chinense. Variables

Source

Net photosynthetic rate (Pn)

Flooding depth (F) Duration of flooding (D) FD

Stomatal conductance (Cs)

Flooding depth (F) Duration of flooding (D) FD

Transpiration rate (Tr)

Flooding depth (F) Duration of flooding (D) FD

Intercellular CO2 concentration (Ci)

Flooding depth (F) Duration of flooding (D) FD

Df

F-values

3 5

75.075 90.314

p-values <0.01 <0.01

14

15.631

<0.01

3 5

41.124 35.042

<0.01 <0.01

14

7.317

<0.01

3 5

53.431 86.052

<0.01 <0.01

14

12.35

<0.01

3

110.542

<0.01

5

148.176

<0.01

14

25.062

<0.01

photosynthesis rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and inter-cellular CO2 concentration(Ci) (Table 2). The influence of different flooding depths on Pn, Tr, Gs and Ci varied with the duration of flooding (Fig. 2). On day 15 after flooding was initiated, there was no significant difference in Pn among the treatments although flooding treatment caused consistently lower photosynthetic rates. All the flooded seedlings showed a significant decline compared to the unflooded seedlings in Pn on day 30 after flooding (P < 0.05), whereas there was not significant difference among flooding treatments. After 90 days of flooding, Pn of all the flooded seedlings was consistently and significantly lower than that of the unflooded controls. Pn of the 1 cm-flooded, 12 cm-flooded, 2 m-flooded seedlings and the unflooded seedlings was 4.5, 3.6, 1.7 and 7.1 lmol m2 s1 respectively, while there was no significant difference between the 1 cmflooded and 12 cm-flooded seedlings. And the degree of decline in Pn of the 2 m-flooded seedlings was the largest, whereas its photosynthesis rate still remained relatively high although decreased by 76% (Fig. 2). After the flooded seedlings were unflooded, photosynthesis rates of all the flooded seedlings rose gradually. Pn of the 1 cmflooded seedlings recovered sooner than that of the 12 cm-flooded seedlings but their difference from the unflooded controls was still significant. After 60 days of recovery growth, new leaves appeared in the 2 m-flooded seedlings and there were no large differences in Pn between the flooded and unflooded seedlings (Fig. 2). Flooding also had significant effects on Tr and Gs. The tendency of changes in Tr and Gs was similar to that of Pn. Average correlation analysis showed that Pn had significantly positive correlation with Gs (r = 0.972, P < 0.05) and Tr (r = 0.974, P < 0.05). Flooding stress also had significant influence on Ci and Ci increased with the increasing duration of flooding. On day 15 of flooding, there were no large differences between the flooded and unflooded seedlings although Ci of all the flooded seedlings increased. On day 30, there was no a significant increase between the 1 cm-flooded, 12 cm-flooded and the unflooded seedlings, whereas Ci of the 2 m-flooded seedlings was significantly higher than that of the unflooded controls. On day 90, Ci of all the flooded seedlings was significantly higher than that of the unflooded controls and Ci of the 2 m-flooded seedlings was increased by 15.4% compared to the unflooded seedlings. After soil drainage (recovery), Ci of all the flooded seedlings decreased gradually. After 60 days of recovery growth, there was no significant difference in Ci between the

Fig. 2. Effects of flooding stress on net photosynthetic rate (Pn), stomatal conductance (Cs), transpiration rate (Tr), and intercellular CO2 concentration (Ci) of D. chinense seedlings (Mean + SE).

flooded and unflooded seedlings (Fig. 2). The effects of flooding treatments on Ci were quite the contrary to those on Pn, Gs and Tr. Average correlation analysis indicated that Ci showed significantly negative correlation with Pn (r = 0.959, P < 0.05), Gs (r = 0.962, P < 0.05) and Tr (r = 0.967, P < 0.05). F0 showed before flooding; F15, F30 and F90 showed 15, 30 and 90 days after flooding respectively; R15 and R60 showed 15 and 60 days after recovery growth. Values without common superscripts in each column are significantly different at P < 0.05 using Duncan’s multiple range test.

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Table 3 Analysis of ANOVA of leaf pigments content and their ratio in leaves of the seedlings of D. chinense. Variables

Source

Df

F-values

p-values

Chl a

Flooding depth (F) Duration of flooding (D) FD

3 5 14

119.73 145.52 18.73

<0.01 <0.01 <0.01

Chl b

Flooding depth (F) Duration of flooding (D) FD

3 5 14

379.00 307.59 47.96

<0.01 <0.01 <0.01

Chl a/Chl b

Flooding depth (F) Duration of flooding (D) FD

3 5 14

19.63 48.49 7.06

<0.01 <0.01 <0.01

Chl (a + b)

Flooding depth (F) Duration of flooding (D) FD

3 5 14

190.55 218.78 28.42

<0.01 <0.01 <0.01

3.3. Effects of flooding stress on leaf pigments content and their ratio ANCOVA showed that there was a significant effect of the flooding duration, flooding depth and their interaction on the leaf chlorophyll a (chl a), chlorophyll b (chl b), their ratio (chl a/chl b) and chlorophyll (a + b) [chl (a + b)] (Table 3). The influence of different flooding depths on the leaf chl a, chl b, chl a/chl b and chl (a + b) varied with the duration of flooding (Fig. 3). The change trend of influence of flooding on chl a, chl b, chl a/ chl b and chl (a + b) was consistent with each other. On day 15 of flooding, there were no obvious differences in chl a, chl b, chl a/chl b and chl (a+b) between the flooded and unflooded seedlings, but chl a, chl b, chl a/chl b and chl (a + b) of all the flooded seedlings were significantly lower than those of the unflooded controls with the prolonged flooding duration. On day 90, chl a of the 1 cmflooded, 12 cm-flooded and 2 m-flooded seedlings was significantly decreased by 53.7%, 56.4% and 68.5% compared to the unflooded controls, chl b by 35.4%, 37.1% and 41.4%, chl a/chl b by 28.6%, 28.1% and 49.6% and chl (a + b) by 48.7%, 51.2% and 61.3%, respectively. F0 showed before flooding; F15, F30 and F90 showed 15, 30 and 90 days after flooding respectively; R15 and R60 showed 15 and 60 days after recovery growth. Values without common superscripts in each column are significantly different at P < 0.05 using Duncan’s multiple range test. After soil drainage (recovery), chl a, chl b, chl a/chl b and chl (a+b) of all the flooded seedlings rose gradually with the prolonged recovery duration. On day 60 after recovery growth, there were no large differences in these indexes between the flooded and unflooded seedlings (Fig. 3). The tendency of changes in chl a, chl b, chl a/chl b and chl (a + b) was similar to that of Pn. Average correlation analysis demonstrated that Pn had significantly positive correlation with chl a (r = 0.985, P < 0.05), chl b (r = 0.972, P < 0.05) and extremely significantly positive correlation with chl (a + b) (r = 0.992, P < 0.01). Fig. 3. Effects of flooding stress on leaf pigments content and their ratio of D. chinense seedlings (Mean ± SE).

4. Discussion 4.1. Effects of flooding stress on morphological characteristics The growth status and survival rate under flooding stress can be regarded as one of important indexes in plant flood tolerance [29,30]. In the present study, although the flooded D. chinense seedlings manifested different growth characteristics under the different flooding depths and durations, the survival rate of all the flooded seedlings under different flooding treatments reached 100%. The 1 cm-flooded and 12 cm-flooded seedlings grew better than the 2 m-flooded seedlings as mainly manifested in the growth

of plant height, stem diameter, adventitious roots and epicormic shoots. After 90 days of flooding, the 2 m-flooded seedlings had no new epicormic shoots occurring on the stems, their growth of plant height and diameter and expansion of leaves were suppressed, and premature leaf abscission and senescence were observed (Table 1 and Fig. 1). As far as the 1 cm-flooded and 12 cm-flooded seedlings were concerned, these seedlings subjected to soil flooding could be able to maintain photosynthetic and growth rates similar to those of non-flooded seedlings. It is obviously advantageous for them under flooding stress to adapt

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to standing flooding conditions for more photosynthates and energy was allocated to the growth of aerial part of seedlings [31]. Because of the smaller stomatal opening, lower light intensity and slower CO2 diffusion rate in water, the submerged plants may only maintain lower underwater photosynthetic rate [32,33]. On the one hand, the photosynthetic production of flooded plants declines and, on the other, a decline in their utilization of nutrients, which is, however, a key factor that determines the plant under flooding stress to survive or not [17]. Therefore D. chinense seedlings may reduce their utilization of nutrients and growth rate to adapt to the flooding conditions. Moreover, according to the judgment criterion on which plant flood tolerance is based by Loucks and Keen [34], plants that can survive more than 50 days of flooding stress are supposed to be considered as an extremely flood-tolerant species. Thus, D. chinense appears to be a relatively high flood-tolerant species. Flooding results in poor soil aeration and depletion of oxygen in rhizosphere of plants, thus adversely affects water and mineral nutrient uptake in plants and transport, and induces an inhibition of shoot growth and premature leaf senescence and abscission [35]. Adventitious rooting is one of important adaptive mechanisms of wetland plants for replacing existing roots that have been killed or whose function is impaired by stressed environments [10]. In the first 15 days of flooding, a few adventitious roots were observed on the submerged portions of stems of D. chinense seedlings above soil line and the submerged portions of stems around the water levels began to swell, which may be an important adaptive response in flooded D. chinense seedlings to soil flooding. With the increasing duration of flooding, more adventitious roots occurred, the number increased and a great number of stem lenticels appeared in swollen stem base. Formation of hypertrophied lenticels and adventitious roots is one of typically morphological adaptations for the flood-tolerant woody plants to flooding stress [20,36]. Formation of hypertrophied lenticels on submerged parts of stems of woody plants under flooding stress facilitates the transport of the oxygen to the roots in the flood water because hypertrophied lenticels are a pathway through which gases, in particular oxygen, can diffuse through the living cells of the bark [4,18,19, 37–39]. Furthermore, the adventitious roots are very important for plants under root hypoxia or anoxia to obtain oxygen from the environment because adventitious roots may directly get oxygen from the surroundings and internally transport oxygen through aerenchyma. Aerenchyma provides a low-resistance internal pathway for gas exchange between the plant parts above the water and the flooded tissues, and improves the internal supply of oxygen. In addition, the adventitious roots supply the plants with enough water, minerals and hormones to maintain plant growth in flooding stress [40,41]. Thus, as far as formation of adventitious roots and hypertrophied lenticels was concerned, soil flooding led to D. chinense morphological adaptations to flooding stress, which showed that D. chinense is, to a certain degree, a high flood-tolerant species in wetlands and riparian areas. 4.2. Effects of flooding stress on photosynthetic characteristics Leaf blade is a plant organ that makes the most extensive contact with external air environment and also a main place of photosynthesis and transpiration in higher plants. So the damage plants suffer under abiotic stress (low temperature, arid weather, flooding, etc.) can always be reflected in leaves, while the most convenient, acutest and least harmful method to investigate the degree of leaf damage is the measurement of leaf photosynthesis changes. Thus, leaf photosynthesis changes are often taken as responses to environmental stresses and evaluation of plant resistance to them. Soil flooding generally is followed by a relatively rapid decrease in the rate of photosynthesis and stomatal conductance in many flood-

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tolerant woody plants. Several studies have shown that photosynthesis was appreciably reduced within hours to a few days after flooding was initiated, but with the prolonged duration of flooding, the closed stomata gradually reopened and the rate of photosynthesis slowly increased; however, the capacity for stomatal reopening varied with species and the duration of flooding [4,42–44]. In the present study, on day 15 after initial of flooding, all the flooded D. chinense seedlings showed no significant decline in net photosynthesis rate, transpiration rate and stomatal conductance. On day 30, all the flooded seedlings showed a significant decline in them compared to the unflooded controls (P < 0.05). On day 90, they further declined, but still maintained at a relatively higher level and there was significant positive correlation among net photosynthesis rate, transpiration rate and stomatal conductance (P < 0.05). The results in the present study were a little different from those in the previous studies partly due to a lack of early flooding dynamic monitoring. The previous studies also suggested that much of the early reduction in the rate of photosynthesis of flooded plants was correlated with stomatal closure, resulting in decreased CO2 absorption by leaves [11,45]. The decline of stomatal conductance would reduce inter-cellular CO2 concentration and the reduction of photosynthetic substrate would contribute to a decline of net photosynthesis rate [46]. Schulze thought a decrease in net photosynthesis rate not only depends on stomatal factors but also on non-stomatal factors [47]. According to Farquhar and Sharkey [46], a decline in photosynthesis rate would be mainly attributed to the non stomatal limitation only if both photosynthesis rate and inter-cellular CO2 concentration decline with the increase of stomatal limitation value (Ls). On the contrary, but if inter-cellular CO2 concentration has a contrary change trend to that of net photosynthesis rate with the decline of Ls, the decline of photosynthesis rate should be attributed to non-stomatal factors, namely, a decline in photosynthetic activity of mesophyll cells, e.g., lower mesophyll carboxylation efficiency, reduced ribulose-1,5-bisphosphate (RuBP) regeneration, poorly photosynthetic electron transport, or a reduced amount of functional Rubisco, etc. Net photosynthesis rate of D. chinense seedlings was appreciably reduced with the prolonged duration of flooding, while inter-cellular CO2 concentration did not decline with the increasing duration of flooding and rose instead with the decline of Ls. This showed that much of the early decrease in photosynthesis of the flooded D. chinense seedlings was attributed to stomatal inhibition, but as flooding continued, nonstomatal limitations progressively dominated. That is to say, short periods of flooding reduced photosynthesis D. chinense seedlings largely by reducing stomatal conductance; however, long-term flooding reduced not only stomatal conductance but also photosynthetic activities of mesophyll cell. According to the present study, during prolonged periods of flooding, photosynthesis rate of all the flooding plants to varying degrees declined compared to the unflooded controls, whereas the decline in photosynthesis rate under flooding is more attributed to inhibitory effects on the photosynthetic process. Meanwhile, more and more adventitious roots and stem lenticels facilitate gas exchange between the interior flooded tissues and atmosphere, improve the internal supply of oxygen, and further contribute to photosynthetic electron transport, photophosphorylation and RuBP regeneration to maintain certain photosynthetic production, which is consistent with the result of Iwanaga and Yamamoto that the formation of adventitious roots could recover the reduced photosynthesis rate and stomatal conductance [18]. Chen et al. also reported that the maintenance of relatively high photosynthesis was an important adaptation for flood-tolerant species in flooded environments [9]. Thus, as far as photosynthetic characteristics was concerned, D. chinense was found to acclimate to the prolonged duration of flooding (90 d), which showed that D. chinense is a relatively high flood-tolerant species.

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Under flooding stress, chlorophyll (chl) content of plants would change, which was chiefly manifested in premature leaf chlorosis, senescence and abscission [48]. It was reported that the value of Chl a/Chl b was taken as an index of the relative amount of reaction centres (RCs) of the photosystems and light-harvesting complexes (LHCs) of thylakoids [49,50]. Mishra et al. found the decline of chl content and the value of Chl a/ Chl b in flooded rice influenced the photochemical efficiency of PSII (photosystemII) as indicated by the loss in oxygen evolving complex (OEC), DCPIP(2,6-dichlorophenol indophenol) photoreduction and the quantum yield of PS2 [49]. In the present study, the decline of Chl a, Chl b and the value of Chl a/Chl b had a significant positive correlation with the decline of photosynthesis rate (P < 0.05). Thus, the decrease of the value of Chl a/Chl b may suggest that the flooding stress contributed to higher degree of degradation in RCs than that in LHCs because Chl a exists more in RCs while Chl b in LHCs [51]. This further supported the above hypothesis that the decline of net photosynthesis rate could be attributed to the decline in photosynthetic activity of mesophyll cells. After flooded plants were unflooded, Chl a, Chl b, Chl a/Chl b and photosynthesis rate in the flooded D. chinense seedlings rose gradually and showed no significant difference compared to that in the unflooded controls after 60 days of recovery, which could be one of reasons that the flooded D. chinense seedlings could maintain normal photosynthesis characteristics. From the above results, we concluded that D. chinense can adapt to long periods of flooding via the morphologic adaptations such as the formation of adventitious roots, stem lenticels, etc and the decline in physiological activities such as proper reduction of photosynthesis in order to reduce the utilization of energy, which showed that D. chinense is relatively high flood-tolerant to flooding. D. chinense is a dominant shrub species in riparian zones along the Yangtze River in the Three Gorges Reservoir Region and the branches of Yangtze River, which has a certain adaptive ability to the fluctuation of water levels in natural flood season for thousands of years [52]. According to the long-term field observations of our research team in flooded season, D. chinense is widely distributed in riparian zone with different altitude from 100 to 200 m above sea level which makes D. chinense submerged or partially submerged under flooding stress [52]. But, after the flood recedes, D. chinense survives the seasonal flooding and recovers its normal growth and development. Moreover, according to the present results about morphological and photosynthetic adaptations, it could be concluded that D. chinense can survive the long period flooding and thrive in a wide area of the new riparian areas. After Three Gorges Reservoir is constructed, new riparian areas are formed along the Yangtze River in the Three Gorges Reservoir Region between the altitude from140 to175 m where is flooded in Autumn and Winter while is drought in spring and summer seasonally [53]. Since most of the native plant species in riparian zones were submerged permanently, what’s the most important thing is ecological restoration and reconstruction of vegetation in the new riparian areas. The present study designed simulated floodings with different flooding depths and flooding durations to understand the responses of D. chinense to flooding stress and it definitely showed that D. chinense was capable of adapting to any kinds of flooding in riparian areas in Three Gorges Reservoir Region. Therefore, D. chinense could be taken as an excellent candidate species in the re-vegetation of riparian zones in Three Gorges Reservoir Region. However, whether D. chinense can adapt to longer flooding needs to be further studied. Acknowledgements The research was supported by key projects in the National Science & Technology Pillar Program in the eleventh five-year plan (2006BAC10B01).

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