Effects of water-stress on growth and physiological changes in Pterocarya stenoptera seedlings

Scientia Horticulturae 190 (2015) 11–23

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Effects of water-stress on growth and physiological changes in Pterocarya stenoptera seedlings夽 Liping Xu a,b,1 , Yali Pan a , Fangyuan Yu a,∗ a b

College of Forestry, Collaborative Innovation Center of Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, Jiangsu 210037, China Nantong Polytechnic College, Nantong, Jiangsu 226001, China

a r t i c l e

i n f o

Article history: Received 28 December 2014 Received in revised form 29 March 2015 Accepted 31 March 2015 Keywords: Water stress Survival Osmotic adjustment Afforestation Flood tolerance

a b s t r a c t This study investigated the effects of water stress on the morphology and physiological responses in Pterocarya stenoptera seedlings originating from five provenances. Plants were subjected to water stress treatments for a period of up to 30 days. Under waterlogging stress, root morphology changed and increased numbers of adventitious roots were produced, resulting in enhanced growth. However, under drought stress, seedlings wilted more severely which resulted in plant death after day 9–15 of stress. Observations of external morphological changes revealed that seedlings from the HF (Hefeng) exhibited the least resistance to water stress. All of the other physiological indices, such as root vigor, MDA, soluble nutrition, proline, SOD, and POD, among others, were also affected. The rank of waterlogging-tolerance of plants originating from different provenances, which was evaluated synthetically by employing mathematical models, was: HS(Huoshan) > JN(Jiangning) > JJ(Jiujiang) > XN(Xiuning) > HF. With respect to drought-tolerance, the rank was: JJ > HS > XN > JN > HF. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In modern society, the probability of flooding has been greatly increased due to intentional activities by humans such as the removal of natural vegetation, improvement of drainage systems, overgrazing by cattle, and the straightening of meanders in order to facilitate shipping via river systems (Blom and Voesenek, 1996). When plants are submitted to flooding, their underground organs face a sudden change in their microenvironment which remains hypoxic or even anoxic for relatively long periods of time. The morphological and physiological responses of plants to flooding include: formation of adventitious root (Steffens et al., 2006), leaf dehydration (Domingo et al., 2002), reduction of growth (Ashraf and Arfan, 2005), death of trees (Kozlowski, 1984), among others. There are many factors which can impact the tolerance of plants to flooding stress such as genetics (species) (Kozlowski, 1997), seed source (Ladiges and Kelso, 1977), and the time and duration of exposure to flooding (Kozlowski, 1984).

夽 The article was supported by “the Science and Technology Development Project of Northern Jiangsu” and “the Priority Academic Program Development of Jiangsu Higher Education Institutions”. ∗ Corresponding author. Tel.: +86 25 85427403; fax: +86 25 85427402. E-mail addresses: [email protected] (L. Xu), [email protected] (Y. Pan), [email protected] (F. Yu). 1 Tel.: +86 15162774129. http://dx.doi.org/10.1016/j.scienta.2015.03.041 0304-4238/© 2015 Elsevier B.V. All rights reserved.

Drought is another important abiotic stress which affects plant growth and productivity in the same environment where flooding stress is often experienced. For example, when river levels and precipitation are low, drought is a relevant factor which limits plant growth (Parolin, 2001). As compared to seedlings that were submerged and exposed to flooding stress, patterns of leaf loss and production were similar in seedlings that were subjected to drought (Parolin, 2001). Various metabolic alterations occur in plants that are exposed to water stress conditions. Specifically, changes in membrane structure (Vartapetian, 1991), soluble sugar content (Su et al., 1998), protein synthesis (Bucher et al., 1996), phytotoxic compounds (Janiesch, 1991), antioxidant enzymes (Yordanova et al., 2004), among others, are altered as compared to non-stress conditions. Due to their sessile nature and inability to escape from environmental stresses, plants have evolved many adaptive responses as a means to provide coping mechanisms for survival within dynamic environments (Aroca et al., 2012). In recent years, many studies have focused on characterizing the adaptive strategies of plants to water stress (Blom and Voesenek, 1996; Kozlowski, 2002; Kreuzwieser and Rennenberg, 2014). Similar to the tolerance to drought stress, flood tolerance is a complex trait which involves multiple forms of adaptation on both the physiological and morphological level (Lopez and Kursar, 2003). In China, there is an approximate area of 65.94 × 104 km2 of land which experiences flooding. For the whole land mass of China,

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this occupies approximately 6.6% of its total area and is mainly distributed along middle and lower valleys of the Yangtze and the Yellow Rivers (Wang et al., 2002). Due to the large sector of land area and the deleterious effects of flooding on plant growth and production, it is critical to study and identify optimal tree species that can be planted to protect the ecosystems in these riparian zones, resulting in more sustainable environments for human civilization. Pterocarya stenoptera, Chinese wingnut, belongs to the Juglandaceae family which is widely distributes along the Yangstze and Huaihe Rivers in China. Morphological features of Chinese wingnut include a prominent taproot, prosperous lateral roots, large tree-crowns and gold-ingot samaras. As a result, this tree has been utilized as a multi-purpose tree species which is valued for bio-pesticides, timber, afforestation and ornamental uses. At the present time, multiple studies of Chinese wingnut have documented its biological characteristics (Xu et al., 2002), growth and development patterns (Li et al., 2001; Xu et al., 2002), containerized nursery production techniques (Qu, 2007), bio-insecticides (Cheng et al., 2000; Xiao et al., 2002), and wood characteristics (Xu et al., 2004), among others. Despite this abundance of knowledge and characterization, little is known however regarding the selection of waterlogging-tolerant provenances and drought tolerance of P. stenoptera. Due to these aforementioned gaps in knowledge, it is imperative to determine the flooding and water stress tolerance of Chinese wingnut seedlings originating from different provenances. Identification of optimal plant material can help to facilitate large-scale afforestation efforts for Chinese forests. The present study characterized various morphological and physiological indexes of Chinese wingnut seedlings from five provenances under water stress and normal conditions. These efforts were designed to increase understanding of preliminary alterations of physiological characteristics, to comprehensively evaluate and rank the water stress tolerance of different provenances by employing mathematical models. In summary, these efforts aimed provide a theoretical basis for Chinese wingnut afforestation and reforestation efforts in areas where flooding and drought stress are experienced. 2. Materials and methods 2.1. Plant materials and study site According to the natural distribution of P. stenoptera in China, five provenance seeds were selected from Jiangning Jiangsu (JN), Hefeng Hubei (HF), Xiuning Anhui (XN), Jiujiang Jiangxi (JJ) and Huoshan Anhui (HS). Our study was carried out at a nursery of Nanjing Forestry University, which is located at Xinminzhou, Jingkou district, Zhenjiang City (32◦ 16 N, 119◦ 33 E). This region is characterized as a subtropical wet climate area with an annual average of 230–240 frost-free days, a sunshine duration of 2080 h, a mean yearly temperature of 15.4 ◦ C, precipitation of 1100 mm and a sandy soil profile with a pH of approximately 7.5. 2.2. Seedling culture and experimental designs Seeds of P. stenoptera were collected and stored in a refrigeration house (0–5 ◦ C) from July to October in 2007. They were taken out on 23rd March, 2008 and transported to the nursery on the following day. The seeds were pre-soaked in cold water for 24 h and then on March 25, seeds were sown in the field with coverage provided by rice straw. After sowing, the developing seedlings were treated with normal field management best practices. Strong seedlings, based upon optimum uniformity of size and development, were transplanted into nutrition pots

(10 cm × 12 cm) for continuation of growth culturing. The pots were primarily filled with a mixture of sandy loam and peat (1:1). In addition, perlite and compound fertilizer were mixed uniformly as a substrate with the following specifications: weight 0.80 g, porosity 69.71%, pH 6.76, organic matter 5.69%, hydrolysable nitrogen 0.22 mg kg−1 , available phosphorus 34.56 mg kg−1 , and available potassium 87.92 mg kg−1 . During the early days of seedling transplantation, dead seedlings were replaced with healthy ones and watered a single time to ensure that there was a single healthy seedling per pot. On 10 July 2008, the seedlings were placed into a plastic greenhouse for continued growth and maintenance. When the healthy and stably growing seedlings reached 20–25 cm in height and root lengths of 15–20 cm, the treatments were initiated. The experiments lasted for a period of one month from July 23 to August 24 under semi-controlled environmental conditions (only water content was controlled). The water-stress experiment was designed with a randomized block design consisting of four groups. The groups were as follows and consisted of three replications: (1) Control group (CK). Twenty-five seedlings were not flooded and soil water content was maintained at 75% of field capacity. (2) Drought group (D). Fifteen seedlings were sufficiently watered until the soil water content was less than 75% of field capacity. At this point, the waterstress experiments commenced and this group of seedlings was not rewatered and the plants were maintained under continuous drought. (3) Submerged group (F-5). Twenty-five seedlings were flooded at 5–7 cm above the ground level. (4) Waterlogged group (F-0). Twenty-five seedlings were flooded at 0 cm above the ground level. In order to create the flooding treatment, the root environment for the plants was flooded by placing the seedling pots inside larger plastic containers that were filled with tap water. Water was periodically added whenever the water level decreased due to evaporative water loss. Due to the small size and young age of seedlings, water loss due to transpiration was negligible. During the course of the experimentation, we monitored morphological changes and survival of seedlings. In addition, we randomly sampled plant material into sealing plastic bags at 3 (July 25), 6 (July 28), 9 (July 31), 15 (August 6), 22 (August 13) and 30 (August 21) days. Sampling of plant material for the drought group (D) ceased at 15 d because the majority of the seedlings within this group were nearly dead. After sampling, the cell membrane permeability of some freshly harvested leaves was immediately determined. At the same time, additional replicate leaf samples were simultaneously stored in an ultra low freezer in order to provide material for subsequent measurements of additional indexes which were measured in triplicate. 2.3. Measurements 2.3.1. Seedling growth and survival rates Seedling growth was observed and recorded every day, including whether the normal growth of leaves and positions and numbers of adventitious roots. Seedling survival rates were recorded 30 days after the initiation of water stress treatments. 2.3.2. Root vigor Root vigor was evaluated at the 30th day in the flooding process by using the triphenyltetrazolium chloride (TTC) method as previously described (Zhang and Qu, 2005). 2.3.3. Cell membrane permeability Relatively electrical conductivity is an indicator of membrane cell integrity. Two grams of fresh leaves from the same position were washed three times with deionized water, and used for

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measuring relatively electrical conductivity following the procedure as described by Zwiazek and Blake (1990) and Renault et al. (1998). 2.3.4. Malordialdehydrade (MDA) content analysis A total of 0.5 g of frozen leaves was ground using for MDA content according the thiobarbituric method as previously described by Li (2000). 2.3.5. Soluble protein and sugar content analysis A total of 0.5 g of frozen leaves was used to determine protein content according to the Bradford method (Bradford, 1976). For the determination of soluble sugars, a total of 0.3 g of randomly selected frozen leaves was used following the Anthrone colorimetry method (Graham and Smydzuc, 1965). 2.3.6. Antioxidant enzyme activity Superoxide dismutase (SOD, EC 1.15.1.1) and Peroxidase (POD) activities were measured by the nitroblue tetrazolium reduction method (Beauchamp and Fridovich, 1971) with slight modifications. Specifically, 0.5 g of randomly selected frozen leaf tissue was extracted using a pre-chilled mortar and a pestle in 5 mL of 50 mM pH7.8 PBS. The extract was centrifuged for 20 min and stored at 4 ◦ C. The SOD and POD activities were determined by measuring the absorbance at 560, 470 nm against the control, respectively. 2.3.7. Proline content A total of 0.5 g of randomly selected frozen leaf tissue was used to determine proline content using the ninhydrin colorimetry (Arbona et al., 2003). 2.4. Statistical analysis The statistical significance of the results was evaluated by using the analysis of variance (ANOVA) method, followed by a Duncan’s multiple comparisons test using the SPSS 13.0 software (SPSS, Chicago, IL, USA). In this study, we utilized correlation analysis and subject function to comprehensively evaluate the tolerance of seedlings to water stress (Wang and Song, 1988; Hu, 2007). The positive correlation indices with water stress tolerance were calculated through Formula (1), whereas the negative correlations were calculated by using Formula (2). Lastly, Xi was calculated through Formula (3). The formulas were as follows: u(Xi ) =

Xij − Xmin Xmax − Xmin

u(Xi ) = 1 −

Xij − Xmin Xmax − Xmin

Xi =

u(Xi ) n

Fig. 2. Effects of 30 days water stress on the root vigor in different provenance of P. stenoptera seedlings. Bars are s. e. (df = 4, F0.01 = 24.883). P, F, and P × F indicate the effects of provenances, flooding treatment, and their interactions. Significant levels were indicated by **P < 0.01 and *P < 0.05.

formed hypertrophied lenticels and adventitious roots in the submerged part of the shoots just above the water surface after 3, 9 days, respectively. By day 30 of the stress treatment, the seedlings were growing well. In relative comparison to the controls, the heights within this group were less than controls. However, the same aforementioned structures were not observed in the F-0 seedlings from 5 provenances and leaf wilting was observed at day 3 in the D seedlings. By day 15, these D seedlings were nearly dead. After exposure to the water stress, seedling survival rates of JN, HS, JJ were approximately 100%, whereas HF seedlings survival rate was 74.7%, 77.3%, in F-5 and F-0, respectively (Fig. 1). During the period of drought stress (D), some seedlings died and the rank of survival rate for the five provenances was JN 24.4% > HS 20.0% > XN 17.8% > JJ 13.3% > HF 6.7%. 3.2. Physiological changes

(1) (2)

where u(Xi ): values of membership degree, u(Xi ) ∈ [0, 1]; Xij : no. j index of no. i provenance; Xmax , Xmin : maximum and minimum of some index of all tested provenances.



Fig. 1. Effects of water stress on seedling survival rate in different provenance of Pterocarya stenoptera seedlings.

(3)

where Xi : mean value of membership degree, the higher the Xi value, the greater the level of water stress tolerance. 3. Results 3.1. Growth changes Water-stress greatly affected the growth of the stems, roots and leaves of P. stenoptera from different provenances. The waterlogged seedlings (F-5), with the exception for the HF provenance,

3.2.1. Root vigor The results for the analysis of root vigor after 30 days of waterstress are shown in Fig. 2. In comparison to CK, under F-5 and F-0 treatments, root vigor was inhibited in all provenances. The lowest reduction of root vigor was observed in the HF provenance. Specifically, there was a reduction of 82.0% and 77.0%, respectively. The root vigor of JN and XN seedlings in F-5 and F-0 was less than that in CK, but was maintained at a high level. As a result, we concluded that seedlings of the two provenances possessed greater tolerance to flooding stress. With the exception for HF, the root vigor of seedlings in F-5 was greater than that in F-0. This may have possibly resulted from the occurrence of some adventitious roots on stems at the water surface in the F-5 treatment. Analysis of variance indicated significant differences in root vigor among the different provenances, water-stress levels, and their mutual interactions (P < 0.01, Fig. 2). Multiple comparisons (Table 1) showed that there were pronounced differences in root vigor among all of the provenances and water-stress levels; with the exception where no significant differences were observed between the HF and HS provenances and the HF and JJ provenances.

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Table 1 Duncan’s multiple comparisons for root vigor in different provenance of P. stenoptera seedling after water stress. Data followed by capital and small letters means significant difference at 0.05 and 0.01 levels, respectively. The same is below. Provenance

Mean value %

Flooding treatment

Mean value %

JN HF HS JJ XN

2.5838Cc 1.7893Aab 1.7449Aa 2.1393Bb 3.1589Dd

F-5 F-0 CK

1.7665Bb 1.5455Aa 3.4549Cc

3.2.2. Relative electrical conductivity Relative electrical conductivity measurements are used as an accurate estimation for the level of cell membrane integrity in plant tissue. As a consequence of water stress, increases in electrolyte leakage were first observed and the relative electrical conductivity was subsequently reduced (Fig. 3). After 9 days of water stress, the relative electrical conductivity of provenances in flooding treatments was significantly larger than that in CK. And the highest increase was recorded for the HS provenances, up to 55.4% and 54.9% in F-5 and F-0 treatments, respectively. The results indicated that membrane integrity could be damaged at this time point. After 15 days of water stress, the electrolyte leakage for all provenances reached the maximum levels, indicating an elevated amount of membrane damage. The relative electrical conductivity growth of flooding treatments in comparison with CK was not big except for the HF provenance. After a 22 day period of water stress, the relative electrical conductivity of provenances in flooding treatments began to drop, but still remained at a higher level than what was observed in CK. This was especially true for the HF provenance in F-5 which was twice the level as what was observed in CK. With the exception of the HF provenance, after 30 days of water stress, only a small increase in relative electrical conductivity was observed for the 5 provenances. During the whole duration of the stress, the electrolyte leakage of the HF provenance in flooding treatments was always higher than that of other 4 provenances. These data clearly indicate that there was more significant damage to membrane integrity for the HF provenance.

Analysis of variance data revealed significant differences of relative electrical conductivity among the different provenances, the level and duration of water-stress and their mutual interactions (P < 0.01); with exception for mutual interactions between flooding treatment and duration (P < 0.05). The results of multiple comparisons revealed pronounced differences in membrane integrity among the provenances, water-stress levels and water-stress duration. At the same time, membrane damage was positively correlated with the level of water-stress. The highest levels of damage were observed initially and the plants subsequently showed a reduction in stress as the duration of the stress increased (Table 2). 3.2.3. MDA contents MDA levels were quantified in leaf tissue of the 5 provenances as a method to estimate the level of oxidation damage in response to water-stress (Fig. 4). After 3 days of stress, the MDA contents of provenances within the treatment groups were higher than those observed in CK. These data indicate that membrane lipids in leaves were peroxidised at that time point of the stress treatment. The MDA contents of the HF provenance were higher than the other provenances. As compared with CK, the increasing range of the MDA contents of the JN provenances in the water stress treatments was higher than the other provenances. The results suggest that the HF provenance experienced the greatest amount of cell membrane damage and that the JN provenance was the most sensitive to the environment. After 6 days of stress, the MDA contents in stress treatments were also higher than controls, and in both F-5 and F-0 larger than in D.

Fig. 3. Effects of water stress on membrane permeabilities in leaves of different provenance of P. stenoptera seedlings. Bars are s. e. (df = 4, F0.01 = 3.886) (a, b, c and d indicate after 9, 15, 22 and 30 d water stress, respectively). P, F, T, P × F, P × T, T × F and P × F × T indicate the effects of provenances, flooding treatment, flooding time and their interactions. Significant levels were indicated by **P < 0.01 and *P < 0.05. The same is below.

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Table 2 Duncan’s multiple comparisons for membrane permeabilities in leaves of different provenance of P. stenoptera seedling after water stress. Provenance

Mean value %

Flooding treatment

Mean value %

Flooding time (d)

Mean value %

JN HF HS JJ XN

28.4141Cc 34.7262Dd 26.6880Bb 22.9830Aa 23.9454Aa

F-5 F-0 CK

30.9789Cc 27.7098Bb 23.3549Aa

9 15 22 30

24.7389Bb 23.7509Dd 28.9279Cc 21.9741Aa

In addition, with the exception of the HF provenance, the MDA contents at 6 days were slightly higher than what were observed at 3 days. After 9 days subsequent to water stress, compared with 6-day stress, the MDA contents increased slightly. And the MDA contents in treatment groups were higher than those in controls, in drought groups higher than those in flooding treatments, with the exception of the JJ provenance. Afterwards, the continuous drought resulted in death of the seedlings, thus sampling for the drought treatment ceased. At day 15, the MDA content of the provenances in stress plants was higher than the controls, with the exception of the HS provenance. In response to flooding treatments, the relative increase of leaf MDA was not high, with the exception for the HF provenance which increased 38.8% and 33.4%, respectively. Interestingly, the variation tendency of MDA content of provenances at 22 days was similar to that observed at 15 days and the general MDA levels were less than those at 15 days. The MDA contents in the

flooding treatments of the JN, HS and JJ provenances were slightly different, whereas, the MDA levels of the HF and XN provenances exhibited higher than those of the controls. After 30 days of stress, the differences of the MDA content between the treatments and controls were not significant. With respect to the JN provenance, the MDA content decreased. On the contrary, the MDA content of the HF provenance in F-5 and F-0 was higher than controls, up to 39.7% and 34.0%, respectively. Analysis of variance detected significant differences in the MDA content among the different provenances, water stress levels, water stress duration and their mutual interactions (P < 0.01). The Duncan’ s Multiple Range test demonstrated that: at P < 0.05 significant differences were detected in MDA content among different provenances, treatments and stress durations. At P < 0.01, significant differences were observed in MDA content between provenances, with the exception between JN and HS. In addition,

Fig. 4. Effects of water stress on MDA contents in leaves of different provenance of P. stenoptera seedlings (a, b, c, d, e and f indicate after 3, 6, 9, 15, 22 and 30 d water stress, respectively). The same below.

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Table 3 Duncan’s multiple comparisons for MDA contents in leaves of different provenance of P. stenoptera seedling after water stress. Provenance

Mean value (␮mol g−1 FW)

Flooding treatment

Mean value (␮mol g−1 FW)

Flooding time (d)

Mean value (␮mol g−1 FW)

JN HF HS JJ XN

1.6709Cc 2.4796Dd 1.6687Cc 1.6101Bb 1.5458Aa

F-5 F-0 CK D

1.8598Cc 1.8856Dd 1.7339Aa 1.7790Bb

3 6 9 15 22 30

1.7879Cc 1.8271Dd 1.8806Fe 1.8680Ee 1.7368Aa 1.7532Bb

significant differences were observed between treatments and the duration of stress, with the exception for day 9 and 15 of stress (Table 3). 3.2.4. Proline content The pattern of change in leaf proline content is shown in Fig. 5. Under continuous drought conditions, proline levels in leaf tissue rose rapidly. After 3 days of stress, the proline levels of dried seedlings were higher as compared to controls. In particular, the XN exhibited the highest accumulation which was up to 1.31 times greater than the control. Even the HF provenance, which exhibited the lowest increase, reached a level that was 0.41 times greater than the control. At the 6th day of the stress treatment, proline levels further increased. The increase value rank of provenances above the controls was JN 5.03 > XN 3.81 > HS 2.18 > HF 1.54 > JJ 1.03. At day 9 of the stress period, leaf proline levels rose sharply, as compared with controls. Under these conditions, the rank of provenances was JN 1464.3% > XN 795.3% > JJ 575.3% > HS 527.3% > HF 210.6%. In relative comparison to the controls, leaf proline content during the F-5 and F-0 treatments decreased initially, then increased and decreased again; reaching the lowest level at day 15 of stress treatment. After 3 days of stress, as compared with controls, proline levels of provenances exhibited some reduction, with the exception for the HF provenance which increased. The highest percent decline was the XN provenance, with up to 3.51% and 19.0% in F-5 and F-0, respectively. After 6 days of stress, the leaf proline of the HF provenance was still elevated and the speed of the accumulation was less than what was observed at day 3. In contrast, the proline content of the other provenances began to drop and was less than the controls. After 9 days of treatment, proline levels further reduced to levels that were lower than controls. The percent decline in F-5 was larger than what was observed with F-0. After 15 days of stress, comparisons of proline levels to controls revealed decrease, with the exception of the HF provenance. After 22 days of stress, proline levels were slightly higher than what was observed at day 15. After 30 days of stress treatment, there were no observable changes in patterns for proline content and little differences were observed between the treatments of provenances. Analysis of variance detected significant differences in the proline content among the different provenances, water stress levels, water stress duration and their mutual interactions (P < 0.01). The Duncan’ s Multiple Range test detected significant differences among the provenances, with the exception between the JJ and XN provenances (also at P < 0.05), water stress levels (except between F-5 and F-0 treatments), and water stress duration (P < 0.01) (Table 4). 3.2.5. Soluble protein and sugar contents Flooding stress resulted in a general reduction of soluble protein content of different provenances with prolonged duration of the stress, whereas, drought stress did not appear to have a significant effect on the levels of soluble proteins. As shown in Fig. 6, the HF provenance had less soluble protein content than the other provenances, indicating that it is more sensitive to water stress.

After 3 days of stress, all provenances maintained high levels of soluble protein, although the values from the different provenances across the various treatments were not significantly different. After 6 days of flooding treatments, the soluble protein levels of the provenances decreased and were less than those of controls with the exception of the HF provenance. In contrast to under flood, under 6-day continuous drought, the soluble protein levels of the provenances were higher than those of controls and similar to those observed at day 3, except for the JN provenance. After 9 days flooding stress, the leaf soluble protein contents of provenances were sharply reduced to levels lower than those of controls. The JN provenance exhibited the largest percent decline which was up to 48.8% and 44.4% in F-5 and F-0, respectively. The HF provenance exhibited the smallest percent decline and this provenance in particular had contents that were higher than other provenances. Collectively, the observed decline of soluble proteins indicates that protein synthesis may be inhibited or protein turnover is occurring more rapidly under the water stress conditions. As a result, compatible solutes, such as proline, accumulated within cells to increase osmotic adjustment and enhance the tolerance of seedlings to flooding stress. At day 15 and 22, soluble proteins in leaves of provenances under flooding stress continued to decrease, although the range of decline was small. As a result, at day 30 of the stress treatment, little differences were observed in the leaf soluble proteins of the JN, HS, JJ and XN provenances between flooding stressed samples and controls. These data indicate that the seedlings acquired some level of tolerance to water stress conditions. As a result of the water stress treatment, Fig. 7 shows that leaf soluble sugar levels of provenances generally increased within a small range. Comparisons across the provenances revealed that the sugar contents within the HF provenance were lower than the other provenances. After 3 days of stress, the leaf soluble sugar levels of provenances in stressed plants were higher than those within controls. As compared to drought stressed samples, the flooding treatment resulted in higher levels of soluble sugars. When comparing across provenances, the HS was the highest amongst the other provenances for soluble sugars. Collectively, these data indicate that the HS provenance may be useful material for flood resistance. We can conclude that the enhanced tolerance to flood resistance resulted from the accumulation of soluble sugars, a reduction of cellular osmotic potential and increased water holding capacity. After a period of 9 days, comparisons to controls revealed elevated levels of soluble sugars in stress treatments. At day 30 of stress treatment, greater levels of soluble sugars were observed in F-0 as compared to F-5, indicating improved osmotic adjustment under the F-0 treatment. Analysis of variance detected significant differences in the soluble protein and sugar contents among the different provenances, water stress levels, the duration of water stress and their mutual interactions (P < 0.01). The Duncan’ s Multiple Range test revealed (Table 5) significant differences in the soluble protein contents among the provenances, water stress levels, and water stress duration; with the exceptions between at day 30 and 15 and at day 30 and 22 (P < 0.01). Regarding soluble sugar contents, significant differences were observed among

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Fig. 5. Effects of water stress on proline contents in leaves of different provenance of P. stenoptera seedlings.

Table 4 Duncan’s multiple comparisons for proline contents in leaves of different provenance of P. stenoptera seedling after water stress. Provenance

Mean value (mg g−1 FW)

Flooding treatment

Mean value (mg g−1 FW)

Flooding time (d)

Mean value (mg g−1 FW)

JN HF HS JJ XN

2.0404Dd 1.5194Bb 1.3669Aa 1.6096Cc 1.8083Cc

F-5 F-0 CK D

0.8598Aa 0.9543Ba 1.2439Cb 5.8784Dc

3 6 9 15 22 30

1.5324Dd 2.5601Ee 2.7302Ff 0.5871Aa 1.3052Cc 1.0535Bb

the provenances, with the exception between the HS and XN provenances (also at P < 0.05); water stress levels, with the exception between F-5 and F-0; and the duration of water stress, with the exception between day 3 and 15, at day 6 and 15 and at day 22 and 30 (P < 0.01) (Table 6).

3.2.6. SOD and POD activity As shown in Fig. 8, under flood stress (F-5), the activity of SOD in leaves of the provenances initially increased and then declined. With the exception of the HF provenance, SOD activity was maintained at a high level with a maximum at day 6, a minimum at

Table 5 Duncan’s multiple comparisons for protein contents in leaves of different provenance of P. stenoptera seedling after water stress. Provenance

Mean value (mg g−1 )

Flooding treatment

Mean value (mg g−1 )

Flooding time (d)

Mean value (mg g−1 )

JN HF HS JJ XN

66.9401Dc 79.9204Ed 66.4472Cc 65.0127Bb 61.8270Aa

F-5 F-0 CK D

60.6059Aa 62.6171Ba 71.0913Cc 87.5847Dd

3 6 9 15 22 30

86.1052Ee 82.5423Dd 62.9895Cc 55.0368Aa 55.6437Bb 55.4239ABb

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Fig. 6. Effects of water stress on soluble protein contents in leaves of different provenance of P. stenoptera seedlings.

day 22, and an increase at day 30; which was still less than day 3. These data indicate that SOD is capable of controlling the damage caused by flooding stress within a certain range. In the F-0 treatment group, similar trends were observed but the low point of SOD activity occurred at day 15. In the drought groups, the leaf SOD activities of the provenances decreased and a large range of reduction was observed; showing levels 8–9 times higher at day 3 than at day 9. These data indicate that the capability of plants to combat oxidative stress failed as the stress treatment progressed. In addition, these data highlighted that there was an imbalance between the generation and elimination of active oxygen under continuous drought stress treatments. Additionally, under water stress, the leaf SOD activities of the HF provenance were less than that of the other provenances. These data suggest that the capability to minimize damage as a result of oxidative stress is reduced in the HF provenance; providing correlative evidence to support the

observation that this provenance exhibited the lowest tolerance to water stress. In response to water stress, no consistent pattern of POD activity was observed in the different provenances (Fig. 9). In the drought stress treatments, the leaf POD activities of different provenances were relatively stable and tended to have higher activities in relative comparison to controls. After 9 days of drought stress, the POD activities of the provenances were greater than what was observed in flooding treatments. Different ranges of increases were detected and the highest increase (347.7%) was observed for the JN provenance, as compared with controls. These data support the hypothesis that more free radicals accumulated in seedlings resulting in oxidative stress and perturbation of membrane integrity; consequently resulting in a loss of cellular water, turgor pressure and finally the wilting of plants. Under flooding stress, the leaf POD activities of the HF provenance increased in direct relationship to the length

Table 6 Duncan’s multiple comparisons for soluble sugar contents in leaves of different provenance of P. stenoptera seedling after water stress. Provenance

Mean value (mg g−1 FW)

Flooding treatment

Mean value (mg g−1 FW)

Flooding time (d)

Mean value (mg g−1 FW)

JN HF HS JJ XN

0.3006Bb 0.2538Aa 0.3121Cc 0.3301Dd 0.3076Cc

F-5 F-0 CK D

0.3155Cc 0.3215Dc 0.2685Aa 0.2985Bb

3 6 9 15 22 30

0.2867Bb 0.2768Aa 0.3124Cc 0.2812ABab 0.3334Dd 0.3286Dd

L. Xu et al. / Scientia Horticulturae 190 (2015) 11–23

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Fig. 7. Effects of water stress on soluble sugar contents in leaves of different provenance of P. stenoptera seedlings.

of the stress duration; showing a tendency of higher activities as compared to controls (with the exception of day 6). At day 22, the range for the increase of activity was especially large in F-5 and F0 in relation to controls, with increases of activities of 165.4% and 30.2%, respectively. On the other hand, POD activities from other provenances were generally lower than controls. Significant differences were detected for both SOD and POD activities among the different provenances, water stress levels, water stress duration and their mutual interactions (P < 0.01). The Duncan’ s Multiple Range test confirmed significant differences of SOD activities among the provenances, water stress levels (with the exception between F-0 and CK which increased in direct relation to soil water content), and water stress duration (with the exception at day 9 and 30 (P < 0.01) (Table 7). Multiple comparison analysis revealed significant differences of POD activities among different provenances, treatments, and the duration of water stress duration (with the exception of day 6 and 22), according to (P < 0.01) (Table 8).

positive correlation of seedling survival percent with root vigor, soluble sugar content and SOD activity. In addition, a significant positive correlation was observed with MDA content and POD activity in the F-5 treatments; as well as in F-0 groups. Furthermore, a significant positive correlation was also observed with biomass and proline content. Collectively, these data suggest that the improvement of root vigor, leaf soluble content and SOD activity was useful to increase seedling survival percent. In contrast, however, it was unfavorable that the accumulation of MDA resulted in the damage of membrane structures. Tables 10 and 11 show the synthetic evaluation on the water stress tolerant membership grade of different P. stenoptera provenances. From Table 10, the rank of waterlogging-tolerance of different P. stenoptera provenances was HS > JN > JJ >XN > HF, and from Table 11, the rank of drought tolerance was: JJ > HS > XN > JN > HF.

3.3. Correlation analysis and synthetic evaluation on water stress tolerant membership grade of different P. stenoptera provenances

4.1. Effects of waterstress on root system

After exposure to water stress conditions, the percentage of seedling survival can serve as an accurate indicator of waterstress tolerance. Under water stress, a significant correlation was observed between percent seedling survival and other indices (Table 9). In the F-5 treatment groups, there was a significant

4. Discussions

As a consequence of flooding, soils typically become anoxic which is an environmentally stressful condition that compromises plant function (Lopez and Kursar, 2003). The anoxic environment first acts on root systems and affects their biochemistry and physiological activities (Larcher, 1995), such as root vigor (Ahmed et al., 2002). Different types of flooding conditions are known to

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L. Xu et al. / Scientia Horticulturae 190 (2015) 11–23

Fig. 8. Effects of water stress on SOD activities in leaves of different provenance of P. stenoptera seedlings.

Table 7 Duncan’s multiple comparisons for SOD activities in leaves of different provenance of P. stenoptera seedling after water stress. Provenance

Mean value (␮g−1 FW)

Flooding treatment

Mean value (␮g−1 FW)

Flooding time (d)

Mean value (␮g−1 FW)

JN HF HS JJ XN

77.6607Dd 22.8489Aa 68.1525Bb 75.1109Cc 88.0800Ee

F-5 F-0 CK D

78.6427Cc 62.8998Bb 62.5839Bb 57.5809Aa

3 6 9 15 22 30

83.2349Dd 93.4501Ee 59.3446Cc 41.8830Aa 51.4942Bb 57.9089Cc

influence and determine the number and characteristics of adventitious roots (Visser et al., 1996). As a result, the determination of root vigor can serve as a good indicator plant stress tolerance. In this study, the values of root vigor in flooding treatments were less than controls. The rank of different provenances was XN > JN > JJ > HS > HF, which was in good accordance with the

growth and adventitious roots that are produced in Chinese wingnut seedlings. These data indicate that the root vigor of the flood-tolerant Chinese wingnut seedlings would gradually decreased under flooding. In addition, the production of adventitious roots on the submerged portion of stems played a positive role in the normal growth (Armstrong et al., 1994). This reflected both

Table 8 Duncan’s multiple comparisons for POD activities in leaves of different provenance of P. stenoptera seedling after water stress. Provenance

Mean value (␮g−1 min−1 FW)

Flooding treatment

Mean value (␮g−1 min−1 FW)

Flooding time (d)

Mean value (␮g−1 min−1 FW)

JN HF HS JJ XN

707.9899Dd 1146.0001Ee 452.0101Aa 652.0570Cc 588.1139Bb

F-5 F-0 CK D

687.0932Cc 543.3068Aa 662.9989Bb 1175.9891Dd

3 6 9 15 22 30

906.7989Ee 652.8945Bb 685.8990Cc 656.0405Aa 636.3189Bb 772.2398Dd

L. Xu et al. / Scientia Horticulturae 190 (2015) 11–23

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Fig. 9. Effects of water stress on POD activities in leaves of different provenance of P. stenoptera seedlings.

Table 9 Correlation analysis among seedling survival, physiological and biochemical indices of P. stenoptera under water stress. Root vigor Root vigor Membrane permeability MDA content Proline content Soluble protein content Soluble sugar content SOD activity POD activity Survival rate

Membrane permeability 0.088

−0.326 −0.762** −0.475 −0.816** 0.318 0.649** −0.755** 0.782**

0.331 0.495 0.348 −0.068 0.025 0.369 −0.333

MDA content

Proline content

Soluble protein content

Soluble sugar content

SOD activity

POD activity

Survival rate

−0.826** −0.056

−0.185 0.476 0.188

−0.794** −0.005 0.994** 0.203

0.832** −0.059 −0.897** −0.151 −0.918**

0.631** −0.154 −0.786** −0.104 −0.802** 0.754**

−0.735* 0.130 0.957** 0.283 0.983** −0.915** −0.826**

0.798** 0.086 −0.996** −0.114 −0.995** 0.911** 0.794** −0.956**

0.833** 0.994** −0.675** −0.735** 0.995** −0.995**

0.817** −0.647** −0.313 0.838** −0.844**

−0.631** −0.751** 0.996** −0.956**

0.398 −0.621* 0.676**

−0.736** 0.713**

−0.989**

*

P < 0.05. P < 0.01. Upper right corner is correlation coefficient of indices of F-5 treatment, whereas the lower left corner is that of F-0 treatment. **

the inhibition of growth for the original roots, as well as the induced compensatory growth of adventitious roots in response to flooding (Steffens et al., 2006). It has been previously reported that adventitious root formation is related to hormones (Steffens et al., 2006; Visser and Voesenek, 2004). Regarding Chinese wingnut, additional studies are warranted in the future to further understand the effect exogenous growth regulators on adventitious root formation and its relationship in the improvement of survival rates under water stress conditions.

4.2. Effects of water stress on membrane structure and MDA content It is well understood that soil waterlogging exerts deleterious effects to plants and alters membrane integrity (Kolb et al., 2004). Relative electrical conductivity, which is a measure of cell integrity and cell membrane leakiness, has been considered as a screening criterion for water tolerance. Additionally, MDA can serve as a marker for lipid peroxidation (Xu and Zhou, 2006). In this

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Table 10 Synthetic evaluation on water-tolerant membership grade of different P. stenoptera provenances. Indices

Membrane permeability MDA content Soluble protein content Soluble sugar content Proline content POD activity SOD activity Comprehensive evaluation Rank of evaluation

Provenances JN

HF

HS

JJ

XN

0.362 0.490 0.463 0.334 0.573 0.367 0.396 0.427 2

0.418 0.460 0.364 0.429 0.239 0.325 0.314 0.363 5

0.527 0.451 0.377 0.517 0.603 0.267 0.316 0.437 1

0.536 0.555 0.322 0.449 0.551 0.310 0.202 0.415 3

0.458 0.430 0.260 0.524 0.404 0.278 0.341 0.387 4

Table 11 Synthetic evaluation on drought-tolerant membership grade of different P. stenoptera provenances. Indices

MDA content Soluble protein content Soluble sugar content Proline content POD activity SOD activity Comprehensive evaluation Rank of evaluation

Provenances JN

HF

HS

JJ

XN

0.615 0.582 0.590 0.471 0.353 0.615 0.536 4

0.418 0.383 0.367 0.494 0.510 0.518 0.449 5

0.432 0.440 0.622 0.510 0.606 0.647 0.542 2

0.519 0.432 0.539 0.609 0.492 0.678 0.545 1

0.593 0.486 0.643 0.604 0.370 0.544 0.538 3

study, with a prolonged duration of flooding stress, the leaf MDA content and membrane permeability of the JN, HS, JJ, and XN provenances initially increased and then subsequently decreased. This early accumulation of MDA was in good accordance to previously results, as described by Arbona et al. (2008). A strong correlation was found between the MDA content and membrane permeability, indicating that short-term stress damaged cell membranes of Chinese wingnut. Seedlings of provenances gradually adapted to the conditions at day 9 of stress. In addition, the MDA content of the HF provenance exhibited the highest increase as compared to the other provenances. These data indicate that the HF provenance was less submergence tolerant due to its weak ability to minimize membrane lipid peroxidation under stress conditions. Drought stress can also induce the accumulation of MDA, resulting in cell injury (Correia et al., 2006); which is consistent to our results within this study. The leaf MDA content of the HF provenance under drought conditions was far higher than that of the other provenances, indicating that the HF provenance had the lowest drought tolerance overall relative to the other provenances. 4.3. Effects of water stress on osmotic adjustment Osmoprotection is a key adaptive strategy that plants employ, especially under severe water stress conditions, to offset the deleterious effects imposed by desiccative stress (Hasegawa et al., 2000). In order to ensure osmotic adjustment, many compatible solutes can accumulate to relatively high levels in plants without deleterious effects (Vendruscolo et al., 2007); such as proline, soluble sugar and protein, among others. Pociecha (2013) hypothesized that plants most likely accumulate additional carbohydrate reserves which allows them to become more stress tolerant and capable of surviving periods of stress. Our results were in good accordance with the aforementioned solutes, which increased overall during the treatments. It is important to note, however, that significant differences among the provenances were observed. We hypothesized that chemical synthesis was first inhibited and that macromolecular proteins proceeded to breakdown and form soluble micro molecules in order to improve osmotic pressure and to consequently avoid water loss.

On the other hand, in our study, we hypothesize that alterations in a metabolic pathway occurred in order to remove the inhibition of proline synthesis and consequently result it the accumulation of proline. The synthesis of proline replaces soluble sugars bit by bit in order to maintain osmotic pressure. These soluble sugars returned to some extent, as determined by the strength of adaptability to stress. 4.4. Effects of water stress on antioxidant enzymes Under severe water stress, plants have evolved defense mechanisms such as enzymatic antioxidant systems to destroy reactive oxygen species that are generated under the stress conditions (Blokhina et al., 2003). POD and SOD are two antioxidant enzymes which are induced under water stress conditions. Similar to the results observed by Arbona et al. (2008), our data confirmed that SOD activity was enhanced as soil water content increased. On the other hand, POD was progressively negatively correlated to survival percentage and positively correlated with MDA; which is not beneficial for plant growth. These data were in contrast to those observed by Ennajeh et al. (2009). Our data demonstrated that there was some species specificity for POD activity. Specifically, higher levels of POD were observed in the HF provenance which did not exhibit good plant growth under droughty conditions. Further experimentation is warranted to fully elucidate the relationship of POD as an adaptive mechanism for plant growth under stress conditions. 4.5. Synthetic evaluation on water-stress tolerance In our study, indices at day 3, 6, 9, 15, 22 and 30 of flooding and drought stress treatments of the provenances were synthetically evaluated via membership function. These efforts revealed water stress tolerance information of different indices and overcame defects of evaluation through one or two indices. Hence, the rank of waterlogging-tolerance of P. stenoptera different provenances was HS > JN > JJ > XN > HF and the rank of drought-tolerance: JJ > HS > XN > JN > HF.

L. Xu et al. / Scientia Horticulturae 190 (2015) 11–23

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