Plant Physiology and Biochemistry 95 (2015) 57e64

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Research article

Genotypic variation in growth and metabolic responses of perennial ryegrass exposed to short-term waterlogging and submergence stress Mingxi Liu a, Yiwei Jiang b, * a b

Department of Grass Science, Hunan Agricultural University, Changsha, 410128, PR China Department of Agronomy, Purdue University, West Lafayette, IN, 47907, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 March 2015 Received in revised form 17 June 2015 Accepted 6 July 2015 Available online 9 July 2015

Physiological mechanisms of waterlogging (WL) and submergence (SM) tolerance are not well understood in perennial grasses used for turf and forage. The objective of this study was to characterize growth, antioxidant activity and lipid peroxidation of perennial ryegrass (Lolium perenne) exposed to short-term WL and SM. ‘Silver Dollar’ (turf-type cultivar), ‘PI418714’ (wild accession), ‘Kangaroo Valley’ (forage-type cultivar) and ‘PI231569’ (unknown status) varying in growth habits and leaf texture were subjected to 7 d of WL and SM in a growth chamber. Plant height was unaffected by WL but was significantly reduced by SM for all grasses except PI418714. The SM treatment caused greater reductions in leaf chlorophyll and total carotenoid concentrations. Substantial declines in water-soluble carbohydrate concentrations were found in the shoots and roots under SM, particularly in Kangaroo Valley and PI231569, two relatively fast-growing genotypes. Significant increases in malondialdehyde concentrations were noted in the shoots and roots of all genotypes exposed to WL and SM, but to a greater extent in Kangaroo Valley and PI231569 under SM. Shoot activities of catalase (CAT) and peroxidase (POD) increased under SM, more pronounced in Silver Dollar and PI418714, two relatively slow-growing genotypes. Waterlogging or SM stresses decreased root activities of superoxide dismutase, CAT, POD and ascorbate peroxidase, especially for Kangaroo Valley and PI231569. The results indicated that maintenance of antioxidant activity and carbohydrate and minimization of lipid peroxidation could contribute to better waterlogging or submergence tolerance of perennial ryegrasses. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Antioxidant metabolism Lolium perenne Fast and slow growing Water excess Water-soluble carbohydrate

1. Introduction Flooding stress occurs to the plants due to frequent, heavy rainfall or over-irrigation followed by slow drainage. As a result, plants may be exposed to waterlogged or submerged conditions. Waterlogging is defined as the saturation of the soil with water around the roots, while submergence describes the condition in which the whole plant is completely covered by water. Either form of excess water stress can negatively affect plant growth and physiology. The capability to adapt to excess water conditions is crucial for increasing survival of the plants, particularly for those plants growing in coastal and flood-prone plains where frequent

Abbreviations: APX, ascorbate peroxidase; CAT, catalase; Chl, chlorophyll; HT, plant height; MDA, malondialdehyde; POD, peroxidase; SM, submergence; SOD, superoxide dismutase; WL, waterlogging; WSC, water-soluble carbohydrate; Car, total carotenoid. * Corresponding author. E-mail address: [email protected] (Y. Jiang). http://dx.doi.org/10.1016/j.plaphy.2015.07.008 0981-9428/© 2015 Elsevier Masson SAS. All rights reserved.

flooding stress may occur. Responses of plant growth and leaf color to flooding stress vary with stress intensity and plant species. Waterlogging reduced shoot and root dry weight in cool-season perennial grass species including creeping bentgrass (Agrostis stolonifera) (Huang et al., 1998; Jiang and Wang, 2006) and Kentucky bluegrass (Poa pratensis) (Wang and Jiang, 2007). In creeping bentgrass, a decline in turfgrass quality occurred even when the water level was maintained at 15- or 1-cm below the soil surface under cool temperatures (Jiang and Wang, 2006). However, waterlogging stimulated plant growth in the tolerant warm-season perennial grass species such as Knotgrass (Paspalum paspaloides) and spiny mudgrass (Pseudoraphis spinescens), while reductions in growth were observed in the intolerant seashore paspalum (Paspalum vaginatum) and centipedegrass (Eremochloa ophiuroides) (Zong et al., 2015). A study by Barney et al. (2009) found that lowland ecotypes of switchgrass (Panicum virgatum) had higher tiller production and length, leaf area and biomass than the upland ecotypes under flooded conditions, demonstrating growth variations of

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different types of grasses in response to excess water conditions. Reduced concentrations of leaf chlorophyll and shoot water soluble carbohydrate were also found in creeping bentgrass (Jiang and Wang, 2006) and Kentucky bluegrass (Wang and Jiang, 2007), but to a much lesser extent in the tolerant cultivars. Submergence stress either inhibits or enhances plant growth, depending on type of species and survival strategy (Bailey-Serres and Voesenek, 2008). Under submersion, some plant species or ecotypes elongated their leaf blades to above the water, while others showed minimum growth possibly for the purpose of conserving energy (Bailey-Serres and Voesenek, 2008; Perata and Voesenek, 2007; Yu et al., 2012). Enhanced shoot elongation, leaf blade and tiller production has been observed in flooding tolerant perennial grass species (Banach et al., 2009;Barney et al., 2009; Mollard et al., 2008). However, in rice (Oryza sativa), submergence tolerance was achieved by minimizing shoot elongation and by increasing dry matter weight underwater (Kawano et al., 2009). In addition, some cultivars or ecotypes within a plant species exhibited little or no changes in leaf growth (Yu et al., 2012). Collectively, variable growth responses and changes of chlorophyll and carbohydrate status are essential for flooding tolerance of a particular plant to waterlogged or submergence stress. Diverse physiological responses to waterlogged or submerged conditions have been found in different plant species (Bailey-Serres and Voesenek, 2008; Wang and Jiang, 2007; Zong et al., 2015). One of the fundamental metabolic changes under excess water stresses is antioxidant metabolism. Waterlogging or submergence stress may increase production of active oxygen species (ROS) such as superoxide (O2
) and hydrogen peroxide (H2O2), which can cause lipid peroxidation and oxidative damages to the plant cell (Mittler, 2002). Plants have evolved enzymatic defense systems to protect cells against oxidative injury by removing, decomposing or scavenging ROS. In this system, superoxide dismutase (SOD) plays a central role in catalyzing the dismutation of O2 $
to H2O2 and oxygen (Bowler et al., 1992), and then H2O2 can be decomposed by several pathways including CAT, POD and ascorbate peroxidase (APX). Qi et al. (2014) reported that ROS detoxification is one of the primary mechanisms for surviving waterlogging for a hybrid of baldcypress (Taxodium distichum) and montezuma cypress (Taxodium mucronatum). It has been found that root SOD activities increased in both tolerant and intolerant cultivars of creeping bentgrass, but to a greater extent in the tolerant cultivar exposed to waterlogging stress (Wang and Jiang, 2007). Similarly, the increased leaf and root activities of SOD and POD were higher in the waterlogging tolerant knotgrass and spiny mudgrass than that of sensitive seashore paspalum and centipedegrass (Zong et al., 2015). However, the decreased and unchanged activities of SOD, CAT, POD, and APX to waterlogging or submergence have also been found in different plant species (Ahmed et al., 2002; Arbona et al., 2008; Lin et al., 2004; Tan et al., 2010; Wang and Jiang, 2007). The results suggest that responses of antioxidant enzymes to flooding stress are complex, varying with plant species, cultivars and stress intensity. Along with growth and other physiological parameters, differential responses of antioxidant enzymes to waterlogging and submergence stress are not fully understood, especially in perennial grasses. Perennial ryegrass is a popular and important cool-season turf, forage and pasture grass that is widely used around the world. This species adapts to well-drained soil conditions, but some cultivars and wild accessions differed in submergence tolerance (Yu et al., 2012). Little is known about physiological mechanisms associated with waterlogging or submergence tolerance of perennial ryegrasses varying in growth habits, leaf texture and flooding response. The objective of this study was to characterize growth, antioxidant activity and lipid peroxidation of perennial ryegrasses

exposed to short-term waterlogging and submergence stresses. We hypothesized that perennial ryegrass accessions differing in growth habits significantly varied in carbohydrate and antioxidant metabolism to waterlogging and submergence stress. Through investigation of shoot and root metabolism in different genotypes, the study would reveal the mechanisms of grass adaptation to saturated or flooded soils. 2. Materials and methods 2.1. Plant materials and growth conditions Silver Dollar is a turf-type commercial cultivar developed from the Turf-Seed Company (Gervais, Oregon, USA). Kangaroo Valley is a forage-type variety. Along with accessions PI231569 (uncertain status) and PI418714 (wild), they were obtained from the USDA National Plant Germplasm System at the Western Regional Plant Introduction Station in Pullman (Washington, USA). These four perennial ryegrasses vary in growth habits, leaf texture, color and flooding responses (Yu et al., 2012). The seeds were sown in sand in plastic pots (4-cm diameter, 9-cm deep) in a greenhouse on 20 November 2013 at Purdue University, West Lafayette, IN, USA. On April 7, 2014, each genotype was propagated into the same-size pots through tillers, and each pot contained 8e10 tillers. Tiller plants were watered as needed with 1/2 Hoagland solution and cut twice a week to 7 cm for Kangaroo Valley, PI231569 and PI418714 and to 6 cm for Silver Dollar. The average air temperatures and photosynthetic photon flux density (PPFD) in the greenhouse were 20  C ± 1.5  C and 450 mmol m
2 s
1, respectively. On 23 May 2014, the grasses were transferred to a growth chamber at 20  C/15  C, with a 12 h photoperiod of 500 mmol m
2 s
1 for 7 d before stress treatments were imposed. 2.2. Waterlogging and submergence treatments Waterlogging and submergence treatments started on 30 May 2014 and ended on 06 June 2014 when severe damage and loss of leaf color occurred to the submerged grasses (visual observation). Waterlogging treatment was imposed by placing the pots into plastic containers (58-cm length  35-cm width  28-cm depth) and tap water (pH ¼ 6.5) was added to the containers until the water level was at the soil surface of each pot. Submergence stress was imposed by submerging the grass pots in same-size containers with tap water. The water level was kept at 8 cm above the grass canopy. No nutrients were supplied to the plants and water was not changed during the period of stress treatments (7 d). Algae were removed if they accumulated. The control pots were well-drained and received normal watering. 2.3. Measurements Plant height (HT) in each pot was recorded to identify growth during the period of stresses. At the end of 7 d, plants were harvested and roots were washed free of sand. A portion of the shoots and roots was immediately frozen in liquid nitrogen and stored at
80  C until further use. A portion of the leaves were randomly selected for chlorophyll (Chl) and carotenoid (Car) extraction. Leaf Chl and Car were extracted by soaking approximately 50 mg leaf samples in 15 mL dimethyl sulfoxide (DMSO) in the dark for 48 h. The absorbance was then read at 665 nm, 649 nm and 480 nm and concentrations of Chl and Car were calculated using the method of Wellburn (1994). Total water-soluble carbohydrate (WSC) concentration was measured using the anthrone method (Koehler, 1952) with some modifications (Yu et al., 2012). Briefly, WSC was extracted from

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approximately 20 mg fine powder of dry shoot or root tissues with 1 mL double distilled water. The extract was shaken for 10 min and centrifuged at 11 000  g for 10 min, and the supernatant was collected. The extraction was repeated three more times and the supernatant was pooled. A 1 mL aliquot of extract was mixed with 7 mL freshly prepared anthrone [200 mg anthrone þ 100 mL 72% (w/w) H2SO4] and placed in a boiling water bath for 8 min. After cooling, the absorbance at 625 nm was read. The standard curve was made using glucose in a range of 0e300 mg mL
1. To extract the soluble protein, frozen shoot and root tissues were ground into fine power using liquid nitrogen. Approximately 50 mg shoot or 100 mg root powder was mixed with 1 mL of extraction buffer (50 mM potassium phosphate, 1 mM ethylenediaminetetraacetic acid [EDTA], 1% polyvinylpyrrolidone [PVP], pH 7.8). The mixture was centrifuged at 15 000  g for 2  15 min at 4  C, and the supernatant was collected for enzyme assay. The protein content was determined using the method of Bradford (1976). The activities of SOD, CAT, POD and APX were assayed by using the methods of Zhang and Kirkham (1996) with minor modifications (Wang and Jiang, 2007). Total SOD activity was measured by recording the rate of p-nitro blue tetrazolium chloride (NBT) reduction in absorbance at 560 nm. The assay medium contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 mM pnitro blue tetrazolium chloride (NBT), 2 mM riboflavin, 0.1 mM EDTA, and 20e40 mL enzyme extract. A reaction mixture was illuminated under 80e90 mmol m
2 s
1 for 10 min. The reaction mixture lacking of enzyme developed maximum color as maximum reduction of NBT. The additional reaction mixture serving as the control was placed in the dark. One unit of SOD activity was defined as the amount of enzymes that caused 50% inhibition in the rate of NBT reduction. The activity of APX was assayed by recording the decrease in absorbance at 290 nm for 1 min. The 1.5 mL reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbic acid, 0.1 mM EDTA, 0.1 mM H2O2, and 0.15 mL enzyme. The reaction was started with the addition of 0.1 mM H2O2. The activity of POD was determined by an increase in absorbance at 470 nm for 1 min. The assay contained 50 mL 20 mM guaiacol, 2.83 mL of 10 mM phosphate buffer (pH 7.0), and 0.1 mL enzyme extract. The reaction was initiated by adding H2O2. The activity of CAT was determined by the decline in absorbance at 240 nm for 1 min. The assay contained 50 mM phosphate buffer (pH 7.0), 15 mM H2O2 and 0.1 mL enzyme extract. The reaction was initiated by adding enzyme extract. Lipid peroxidation was measured in terms of malondialdehyde (MDA) content (an end product of lipid peroxidation) (Dhindsa et al., 1981), with some modifications. A 0.5 mL aliquot of supernatant was mixed with 2 mL of 20% trichloroacetic acid containing 0.5% thiobarbituric acid. The mixture was heated at 95  C for 30 min, quickly cooled, and then centrifuged at 10,000 g for 10 min. The absorbance was read at 532 and 600 nm (Health and Packer, 1968). The concentration of MDA was calculated using an extinction coefficient of 155 mm
1 cm
1. 2.4. Statistical data analyses The experiment was a split plot design with three treatments (control, waterlogging and submergence). Each genotype was replicated three times (three pots) for all treatments. The pots were randomly assigned into the containers within the control, waterlogged or submerged treatments. The analysis of variance and Fisher's protected least significant difference mean separation tests were performed using Statistical Analysis System (SAS Institute Inc. Cary, NC, USA). The root MDA, root POD and leaf WSC data were log transformed prior to analysis. Results were presented as back transformed means for the measurements above.

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3. Results 3.1. Treatment effects Significant treatment and accession effects were found for all parameters. Compared to the control, waterlogging (WL) did not change HT, Car, and shoot WSC, but significantly reduced Chl and root WSC and increased MDA in both shoots and roots (Table 1). Unlike WL treatment, submergence (SM) significantly decreased HT, Car, and shoot WSC to 33.7%, 43.8%, and 74.3%, respectively, compared to the control (Table 1). Significant differences in HT, Chl, Car, shoot WSC, root WSC, shoot MDA and root MDA were found between WL and SM, with more severe reductions in HT, Chl, Car, and shoot and root WSC and more dramatic increases in shoot and root MDA identified under SM than under WL, compared to the control (Table 1). Waterlogging decreased shoot and root activities of all antioxidant enzyme tested in this study for shoot CAT, whereas an increased CAT was noted in shoot exposed to WL (Table 2). Submergence decreased shoot and root activities of SOD and root activities of CAT, POD and APX, increased shoot activities of CAT and POD and did not affect shoot APX activity, compared to the control (Table 2). However, significant differences in shoot and root activities of antioxidant enzyme were also observed between WL and SM. 3.2. Differences among accessions Across all treatments, four accessions significantly differed in all parameters measured in the study. PI231569 had the highest HT, followed by Kangaroo Valley and PI418714; Silver Dollar had the smallest HT (Table 3). The significantly higher values of Chl and Car were found only in Silver Dollar, not in the other three accessions. Silver Dollar has the highest shoot WSC, while the highest root WSC was found in PI418714. There were significant differences in shoot and root MDA among the four accessions, with the highest shoot and root values both shown in PI231569, followed by Kangaroo Valley. Silver Dollar had the lowest accumulation of MDA in both shoot and roots, compared to other accessions. Silver Dollar and PI418714 had higher shoot SOD than Kangaroo Valley and PI231569, while Silver Dollar had the highest root SOD than other accessions (Table 4). Silver Dollar also had higher activities of shoot and root CAT than other accessions. PI418714 exhibited the highest shoot POD and APX activities, while Kangaroo Valley and PI231569 showed the highest root POD activities. Root APX activities remained unchanged among Kangaroo Valley, PI418714 and Silver Dollar, but were higher in these three accessions than in PI231569. 3.3. Responses of individual accessions to treatments The absolute growing HT under treatments was unaffected by WL but was significantly reduced by SM for all grasses except Table 1 Treatment differences in plant height (HT), chlorophyll concentration (Chl), carotenoid (Car) concentration, water-soluble carbohydrate concentration for shoot (SWSC) and root (RWSC), and malondialdehyde concentration for shoot (SMDA) and root (RMDA) across all accessions of perennial ryegrass. Treatment

HT

Chl

cm

mg g
1 mg g
1 mg g
1 mg g
1 nmol g
1 nmol g
1 FW FW DW DW FW FW

Control 8.6 a 2.6 a Waterlogging 9.1 a 2.3 b Submergence 5.7 b 1.2 c

Car

0.48 a 0.46 a 0.27 b

SWSC

52.2 a 55.2 a 13.4 b

RWSC

21.8 a 13.7 b 9.6 c

SMDA

5.4 c 8.1 b 9.6 a

RMDA

5.6 c 10.6 b 12.3 a

Means followed by the same letter in each column for a given treatment are not significantly different at P < 0.05.

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Table 2 Treatment differences in activity of superoxide dismutase for shoot (SSOD) and root (RSOD), catalase for shoot (SCAT) and root (RCAT), peroxidase for shoot (SPOD) and root (RPOD), and ascorbate peroxidase for shoot (SAPX) and root (RAPX) across all accessions of perennial ryegrass. Treatment

SSOD

RSOD

SCAT

Unit mg
1 protein Control Waterlogging Submergence

271.6 a 224.3 c 255.5 b

RCAT

SPOD

RPOD

SAPX

RAPX

0.17 b 0.13 c 0.28 a

0.14 a 0.07 c 0.09 b

5.2 a 4.4 b 5.0 a

1.2a a 0.76 c 0.89 b

mmol mg
1 protein min
1 100.0 a 75.7 b 64.8 c

77.4 c 95.4 b 140.2 a

18.4 a 13.4 b 9.2 c

Means followed by the same letter in each column for a given treatment are not significantly different at P < 0.05.

Table 3 Accession differences in plant height (HT), chlorophyll concentration (Chl), carotenoid (Car) concentration, water-soluble carbohydrate concentration for shoot (SWSC) and root (RWSC), and malondialdehyde concentration for shoot (SMDA) and root (RMDA) across control and stress treatments. Accession

Silver Dollar PI418714 Kangaroo Valley PI231569

HT

Chl

Car

SWSC

RWSC

SMDA

RMDA

cm

mg g
1 FW

mg g
1 FW

mg g
1 DW

mg g
1 DW

nmol g
1 FW

nmol g
1 FW

2.6 1.9 1.9 1.8

0.50 0.38 0.37 0.36

38.8 32.5 30.1 31.3

14.8 17.9 14.2 13.3

6.6 7.1 7.7 9.5

8.0 9.0 9.0 9.9

4.6 7.3 9.0 10.3

c b ab a

a b b b

a b b b

a ab b b

ab a b b

d c b a

c b b a

Means followed by the same letter in each column for a given treatment are not significantly different at P < 0.05.

Table 4 Accession differences in activity of superoxide dismutase for shoot (SSOD) and root (RSOD), catalase for shoot (SCAT) and root (RCAT), peroxidase for shoot (SPOD) and root (RPOD), and ascorbate peroxidase for shoot (SAPX) and root (RAPX) across control and stress treatments. Accession

SSOD

RSOD

Unit mg
1 protein Silver Dollar PI418714 Kangaroo Valley PI231569

290.5 296.7 220.4 194.3

a a b c

SCAT

RCAT

SPOD

RPOD

SAPX

RAPX

0.19 0.24 0.16 0.18

0.08 0.10 0.11 0.11

5.1 5.6 5.0 3.8

1.07 1.06 0.96 0.74

mmol mg
1 protein min
1 92.6 80.5 77.9 69.8

a b b c

126.7 105.7 81.4 103.5

a b c b

17.2 12.2 13.5 11.9

a bc b c

b a c bc

c b a a

ab a b c

a a a b

Means followed by the same letter in each column for a given treatment are not significantly different at P < 0.05.

PI418714, compared to their respective control (Fig. 1). The Chl and Car remained unchanged under WL but was reduced under SM for all accessions (Fig. 1). Chl was reduced by 48%, 55%, 56% and 59% and Car was reduced by 41%, 50%, 35% and 48% for Silver Dollar, PI418714, Kangaroo Valley and PI231569, respectively. Waterlogging did not change shoot WSC concentrations, but shoot SWC was reduced by 65%, 66%, 84% and 80% for Silver Dollar, PI418714, Kangaroo Valley and PI231569 under SM, respectively (Fig. 2). Root WSC was unaffected for Silver Dollar under both WL and SM treatments, but decreased for PI418714 under SM, but not under WL. Root WSC was reduced by 50% for Kangaroo Valley and 47% for PI231569 under WL, compared to the control. Further reductions in root WSC were found in Kangaroo Valley (71%) and PI231569 (63%) exposed to SM. WL or SM significantly increased MDA concentrations in the shoots and roots for all genotypes (Fig. 3). Specifically, shoot MDA concentrations increased 21% and 42% for Silver Dollar, 41% and 56% for PI418714, 50% and 71% for Kangaroo Valley and 87% and 145% for PI231569 exposed to WL and SM, respectively. Root MDA concentrations increased 59% and 75% for Silver Dollar, 75% and 128% for PI41871, 88% and 100% for Kangaroo Valley and 140% and 177% for PI231569 under WL and SM, respectively (Fig. 3). Waterlogging significantly decreased shoot SOD activities for all genotypes, while SM reduced SOD activities in Silver Dollar and PI231569 but increased SOD activities in PI418714 and Kangaroo Valley (Fig. 4). For roots, SOD activities were not affected by WL in Silver Dollar and PI418714 but were reduced in Kangaroo Valley and PI231569. However, SM caused reductions in SOD activities for all genotypes, to a greater extent in Kangaroo Valley and PI231569. The SOD activities were reduced by 16%, 24%, 39% and 60% under SM for

Silver Dollar, PI418714, Kangaroo Valley and PI231569, respectively. Shoot CAT activities remained unchanged in all genotypes exposed to WL but significantly increased 70% for Silver Dollar, 134% for PI418714 and 137% for Kangaroo Valley under SM, compared to the control (Fig. 5). Both WL and SM significantly decreased root CAT activities. Root CAT activities were reduced by 27% and 36% for Silver Dollar, 23% and 50% for PI418714, 24% and 53% for Kangaroo Valley and 35% and 63% for PI231569 exposed to WL and SM, respectively. The shoot POD activities were reduced by WL in Silver Dollar and PI231569, but was unaffected in Kangaroo Valley and PI418714 (Fig. 6). However, SM dramatically increased shoot POD activities to 127%, 62%, 69% and 33% for Silver Dollar, PI418714, Kangaroo Valley and PI231569, respectively. Root POD activities were unaffected by both WL and SM for Silver Dollar, but significantly decreased 41% and 51% for PI418714, 50% and 14% for Kangaroo Valley, and 67% and 44% for PI231569 exposed to WL and SM, respectively. The activities of APX in the shoots were unaffected by WL in Silver Dollar and PI418714, but decreased 18% in Kangaroo Valley and 29% for PI231569 (Fig. 7). Under SM, shoot APX activities increased 22% for Silver Dollar and 16% for PI418714, but decreased 18% for Kangaroo Valley and 32% for I231569. Root APX activities remained unchanged in Silver Dollar under both WL and SM, but were reduced by 34% and 22% for PI418714, 26% and 27% for Kangaroo Valley, and 68% and 43% for PI231569 exposed to WL and SM, respectively. 4. Discussion Four perennial ryegrasses naturally vary in growth habits, leaf

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Fig. 2. Shoot and root concentrations of water-soluble carbohydrate (WSC) concentration as affected by 7 d of waterlogging (WL) and submergence (SM) in perennial ryegrass genotypes. Comparisons are made among the control (CL), WL and SM treatments for a given genotype. Means followed by same letter are not significantly different at P < 0.05 within a genotype for a given treatment. Bars indicate standard deviation.

Fig. 1. Plant height (HT), leaf chlorophyll (Chl) and total carotenoid (Car) concentrations as affected by 7 d of waterlogging (WL) and submergence (SM) in perennial ryegrass genotypes. Comparisons are made among the control (CL), WL and SM treatments for a given genotype. Means followed by same letter are not significantly different at P < 0.05 within a genotype for a given treatment. Bars indicate standard deviation.

texture and color, with Silver Dollar and PI418714 being slowgrowing types and Kangaroo Valley and PI231569 being fastgrowing types (Table 1 and Fig. 1). Submergence generally decreased leaf elongation and chlorophyll in this study. There are a variety of plant growth responses to flooding stress. When plants are exposed to SM, some ecotypes quickly elongate the leaf blade (escape type) and some ecotypes show minimum growth (quiescence type) (Bailey-Serres and Voesenek, 2008; Perata and Voesenek, 2007), while some other plants exhibit moderate growth that falls between the escape and quiescence types (Yu et al., 2012). The behaviors of both escape and quiescence types can be survival strategies, depending on factors such as depth of water and duration of a stress. Escape-type plants may survive in shallow floodwater by rapidly elongating their leaves but may not survive if the water depth is too deep. In this study, water depth was 8 cm above the plants but no leaf elongation to above the water level was observed for all genotypes, even for the relatively fast-growing Kangaroo Valley and PI231569. The fast-growing grasses could completely

Fig. 3. Shoot and root concentrations of malondialdehyde (MDA) concentration as affected by 7 d of waterlogging (WL) and submergence (SM) in perennial ryegrass genotypes. Comparisons are made among the control (CL), WL and SM treatments for a given genotype. Means followed by same letter are not significantly different at P < 0.05 within a genotype for a given treatment. Bars indicate standard deviation.

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Fig. 4. Shoot and root activities of superoxide dismutase (SOD) as affected by 7 d of waterlogging (WL) and submergence (SM) in perennial ryegrass genotypes. Comparisons are made among the control (CL), WL and SM treatments for a given genotype. Means followed by same letter are not significantly different at P < 0.05 within a genotype for a given treatment. Bars indicate standard deviation.

Fig. 5. Shoot and root activities of catalase (CAT) as affected by 7 d of waterlogging (WL) and submergence (SM) in perennial ryegrass genotypes. Comparisons are made among the control (CL), WL and SM treatments for a given genotype. Means followed by same letter are not significantly different at P < 0.05 within a genotype for a given treatment. Bars indicate standard deviation.

Fig. 6. Shoot and root activities of peroxidase (POD) as affected by 7 d of waterlogging (WL) and submergence (SM) in perennial ryegrass genotypes. Comparisons are made among the control (CL), WL and SM treatments for a given genotype. Means followed by same letter are not significantly different at P < 0.05 within a genotype for a given treatment. Bars indicate standard deviation.

Fig. 7. Shoot and root activities of ascorbate peroxidase (APX) as affected by 7 d of waterlogging (WL) and submergence (SM) in perennial ryegrass genotypes. Comparisons are made among the control (CL), WL and SM treatments for a given genotype. Means followed by same letter are not significantly different at P < 0.05 within a genotype for a given treatment. Bars indicate standard deviation.

M. Liu, Y. Jiang / Plant Physiology and Biochemistry 95 (2015) 57e64

consume the carbohydrates prior to growing leaves above the water, which may cause death of the plants. A greater decline in WSC in both shoots and roots in Kangaroo Valley and PI231569 suggested that these two genotypes might not be able to survive deep submersion due to a rapid consumption of carbohydrates to sustain growth (Fig. 2). Since SM can severely restrict photosynthesis (Sand-Jensen, 1989), diminished shoot carbohydrate was found under SM when compared to the control and WL treatment. It can be disadvantageous to plant survival when carbohydrate becomes limited and less energy is produced under stress, particularly for fast-growing grasses. Depletion of carbohydrates during submergence is considered a major factor influencing survival of submerged rice (Setter and Laureles, 1996). Negative effects of shoot elongation on submergence tolerance have been found in other plant species (Jackson and Ram, 2003; Setter and Laureles, 1996). Silver Dollar can be classified as a quiescence type because of its slow growth. It maintained relatively higher WSC under SM, indicating that these plants could minimize growth by conserving energy under SM conditions. In addition, chlorophyll stability is known to be associated with stress tolerance, and a decrease in chlorophyll concentration may be the result of increased chlorophyllase activity under submergence (Ella and Ismail, 2006). Although the Chl of Silver Dollar was affected by WL and SM similarly to other genotypes, this accession was still able to maintain higher shoot and root WSC. It seems that variation of WSC, Chl and HT among genotypes provides an important basis for studying metabolic responses of the plants to flooding stress. Flooding stress induces oxidative damage at the cellular level and activates lipid peroxidation, thus damaging membrane integrity (Blokhina et al., 2003). Lipid peroxidation is an indicator of the prevalence of free radical reaction in tissues (Halliwell and Gutteridge, 1989), and MDA is the end product of lipid peroxidation. It has been found that MDA concentration increased in plants under waterlogging (Candan and Tarhan, 2012; Simova-Stoilova et al., 2012; Tang et al., 2010) and submergence (Damanik et al., 2010; Tan et al., 2010) and also either remained unchanged or increased and then deceased under soil flooding, depending on genotypes and duration of stress (Damanik et al., 2010; Celik and Turhan, 2011; Wang and Jiang, 2007). Our results demonstrated that Silver Dollar, with the smallest height, showed the least amount of increase in shoot and root MDA concentrations under both stresses, especially compared to PI231569, with the highest plant height. The findings suggested that slow-growing plants such as Silver Dollar might suffer to a lesser degree from the oxidative damage caused by WL or SM. The changes of MDA may be also related to alterations of antioxidant enzymes for both shoot and roots under WL or SM. Flooding alters antioxidant metabolism in the plants. The increased, decreased and unchanged SOD activities were found in different plant species subjected to waterlogging stress (Ahmed et al., 2002; Arbona et al., 2008; Balakhnina et al., 2010; Lin et al., 2004; Tan et al., 2010; Wang and Jiang, 2007). For example, root SOD activities of creeping bentgrass increased in both tolerant and sensitive cultivars, but to a greater extent in the tolerant cultivar under waterlogging (Wang and Jiang, 2007). Zong et al. (2015) reported that waterlogging tolerant knotgrass had higher root SOD activities but the intolerant centipedegrass had lower root SOD activities when grasses were exposed to a longer period of waterlogging stress (30 d). Our results supported a role of SOD in flooding tolerance in other perennial grass species. Lesser reductions in root SOD activities in Silver Dollar and PI418714 and more reductions in Kangaroo Valley and PI231569 under WL or SM stress suggested that maintenance of root SOD activities contributed to waterlogging or submergence tolerance, especially for slow-growing perennial ryegrass genotypes.

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Antioxidant enzymes such as CAT, POD and APX decompose H2O2 at different subcellular locations, which could contribute to flooding tolerance of the plants. Differential responses of activities of antioxidant enzymes have been found in plant species exposed to waterlogging or submergence (Arbona et al., 2008; Damanik et al., 2010; Lin et al., 2004; Tan et al., 2010; Wang and Jiang, 2007; Zong et al., 2015). In maize (Zea mays), CAT was the most important H2O2 scavenging enzyme in leaves, while APX seemed to play a key role in roots exposed to waterlogging (Tang et al., 2010). Tolerance of rice plants to submergence stress might be proven by increased the capacity of antioxidative system (Damanik et al., 2010). However, root POD activities of creeping bentgrass did not differ between the control and waterlogged plants in both tolerant and intolerant genotypes (Wang and Jiang, 2007). Zong et al. (2015) reported that increased root POD activities were associated with waterlogging tolerance of warm-season turfgrass, especially under an extended period of stress. Under SM, larger increased shoot activities of CAT, POD and APX in Silver Dollar and PI41874, lesser reductions in root activities of CAT, POD and APX in Silver Dollar, as well as more dramatic declines in root activities of CAT, POD and APX shown in PI231569, suggested a positive relationship between CAT, POD and APX activities and submergence tolerance of perennial ryegrasses varying in growth habits. 5. Conclusions Waterlogging did not affect HT, Car and shoot WSC, but SM caused more reductions in HT, Chl, Car, and WSC. Substantial declines in WSC under SM and increases in MDA under WL or SM were shown in all genotypes, but to a lesser degree in Silver Dollar and more severe in PI231569. Shoot activities of CAT and POD increased under SM, more pronounced in Silver Dollar and PI418714. Root activities of SOD, CAT, POD, and APX decreased under WL or SM, especially for Kangaroo Valley and PI231569. The results indicated that maintenance of antioxidant activity and carbohydrate and minimization of lipid peroxidation could contribute to WL or SM tolerance of perennial ryegrasses, especially for the slow-growing genotypes. Author contributions M. Liu and Y. Jiang conceived and designed the experiment. M. Liu conducted the experiment. M. Liu and Y. Jiang analyzed data and wrote the manuscript. The authors read and approved the paper. Acknowledgments The authors would like to thank Dr. Zijian Sun for assisting in sample collection and Yu Cui for assisting in growing grasses. This research was supported by the Midwest Regional Turfgrass Foundation at Purdue University. References Ahmed, S., Nawata, E., Hosokawa, M., Domae, Y., Sakuratani, T., 2002. Alterations in photosynthesis and some antioxidant enzymatic activities of mungbean subjected to waterlogging. Plant Sci. 163, 117e123.  pez-Climent, M.F., Pe rez-Clemente, R.M., Go  mezArbona, V., Hossain, Z., Lo Cadenas, A., 2008. Antioxidant enzymatic activity is linked to water-logging stress tolerance in citrus. Physiol. Plant 132, 452e466. Bailey-Serres, J., Voesenek, L.A.C.J., 2008. Flooding stress: acclimations and genetic diversity. Annu. Rev. Plant Biol. 59, 313e319. Balakhnina, T.I., Bennicelli, R.P., Ste˛ pniewska, Z., Ste˛ pniewski, W., Fomina, I.R., 2010. Oxidative damage and antioxidant defense system in leaves of Vicia faba major L. cv. Bartom during soil flooding and subsequent drainage. Plant Soil 327, 293e301. Banach, K., Banach, A.M., Lamers, L.P.M., De Kroon, H., Bennicelli, R.P., Smits, A.J.M., Visser, E.W., 2009. Differences in flooding tolerance between species from two

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