Response of Piper Species to water stress K. S. Krishnamurthy1, S. J. Ankegowda2 and K. V. Saji1 Abstract




Дата канвертавання25.04.2016
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Response of Piper Species to water stress
K.S. Krishnamurthy1, S.J. Ankegowda2 and K.V. Saji1
Abstract:Experiments conducted at Indian Institute of Spices Research, Calicut to assess the relative tolerance of different Piper species viz. P. nigrum, P. colubrinum, P. chaba, P. longum and P. hymenophylla to water stress revealed that P. colubrinum is very susceptible while P. longum and P. chaba were on par with Piper nigrum accessions. The various parameters studied included relative water content, cell membrane leakage, catalase, peroxidase, super oxide dismutase (SOD) and acid phosphatase activities. Susceptible varieties showed very high activities of peroxidase during stress while catalase activity was generally higher in tolerant varieties. Acid phosphatase activity decreased during stress.
Black pepper (Piper nigrum L.), the king of spices is grown for its spicy berries. It belongs to the family piperaceae. It is mostly grown in Western Ghats of India. Pepper is a shade loving plant and needs a support to cling on. The black pepper has a great export potential and the contributory factor is the presence of chemical piperine in it. In India, black pepper is generally grown in home stead gardens as a rainfed crop. Though the average rainfall in pepper growing areas is well above 2000 mm, the distribution is not uniform. The rainfall received during December to May is very negligible. Hence, the crop suffers due to severe soil moisture shortage during these months. Some of the other species in Piper genus are P. colubrinum, P. chaba, P. longum and P. hymenophylla. Pepper suffers due to soil moisture shortage during summer months. Evaluating these Piper species to water stress gives some knowledge on the stress coping ability of these species and if found tolerant, can be used for inducing tolerance in Piper nigrum either through conventional or through biotechnological approach. Though other Piper species are not grown for piperine purpose, Piper chaba and Piper longum have great medicinal uses.
Many physiological and biochemical parameters are used to evaluate black pepper for drought tolerance. These include relative water content, cell membrane leakage, catalase, peroxidase and super oxide dismutase activities, etc (Krishnamurthy et al., 2000). Activities of all these enzymes have been shown to increase during water stress in coconut and the tolerant varieties maintained higher activities than the susceptible ones (Chempakam et al., 1993). The extent of oxidative stress in a cell is determined by the amounts of superoxide, H2O2 and hydroxyl radicals. Therefore, the balance of SOD, ascorbate peroxidase and catalase activities will be crucial for suppressing toxic reactive oxygen species levels in a cell (Apel and Hirt, 2004).
It has been shown that membrane leakage and relative water content can be used to screen black pepper germplasm for drought tolerance (Krishnamurthy et al., 1998). The objective of the present experiment was to investigate the biochemical and physiological changes in Piper species when subjected to water stress and probability of utilizing some of these parameters to distinguish between susceptible and tolerant types. Tolerance trait if found in any one

these species can be transferred to high yielding P. nigrum types either through conventional or through molecular approaches.


Materials and methods
Piper species viz, P. chaba, P. colubrinum, P. hymenophylla , P. longum and Piper nigrum varieties Panniyur-1, Sreekara, Acc 1618 and 1567 were selected for the study. The rooted cuttings (8 nos. each) of these selected species and varieties were planted in pots of 12 inch diameter and allowed to establish for six months. The potting mixture contained forest soil, sand and farm yard manure in 3:1:1 proportion. Moisture stress was imposed by withholding irrigation. The stress treatment continued till the plants started showing wilting symptoms. Before induction of stress, plants were irrigated to field capacity daily for 5-6 days. Observations on relative water content (RWC), cell membrane leakage, catalase, peroxidase, super oxide dismutase and acid phosphatase activities were recorded under control condition (irrigated) and 5 and 10 days after stress induction. RWC and cell membrane leakage were calculated as per the standard procedures.
Preparation of enzyme extracts:
All extractions were done under ice cold conditions using a pre chilled pestle and mortar. The procedure used by Dhindsa et al., 1981 was followed with minor modifications for assaying catalase, peroxidase and SOD enzyme activities. Youngest fully matured leaves were collected and both the surfaces were cleaned thoroughly by rubbing with cotton to remove the particles adhering to the leaf surface. 0.5 g of the leaf material was ground in 5 ml of 0.1 M phosphate buffer (pH 7.2) containing 5 % PVP, 0.2 M ascorbic acid and 0.1 % sodium metabisulphite. The homogenate was filtered through 4 layers of cheese cloth and centrifuged at 10000 rpm for 20 minutes. The supernatant was used to assay catalase, peroxidase and SOD activities. For acid phosphatase activity, the samples were extracted as per Sadasivam and Manickam, 1992 and 0.5 ml of the supernatant was used for assay.
Enzyme assays
Catalase was assayed by measuring the initial rate of disappearance of hydrogen peroxide according to Dhindsa et al., 1981 with little modifications. Peroxidase activity was assayed as per Putter, 1974. SOD activity was assayed by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazolium as per Dhindsa et al., 1981 with little modification. Acid phosphatase was assayed as per Sadasivam and Manickam, 1992. Optical density of 0.1 was considered as one activity unit.
Results and discussion
In all the Piper species, with stress intensity, relative water content decreased while cell membrane leakage increased. Cell membrane leakage was negatively correlated with relative water content.

Figure 1 shows the relative water content (RWC) of Piper species in relation to water stress. Relative water content decreased in all Piper species with increasing stress intensity. Relative water content among the species was on par under irrigated condition. After 5 days of stress, Piper colubrinum showed lowest RWC (74.4 %). P. chaba maintained highest RWC (83.8 %) which was on par with P. longum, Acc 1567 and Sreekara. After 10 days of stress induction, the trend was similar. Piper colubrinum showed the lowest value (47.4 %). Highest value was noticed in Sreekara (66.5 %) which was on par with P. chaba, P. longum and Acc 1567.


Fig. 1: Relative water content of Piper Species as infuenced by water stress



1 = P. longum

2 = P. chaba

3= P. colubrinum

4 = P. hymenophylla

5= Sreekara

6 = Acc 1567

7 = Acc 1618

8 = Panniyur -1

Membrane leakage increased with stress intensity (Table 1). The leakage among the species was on par under control. But there was significant difference among the species for membrane leakage during stress period. Membrane leakage percentage ranged from 4.9 (Sreekara) to 6.0 (P. hymenophylla) under control condition while it ranged from 9.1 to 13.3 after 5 days and from 12.1 (P. longum) to 19.8 % (P. colubrinum) after 10 days of stress induction. Highest leakage was recorded in P. colubrinum followed by P. hymenophylla. Rest were on par.


Table 1: Relative water content and cell membrane leakage in different Piper species as affected by water stress


Piper Sp.

Relative water content (%)

Membrane leakage (%)

Control

5 DASI

10 DASI

Control

5 DASI

10 DASI

P. longum

91.2

83.7

66.4

5.35

9.60

12.10

P. colubrinum

90.3

76.5

47.4

5.70

13.30

19.80

P. chaba

92.1

83.8

65.9

5.05

9.10

12.70

P. hymenophylla

90.7

80.6

53.6

6.00

11.30

14.40

Sreekara

91.9

80.9

64.5

4.90

10.50

13.50

Acc 1567

92.7

83.0

65.1

5.00

9.30

13.00

Acc 1618

92.3

81.2

61.4

5.30

10.00

13.60

Panniyur-1

93.1

81.7

62.9

5.60

9.40

14.10

CD (5 %)

NS

1.5

2.6

NS

1.25

1.65

Protein content decreased due to water stress in all the species (Table 2). In general, P. nigrum (Acc 1567, 1618, Sreekara and Panniyur-1) showed higher protein content than the rest both under control and stress conditions. Piper colubrinum showed the least protein content. Protein content ranged from 4.96 to 2.83 mg g-1 fresh weight. Highest protein content was observed in Panniyur -1 (4.96 m mg g-1 fresh weight) which was on par with other P. nigrum accessions (Acc 1567, 1618 and Sreekara) followed by P. chaba, P. longum and P. hymenophylla and P. colubrinum which were on par. Even after 5 and 10 days of stress, P. nigrum accessions showed higher protein content than the rest of Piper sp. After 5 days of stress, values ranged from 4.37 (Panniyur -1) to 2.2 (P. colubrinum) and after 10 days, from 3.41 (Panniyur -1) to 1.79 (P. colubrinum) mg g-1 fresh weight.


Peroxidase activity increased during water stress in all the species. Piper colubrinum maintained very high activity both under control and stress conditions. Activity ranged from 4.74 to 8.11 under control, 8.5 to 38.8 and from 6.4 to 20.9 activity units (a.u) mg protein-1 after 5 and 10 days of stress respectively. After 10 days of stress, peroxidase activity reached back to almost control activity levels in all the species. All other Piper species showed similar values as that of Piper nigrum. Among Piper nigrum varieties, Acc 1567 showed highest activity both under control as well as 10 days after stress ( 6.48 & 7.60 a.u. mg protein-1) while after 5 days of stress, Acc 1618 showed highest activity (15.4 a.u. mg protein-1). The lowest activity was noticed in Piper chaba (4.72 a.u.). followed by Sreekara (4.74 a.u.) during control while after 10 days of stress, the lowest activity was again noticed in Piper chaba and Panniyur –1 (6.4 a.u.) which was on par with the rest except P. colubrinum which showed highest activity (20.9 a.u.). Under control conditions, P. colubrinum showed highest activity followed by Acc 1567 and the rest were on par. After 5 days of stress, P. colubrinum maintained the highest activity followed by Acc 1618 and 1567 and the lowest in Panniyur -1.

Table 2: Protein content and peroxidase activity in Piper species as affected by water stress




Piper Sp.

Protein content (mg g f.w-1)

Peroxidase activity (a.u. mg protein-1)

Conrol

5 DASI

10 DASI

Control

5 DASI

10 DASI

P. longum

3.15

2.59

2.08

5.72

12.80

7.10

P. colubrinum

2.83

2.20

1.79

8.11

38.80

20.90

P. chaba

3.36

2.69

2.13

4.72

10.40

6.40

P. hymenophylla

2.92

2.23

1.88

5.09

8.50

7.00

Sreekara

4.29

3.63

3.15

4.74

11.70

6.45

Acc 1567

4.59

4.03

3.38

6.48

13.75

7.60

Acc 1618

4.87

4.11

3.23

5.62

15.40

6.80

Panniyur-1

4.96

4.37

3.41

5.06

10.40

6.40

CD (5 %)

0.7

0.9

0.8

1.1

1.4

1.3

Catalase activity decreased during water stress in all the species (Table 3). Activity was highest in Piper colubrinum under control (3.54 a.u. mg protein-1) but in early as well as severe stress, P. longum showed highest activity (1.32 in early and 1.05 a.u. mg protein-1) in severe stress). There was no significant difference in catalase activity among Piper colubrinum, P. chaba and P. hymenophylla but activity of P. nigrum accessions differed significantly from the rest both under control and stress condition. Piper nigrum accessions showed lowest activity and the activity was on par among the accessions.


Super oxide dismutase activity did not change much during control as well as early stress in all the Piper species (Table 3) but under severe stress, it showed a decrease in all the species. Piper nigrum accessions showed significantly higher SOD activity in general compared to other Piper species both under control as well as under stress conditions. Among other Piper sp, the activity of SOD was high in Piper colubrinum during control which was on par with P. nigrum accessions but activity reduced at a greater rate than P. nigrum accessions during stress. Acc 1618 showed highest activity both under control and stress conditions while the lowest activity was recorded in P. hymenophylla.
Table 3: Catalase and SOD activity as affected by water stress in different Piper species


Piper Sp.

Catalase activity (a.u. mg protein-1)

SOD activity (a.u. mg protein-1)

Conrol

5 DASI

10 DASI

Control

5 DASI

10 DASI

P. longum

3.30

1.32

1.05

2.72

2.80

2.35

P. colubrinum

3.54

1.10

0.89

3.27

2.68

2.15

P. chaba

2.89

0.93

0.94

2.61

2.96

2.41

P. hymenophylla

2.28

0.75

0.59

2.36

2.16

1.95

Sreekara

1.28

0.33

0.25

3.38

3.46

3.05

Acc 1567

1.70

0.28

0.19

3.09

2.98

2.68

Acc 1618

1.41

0.29

0.21

3.40

3.52

3.05

Panniyur-1

1.31

0.28

0.22

3.32

3.17

2.98

CD (5 %)

0.80

0.42

0.30

0.64

0.58

0.91

Acid phosphatase activity decreased with stress intensity in all the species (Fig 2). Sreekara maintained highest activity both under control and stress condition followed by Piper colubrinum which were on par but the rate of decrease was more in Piper colubrinum than Sreekara. The activity ranged from 1.7 to 4.1 during control, from 1.4 to 3.5 after 5 days of stress and from 1.1 to 2.4 a.u mg protein-1 after 10 days of stress induction. Piper longum and Piper chaba showed the lowest activity both during control and stress condition. Compared to Piper colubrinum and Sreekara, the rate of reduction in activity during stress was less in other accessions.


Among different Piper species, Piper nigrum is the one which has great export value. Piper longum and Piper chaba have medicinal uses and have been common medicines for various disorders of humans in traditional Indian families. Piper longum and Piper chaba are little hardy and can be cultivated with minimum management practices while Piper nigrum needs lot of care. P. colubrinum is better suited for marshy areas but has a great potential for utilizing it as a root stock for P. nigrum for controlling the deadly Phytophthora foot rot disease as P. colubrinum is found to be resistant to Phytophthora disease.
The objective of the present study was to explore the drought tolerance characteristics of the selected Piper species so that the same may be exploited for crop improvement of black pepper. The results revealed that P. colubrinum is very susceptible while P. longum and P. chaba are no better than P. nigrum for drought prone areas.

1 = P. longum

2 = P. chaba

3= P. colubrinum

4 = P. hymenophylla

5= Sreekara

6 = Acc 1567

7 = Acc 1618

8 = Panniyur -1

Relative water content and membrane leakage values showed that though P. longum and P. chaba maintained higher relative water content and lower membrane leakage values than P. nigrum at the same level of stress, the values are on par. This shows that these species are not better than P. nigrum in terms of drought tolerance. It has been shown in black pepper that relative water content and membrane leakage can be used to screen germplasm for drought tolerance (Krishnamurthy et al., 1998). It has been reported that the more drought tolerant tifdwarf Bermuda grass had high levels of relative water content and low levels of relative electrolyte leakage where as the susceptible carpet grass showed rapid decline of relative water content and rise in electrolyte leakage (Shaoyun et al., 2003). Relative water content is related to the water potential of the same tissue though the relationship is dependent on species and stages of growth. RWC declined during drought progression in tomato (Rahman et al., 1999) and in fescue and the severity of reduction varied with cultivars (Huang and Gao, 1999). Similar results have been reported in black pepper accessions (Krishnamurthy et al., 1998; 2000). Membrane leakage also showed very high correlation with drought tolerance in black pepper (Krishnamurthy et al., 2000), in Bermuda grass (Shaoyun et al., 2003) and the usefulness of this parameter in screening wheat cultivars for drought tolerance has been established by Ebercon, 1981.



Activities of antioxidant enzymes viz. catalase, peroxidase and super oxide dismutase as screening parameters for drought tolerance has been established in many crop plants (Dhindsa et al., 1981; Rahman et al., 1999; Chempakam et al., 1993; Krishnamurthy et al., 2000; Chowdhury and Choudhuri, 1985). Transient rise in activity of superoxide dismutase and prolonged increase in activities of catalase and peroxidase were noticed in drought tolerant tifdwarf grass while the susceptible carpet grass showed continuous increase in SOD activity, transient rise of catalase and a reduction of peroxidase activity leading to H2O2 accumulation and thus the injury (shaoyun et al., 2003). SODs act as first line of defense against reactive oxygen species, dismutating superoxide to H2O2. Ascorbate peroxidase, glutathione peroxidase and catalase subsequently detoxify H2O2.
Among different Piper species, Piper colubrinum showed highest peroxidase activity (five fold increase in activity under stress condition) but among all the species, it wilted very fast. This implies that the increase in peroxidase activity in P. colubrinum under stress condition is not related to tolerance but it may be an indicator of severity of stress in that species.
Contrary to the findings in coconut by Chempakam et al., 1993, in black pepper catalase activity decreased after imposing stress. All the Piper nigrum accessions showed lesser activity than other species both under irrigated as well as stressed conditions. Catalase is indispensable for oxidative stress tolerance because transgenic tobacco plants with suppressed catalase have reactive oxygen species in response to both abiotic and biotic stresses (Very and Sentenac, 2002).
In black pepper, SOD activity was not altered much during initial stages of stress but decreased during severe stress. Interestingly, P.nigrum accessions showed higher SOD activity than other Piper species except P. colubrinum (which was on par with P. nigrum) while the reverse trend was observed for catalase activity. Expression of ascorbate peroxidase isozymes has been noticed in drought tolerant spinach by Yoshimura et al., 2000. In tissue cultures of four wheat varieties, after the mannitol treatment, catalase increased and peroxidase and SOD decreased in the drought tolerant varieties (Kocsy et al., 1991). In tobacco, over expression of chloroplast SOD to chloroplasts did not alter tolerance towards oxidative stress which suggests that other antioxidant mechanisms might be limiting (Allen, 1995). Expression of high rate of glutathione reductase in drought resistant maize strain has been reported by Pastori and Trippi, 1992.
In five tomato varieties with varying drought tolerance, water stress for 7 days increased the acid phosphatase activity and the increase was more in susceptible varieties (Thakur and Thakur, 1993). Acid phosphatase activity decreased with water stress during seed filling in beans (Pasin et al., 1991). In the present investigation also, acid phosphatase activity decreased with stress intensity in black pepper. Piper colubrinum as well as all the P. nigrum accessions had higher activity than the rest.
Piper colubrinum which is resistant to Phytophthora foot rot disease showed high initial activity of all the three antioxidant enzymes and also acid phosphatase activity though under stress condition, the activity levels were significantly altered. Even other Piper species have also higher levels of disease tolerance compared to P. nigrum. Hence, these antioxidant enzymes may play a major role in disease tolerance than water stress tolerance in these species. There are reports highlighting the importance of these antioxidant enzyme activities in relation to disease tolerance (Klessig et al., 2000; Mittler et al., 1999). It is interesting to study the role of these enzymes in inducing disease tolerance in pepper also.
Acknowledgement:
Authors thank the Director, Indian Institute of Spices Research for providing the facilities.
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1 Indian Institute of Spices Research, Marikunnu P.O., Calicut 673 012

2 Cardamom Research Centre, IISR, Heravanadu P.O., Appangala, Madikeri 571 201




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