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ProtoplasmaAn International Journal of Cell Biology ISSN 0033-183XVolume 249Number 1 Protoplasma (2011) 249:75-87DOI 10.1007/s00709-011-0263-8

Response of two mustard (Brassica junceaL.) cultivars differing in photosyntheticcapacity subjected to proline

Arif Shafi Wani, Mohammad Irfan,Shamsul Hayat & Aqil Ahmad

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ORIGINAL ARTICLE

Response of two mustard (Brassica juncea L.) cultivarsdiffering in photosynthetic capacity subjected to proline

Arif Shafi Wani & Mohammad Irfan & Shamsul Hayat &Aqil Ahmad

Received: 28 August 2010 /Accepted: 13 January 2011 /Published online: 12 February 2011# Springer-Verlag 2011

Abstract The present paper deals with the effect ofexogenous application of proline as a shotgun approachon growth, photosynthesis, and antioxidative system in25-day-old plants of two different cultivars of Brassicajuncea L. (Varuna and RH-30) under natural conditions.Exogenous application of proline significantly increasedplant growth, photosynthetic rate, and the activities ofantioxidant enzymes, compared with untreated seedlings.Pre-sowing seed soaking in 20 mM proline, for 8 h, provedbest among all the other concentrations used.

Keywords Antioxidant response . Brassica .

Photosynthesis . Proline . Shotgun screening

Introduction

Plants during their lifecycle experience various environ-mental stresses such as drought, salinity, temperatureextremes, toxic metal ion concentration, and UV radiations.All these stressors result in the restriction of plant growthand productivity to varying degrees depending upon theextent of the stress (Kaul et al. 2008). In order to cope withthese environmental stresses, plants have evolved certainadaptive mechanisms. One such mechanism includes theosmotic adjustment of cells by accumulating large amountsof low molecular mass compounds called compatiblesolutes particularly proline (Rains 1989; Ashraf 1994; Ali

et al. 1999; Rhodes et al. 1999; Ozturk and Demir 2002;Hsu et al. 2003; Kavi Kishore et al. 2005).

Proline, an essential amino acid, is ubiquitously distrib-uted in all plants and has multiple metabolic roles(Szabados and Savoure 2009). Besides acting as anosmolyte for osmotic adjustment (Ahmed et al. 2010a), itstabilizes sub-cellular structures (membranes and protiens),scavenging free radicals and buffering redox state understress conditions (Ashraf and Fooland 2007). It may alsofunction as protein compatible hydrotrope (Srinivas andBalasubramanian 1995), alleviating cytoplasmic acidosisand maintaining appropriate NADP+/NADPH ratios com-patible with metabolism (Hare and Cress 1997). It alsoserves as organic nitrogen reserve that can be utilizedduring recovery (Hare et al. 1998). On being relieved fromthe stress, it is metabolized providing sufficient reducingagents that support mitochondrial oxidative phosphoryla-tion and generation of ATP for recovery from stress andrepairing of stress induced changes (Hare and Cress 1997;Hare et al. 1998).

The precursor of proline biosynthesis is L-glutamic acid.Two important enzymes, pyrroline-5-carboxylate synthetase(P5CS) and pyrroline-5-carboxylate reductase, play a vitalrole in proline biosynthesis (Delauney and Verma 1993).Transgenic tobacco plants are known to over express P5CSto increase proline concentration and resistance to bothdrought and salinity stresses (Ashraf and Fooland 2007).

Exogenous application of proline plays an important rolein enhancing plant stress tolerance (Claussen 2005; Aliet al. 2007; Ashraf and Fooland 2007) as it protects proteinstructure and membranes from damage and reduces enzymedenaturation (Iyer and Chaplan 1998; Rajendra Kumaret al. 1994; Saradhi et al. 1995; Smirnoff and Cumbes1989). It may also act as a regulatory or signaling moleculeto activate a variety of responses (Maggio et al. 2002).

Handling Editor: Bhumi Nath Tripathi

A. S. Wani :M. Irfan : S. Hayat (*) :A. AhmadPlant Physiology Section, Department of Botany,Aligarh Muslim University,Aligarh 202002 Uttar Pradesh, Indiae-mail: shayat@lycos.com

Protoplasma (2012) 249:75–87DOI 10.1007/s00709-011-0263-8

Author's personal copy

Tab

le1

Effectof

prolineon

root

freshweigh

t(m

g)of

high

andlow

photosyn

thesizingcultivars

ofBrassicajuncea

(Varun

a&

RH-30)

Soaking

duratio

nof

proline(m

M)

4h

8h

12h

010

2030

4050

010

2030

4050

010

2030

4050

V1

314.15

±19

.93

474.36

±31

.24

499.12

±37

.25

484.10

±31

.51

408.39

±31

.05

380.12

±25

.78

319.46

±26

.23

501.55

±37

.88

549.05

±38

.06

517.52

±31

.79

442.62

±31

.02

408.90

±26

.46

321.21

±19

.89

401.08

±26

.36

517.14

±37

.89

504.29

±35

.01

398.30

±27

.10

366.17

±22

.74

V2

301.41

±20

.15

434.03

±31

.74

458.14

±32

.35

446.08

±31

.93

376.76

±25

.77

358.67

±26

.57

305.75

±20

.66

455.56

±31

.74

492.25

±38

.38

473.51

±32

.07

394.41

±25

.90

373.01

±26

.41

314.92

±20

.38

481.82

±32

.43

494.42

±35

.39

472.38

±31

.52

377.90

±26

.24

359.00

±22

.65

LSD

at5%

Variety

(V)

=6.62

11.93

0.13

Con

centratio

n(C)

=11.47

20.66

13.57

V×C

=2.37

1.22

19.19

Tab

le2

Effectof

prolineon

root

dryweigh

t(m

g)of

high

andlow

photosyn

thesizingcultivars

ofBrassicajuncea

(Varun

a&

RH-30)

Soaking

duratio

nof

proline(m

M)

4h

8h

12h

010

2030

4050

010

2030

4050

010

2030

4050

V1

35.00

±2.34

51.80

±2.92

54.95

±2.91

52.85

±3.49

44.45

±2.34

42.70

±2.66

35.64

±2.67

57.02

±3.23

59.91

±3.96

57.73

±3.77

47.04

±2.99

45.26

±2.74

35.93

±2.21

58.56

±3.47

57.88

±3.98

52.86

±3.06

46.38

±2.86

41.31

±2.63

V2

30.01

±1.85

42.01

±2.81

45.61

±2.77

43.51

±2.91

36.91

±2.76

35.71

±2.91

30.54

±1.81

45.06

±2.90

49.47

±2.87

46.81

±2.85

39.09

±2.53

37.56

±2.60

30.91

±1.97

47.91

±2.97

47.36

±3.44

44.77

±3.37

38.63

±2.35

35.23

±2.33

LSD

at5%

Variety

(V)

=1.03

1.57

1.10

Con

centratio

n(C)

=1.79

2.72

1.90

V×C

=2.54

2.08

2.69

76 A.S. Wani et al.

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Exogenous application of proline enhances gas exchangeattributes like net CO2 assimilation rate, transpiration rate,and stomatal conductance (Ali et al. 2007). It upregulatesthe level of stress protective proteins (Khedr et al. 2003)and reduces oxidation of lipid membranes (Okuma et al.2004). However, its effects/actions are concentration-de-pendent (Ashraf and Fooland 2007). Proline can beexogenously applied to plants in three different ways suchas through root application, foliar spray, and pre-sowingseed treatment (i.e., shotgun approach) (Ashraf et al. 2008).There is enough literature available about the action ofproline applied through foliar spray (Claussen 2005; Ali etal. 2007; Ashraf and Fooland 2007), but there is littleinformation available on the effect of exogenous prolineapplication as shotgun approach (i.e., seed soaking) inplants under normal growth conditions. Apart from bene-ficial effects of proline, its higher concentration could haveharmful or deleterious effects on plant growth andmetabolism (Ehsanpour and Fatahian 2003; Nanjo et al.2003). It is, therefore, necessary to determine the optimalconcentration of proline that has beneficial effects indifferent plant species.

Among the human consumables, oil crops share theimportant part. Indian mustard is one of the important oilcrops of India. Efforts have been made time to time toutilize the plants’ own natural products to screen the bettercultivars and to enhance the crop production utilizingvarious methodologies and approaches under “treatment–response strategy.” Some of these plant natural productshave also been recognized as plant hormones. Keeping thisin view, the present experiment was designed to investigatethe effects of sole treatment of multifunctional metaboliteproline in young mustard seedlings as shotgun approach.Two cultivars of Indian mustard Varuna and RH-30 havebeen analyzed considering growth, photosynthetic activity,and antioxidant enzymes as the marker parameters. Sec-ondly, the objective of the experiment was also to explorewhich concentration of proline proves more effective inenhancing these parameters. The promising cultivar linescan be exploited further for use in selection and breedingprograms to make further advancement in stress tolerance.

Materials and methods

Plant material and treatments

Healthy seeds of Brassica juncea cultivars Varuna (highphotosynthesizing) and RH-30 (low photosynthesizing)(Mobin and Khan 2007) were obtained from Chola BeejBandhar, Aligarh. The seeds were surface sterilized with0.01% mercuric chloride solution and washed two to threetimes with double distilled water (DDW). The sterilizedT

able

3Effectof

prolineon

root

leng

th(m

g)of

high

andlow

photosyn

thesizingcultivars

ofBrassicajuncea

(Varun

a&

RH-30)

Soaking

duratio

nof

proline(mM)

4h

8h

12h

010

2030

4050

010

2030

4050

010

2030

4050

V1

15.30

±1.07

23.71

±1.56

26.01

±1.67

24.93

±1.90

22.64

±1.62

21.87

±1.58

15.54

±1.06

25.01

±1.77

27.97

±1.75

26.10

±1.59

25.17

±1.73

22.99

±1.38

15.89

±1.07

27.80

±1.74

27.18

±1.88

24.96

±1.99

22.56

±1.55

19.06

±1.37

V2

14.40

±1.09

21.31

±1.53

23.04

±1.56

22.03

±1.69

19.72

±1.59

17.85

±1.50

15.05

±1.18

23.02

±1.77

26.08

±1.61

24.08

±1.56

21.67

±1.35

19.56

±1.20

15.27

±1.06

22.90

±1.65

24.89

±1.75

22.59

±1.55

19.01

±1.20

17.40

±1.07

LSD

at5%

Variety

(V)

=1.06

0.61

0.91

Con

centratio

n(C)

=1.84

1.06

1.58

V×C

=0.72

1.50

2.10

Response of two mustard cultivars 77

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seeds of both cultivars were soaked in graded concentrations ofproline dissolved in DDW (0, 10, 20, 30, 40, and 50mM) for 4,8, and 12 h. Such a method is known as shotgun approach(Ashraf et al. 2008). The soaked seeds were sown in plasticpots of 6 in. diameter. These pots were placed in net houseunder natural conditions. Twenty-five-day-old plants weresampled with intact roots to assess the following parameters.

Root fresh weight, dry weight, and length per plant

Plants were removed along with soil and dipped in water todislodge the adhering soil particles without injuring theroots. The roots were cut from plants and blotted. Theblotted roots were weighed to record their fresh weight andplaced in an oven (80°C for 72 h). The samples wereweighed again to record their dry weight. The weight wasexpressed in milligrams. The length of the roots wasmeasured by metric scale and expressed in centimeters.

SPAD chlorophyll

SPAD chlorophyll meter (SPAD-502, Konica MinoltaSensing, Inc., Japan) was used to assess SPAD value ofchlorophyll in fresh leaves.

Photosynthetic attributes

The photosynthetic attributes (net photosynthetic rate, stoma-tal conductance, transpiration rate, water use efficiency, andinternal carbon dioxide concentration) were measured by infrared gas analyzer (IRGA, LICOR 6400, Lincoln NE, USA) inwell expanded upper third leaf between 1100 and 1200hours under bright sunlight. The atmospheric conditionsduring measurement were photosynthetically active radia-

tion, 1,016±6 μmol m−2 s−1, relative humidity 60±3%,atmospheric temperature 22±1°C, and atmospheric CO2,360 μmol mol−1. The ratio of atmospheric CO2 tointercellular CO2 concentration was constant.

Extraction and estimation of antioxidative enzymes

Fresh leaf tissue (0.5 g) was homogenized in 5 ml 50 mMphosphate buffer (pH 7.0) containing 1% PVP. Thehomogenate was centrifuged at 15,000 rpm for 10 min,and the supernatant was used as enzyme source. Theextraction process was carried out at 4°C.

Peroxidase and catalase (CAT) were assayed by adaptingthe procedure given by Chance and Maehly (1955). CATactivity was assessed by titrating the reaction mixtureconsisting of phosphate buffer (pH 6.8), 0.1 M H2O2,enzyme extract, and 2% H2SO4 against 0.1 N KMnO4.

Reaction mixture for peroxidase (POX) consisted ofpyragallol phosphate buffer (pH 6.8), 1% H2O2, andenzyme extract. Change in absorbance, due to catalyticconversion of pyragallol to perpurogallin, was noted at aninterval of 20 s for 2 min at 420 nm. A control set wasprepared by using DDW instead of enzyme extract.

The activity of superoxide dismutase (SOD)was assayed bymeasuring its ability to inhibit the photochemical reduction ofNBT using the method of Beauchamp and Fridovich (1971).Reaction mixture containing 50 mM phosphate buffer(pH 7.8), 13 mM methionine, 75 μM NBT, 2 μM riboflavin,0.1 mM EDTA, and 0–50 μl enzyme extract was placedunder 15 W fluorescent lamp. The reaction was initiated byswitching on the light and was allowed to run for 10 min. Thereaction was stopped by switching off the light. The amountof enzyme which causes 50% inhibition in photochemicalreduction of NBT was considered as one enzyme unit.

Source ofvariation

Degree offreedom

Root fresh weight Root dry weight

4 h 8 h 12 h 4 h 8 h 12 h

Cultivar 1 8570.24 14915.76 15.60 563.26 730.89 578.64

Treatment 5 26507.73 36123.01 32582.18 268.60 403.86 391.00

Interaction 5 198.08 331.38 2565.01 5.34 9.75 7.80

Error 22 83.26 269.88 116.44 2.04 4.68 2.30

Table 4 Mean squares fromANOVA of data for fresh anddry weight of root of high andlow photosynthesizing cultivarsof Brassica juncea(Varuna and RH-30)

Source ofvariation

Degree offreedom

Root length SPAD chlorophyll

4 h 8 h 12 h 4 h 8 h 12 h

Cultivar 1 64.85 44.44 59.13 654.58 600.73 452.41

Treatment 5 72.00 100.18 104.05 257.95 411.07 448.99

Interaction 5 1.56 1.89 3.34 5.50 7.63 6.90

Error 22 2.14 0.71 1.58 0.0045 0.0014 0.0067

Table 5 Mean squares fromANOVA of data for root lengthand SPAD Chlorophyll of highand low photosynthesizingcultivars of Brassica juncea(Varuna and RH-30)

78 A.S. Wani et al.

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Determination of proline content

The proline content of fresh leaves and root samples wasdetermined by adapting the method described by Bates etal. 1973. Samples were extracted in sulphosalicylic acid. Tothe extract, equal volumes of glacial acetic acid andninhydrin solution were added. The sample was incubatedat 100°C for 1 h after which the reaction was terminated inthe ice bath followed by the addition of 5 ml toluene. Theabsorbance of aspirated toluene layer was read at 528 nmon spectrophotometer.

Data analysis

The experiment was conducted according to simple randomblock design. Data were statistically analyzed usinganalysis of variance (two-way ANOVA) by SPSS (ver. 10;

SPSS Inc., Chicago, IL, USA). The least significantdifference was calculated for the significant data at P<0.05.

Results

Root fresh weight, dry weight, and length per plant

Plants raised from the seeds given pre-sowing seedtreatment (shotgun approach) in graded concentration ofproline for different soaking durations showed significantincrease in root fresh weight, dry weight, and length perplant. Increase was more pronounced in Varuna than in RH-30 at 20 mM proline concentration (8 h). Proline (20 mM)increased root fresh weight, dry weight, and length perplant by 70%, 68.09%, and 79.98% in Varuna and 61%,62%, and 73.20% in RH-30, respectively, over their

Plate 1 The treatment with dif-ferent concentrations of prolineas shotgun approach increasedthe root proliferation (rootlength and fibration)

Response of two mustard cultivars 79

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controls. The concentrations, 10 and 20 mM linearlyincreased these parameters. However, 30, 40, and 50 mMgenerated values that were near the control (Tables 1, 2, 3,4, and 5; Plate 1).

SPAD chlorophyll

The SPAD value of chlorophyll linearly increased withproline concentration in all soaking durations. Varunaseeds soaked for 8 h in 20 mM had the plants whosechlorophyll content increased by 81%, whereas in RH-30, the increase was 72% with respect to control(Fig. 1a; Table 5).

Photosynthetic attributes

Significant increase in photosynthetic attributes occurred byproline application. Increase in net photosynthetic rate (Pn),stomatal conductance (gs), internal carbon dioxide concen-tration (Ci), transpiration rate (E), and water use efficiency(WUE) occurred in both varieties at 20 mM proline for 8 h.It was more pronounced in Varuna than in RH-30. Pn

increased by 72.80% and 65% (Fig. 1b); gs by 70% and61% (Fig. 1c); Ci by 74% and 66% (Fig. 1d), transpirationrate by 77.91% and 74% (Fig. 2a), and WUE by 59% and53% (Fig. 2b) in both varieties, over their respectivecontrols (Tables 6, 7, and 8).

Fig. 1 Effect of proline onSPAD Chlorophyll (a), net pho-tosynthetic rate (b), stomatalconductance (c), and internalCO2 concentration (d) of highand low photosynthesizing cul-tivars of B. juncea (Varuna &RH-30)

80 A.S. Wani et al.

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Antioxidant enzymes

Enzyme activity increased in response to the proline in bothvarieties. Much increase occurred at 20 mM supplied for8 h. Varuna showed higher enzyme activity than RH-30 in

response to the treatment. SOD activity increased by 51%in Varuna and 49% in RH-30 over their respective controls(Fig. 2c). POX and CAT activity increased by 55% and66.9% in Varuna and 47% and 58% in RH-30, respectively,over their controls (Figs. 2d, 3a; Tables 8 and 9).

Fig. 2 Effect of proline ontranspiration rate (a), water useefficiency (b), superoxide dis-mutase (c), and peroxidase (d)activities of high and low pho-tosynthesizing cultivars of B.juncea (Varuna & RH-30)

Source of variation Degree of freedom Pn gs

4 h 8 h 12 h 4 h 8 h 12 h

Cultivar 1 59.93 64.72 56.40 0.009 0.010 0.010

Treatment 5 8.36 13.46 14.83 0.004 0.008 0.009

Interaction 5 0.51 0.52 0.54 0.0002 0.0001 0.0002

Error 22 0.002 0.006 0.0005 0.000 0.000 0.0000

Table 6 Mean squares fromANOVA of data for net photo-synthetic rate (Pn) and stomatalconductance (gs) of high andlow photosynthesizing cultivarsof Brassica juncea(Varuna and RH-30)

Response of two mustard cultivars 81

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Proline content

The leaves of Varuna possessed higher quantity of prolineas compared to RH-30. However, the pattern of response totreatment in both varieties was similar. At 20 mM prolinefor 8 h, leaves possessed high proline content but as theconcentration increased above 20 mM, the proline contentdecreased with respect to controls in both varieties. Theproline content increased by 67.5% and 60%, respectively,in Varuna and RH-30 at 20 mM proline for 8 h over theirrespective controls (Fig. 3b; Table 10).

The roots possessed lower proline content than theleaves in both varieties. The roots showed the similarresponse as that of the leaves. The root proline contentincreased by 40% and 35% in both varieties at 20 mMproline concentration for 8 h soaking duration with respectto their controls (Fig. 3c; Table 10).

Discussion

It is now an established fact that proline accumulationprovides energy for growth of plant and stress toler-ance. Of different concentrations used for pre-sowingtreatment of B. juncea seeds, 20 mM proline concentra-tion for 8 h duration was found to be most effective inpromoting growth and activities of antioxidant enzymesin mustard plants. Growth response was more prominentin Varuna than in RH-30 to exogenous proline application(Tables 1, 2, 3, 4, and 5). These findings confirm theargument of Garg (2003) that genotypes of the samespecies may vary in their responses to exogenous proline.The increase in growth affected by proline was inconformity with Cnoska and Hanson (1991) and Yancey

(1994) where exogenous application of proline providedosmoprotection in cells of plant tissues. Proline at lowerconcentration (20 mM for 8 h seed soaking) proved to bebeneficial for plant growth in contrast to higher concen-trations in both the varieties. These findings were similarto the observations of Jain et al. (2001) where low prolineconcentration effectively alleviated salinity induced de-cline in fresh mass in Arachis hypogea as compared tohigher concentration. However, the inhibitory effect ofhigher concentration of proline was more prominent inRH-30 than in Varuna. Moreover, it has been reported theexogenous application of proline to the culture mediumincreased dry weight and also induced free prolineaccumulation in the callus of alfalfa (Ehsanpour andFatahian 2003) and strawberry (Gerdakaneh et al. 2010).The treatment with different concentrations of proline asshotgun approach increased the root proliferation (rootlength and fibration) as evident from Plate 1.

Effectiveness of proline applied through shotgun methoddepends on the type of species, tissue of application andconcentration used. An explanation for the beneficial effectof lower concentration of proline is attributed to the factthat it activated a cycle of cytosolic proline synthesis fromglutamate and mitochondrial proline degradation whichsimultaneously provided NADP+ to drive cytosolic purinebiosynthesis and reducing equivalents for mitochondrialADP phosphorylation (Hare 1998). Arabidopsis geneinduction by exogenous proline encoding proline dehydro-genase (Kiyosue et al. 1996) is consistent with thishypothesis. However, at higher levels of exogenous prolinefeedback inhibition of P5CS (Garcia-Rios et al. 1997;Zhang et al. 1995) blocked the biosynthetic portion of thiscycle and thereby inhibiting organogenesis in Arabidopsis(Hare et al. 2001).

Source ofvariation

Degree offreedom

Ci E

4 h 8 h 12 h 4 h 8 h 12 h

Cultivar 1 6006.19 20736.00 21904.00 1.17 1.71 2.41

Treatment 5 22462.48 33488.13 29705.33 2.00 4.25 3.31

Interaction 5 406.49 456.66 867.46 0.01 0.02 0.06

Error 22 2.84 3.93 4.96 0.0008 0.001 0.0006

Table 7 Mean squares fromANOVA of data for internalCO2 concentration (Ci) andtranspiration rate (E) of high andlow photosynthesizing cultivarsof Brassica juncea(Varuna and RH-30)

Source of variation Degree of freedom WUE SOD

4 h 8 h 12 h 4 h 8 h 12 h

Cultivar 1 0.003 0.003 0.002 1122.25 729.00 702.25

Treatment 5 0.004 0.009 0.009 1294.05 2446.80 1154.05

Interaction 5 0.0001 0.0002 0.0001 16.45 40.20 38.05

Error 22 0.000 0.000 0.000 4.09 5.70 10.24

Table 8 Mean squares fromANOVA of data for WUE andSOD of high and low photo-synthesizing cultivars of Brassi-ca juncea (Varuna and RH-30)

82 A.S. Wani et al.

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As proline acts as a membrane stabilizer, its exogenousapplication at lower concentration results in the rapiduptake coupled with its de novo synthesis (Santos et al.1996; Zhu et al. 1990); therefore, this increases theendogenous proline level which is in conformity with theobserved increase (Fig. 3b, c; Table 10). However,application of higher doses of proline imposes check onits biosynthesis through feedback inhibition thereby de-

creasing the endogenous proline content (Zhang et al. 1995;Garcia-Rios et al. 1997). The level of proline content ismuch higher in leaves than in roots because leaves arecontinuously exposed to the environmental stress ascompared to roots. Reactive oxygen species (ROS) contin-uously produced by plants as bi product of metabolicpathways (Foyer and Harbinson 1994) plays an importantrole in providing protection against harmful pathogens

Fig. 3 Effect of proline oncatalase activity (a), leaf prolinecontent (b), and root prolinecontent (c) of high and lowphotosynthesizing cultivars of B.juncea (Varuna & RH-30)

Source of variation Degree of freedom POX CAT

4 h 8 h 12 h 4 h 8 h 12 h

Cultivar 1 6.00 5.06 12.81 7310.19 9312.41 1260.19

Treatment 5 17.57 21.11 17.46 25944.75 61838.21 35603.28

Interaction 5 0.53 0.48 0.91 134.89 328.17 388.36

Error 22 0.01 0.01 0.01 22.69 62.81 34.72

Table 9 Mean squares fromANOVA of data for POX andCAT enzyme activities of highand low photosynthesizingcultivars of Brassica juncea(Varuna and RH-30)

Response of two mustard cultivars 83

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(Alvarez and Lamb 1997; Doke 1997; Bolwell et al. 2002).But excessive levels of ROS results in oxidative damage tonucleic acids and degradation of chlorophyll pigments, etc.(Fridovich 1986; Davies 1987; Imlay and Linn 1988;Schutzendubel and Polle 2002). Thus, ROS generationshould remain within compatible limits. These ROS arescavenged by different antioxidant defense mechanisms(Alscher et al. 1997) which include activation of antioxi-dant enzymes and production of defense molecules viz.proline, glycine betaine, etc. Proline has been reported to beresponsible for scavenging of ROS and other free radicals(Smirnoff and Cumbes 1989; Bohnert et al. 1995; Noctorand Foyer 1998; Niyogi 1999; Hong et al. 2000; Okuma et

al. 2004). Exogenous application of proline reduces cellularROS level indicating its free radical scavenging potential(Cuin and Shabala 2007), which also confers enhancedactivity of antioxidant enzymes, i.e., CAT, POX, and SOD(Hoque et al. 2007; Ahmed et al. 2010b). In the presentstudy, the exogenous application of lower concentration(20 mM proline for 8 h soaking duration) resulted inincreased activity of these antioxidant enzymes (Figs. 2c,3a, b; Tables 8 and 9; being more in Varuna while lesser inRH-30). This is further confirmed by the findings of Cuinand Shabala (2007) and Ashraf and Fooland (2007) whereproline increased activities of these enzymes by acting atthe level of transcription and/or translation. It has also been

Source of variation Degree of freedom Leaf proline content Root proline content

4 h 8 h 12 h 4 h 8 h 12 h

Cultivar 1 5.75 3.42 11.33 8.00 5.95 4.00

Treatment 5 89.72 194.37 168.89 11.02 15.06 20.30

Interaction 5 0.14 0.28 0.37 0.09 0.05 0.02

Error 22 0.16 0.20 0.24 0.02 0.01 0.02

Table 10 Mean squares fromANOVA of data for prolinecontent of leaf and root of highand low photosynthesizingcultivars of Brassica juncea(Varuna and RH-30)

Fig. 4 Correlation coefficientvalues between net photosyn-thetic rate and SPADChlorophyll

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reported that exogenous proline induced total peroxidaseactivity in rice seedlings (Chen and Kao 1995).

It is also evident from the present experiment that thephotosynthesis increased with the application of exogenousproline in both the varieties being more prominent inVaruna followed by RH-30. Since photosynthetic capacityis heavily dependent on the stomatal movement and themetabolism (protein associated with PSI and PSII andchlorophyll) (Lawlor and Cornic 2002; Athar and Ashraf2005), the present study suggests that exogenous prolineapplication causes increase in stomatal conductance therebysub stomatal accumulation of carbon dioxide leading toincreased net carbon dioxide assimilation rate (Figs. 1b–d,2a, b; Tables 6, 7, and 8). These results reveal thatphotosynthetic enhancement primarily correspond to in-creased stomatal conductance and higher carbon dioxidediffusion rate within the leaves to favor higher photosyn-thetic rate. These results correspond to the findingsprocured by Ahmed et al. (2010b) in young olive plants.The chlorophyll content also increased in both varieties inresponse to exogenous proline. It was more prominent inVaruna than in RH-30 (Fig. 1a; Table 5). Being membrane-bound, the stability of chlorophyll molecule highly dependson membrane integrity which is maintained by proline as itacts as membrane stabilizer (Ashraf and Fooland 2007).The increase in chlorophyll content by the application ofthe proline will naturally result in higher photosyntheticrate. Chlorophyll enhancement showed positive correlationwith net photosynthetic rate at all the concentrations ofproline soakings as evident from correlation graph (Fig. 4).This increase in chlorophyll content in the presentexperiment is similar to the earlier findings in Vicia faba(Gadallah 1999), wheat (Waseem et al. 2006), canola(Kauser et al. 2006), and maize (Ashraf et al. 2007).

Conclusion

The critical assessment of the present investigation providessome important clues about the role of proline when giventhrough shotgun approach in mustard plants. It may besummarized as: (a) at a lower concentration (20 mM),proline favored most of the parameters studied, includingantioxidants, whereas, the higher concentrations (30, 40,and 50 mM) proved inhibitory; (b) the photosyntheticfeedback in genotypes selected in present study provided anadded advantage to the growth and physiological attributes.However, again, the correct duration of soaking (here 8 h)along with appropriate concentration (20 mM) of thetreatment is prerequisite for the fruitful results. Further-more, the promoting effects of lower concentration dose ofproline (20 mM) was more prominent in Varuna ascompared to RH-30.

Conflict of interest The authors declare that they have no conflict ofinterest.

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