Indian Journal of Experimental Biology Vol. 38, May 2000, pp. 483-487

Comparison of protein profiles and enzymes in non-mycorrhizal and mycorrhizal roots of Pennisetum pedicellatum

C Rarnesh, P Chellappan & A Mahadevan*

Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 60 025, Indi a

Received 23 September 1999; revised 20 Janua ry 2000

Pennisetum pedicel/a tum pl ants were inocul ated with Glomus mosseae, G. aggregatum and Gigaspora margarita . There were both quant itat ive and qualitative changes in the protein pattern of inoculated plants. Gi. margarita induced increase in protein in the plants. Acid phosphatase, alkaline phosphatase, superoxide dismutase and chitinase activities were high at the beginning of infection, but declined as the infection advanced. Gi. margarita was an efficient fungus in enhancing enzyme activity and proteins in roots compared with G.mosseae and G.aggregatum. Protein profile revealed the presence of 12 peaks in mycorrhizal plants compared with 8 in nonmycorrhizal plants.

Vesicular-arbuscular mycorrhizal (VAM) fungi are ubiquitous in distribution and occur on almost all vascular plants1

• Mechani sm of recognition is not known, however important biochemical changes occur. Interaction between fungus and plant cell takes place at both extracellular and intracellul ar levels. At intercellul ar level, interface between arbuscular membrane and plant cell represents the most important structure in symbiosis because it is a major site of nutrient exchange and the place where the most intimate contact between fungal and plant cell exists. Interface contains matrix that has high enzymatic activity. Alkaline phosphatase was present in G. mosseae colonized onion plant during growth phase2

. Acid ~-glycerophosphatase was located in the lysed hyphae of G. mosseae and matrix3 Peroxidase activity was absent in the matrix and host walls which were intact with the fungus. Maximum ATPase acti vi ty was associated with the finest arbuscule tips and not with young or new ly senescing arbuscules of G. mosseae coloni zed onion plants4

. Spec ific fungal enzyme activity such as alkaline phosphatase5

changed during infect ion . Presence of superox ide dismutase (SOD) in G.

mosseae colonized Trifolium pratense roots has been reported6

. Oxygen free radicals might be implicated in mycorrhization process, since synthes is of new SOD isozyme takes place after the formation of mycorrhizal symbiosis. Pathogen related (PR) prote ins involved in the defense response of plants include acid and basic chitinases and ~-I ,3-gluca-

*Correspondent author

nases which are antimicrobial hydrolases7. Chitinase

isozymes different from those related to plant defen se response to root pathogens are activated in the mycorrhizae infected plants in addition to constitutive root enzymes8

·9

. The mycorrhiza-related chitinase isozyme is weakly active in incompatible interaction . 1 10 p . d 111 myc- mutants . rotem patterns an enzymes such as acid phosphatase, alkaline phosphatase, superoxide dismutase and chitinase were analysed in mycorrhizal and non-mycorrhizal roots of Pennisetum pedicellatum roots in order to understand whether any specificity exi sts in the induction of proteins and enzymes.

Materials and Methods P. pedicellatum seeds were obtained from Fodder

Agriculture Research Station, Ambattur, Chennai , Tamil Nadu and grown in sterilized fi e ld soi l in pots ( 16x 10x 10 em) . In each pot, 5 plants were grown and inoculated after 10 days with 50 spores of Glomus mosseae, G. aggregatum and Gigaspora margarita, obtained from our culture collection. Spores were multiplied in Rhodos grass (Chloris guayana). Once in 15 days, Hoagland solution ( I 00 mL) 11 was added to each pot . At regular intervals, the roots were collected and evaluated for mycorrhizal infection 12

.

Extraction of protein-Infected roots ( I g) were collected, powdered using liquid nitrogen and extracted (I :5 w/v) with K-phosphate buffer (0. I M, pH 7.2) containing 0.15 M, NaCI; 3 mM, KCl ; 5 m!vf, isoascorbic acid and 2% polyvi nyl polypyrrolidone at 4°C for 2 hr with stirring. Extracts were filtered through cheese cloth and centrifuged at 20,000 g for

484 INDIAN 1 EXP BIOL, MAY 2000

20 min. Equal volume of chilled acetone was added, kept at 4°C overnight and centrifuged at 20,000 g for 20 min. The pellet was suspended in phosphate buffer (50 rnM, pH 7 .8) and dialyzed against phosphate buffer (5 mM, pH 7.8) for 24 hr. The dialyzed solution was concentrated and dissolved in water (5 mL) 11 . Protein level in samples was estimated 13.

Root tissue (I g) was cut to pieces ( 1-2 mm), ground in liquid nitrogen in a cold room (4°C) and added tris-HCI (5 mL, pH 7.2). Extract was squeezed through 3 layers of cheese cloth, centrifuged at 20,000 gat 4°C for 20 min used the clear supernatant as enzyme source.

Estimation of enzymes- To estimate acid phosphatase and alkaline phosphatase, enzyme extract (0.2 mL) was pipetted out in a test tube to which 0.4 mL of p-nitrophenol phosphate solut ion was added and made up to 3 mL with distilled water. The mixture was incubated at 37°C for 30 min and terminated the reaction by adding I mL of I M NaOH. Absorbance was read at 410 nm in a spectrophotometer. Enzyme activity was calculated by calibrating OD value with nitrophenol standard graph 11 . The method of estimation of acid phos­phatase11 was followed with s light modification . Tri s­HCI buffer (0.25 M, pH 9.7) was used instead of 0.025 M acetate buffer (pH 5.8) for alkaline phosphatase estimation 11 .

Superoxide dismutase (SOD) was estimated according to Marklund and Mark lund 14 . Roots (I g) were homogenized in ice cold sodium tetraborate buffer (0.2 M, pH 7.4) containing polyvinyl pyrrolidine (25 %) . The homogenate was centrifuged at 10000 g for 10 min . The supernatant was collected, precipitated with equal volume of acetone and stored at 4°C. It was again centrifuged at 10000 g for 3 min. The pellet was collected, resuspended in I 0 mL of sodium phosphate buffer (0.02 M, pH 6.4) and used as enzyme source. For enzyme assay, I mL of enzyme extract, sodium phosphate buffer (2 mL) and pyrogallol (3 mM, 1.5 mL) were added and absor­bance was measured at 420 nm in a spectrophoto­meter (Beckman- DU40).

To estimate chitinase activity, the reaction mixture contained I mL of 0.1 % colloidal chitin in sodium acetate buffer (0.05 M, pH 5) and I mL of enzyme and incubated at 3°C for 2 hr. Substrate and enzyme blank were used as control. The reaction was terminated by adding 0.1 mL of 0.08 M, potassium tetraborate (pH 9.2) to 0 .5 mL of reaction mixture and boiled in a water bath for 3 min. N-acetylglucosamine

was estimated by the method of Reissig et a/15•

Gel separation of proteins-Separation of proteins was made using sodium dodecyl sulphate polyacryl­amide gel electrophoresis (SDS-PAGE) 16 using a vertical slab gel electophoresis apparatus (Balaji Scientific, Chennai) . Each lane recei"ved 100 J..Lg of protein. The gels were scanned in a scanning densitometer model GS300 (Hoefer Scientific Instruments, USA). The experiments were repeated 4 times and the results were reproducible.

After 15 days, intercellular and intracellular hyphae of G. mosseae, G.aggregatum and Gi. margarita were observed in th~ roots of P. pedicellatum. By 30th day, colonization of fungi was well established and continued till 60th day.

Results and Discussion Proteins-Root protein level (expressed as mg

protein per g root dry weight) increased in the mycorrhizal plants compared with non- mycorrhizal plants from 30 days of infection. Protein level was maximum in the plants collected from 45 to 60 days in all the treatments which decreased later (Fig. I). Gi. margarita inoculated P. pedicellatum plants exhibited higher level of protein than non-mycorrhizal and other mycorrhizal inoculated plants. G. aggregatum. induced more protein in roots than G. mosseae. In general mycorrhizal roots contained significantly high level of protein . Analyses of proteins present in both mycorrhizal and non- mycorrhizal ·P. pedicellatum roots revealed that the level was higher in Gi. margarita inoculated P. pedicellarum plants at 60th day than in the plants infected by G. mosseae and G. • aggregatum. Polypeptides in Gi. margarita infected roots of P. pedicellatum were more than in non­mycorrhizal roots. Accumulation of proteins in the mycorrhizal roots is reported in many plants and are referred to as mycorrhizins 17. This is one of the responses of the host to infection . Indeed we found 2 new polypeptides, p5 and pI 0 in the mycorrhizal

5.---------------------·----------. •contr~l

:C 4 ~ G mosseae -~ D G aggregatum il: 3 E2l Gi margarita

~ E 2

·=

Fig. !-Protein level in the mycorrhizal and non-mycorrhizal P. pedicr·l/atttlll roots

RAMESH et al.: PROTEIN PROFILES & ENZYMES IN MYCORRHIZAL PLANTS 485

roots and these must be considered mycorrhizins. Polypeptide having a molecular weight 35 KDa was exclusively present in the mycorrhizal roots and it was implicated in the mycorrhization process 18

.

Proteins were separated by SDS-PAGE and bands of different molecul ar weights were recorded (Fig. 2). Quantity of polypeptide pI was high in the mycorrhizal roots, whereas it was low in non­mycorrhizal roots. Polypeptides p2, p3 and p4 were recorded in both mycorrhizal and non-mycorrhizal roots. Polypeptide p5 was present only in mycorrhizal roots . Polypeptides p6 and p7 were present at the same concentration in both mycorrhizal and non­mycorrhizal roots. Polypeptide pI 0 was present only in the mycorrhizal roots.

Densitometer scanning of gels revealed that 12 peaks were present in the extract from mycorrhizal plants and 8 peaks in non-mycorrhizal plants . Peaks pI , p2, p5 , and pI 0 were prominent only in the extracts of mycorrhizal plants (Fig. 3). Level of separation of proteins isolated from G. mosseae and G. aggregatum infected roots was less compared with Gi. margarita. The protein profile of Gi. margarita colonized P. pedicellatum roots showed difference in polypeptides 19

. Polypeptides of both low and high molecular wei ght were reported in G. mosseae

MW M a b ---~-·--------

kDa

97

66

4 5

29 14

Fig. 2-SDS PAGE showing protein patten of Gi. margarita colonized P.pedicellatum root [M-Marker; a- Inoculated with Gi. margarita; b-Control.]

colonized soybean20. Ten polypeptides called

ectomycorrhizins were reported in Eucalyptus­Pisolithus tinctorius ectomycorrhizal symbiosis but not in the free living mycelium and non-infected roots2 1

. Clearly, synthesis of new polypeptides is a response of host to mycorrhizal fungi .

Enzyme activity: acid and alkalline phospha­tases-At the early stage of roots colonized by Gi. margarita, acid phosphatase activity decreased, compared with non- mycorrhizal plants. After 60 days, the activity decreased in mycorrhizal and non­mycorrhizal plants (Fig. 4A) . However, Gi.margarita inoculated plants displayed significantly high acid phosphatase activity compared with G. mosseae and G.aggregatum. Alkaline phosphatase activity dec­reased in the roots of plants collected on 30th day however, it increased in mycorrhizal inoculated plants (Fig. 4B). The enzyme activity declined in older plants. In Gi. margarita inoculated plants, alkaline phos­phatase was significantly high.

Mycorrhizal fungi improve P availability by solubilizing inorganic P (Ref. I) . Solubilization of P is mediated through release of organic acids and phosphatases22

. Acid and alkaline phosphatases significantly increased in mycorrhizal infected P. pedicellatum plants of 45 to 60 days especially in Gi. margarita inoculated plants. After 60th day, the activity declined. It has been reported that acid and alkaline phosphatases mcrease m mycorrhizal

250

200

E c

0 00 150 V'l

70 Ill ., ....

0 · -c c: "' :::> ~ "' 100 .0

<

50

' I I I I

2

,' ' I

' ' ' ' ' I , I I

-Control ---- Inoculated with

Gi margarita

,,

0~=-£/ __ ~~----~~-----r~----~ 200 600

Length of gel (mm)

Fig. 3- Densitometer scanning of SDS gel showing protein pattern of Gi. margarita colonized P. pedicillatum roots

486 INDIAN J EX P BIOL, MAY 2000

75 inoculation

90

c

D

Fig. 4-Enzymc activity in the mycorrhizal and non-mycorrhizal P. pedicellatum roots-(A) Acid phosphatase act ivity; (B) alkaline phosphatase act ivity; (C) SOD activity; (D) chitinase acti vity [significant at P<O .OO I]

infected Allium cepa roots23. Increase in ac id

phosphatase in the mycorrhiza l plants is strongly influenced by the fun gus24

.

Superoxide disinutase (SOD)-SOD activity was higher in mycorrhi za l roots than in non-mycorrhiza l roots. The activity increased on 60th day and thereafter it declined (Fig. 4C). Gi. margarita colonized P. pedicellatum roots exhibited signifi­cantly higher SOD activity compared with G. mosseae and G. aggregatum infected pl ants. SOD is involved as an antioxidant protection of the nodule, scavenging the excess of superoxide free radicals generated by increase in 0 2 tension inside the cell

25.

Oxygen free radicals are implicated in mycorrhization process, since synthes is of new SOD isozymes takes place after formation of mycorrhizal symbiosis6

·26

. P. pedicellatum colonized by Gi. margarita showed more SOD activity than by G. mosseae and G. aggregatum.

Chitinase-Maximum activity of chitinase was recorded in the roots of plants inoculated with mycorrhizal fungi after 60 days of inoculation . Gi. margarita was an efficient symbiont in enhancing chitinase activity (Fig. 4D). However, chitinase level in the mycorrhizal infected plants decreased with increase in infection. Chitinase activity in P. pedicellatum roots colonized by G. mosseae, G. aggregatum and Gi. margarita was higher in all the treatments than the control on 60th day. In G. versiforme colonized Allium porrum roots, chitinase activity also increase27

. After the infection was fully established, the specific actiVIty of chitin in mycorrhizal roots was lower than the control roots. One possible explanation is that mycorrhizal infection causes repress ion of chitinase by way of altered hormonal status of root. Chitinase activ ity was higher in the primary rootlets than in older roots ie, it was hi gh in those roots which were the main sites of mycorrhizal infection28

.

Based on our results, we propose that in the first phase of colonization by mycorrhiza l fungus, the roots respond with a typical stress reaction against infection ie, an increase in prote ins and a few enzymes. During the second stage, host-symbiont re lationship led to the reduction of enzymes and protein s. The response is essentiall y similar and no specificity in the induction of enzymes and protei ns exists.

Acknowledgement The author (CR) is thankful to CSIR, New Delhi

for awarding the fellowship. Thanks are also due to DBT, New Delhi for financial assistance to the author (PC).

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