'".,,,,
'tS'tARCH, SOLUBLE SUGARS ~ND AMYL'Ol.YTIC 'ENZTMBS TN'THE
UERMIN ATlNGPIGEONPEA (CAJ:.ifNVS'CA.1AN L.)l'SEEDS
S.N,'SIMRMA AND .R;C. PNNT
Department of Plant Physiology G.B. Pant University qfAgriculture and Technology Pant-nagar (Naini Tal). U.P., India
Received on 1anuary 10, 1978
SUlDfARY
Six 'varieties· ofpigeon pea (Cajanus .cajan L.):seeds were-found to eontain.53 .. S9
.poI'-:eent starch and 2-4 percent soluble sugars of dry -weight. During,theircgurmination ,thetdegradation of starch was mainly due to the ..actiNities of 'phosphorylase..a:wi «~amylase. Electrophoresis revealed only one mo1ecutar form of, amylase and phosphorylase. Two distinct phases of starch depletion· were recognized; ,nrst slow, during which phosphc::rylase activity was at its maximum, ·.and the .seoond, rapid, .which coincided with the maximal activity of «.;,amylase.
IMI'RODUCflON
. Several varieties of pigeon pea (Cajanus cajan L.) are available to the·farmers.,in .ourrountry. Some ofthese' such as Pant A1, Pant-~, tl?ant Aa, Type-21,and UPA~120
are early maturing while others such as Hyd..3Care late maturing. ,Information on the physiology of pigeon pea-seeds is scanty (Meyer -.and PoljakOft'-Mayber, 1975). No information is available on ,the behaviour of amylolytic .enzymes in the seeds of this plant during germination although studied in other legumes (Swain and Dekker, 196.6. Bievenido and Varner 1969; Juliano and Varner, 1969, Tarrago and Nicolas, (976). However, hormone and protein ..nucleic acid metabolism bve been explored in Pigeon pea (Madhava Rao and Rajeshwara Rao, 1974 a, b and 1975). It was decided to study the amylolytic enzymes.in all the above mentioned v.arieties during germina~
tion of thoirseeds mainly to find out the varietal differences.
MATElUALS AND MlrrHoDS
Seeds of pig·~o:l p~:l (C'Jjl'lllS c'Jj:zn L.) we~e the gift of Dr. N.P. Singh Depart _nti~of 1tgtmtmny.
':.t..~'tSfiunlfc:mn·size ·werelWlMhed iillJlIllnningltap -water' ,for l2 hr to facilitate
S.N. SHARMA AND R.C. PANT
and were germinated at 28± 1°C in an incubator in large petridishes lined with thin beds of cotton spread over agar-agar gel. The beds were kept adequately moistend with gJass distilled water. Starting with the 12 hr washed seeds (zero day of germination) various analyses were performed in the cotyledons at an interval of two days. The seedcoats were removed in all cases.
Soluble sugars and starch were determined by the method of Clegg, 1965. For extraction of amylase the method described by Goldstein and 1ennings, 1975 was used with slight modifications. 109 blotted-dry cotyledo:::ls were taken and homogenized for 60 sec. in 5.0 ml precooled 0.05 M Tris HO buffer, pH 7.6, containing 5 mM~ mercaptoethanol in a precooled mortar. The homogenate was centrifuged at 20,000
x
g and the supernatant after adjusting the volume was used as a source of enzyme which was assayed by the method of Bernfeld, 1955 Two aliquots of the supernatant were removed and one of them was heated for 5 minute at 70°C in water bath in the presence of 3 mM Ca~to inactivate !'-amylase and was then cooled to O°C (Bilderback, 1973). Total amylolytic as well as IX-and !'-amylase activities were deter
mined in both of these aliquots. The unit activity is defined as the mg maltose liberated in 3 minutes at 30°C by I ml enzyme extract. The specific activity is ex pressed as units per mg protein.
For extraction of phosphorylase the method described by Tarrago and Nicolas,
1916 was used. Enzyme was assayed by the method described by Whelan, 1955 with
slight modifications. Incubation mixture contained 0.25 mlO.l M G~I-P, 1 mI 0.05 M citrate buffer pH 6.0, soluble starch solution (5g/100 m] in water) 0.25 ml, enzyme 0.05 mI and water 0.5 mI. A blank was prepared by boiling the enzyme at 100°C. The enzyme reaction was initiated by the addition of 0.25 mt G-
f-p
and was interrupted after 10 minute by the addition of 5 mI 5% TCA solution and the mixture was then centrifl,lged. Suitable aliquots were removed for phosphorus determination by the method of Bartlett,. 1959. Theunit activity is defined as the amount of enzyme causing the liberation of 0.1 mg organic phosphate in 10 minutes at 35°C. The specific activity is expressed as units per mg protein.Protein in the enzyme extracts was determined by the method described by
Lowry et 01., 1951. For polyacrylamide gel electrophoresis the method used was as described earlier (Verma and Pant, 1978). Bands of starch degrading enzyme were visualized by the method of Tarrago and Nicolas, 1976.
The data presented here are the averages of those obtained in two ditl'erent sets of experiments.
REsuLTS
Changes in the dry weight, starch and soluble sugars of the cotyledons: In
all
the varieties of pigeon pea. the change in the dry weight consisted of a slow phase of
AMYLOLYTIC ENZYMES IN CAJANUS CAJAN SEEDS 221
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Fig. l. Changes in the starch and dry weight of the cotyledons of different varieties of pigeon pea (Cajanus cajon L.) during germination of seed..,
varied between 53-59% of the dry weight. Starch was found to be degraded slowly
r
during the, initial 2 days followed by a rapid utilization. The residual starch on ther
12th daywas only 3-9% of the original (Fig. 1),The content of soluble sugars present was very low. On the second day (except Hyd-3C where it was maximum on the 4th day) it was maximum. decreased slowly , upto the 4th day followed by a rapid decline. UPA-120, however, did not:fit in
with
this pattern (Fig. 2).
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Fis. 2. Changes in soluble sugars in tilt cotYledbos of different varieties of pigeon pea
(Cajanus cajan L.) during germination of seeds.
Phosphorylase and amylase activity in germinllting pigeon pea seeds.' Amylase
activity in nongetminated seeds was very low. Activity of this enzyme in6l'eased slowly-during the initial 6 days foJlowed by a steep..rise. tin. the 10th day. There was a fall in the enzyme activity in the next two days., Phosphorylase actiYity
was
also low in the nongerminared seeds, increased slowly in the-first two days but reached the maximal level by the 4th day. There was: a slight,. decrease in theactivi~ by the 6th da:; followed by a rapid decline upt9 .l2th day when its· level was lower thaa that found in the nongerminated seeds. 'thus the period of, higher amylase actiMity. coin cided withlthe decrease in the phosphorylase activity. The magnitude of the specific activities of both the enzymes were ~uite different 'in aD ilie six varieties (Fig: 3).Electrophoretic. . stui/y . of stal'ch.degrmlintf enzymes· in-.pigean... _pea..cotyledons
duringgerminatio1t: The-slowest band (band A) couldv.lJe.dcteeted. only in the
presence of Pi. It was insensitive to EDT A and disappeared when the extracts were
heated at 70°C for 5 minute and was identitied as phosphorylase. The middle band
I
(band B) was the most prominent and was present in the gels incubated with starchj
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AMfiOLtttc EN~YMES IN CAJANUs CAlAN Sl!t!DS
223
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Fig. 3. Changes in the activities of amylase and phosphorylase in the ooty1edons of different varieties of pigeon pea (Cajanu!i cajun L,) during germination of seedS.
I
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TII lZFig. 4. The zymogram pattern of the starch degrading enzyDl.e'i (amylase and phosphorylase) of the cotyledons" of pigeon pea (Cajonus cajon L.) varieties, Treatments : J;
Gels in phosphate buffer II. The gels in acetate buffer
m.
Gels in phosphate buffer after heating at 1O"C4D.rS. }{. SHARMA AND R. C. PANt
Changes in the enzyme pattern during germination: The intensity of a-amylase
band was very low in the nongerminatedseeds.however, it showed a progressive in crease as the germination proceedecl. Phosphooyiase band remained almost unchanged during the initia1.six days but disappeared thereafter. The intensity of !,-amylase was v~low and the band could not be photographed: However. this ;band dis appearcd~after the second day of ~ermination.
DISCUSSION
Such fast rates of starch degradation as seen in germinating .pigeon pea ..'Seeds have als& been reported for pea (Juliano and Varner, 1959). for rice (Murata 'Nt al.•
1968)
8.nd
fur lima beans (Hegwood and Gaines, 1974).The result&obtained here indicate that the amylase activity is or' it-type in
an
the varieties. Similar observations have been made in peas (Swain and Dekker, 1966 ; .lnliano and Varner, 1969) for rice (Tanaka and Akazava, 1970). The increase in aamylase ac1iivity during.germinlltion may be de IJOVO synthesis ot:tlienenzyma as report ed for batJity(Filner and Varner, 1967 and Chrispeel& and iVamer;'·I967).
The involvement of phosphorylase in the degradati.n of stareh ..1"eSeA'eS!.in pigeon 'pea cotyledons seems not to be very significant. Both phosphorolytic and ·hydrolytic pathwa)s of starch breakdown are present in thepigfX)n pea \ cotyledons", Similar observations have been made}or pea '(Swain and Dekker. 1966; lnliano and l'arner. 1969).~·The sequence of phosphorolytic pathway present initially followed' by the hydrolytic pathway in germinating seeds seems to be useful in terms of energy.'avail ability'- for germination. Phosphorylase enzyme provides the substrate (G...P) for glycolysW without using energy tbeeause the energy of the glycosidic bond is retained in this reaeti~n. Phosphorylases I, II and IV isolated from com endosperm and embryo respectively are competitively inh~bited by ATP and this inhibition has been
suggested '.as a contr~ meehllnism of starch degradation .during seed germination (Tsai and Nelson, 1968, 1969). Thus ATP concentration may also be a factor of decrease of phosphorylase activity in the pigeon pea cotyledons. The hydrolytic path
way may beinflneneed by hormone levels (Madhava Rao and Rajeshwara Rao. 1974b).
ACKNOWLJIDGEMENT
We thank Dr. K.G. Gollakota, Dean, C.B.S. and H. for his helpful'criticistb of the work throughout the course of this study. The assistantship received by the
~.av.thor from· G.B. Pant University of Agriculture and TeChnology is gratefully
ac~•
.. Bartlett, RG. ('959): .i:'hosphorus assay in column chromatography. J. Bioi; Chern•• 134 ~ 466-68.
Bernfetd., P. (1955). Amylases", and.~. Methods Enzymel .• 1 : 149-58.
AMYLOLYTIC ENZY'MBS IN CAJANUS CAJAN SEIIDS
22S
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-
germinating pea. Plant Physiol., 44 t 886-92.Biiderback .. n.E. (1913). 'A simple 'method to differentiate between 11- and p- ~ylase. Plant
,Mys/ot..
'1 :
594-95.Chrispeels,J.M.and Varner,J.E. (1967). Gibberellic acid enhanCed synthesis-and release· of 11
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--~-l(1914b). Gibberellin like substances in developing and germinatlDg seeds of pilt'l9n pea. Indian J. Rlant Phy&iol •• 17: 65-72.
---'-(1975). Growth, respiration and endogenous ~~s of developS. .
ana>'
germinating pigeon pea. Seed Rneorch. 3: I-tO .. Mayer, A.M. and Poljakoff-Mayber (1975).
The
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n.··
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