Indian Journal of Experimental Biology Vol. 37, October 1999, pp. 995-1000

Naringenin application alters function and metabolism of pea nodules

Veena Jain & Hirnmat S Nainawatee·

Departments of Biochemistry and *Biotechnology & Molecular Biology, CCS Haryana Agricultural University. Hisar 125 004, India

Received 5 February 1999; revised 1 July 1999

Application of naringenin at the time of sowing influenced metabolism of pea (Pisum salivlll11) nodules at various stages of plant growth. Proportion of assimilated 14C was higher in nodules of plants from naringenin treatment as compared to control plants. Activities of enzymes of ammonia assimilation, viz. glutamine synthetase (GS) , glutamate synt hase (GO­GAn, glutamate dehydrogenase and carbohydrate metabolism, viz. phosphoenolpyruvate carboxylase (PEPCase), malate dehydrogenase (MDH), isocitrate dehydrogenase and enzymes of Entner Dudoroff pathway were enhanced by naringenin treatment. Naringenin also increased the acetylene reduction activity of nodules, which positively correlated with increase in GS, GOG AT, PEPCase and MDH activities. However, there was no effect of naringenin application on key enzymes of hexose monophosphate (HMP) pathway, viz. glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.

Flavonoids secreted by legume roots trigger the ex­pression of nod genes of rhizobia l

. This leads to for­mation of root nodules which fix atmospheric dinitro­gen2.3. Regulatory property of f1avonoids is known to be Rhizobium and legume host specific4. Some f1a­vonoids act species specifically as antagonists of nod gene inducers5

.6 while some other also act synergisti­

call/·5. Application of f1avonoids is known to over­come flavonoid Iimitation8 and enhance nodulation status and nodule efficiency in alfalfa

9 and mung­

bean JO. Presence of f1avonoids is also necessary for

functioning of soybean nodules2. The efficiency of

nodule is critically dependent upon its metabolic ac­tivities, particularly the carbohydrate metabolisI'n II and ammonia assimilation l2

• In pea, naringenin, a flavanone was reported to induce nod genes of R. leguminosarum biovar viciae5 and enhance nodula­tion status and nodule efficienc/ 3

. However, the ef­fect of f1avonoids on carbon and nitrogen metabolism does not appear to have been investigated. Results reported here show that sowing time application of naringenin, while enhancing nitrogenase activity, also increases activities of enzymes of nitrogen assimila­tion and carbohydrate metabolism in pea nodules.

Materials and Methods Plant material and growth conditions--5eeds of

pea (Pisum sativum) var. HP4 were surface sterilized

*Correspondent author e-mail : hsn@hau. hry.nic.i n

and treated with R. legumillosarul11 bv . viciae stra in PD 12. The crop was raised in modified Leonard jars, using N-free nutrient solution under aseptic condi­tions in a screen house 1o. The nutrient solution sup­plemented with 10 11M naringenin di ssolved in alka­line water was applied at the time of sowing. Jars were supplied with N-free so lution at an interval of 10 days . From each treatment , four repl icates of two plants were sampled at 54. 68 , 82 and 96 days after sowing (DAS) .

Nitrogen Jixation--Rate of nitrogen fixation was determined by acetylene reducti on assay method l4

.

Nodule bearing excised roots in ten replicates of one plant each, were incubated in acetylene atmosphere (10%, v/v) for I hr at 28°C in 250 ml stoppered glass jars fitted with Subaseals. Ethylene produced was estimated on Shimadzu GC Mini-2 using Porapak N­column.

14 C-partitioning--The whole potted plant was ex­posed for 30 min to 100 I1Ci 14C02 in a perspex chamber with water circulating in its double wall for

. 15 d h 14C "1 d temperature mamtenance ' an t e aSSlnlt ate was allowed to partition for 24 hr. Different plant parts were then dried to a constant weight at 80°e. Samples were extracted thrice with 80% ethanol and pooled extract was centrifuged at 10,000 g for 30 min to obtain the soluble fraction . Radioactivity of soluble fraction of each plant part was measured by LS 180 I Beckman LiqUid Scintillation Counter using naph­thalene based scintillation cocktai I 16.

996 INDIAN 1. EXP. BIOL. , OCTOBER 1999

Enzyme extractioll-Detached noduies were washed free of sand and homogenized in a buffer us­ing pestl e and mortar. Tri s- HCI bu ffer (0 . 1 M , pH 7.5) was used for the extracti on of glutam ine syntha­lase (GS; EC 6.3. 1.2), glutamate synthase (GOGAT; EC 1.4.1.14) and glutamate dehydrogenase (GDH ; EC 1.4.1.2) and for all other enzymes phosphate buffer (0. 1 M, pH 7.4) was used. The homogenate was passed through cheese c loth and centri fuged at 10,000 g for 30 min. The supernatant obtai ned was used for the assay of enzyme. All the operat ions were carried out at 4°C.

Enzyme assays-GS was assayed using g lutamate

as substrate '7 . GOGAT and GDH were assayed fol­

lowing the ox idation of NADH using <x-ketoglutarate with g lutamine and a-ke toglutarate as subslralcs re­spectively' s. Phosphoenol pyruvate carboxylase (PEPCase; EC 4.1 . 1.31) was assayed by measuring the decrease in absorbance at 340 nm l

'). Activity of isoci trate dehydrogenase (ICDH; EC 1. 1. 1.42) was measured following the reduction or ADP+ using isocitrate as subs't rate20 Malate dehydrogenase (MDH; EC 1.1.1.37) was assayed by measur ing de­crease in absorbance due to oxidation of NADH by oxaloacetate21

• Activ ities of g lucose-6-1 hosphate de­hydrogenase (G6PDH; EC 1.1 . 1.49) and 6-

Table I-Effcc t of naringcnin on activities of ni trogenase and ammonia assimi lating cnzymcs in flca nodull; ~

[Valucs are mean ±SD of 10 replicates*/4 replicates**]

Enzymc Days after sowing (DAS ) 54 68

Ni trogenase* C 254.0±20.6 195.3±12.7 N 290.2±24.9 263.7±1 8.9

GS** C 96 .3±7.7 84.0±3.8 N 166 .S±S.6 160.S±10.2

GOGAT** C 46 .6±0.8 13.S±0.6 N 66.3±3. 1 19.7±0 .6

GDH** C 56A±3.8 SO.2±1 .7 N 98.3±6. 1 89.3±3.2

*nmoles C2H4 produced/hr/nodule; **nmoles of product formed/min/mg protein C = control; N = naringenin

100

120 90 1 7S -~

I ~

I 0- t-

90 ,\'" G" < o· '\ ...... 8 <!)

60 0 50 ,~ .. ',\'" <!) :r 0 .... " ().1 0

or. -= <!) <!) 60

-,,' -\'" 0 .. .. , c c "0

.. .-0 .. .. .. OIl

OIl 30 ~ 0 2S u 0 A 0 c ..

~ 30 ... u

u ;,! C C

• . - ... . ,. , 20 30 40

82

17S.2± 11.9 243 .8±1 7.6

72.3±S.S IS 3.2±6.6

6.0±0.S IOA±O.6

42.0± I .O 83 .1±2.3

6.

C 6.

20

!)()

oO. I±o.:l 73 .lJ±o.6

44 .0±2.6 5X .6±4.1

5 .2±OA 7. I±O.S

22 .4± I.S 30.S:,:: IA

6-

,\,'\ 6.

'>:). ",q

30 40

'f, Increa~e in Nitrogenase 'f, Increase in Nit r ogenaH

Fig. l~orrelation between naringenin enhanced nitrogenase activity and activi ties of ammonia ass imil ati ng enzyme. o f flea nodules. [A, GS; B, GOGAT; C, GDH ] .

JAIN & NA INAWATEE: NAR INGENIN & FUNCTION & METABOLISM OF PEA NODULES 997

albumin as a standard prote in . phosphogluconate dehydrogenase (6PGDH ; EC 1.1.1.44) were esti mated by measuri ng the increase in absorbance at 340 nm at 30°C due to reduction of NADP+ (re f. 22) . Entner Dudoroff pathway was as­sayed as combined acti vity of 6-phosphogluconate dehydratase (EC 4 .2. ).) 2) and 2-keto-3-deoxy 6-phosphogluconate aldolase (EC 4. 1.2. 14) by estimat­ing the amount of pyruvate liberated from 6-phosphogluconate2.1. So luble proteins were es timated by the method of Lowry et al. 24 using bovine serum

Results N2 fixation and a /1/I/W/lW assi ll1ila-

lion-Application of naringenin increased the acety­lene reduction activity (ARA) and act iviti es of en­zymes of ammonia ass imil ati on. Th e inc rease in acetyl ene reduction activ ity was 14.2-38.8 % at differ­ent stages of growth Crable I). The maxi mu m in­crease was at 82 DAS stage ;1t whi ch the ARA with

Table 2 - Effect of naringeni n on enzymes of carbohydrate metabolism in pea nodule~ [Valu es are mean ±SD of 4 replicates)

Enzyme act ivity (productlmin/mg protein)

PEPCase" C N

MDHh C N

ICDP'l a

C N

G6PDHh

C N

6PGDH" C N

ED' C N

54

47.8±3.2 60 .3±4A

38 1.8± 14.S 490.1±28A

2 1.7±I A 3 1.2± 1.5

55.5±3.8 64.2±4.2

79.7±5.7 62.5±3 .9

273 .6±12.2 319.5±I S.5

"nmoles or \1 moles of NAD+/NADP+, ' pmoles pyruvate C = control; N = naringeni n

8% ".,:>" 30

[) "i~ " " '" 0 45

:c u D

B 0 20 ~ a. 0 w c: a. c:

30 .. '"

" 0

'" ~ 0 u ~ 10 r.: u

15 A E 0

0

;t- .. 20 30 40

.,. tncr~os~ in Nltro9~nos~

Days after sowi ng (DAS ) 68 ~2

26.2±1.7 27.3± 1.9 34.9±2.1 39.0±2.9

184.1±!3 .5 176.2±1 6.3 249.2± 18.2 236A±I J.2

IS.5±O.7 14A±I .O 25.8±2.3 22.0± 1.7

38.9±2.3 32.5± 1.6 40.2± 1.9 36. 1±2.0

43. 1±2A 45 .5±3.7 35.8±3. 1 43.2±2. 1

In . I±7.1 178.7±7.2 222.2± 12.6 221.1± 12.0

9 60

9-:c 45 0 u -c: . ~ 30 0 II ~

u c: - 15

o 0

I ,..

20 30 40 .,. Incr~os~ in N itroCJ~no~

30

20 ;;

II .. 10 ~

~

u c:

20.3±I.l 25.0± I.~

154.G± I 0.1 17:l.2±14.J

R.O±O.6 10.1 ±O.9

2 1.2±O.9 22.7±O.X

3~U;±2.6

34.2±2.6

I X3A± 13.9 194.8±14.2

Fig. ~orrelation between. naringenin enhanced nitrogenase activity and acti vities of enzymes of carbo hyd rate metaholi sm of [lea nod· ules . ~, PEPCase, B, MDH , C, lCDH, D, ED]

998 INDIAN 1. EXP. BIOL., OCTOBER 1999

Table 3--Effect o f naringenin on distribution of 14C in various part s o f pea plant [Values are mean ±SD of 4 replicates)

Days after sowing (DAS) nmoles o f 14C1plant 54 68 82

Nodul es C N

Shoot C

26.9±2.3 30.8±3.0

61.2±4. 1 86.2±5.0

469.7±3.8

13.3± I. O 19.3± 1.1

. 692.7± 14.7

5.i{±0.4 ~: .I±(U\

N Root

98 .3±7.9 120.2±8.7 668.4±26.4 959.4±21.4

1245.2±30.9 I 560.7±54.2

C N

27.8±3.1 3 1.6±3.4

6 1.5±3.2 18.3± 1.5 17.5± 1.2 I3.X± 1.4 82.9±6. 1

C = control; N = naringenin

and wi thout naringenin was 244 and 175 nmole/hr/nodul e, respec ti ve ly. Among different en­zymes of ammonia ass imilation , the e ffect of narin­genin was maxi mum on GS acti vity in which the max imum increase was 111 .9% and it was at 82 DAS stage. Regress ion analys is showed that naringenin effected increase in GS , GOGAT and GDH ac tivities positive ly corre lated with the increase in nitrogenase act ivi ty by 77 .4, 77 .9 and 71 .7 % respecti ve ly (Fig. I ).

Carbohydrate l1Ietabolism-Naringenin caused in­crease in the acti vit ies of enzymes of ·tricarboxy lic acid cycle, ED pathway and PEPCase, while it e ither sl ightly decreased or had no effec t on the activities of enzymes of hexose monophosphate (HMP) pathway (Table 2). Max imum acti vi ties of a ll the enzymes, both in presence and absence of naringenin treatment were at 54 DAS stage. However, the magn itude of effect of naringenin on the acti vities of different en­zymes differentl y varied w ith the stage of pl ant growth . The maxi mum increase in PEPCase (43.4%) and ICDH (52.8%) acti vit ies was at 82 DAS , while in MDH (36 .2%) it was at 68 DAS stage. The increase in PEPCase and MDH correlated posi ti ve ly, respec­tively by 88 .7 and 77 .6% with the increase in nitroge­nase activity (Fig.2). Ac tiviti es of enzymes of HMP pathway viz. G6PDH and 6PGDH most ly remai ned unaffec ted by naringeni n, except a sli ght increase ( 16.4%) in G6PDH at 54 DAS stage. A lso, there was 110 correlat ion between the acti vities of 6PGDH and G6PDH with the nitrogenase ac ti vity. Naringenin treatment callsed 29. 1 % increase in the activi ty of ED and th is increase correlated positively (76.7%) with the increase in n itrogenase acti vity (Fig. 2).

l-IC-assimilate partitioning-Naringenin applica-

20.2±2.0

4S 45 c: 0

c B 0 o ~ 0/8' ~ I

~I 0 0 I ~ C)'1

/0 . a. I 0

C).'b , /

, ~ 6 a. . , 0

~ ~" ' / <-,'0\ u 0- 30 c: U \II , / C).~ 30 ~ c::~ o"' ... .. · ~ ~

:l U 0

u"O /

, . ;! .<: \II

~ 0 c:: - c:: c::

~

, .- c: c: .. . -.. .- 1 S - A r

\II

\II 0 0 0 .. ~

~

u u c: c -~ .

i

20 30 40

0/. In crease in N itro9rn a~e

Fig. 3-Correlation betweell naringenin enhanced nit rogenas(! acti vit y and 14C incorporation in pea plallls. I A. lIod ules; 13. roots!

tion increased )4C ass imil ates in d ifferent plant parts (Table 3) and this increase was pos iti ve ly corre lated with increase in nitrogenase ac ti v ity (Fig. 3). Narin­genin caused ( 14.5-45. 1 %) inc rease in the level of ) -Ie ass imilate in nodules, the max imum co ncentration (86.2±4.96 nmole) of whi ch was at 68 DAS stage. T he average root to nodule )-IC-ass imi lates rat io for control p lan t was I :0.76, which increased to I :0 .95 in nari ngenin treatment.

Discussion Naringenin, an ind ucer of nod genes of R. lcgullli­

llosarUI11 bi ovar viciae25 is known to enhance nodu l <~~ tion in pea) 3. T he present resu lts indicate the stimu­latory effect of naringenin on nitrogen fixatio n, am­monia assi milation and ) -IC partit ioni ng in nodulated pea plants. The increase in dinitrogen reduction may be lead ing to enhancement in ammoni a ass im ilation as evident by regression analys is.

JAIN & NAINAWATEE : NARINGENIN & FUNCTION & METABOLISM OF PEA NODULES 999

Legume-rhizobium symbiosis in general and nitro­gen fixation in particular is an energy intensive proc­ess. The effect of naringenin on nitrogen fixation was also evident in the flow of '4C-photosynthetic as­~imilates towards nodules. During vegetative phase of growth, nodules and roots received major fraction of 14C-assimilates due to their higher requirement for nodule development and functioning. Recovery of large proportion of 14C-assimilates in the actively growing parts have been reported in several leg­umes26. The level of '4C-assimilates was higher in nodules of plants from naringenin treatment as com­pared to the control plants. The increase in 14C_ assimilates had positive correlation with enhanced ARA of nodules from naringenin treatment. Maxwell et ai.27 showed that incorporation of label in the nod­ules was intimately rel ated to production of ammonia by effective nodule. Increase in nodule efficiency un­der naringenin treatment l3 perhaps necessitated in­creased partitioning of 14C-assimilates into nodules.

Sugars per se are not utilized as carbon source28

and even the peri bacteroid membrane is reported. to be impermeable to sugars2tJ. C4-dicarboxylic acids viz. malate, fumarate and succinate are reported to be important in carbon metaboli sm to provide carbon necessary to reduce nitrogen3o. Thus formation of C4-dicarboxylic ac ids reported here is related to nitrogen fixation by effective nodules . Increase in the activities of PEPCase and MDH have been reported to be coin­cident with nitrogenase during nodule development31 . PEPCase present in nodule cytosol, plays an impor­tant rote in da rk fixation of CO2 in nodul es resulting in the production of oxaloacetate30, which acts as a carbon skeleton for ass imilating NH/ thereby pro­ducing aspartate. Naringenin enhanced activities of PEPCase, ICDH and MDH would result in the for­mation of C4-dicarboxylic acids and thi s could be related to increase in nitrogen fixati on by plants in naringenin treatment. The fix ation 14C0 2 via PEPCase and subsequent incorporati on of label into organic acids is intimately related to production of NH/ by effective nodules " . Activities of enzymes of HMP pathway, vi z. G6PDH and 6PGDH remained unaf­fected , as pea is an amide transporting legume and in such legume, HMP pathway is known to make only minor contribution to the glucose metabolism32

.

The effect of naringenin on the enzymes of ammo­nia assimilation and carbon metabolism seems 10 be complex. Rol e of nod regulators other than control of nod gene expression is known'u . Carbohydrate me-

tabolism and ammonia assimilating enzymes of Rhizobium sp. Vigna are also known to be influenced by nod regulators34. Legume roots exude a variety of nod regulating compounds, thus there is a need to examine their effect in totality on va ri ous aspects of symbiotic nitrogen fixation and plant growth .

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1000 INDI AN J. EXP. SIOL. , OCTOBER 1999

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