Platit Physiol. ('1966) 41, 519-522
Variation of Nicotinamide Adenine Dinucleotide Phosphate Level
in Bean Hypocotyls in Relation to 02 Concentration1
Yukio Yamamoto
Biological Institute, Faculty of Science, Nagoya University, Nagoya, Japan
Received September 2, 1965.
Sionnary. Slices of hypocotyls from 3-day-old seedlings of Vigna(i sesquiipedalis
(L.) Fruwirth in the germination stage were incubated under various gaseous conditions.
The NADP+NADPH level in the hypocotyl slices changed with the oxygen tension. A
high NADP+NADPH level was observed uinider aerobic conlditionis and(I a low NADP -iNADPH level under anaerobic conditions.
The 100 X NADH/NAD+NADH ratio increased greatly unider anaerobic con(litions. In general a low NADP + NADPI-I level corresponded with a high 100 X NADH/
NAD + NADH ratio. On the basis of the results given in the followilg paper, it was
discussed that the slowness of NADH oxidation in hypocotyl tissue (tle to anaerobic
conditions results in the inhibition of NADP formation.
The variation of the NADP+NADPH level wais considered to produce a modification
of the carbohydrate metabolism.
The NADP±NADPH level in Ei. coli cells suspended in glucose solutioni also
changed with the oxygen tension.
We observed in previous work (5, 6) that the
endogenous level of NADP is a rate-limiting factor
and plays a key role in the control of metabolism in
plants. A high NADP concentration was observed
in the growing parts of germinating seeds and in tissue with a low NADH/NAD ratio, whereas a comparatively low NADP level was detected in the storage organ of germinating seeds and in tissue with a
high NADH/NAD ratio (6). Highly anaerobic
conditions may prevail in pulpy tissue such as the
storage organ of germinating seeds, while the growing tissues are likely to be under highly aerobic conlditions (7). It has been suspected that aerobic conlditions, under which NAD is maintained in the oxidized state, m,ay be required1 for the production of
NADP and for the maintenance of a high level of
NADP.
NADP is important in the switch to the Ipentose
phosphate cycle from the EMP2 pathway. Accordingly, fluctuation in the NADP level brought about
by changes in the O., level, if any, may play an iniportant role in the regulationi of carbohydrate metabolism.
The purpose of this work was to ascertaini
whet4her tllere is anyv variation in the level of NADP
l This work was financed by a scientific research
the Ministry of Education.
Abbreviations used: EMP, Embden-Meyerhof-Parnas; CG/CG, percent yield of "4CO;2 from glucose-6-'4C
added per percent yield of 14C02 from glucose-1-14C
added; NMN, nicotinamide mononucleotide; (EC),
(Enzyme Commissioin number).
from
granit
2
wi-th changes in the 02 level and the role of 'NADP
in the control of respiratory metabolismii. The
enzymological mechanism of the variation in the
NADP level is also discussed on the basis oaf the
chemical properties of plant NAD kinase (EC
2.7.1.23) which are reported in the next paper (10).
Preliminary accounts of this work have appeared
elsewhere (8, 9).
Materials and Methods
HApocotvl Slices. Seeds of Vignia sesquipedalis
(L.) Fruwirth were immersed in water at 300 for 12
hours, and then germinated on sand at 300 in the
da,rk. The hypocotyls were removed from 3-day-old
seedlings. They were cut into sections of 0.5 to 0.8
miIm thickness with a razor. and were washed twice
with distilled water.
Inicutbationi of Hvpocotvl Slices iunider Various
Conditions. The sectionis of hypocotyl were blotted
lightly and( tlloroughly ranldomized. Twelve groups
of sections (6 pairs), each of 4 g fresh weight, were
prepared.
The first pair were placed in 20(-ml beakers ani(l
putt in a closed 5-liter contaiiner. A vial containinlg
5 ml of 20 % KOH was l)laced in the contailner to
absorb the respired CO.. and( water was placed in the
bottom of the container to mainitaiin a high relative
humildity (fig 1 A). The second pair were treated
in the same way except that the air in the 5-liter colntainer was replaced by nitrogen gas (fig 1 A). The
third pair were both immersed in 8 ml of 0.025 M
Tris-HCl, pH 7.6. The fourth pair were both im-
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520)
I52L-AN'T PIHYSIOLOGY
I
Gassinig of the I,. coli Suispenisioni. T he E. coti
suspensioni wa's vigorously bubbled alternately with
air and( with nitrogeni for 10-miniute periods at 300.
Two 5-ml aliquots of the cell suispelnsioni were removed at zero timiie ancd thereafter at 10-minutte intervals. Gne of each pair of aliquots was heated at
900 for 2 miiinuttes in the presence of 0.5 ml of N
HCI anid grounild with quiartz sanid while heatinig to
extract the oxidized form of coenizymile. T'he other
aliquiot was treate(l in the same way witlh 0.5 ml of N
Air
B
kI
I
Slices
NaOH to extract the redutced coenzvme.
Eachl hiomiiogenlate was then rapidly transferred to
an ice bath ani(l treate(l in the samiie way as the hypocotvl slices before assav of the coelz-viime concentra-
suspension
tions.
I
eAl
Q!,7 -Glass
)% KOl!
/
20
I(;.
8 ml of
Slices
20
ml
water
ml water
filter
~
0.025
M
Tris-HCl
pH
7.6
1. Apparatus for incubation of lhypocotyl slices.
mersedl in 8 ml of 0.025 Mi Tris-HCI p)H 7.6, which
was thorotughly aerate( with ani air putlmp) (fig 1 B).
All systemis were kept for 14 houirs at 200 in the dark.
'rlie fifth pair were tightly call)e(l in weighing bottles for 2 hoturs at 20° in or(ler to (leterniine the
velocity of redutctioni of NAD.
The sixth pair were immiie(liately homiiogenized
wvilthout incubation for assay of coenizymiie as described belowsr. This value is designated as the zero
time value in table T.
.\t the enid of inctubation.
of the pair of hypocotvl slices in each of the first, second, fifth and
sixtlh systenms kept in a
plase x% as immiiiiediately
grotul(l wvith 2 ml of 0.1 N HCl in a lhomiiogeniizer at
9()° for 2 minutes. The other one of each pair was
treated in the same wxayvwith 2 ml of 0.1 N NaOH.
\\Vith the third anld fourth systemiis which had
been kept in aqueous soltution. I m1l of 1 N HCl was
adIled to 1 group of each pair ancd 1 ml of 1 N NaOH
to the other jtUst before homogenizationi at 900 for
2
miniutes.
The hoiiiogeniates were theni rapidlv transferred
to aln ice bath. Each holmiogenate was adjusted to
PH 7.6 and then 0.5 ml of 0.2 NM Tri,s-HCI, pH 7.6
w\as added. Tlhe total voltumiie of each homogeniate
was iiieastired and the homogeniate was tlhenl cenltrifuged at 10,000 X g for 20 niiiiitites at 30. Eachi
supernatant fraction was storel in the frozen state
180 ) for assav of coenzymne conicenitrationi.
Thle
oxilize(l and re(lutce(l formn.s of the coenzvmes w-ere.
resl)ectively, (letermillel ini the nieutralized acid ain(
alkaline tissue extracts, as rel)orte(l previously (6).
1c. 0li S1isp1e/lsioi.
5scIlicric/li(i oli 8(H1) was
gas
c
bouillon-pel)tone-agar niie(ditlu for 24 hours
at 370 aln(d theni collected and washed w,ith two 100miil portions of 0.35 Mt gluco,se.
'Theni the cells were
susl)enle(ld in 60 ml of 0.35 M1 glutcose. One ml of
this suspension contained 110 mig of )rotein, as calculated from the ODs at 280 anid 260 ni,ux.
grrown oni
Mcasurmceint of the C,,I/C1 Ratio. Twog (fr wt)
of slices of 3-day-old r ignia hypocotvls, 175 unioles
of potassium p)hospjhate pH 5.2 and( the ini(licate(l
amiiounit of NAD)P (table II) in a total voltume of I.0
ml were place(l in 6 Warburg flasks (w hich were
sepaarated ilto 3 p)airs). The first pair of flasks received no NADI) (control ruin) while 0.5 tiniole of
NADP was added to the second pair ail(1 1.5 pnoles
of NADP to the third pair. Each flask was evacutated alwd air was introduced. The proceduire w\as repeate(l twice miiore (vacuumii inifiltration) Faclh
flask was kept for 3 houlrs in a refrigerator (30) to
ensure that the NADP penetrated the tissues. 'Tlhen.
to one of eaclh pair of flasks 24 uiioles of gluicose and
5.0 ,moles (42,000 cpm ) of gluicose-1-14C wvere added,
while the other miemiber of the pair receive(l 24 ,moles
of glucose anld 5.0 ,jimoles (41,500 cpm) of glutcose-
6-14C.
The glucose was added immiinlediately before attaclhing the flasks to the mlaniometers. The flasks were
shaken at 300 for 200 minutes. The atmiosphere in
the flask was air and the cen-ter well contained 0.;
ml of 20 % KOH to absorb the respired CO.. At
the end of the experiments the KOH-K.,CO:
was removed and converted to BaCO., which was
then washed and (Iried and assayed for radioactivitv.
In all the experiments 14C was dletermined( by assay
of Ba'4CO0.
Results
According to an earlier report (6). 3-day-old bypocotyls from the germiiniatinig stage of [`igna seeds
fonrm NADP rapidly. Thluts, in the present work the
slices were p)repare(l from 3(lax 1-old hVpocoty 1s.
They- were incubated tilider various conlditionis as
showIn in table 1. 'T'he NAkl)P+NAkDPU- coniteiit
increase(l greatly in the hvl)ocotvl slices kept in air.
but inicrease(l olnly slightly in those kept tlul(ler initrogeni. A decrease in the colntenit was observe(l in the
systeimi immersed in water, but there was a striking
increase in the system kept in well-aerated water.
In general, hiigh NADPA+NADPH/NAD+
NADH ratios were observed in aerobic conlditionis
and low ratios in anaerobic conditionis.
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YAMAMOTO-NADP LEVEL,
521
AND OXYGEN TENSION
Table I. Effect of Gaseouts Contditions ont the Coenzyvnie Levels iit Vigna Hypocotyl Slices
The incu.bations were for 14 hours at 200. The data are expressed in mrwmoles of coenzymes per g fresh weight of
3-day-old Vigna hypocotyl slices.
Coniditionis
Zero time
In air
In N, gas
Immersed in water
In water + bubbling
Q
16
NAD NADH NAD+ NADP NADPH
NADH
0
1.09
1.15
6.48
6.48
0
2.43
2.43
4.37
3.47
2.08
1.44
2.41
3.71
6312
1.41
0.80
0.69
2.10
0.46
2.27
1.89
1.70
2.57
0.38
Xzz
22.5
R
13
*
2.
0~~~~~~~~~~~~~~
0
/NADPHI/NADP
12
NADP-NADPH
2.24
7.84
3.52
1.26
4.27
NADP +
NADPH
NAD+
NADH
0.35
3.23
0.58
0.60
1.88
NADH
X 100
±
NAD+
NADH
0
0
39.4
32.9
16.7
NADPH
NADP
1.06
1.26
0.69
0.58
1.51
An E. coli suspension was used inistead of hypocotyl slices because it was easier to prepare homogeneWVhen the E. coli suspension
ous samples from it.
was vigorously bubbled alternately with air and with
nitrogen for 10-m-inute periods, the NADP+NADPH
level increased during the air bubbling and decreased
during the niitrogen bubbling and the change in the
level was rapid, as shown in figure 2. The NADPH/
NAI)P ratio increased in aerobic conditions.
The exogenous NADP infiltrated into Vigna hypocotyl slices in vacuo lowered the C6/C1 ratio, as
shown in table II.
Discussion
Time (minutes)
FIG. 2.
Changes in the NADP±NADPH level and
the NADPH/NADP ratio in E. coli B(H) under different gaseous conditions.
Table
II.
on
NADP added
,umoles
0
0.5
1.5
I
Effect of E.rogentoins NADP
4C-Glhcose Dissimilation
Glucose-6-14C
% Yield
2.35
2.38
2.58
Glucose-1-14C
% Yield
2.84
3.52
4.08
CQ/C,
0.83
0.68
0.63
Under anaerobic conditions the 100 X NADH/
NAD±+NADH ratio increased by about 40 %. A
very high velocity of NAD reduction was observed
under anaerobic conditions. When the slices were
tightly capped in a weighing bottle for 2 hours at
200, 75 % of the NAD was reduced. In general, a
low NADP + NADPH level corresponded with a high
100 X NADH/NAD+-NADH ratio. The NADPH/
NADP ratio was several times higher under aerobic
conditions than under anaerobic conditions.
Before evaluating the physiological meaning of
the variation in the NADP+NADPH level with the
gaseous conditions, the rate of change of the
NADP + NADPH level with change in the gaseous
conditions must be determined.
Potter and Niemeyer (4) observed that exogeNADPH acts like NTADP in modifving the glycolysis of animal tissues. The NADP+NADPH
level in bean hypocotyl slices varied with the O2 tensioin. A high NADP+±NADPH level was observed
tunder aerobic conditions and( a low level under anaerobic conditionis. The change of tlle NADP+
XADPH level in E. coli closely followed the change
Exogenous NADP inin the gaseous conditions.
filtratedl into bean hypocotyl slices in vitro lowered
the C,/C1 ratio. This decrease suggests an increased
contributtion of the pentose phosphate cycle to respiratory metabolism. Potter and Niemeyer (4) have
noticed that, in animal 'tissue, the variation of
NADP + NADPH level mav play a role in the
Pasteur effect.
The NADP+NNADPH level in hypocotyl tissues
is determined by the activities of the NADP synthesizing system (NAD kinase) and of the NADP
decomposing system. As reported in the next paper
(10), plant NAD kinase catalyzes NADP formation
from NAD and( ATP, but unlike yeast NAD kinase it
does not catalyze NADPH formation fromi NAD)H
and ATP. Reduced NAD was a very potent competitive inhibitor of NADP formation by plant NAD
kinase. Accordingly, in tissue in wihich reduced
NAD is accumulated, i.e., in anaerobiosis, NADP
formation ceases. The reduction of NAD in anaerobiosis was found to be very rapid (cf. ref. 3).
On the other hand, the rate of decomposition of
the NADP component will be independent of the
nous
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522
PLANT
coniditions. NAD kinase does Inot catalyze
the (lecomiiposition of NAI)P to NAD (9, 10). Three
kin(ds of enzymies. nutcleotidles phosphatase (1), Iiticleoti(le )}yrophosphatase (EC 3.6.1.9.) (2) and
NAD)ase (EC 3.2.2.6) (cf. 1) are known to be reslponsil)le for the decomiposition of NAD)P in plants.
Nticleotide phosphatase and niucleotide pyrophosphatase are highly active in decomposing NADP andl
NAD)PHf1. Accordingly, the rate of decompositioni of
the NA1)P componenit is believed to be almost independent of its redox state or the gaseous coin(litiois.
T'hluts slowving (lown of NADP formation tinder aniaerobic coin(litions resuilts in a lowv NADP level. Table
I shows that a low NADP-F NAI)PH level corresgaseous
l)onllds
with
a
high
100
X
NADH/NAD
-1- NADII
ratio.
Inl gerniunating
1
yigna
root,
NAI.)
was
in the
iimore
thaln in the utl)er
oxi(lize(l state in the tip portion
p)ortion. AIhigh NADP + NADPH concenitration
has been observed in the tip (6). The correlationi
between the redox state of NAY) anid the level of
NA.-\P)1'+ NAD PH in the root is also explained bv the
prol)erties of plant NAD kinase.
Literature Cited
1.
FoirI, G., C. TOG;NOI, AND B. PARISI. 1962. Purification from1 pea leaves of a phosphatase that atBiochimi. Biophys. Acta 62:
tacks nucleotides.
251-60.
PHYSIOLOGY
2. KORNBERG, A. AND W. E. PRICER, JR. 1950. Nucleotide pyrophosphatase. J. Biol. Chem. 182:
763-78.
3. LYNEN, F., G. HARTMANN, K. F. NETTER, AND A.
SCHUEGRAF. 1959. Phosphate turnover anid Pasteur effect. In: Ciba Foundation Symposium,
Regulation of Cell Metabolisnm. G. E. W. Wolstenholme and C. M. O'Connor, eds. Churchill.
London. p 256-76.
4.
POTrER, V. R. AND H. NTIEMEYER. 1959. Role
of
triphosphopyridine nucleotide in the regulation of
glycolysis in a cell-free preparation. In: Ciba
Foundation Symposium, Regulation of Cell Metabolism. G. F. WV. Wolstenholme and C. M.
O'Connior, eds. CChurchill, London. p 230-55.
5. YAMAMOTO, Y. 1961. A metabolic pathway of isocitric acid in cotyledoins of a bean, Vtigia sesqniipedalis. Plant Cell Physiol. 2: 277-89.
6. YAMAMIOTO, Y. 1963. Pyridine niucleotide con1tent
in the higher plant. Effect of age of tissue.
Physiol. 38: 45-54.
Plaint
7. YAMAMOTO, Y. 1963. Enzymological studies onl
seed germination. Advancinig Frontiers of Plant
Sciences 6: 163-86.
8. YAMAM1OTO, Y. 1964. A mechanism of control of
respiration. NAD kinase in higher plants. Planit
Physiol. 39: lxi.
9. YAMAMOTO, Y. 1965. Role of nicotinamide adeniime dinucleotide phosphate in the regulation of
respiratory nmetabolism (in Japanese). Vitamitis
31: 343-55.
10. YAMAMOTO, Y. 1966. NAD kiniase in highler
plants. Plant Physiol. 41: 523-28.
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