Carbohydrate Metabolism of Avocado. II. Formation of
Sugars During Short Periods of Photosynthesis'
R. C. Bean, G. G. Porter, & Barbara K. Barr
Department of Biochemistry, University of California, Riverside, California
M\ithough inannolheptulose andI perseitol have i)een
known as major constituents of avocadlo sugars for
matin years (14) no mlechanismi ftor the synthesis or
accumulation of these seven carbon sugars has been
dlemonstrate(l. Recently miianniolheptulose has been
foun(l to be miiore wi(lely distributed than previously
b)elievel, having been found in leaves of figs (4)
andi alfalfa (15)). Olther lheptoses andl heptuloses
(5, 7. 8. 10. 18) as Nvell as octuloses, octitols. an(l a
nonulose (5, 16, 17) have also been isolated froml
biological miiaterials. Thus, the syntlhesis of these
less commoilnoi sugars has beconie of somilex-lhat wvider
interest in mletabolic investigatioins.
lFracer elxperimnents 1by Nordal aan(l lBeiison (14)
with photosynthesizing avocadlo leaves estai)lished
that a phosphate ester of mannol)eptulose x-as formed
(luring early photosynthesis. This was confirmed
in this laboratorv` (3 ) vith the additional (lemonstration tllat mannolheptulose appears concurrenitly wN-ith
sucrose (duriing photosynthesis in avocado lea-es.
l'erseitol. in these experimiients. seemiie(l to he a
seconi(larv plroduct, (lerive(l fromii mannolheptulose.
ReId(lig and(t MAlcComb (13. 15 ) fe(l various sugars
to alfalfa in attempts to eluci(late tlle miiechanisilm of
Iimantiloheptuilose synthesis. Thex apparently didl not
find(l significant increases in mlaninolheptulose but sonme
sugars gave rise to new lhepttuloses, prestumably by
traanisketolase aIlCI transaldolase r-eactions.
Since mannolheptulose appears to be a nmajor
photosynthetic l)rodluct in avoca(lo leaves, a kinetic
study of the lablele(d intermie(liates in short period
photosynthesis w-ith C' 40. miiighlt aid in eluicidlating
the mechanism of synthesis. This paper reports the
results of studies in avocadlo leaves u.sinig photosynthetic exposure I)eriodls of 10 seconds to 16 nlinutes.
Material & Methods
As in previous work (3 ), freshly picked leaves
from avocado trees (Persea gratissinin Gaertn., var.
Bacon) were used in all experimlents. Disks, 1.1
cm in (liameter, were cut from the leaves un(ler watter
for utse in the photosynthetic chambers.
An apparatus sinmilar to that in figure 1 (differing mainly in having four rather tlhani six leaf
chambers) was constructe(d for simlultanieous exposure of several sample (lisks to identical atmos-
Received Aug. 10, 1962.
280
'U
/
FI(.. 1. Photosyinthetic apparatus. TIhe miiain bodv
of the photosynithetic apparatus (A.) was miade from
plexiglass sheet, (Irille(i for flow passages ani(i the sample
chamiiber areas (B). The sample chambiher caps (C)
were plastic vial caps xx ith a w ire platform to hold the
leaf (lisk sample. Not used in the experiments reported
here xvere the modifiedlcaps (Cc) for continutous flowx.
The caps, containitng the samples wvere firmly heldl in
pllace wvith the adjustable spring clips (D) agaiinst tile
rubber gasket (E). The entrance passages ( F) wxerc
openled by pulling the p)ullgers out so that tile ring sea]
gaskets ,\-ere past the chamlller inlets (H). Tile chamber
venits (J ) x-ere uilplugged so thlat a gentle flowx of air
couldi be l)assed through all the cliamllbers durinlg the preillumilnatiolls period by applying a slight vacuum at the
3-xway stopcock (K). C140., was generated in an evacuated tube attached to the other arnm of K. After 40
mlinutes pre-illumination, tile cliamber vents wvere replugged and( a vacuunm (half an atmosphere) drawn upon
the cliambers monleltarily. The stopcock (K) was
turned to the C1'4.Q tube am(I then a secoind stopcock oIl
the generating system wvas opened to the atmlospiere to
allow air to sweep the C'4Q., into the exposure chambers.
After the proper exposure timies (miniimum of 5 sec)
each sanmple in turn was renloved by closing the valve
for a givenl chamber (ring gaskets on eaci side of cliaulber entry) and the cap, with sample, simply pulled away
from unmder the clip and the sample dropped quickly into
boilimng alcohol. The closinig of each chamber before
saniple removal allowed different tinmes of exposure for
each sample xxithout disturbing the remllainimlg samples.
The lamp was niormally maintained at 12 inches from
the chambers. A heat absorbing unit, consisting of
thermal glass inlnlersed in a circulating water bath was
used between the lamp and the chambers.
BEAN ET AL.-CARBOHYDRATE METABOLISM OF AVOCADO
pheres with provision for renmoving one sample at
a time rapidly without (listurbing the remaining
samples. Samples were placed in the four chambers
and pre-exposed to the light (luring a period of 40
minutes with a stream of air flowing through the
apparatus. Experiments using the Liston-Becker
Infra-Red CO, Analyzer, Model 15, to measure
photosynthetic rates established that 25 to 30 minuites
of illumination was required to attain maximal photosynthesis and steady state. A General Electric
Projector Floo(d lailip (150 wv) provided illumination
at a level of 2500 to 4000 ft-c. Thermlal glass immersed in a circulating water bath was used to absorb the infra-re(d radiation.
Following the exposures to the radioactive atmosphere for periods of 10 seconds to 16 minutes. the
samples were killed in boiling alcohol and extracted
as previously described (1. 3). Counting procedures
and general chromiiatograplhic prccedures have also
been described (1, 2, 3).
The organic moieties of phosphlate esters were
identified by elution of the chromatographic spots
and treatment with alkaline phosphatase followed bv
rechromatography. In some cases, the ester spots
were treated directly with phosphatase on the paper
and then eluted directly onto the second chromatographic sheet by chromatographic transfer.
Rechromatography was required to obtain complete resolution of some of the original components.
Glucose, mannoheptulose. and glycine were coincident
on the original tAvo-dimensional chromatogranms.
These w-ere resolved by elution onto a second filter
paper strip (chromlatographic transfer) and developing w-ith ethyl acetate-pyridine-water (8: 2: 1).
This moved the imannoheptulose and glucose away
fromii the glycine. w-hich renlained at the origin in
this system. The glycine spot was cut off and the
glucose in the glucose-mannolheptulose area was oxi(lize(l to gluconic acid by treating the paper with
fungal glucose oxidase. Development once more
x-ith the ethy-l acetate-pyridine-water solvent separate(d the gluconate and mannoheptulose.
Mlost of the confirmatory identification procedures
for various sugars (in addition to the chromatographic positions) have been presented in previous
papers (1, 3). Oxidation by fungal glucose oxidase
was used as the criterion for glucose identification.
Decarboxylation with ninhvdrin was used for amino
acid confirmation (2). Since organic acids were
characterized only by chromatographic positions in
the usual solvents (water-saturated phenol, butanolacetic acid-wvater) and by rechromatography in pentanol-5 M formic acid (1: 1), their specific identifications are only tentative.
Results
The incorporation of C14-activity into gross fractions of the avocado leaf (lisk is represenited in figure
2. The total quantities of activity fixed by the leaves
varie(l as much as fivefold in the various experiments
281
but the general pattern of uptake remaiinedl very much
alike in all five experiments.
A schematic diagram of the positions of the nmajor
labeled components in the extracts as they appeared
on the chromatograms and radioautograms is given
in figure 3. Despite a careful search for labeled
comiiponents which might give qualitative evi(lence
for new synthetic pathwvays, very few- clues could be
found as to the intermediates in mannoheptulose formation. AMannoheptulose was positively identified
as a phosphate ester overlapping with glucose monophosphate. No evidence could be obtained for labeling of a perseitol phosphate, free perseitol. or other
polvols in these short period experimlelnts. Nor was
it possible to detect activity in significalnt amounts
in pentose phosphates other than ribose and ribulose
although a searclh wvas specifically- made for arabinose5-phosphate which might be a potential acceptor in
a cis- condensation of an abnormal transketolase reaction. No other sugar phosphates were discovered
w-hich mliglht logically be construed to act as intermlie(liates in novel paths leading to the unique conlfigurations of the avocado sugars.
20
it)
16
0
E 12
Qu
.4
0
8
4
0
2
4
6
Time (min)
8
FIc;. 2. Assimilation of C140., in avocado leaf disks
during photosynthesis. 0, Total activit; 0, alcolholsoluible activ it; X, alcohol-insoluble activity.
Some unusual components did appear amiiong the
products in these experiments. In the phosplhate
ester area. component eight (fig 3) was found to contain glucose but it does not corresponcd chromiiatographically with the known phosphate esters. nucleotides. or cyclic phosphates of glucose. This compouncI xwas not found in all experimenits. however.
It is also notable that glucose, rather thani fructose,
ribulose, and sedoheptulose, seemed to be the major
labeled component of the (liphlosphate area altlhough
the others were present.
28
PHYSIOLOGY
PLANT
Table I
I)ifferem-t's betzetent Bean Leaves & .Aocaado Leaves inl
.c- tii itv Distributions after Short Ieriods of
Phjotos,nthjCesis inl C'4O.
Leaf (lisks x-ere exposed and treated as (lescribed ill
21
c~~- J
I
the text. Activities represenit the totals for all sugar
phosphate ester areas by direct count oni clhromiiatogramis of one tenthl of enitire extracts.
or
20
I8I
._
I'7
Plhoto-
16
Sample
CED 1 5
14
Beani leaf
Avocado leaf
Bean leaf
Avocado leaf
13
11
3:
12
syn-ithesis
perio(l
seconds
10
1()
20
20
Act ivi ties
Free
Phosphate
esters
sugars
Cplml
cpml)
150
1140
8810
342 0
14900
44150
400
3440
M
^Le)
c1
17 w22
.
- ~-
'¢zi
-- PHENOL
A
.
.
. -
.
-
-
-
Uw,G. 3. Diagramii of chromatographic positions of
labeled comiiponents in avocado phiotosynthesis in C14O.
Chromatogramiis were on Schleicher and(l Schull No. 589
White Ribboin filter paper developed first in watersattirate(I phenol anid theni twice in butanol-acetic acidwater
52 13 33). 1, (lil)hosphates, iaiinly glucose
with
frtuctose,
ribulose,
sedoheptulose;
aind(
manino-
2,
lieptulose (mno ) phosplhate; 3, glucose nmonopliosphate;
4. se(lolheptulose an(l glucose nmonophosphates: 5, fructose
and g-lucose monophosplites; 6, pentose phosphate; 7,
,glucose and(I galactose phosphates (p)rohal)ly inucleotides)
8,
g-lucose conItaininlg
)..U) ><
Q)
Q
I
I
00
<
p)hosp)lhate
appearing onlly ill sonic
aci(l
10, sucrose: 11,
g!ncose, mannohleptulose ani
0
saml)le series; 9, phosphoglvceric
serinite aIIn(d perseitol; 12,
-lycine: 13.) sedohleptulose 14, fructose: 15, alaninle; 16,
netutral, possibly ribulose 17, glvccric acidi ; 18, milalic
aci(ld 19, citric acid; 20, sUccinlic aci(l; 21, lactic acid
22,
iniidntiiled nieutral comipounds.
4-
A\ ntullmber of neutral coml)onents
ere found(I
d(1id nlot coinlci(le wx ith compounds usually
0
w
which
labeled (lurinlg photosynthesis in otlher plants. Spot
16 ( fig- 3 appears to coinici(le with free rihulose.
se(lol)epttilose
Free
was
(lefinitely i(lelitifie(d
plro(ltict of early1 photosvnthesis
\
as
a
number of slower
111mving
neutral comilponielnts, in low\- coIncentration
activitv, remain unidentified hut may corlresl)ond(
eithier With oilonger carbon chalini ketoses or oligosaccharides.
of
the incorporation of activity inito
the varmsi,) free stigar components (ltiring
se(quenlce is presente(l in figure 4.
a
timle
rapid labeling
fotnII(l.
similar
suIlts
of
tVl)ical
A
very
Assimiilation of C140., inito free stigars ot
the avocado (lurinig short period photosynithesis.
sucrose (ctiurxe 1)
X, mannolheptulose (curve 2)
fructose ( curve 3) A, glucose (cuirve 4) O, sedolleptu-
en
that the ratio
of activitv
in
esters
T
ill
( curve 5).
lose
ocaldo leaf to actix itv inI phosphate esters is
rather high in comparison wN-ithi the proportions found
in other leaves (lurilig short perio(ds of photosynthesis.
ax
This is emphasized in table I which gives conipariof total sugrar atctivit \activity
phosphate ester
for beall leaf (lisks anl( for avoca(lo leaf (lislk-s after
10 or 20 seconids p)hotosy nthesis In t '()
\
i-tl
ItAlo
COli(litiOlis Wx ere identical in these
\xpelrlim'entts. th(
a
free sugars, partictilarlyv ketoses, is
Thle labeling of the phosphate
in figure 5.
experiment is giv
Show
Time (min)
FI(;. 4.
of
analvslis
AnI
16
8
aI
e..e re-
frlee stigar.1s
son
BEAN ET AL.-CARBOHYDRATE METABOLISMI OF AVOCADO
9
6
0~~~
0
x
40
| Seconds
0
4f
Time (min)
FIG. 5. Assiniilation of C1402 into the phosphate
esters of avocado leaves during short period photosynthesis. A, phosphoglyceric acid; 0, diphosphates (mainly glucose); 0, glucose monophosphates; El, sedoheptulose monophosphate; X, mannoheptulose monophosphate;
o. fructose monophosphate; A, nucleotides.
sugar activity in heans anmounts only to about 2 %
of the phosphate ester activity while the free sugar
activity in avoca(los amiiounts to nmore than a third
that of the phosphates.
Discussion
There are a nuiiiber of wvays in which mannoheptulose imiight be formie(d during photosynthesis making
use of variationvs of known reactions and common,
or plausible intermiiedliates. Among these would be:
1. an abinoriiial transaldolase or aldolase reaction
foriining the heptulose with a cis- rather than the
norimial trains- coinfiguration at the point of condensation (9, 19. 20) 2. a trans- rather than normal
cis- configuration resulting from a transketolase condensation: 3. (lirect epimerization of sedoheptulose
or its phosphate ester, at C-4 as suggested by Venkataraman and Racker (20); 4. reduction of sedoheptulose at C-2 (giving the volemitol configuration)
with re-oxidation at C-6 pro(lucing mannoheptulose
with C-1 equivalent to the original C-7 of sedoheptulose. The original intent of this study Nvas to
determine whether a kinetic examination of the
detectable, early products of photosynthesis miglht
283
indicate a choice between these mechaniismiis. It was
found that mannoheptulose phosphate, discovered in
earlier experiments (3. 14), is fornmed very early in
the photosynthetic sequence, although in low quantities. Extrapolation of the mannioheptulose phosphate
labeling curve would indlicate that it is formiied either
simultaneously or very shortly after the other ketose
phosplhates. No evidenice could be found for isotope
in any of the prodlucts w hiclh might be associated
with the alternate re(luction and oxidation of opposite
endls of se(loheptulose so that the inversion pathway
miglht be considere(d of very low probability. Althouglh somiie ainomiiolous phosphate esters were present in somiie experimlenlts. thlese would not seem to
have any apparent specific bearing upoIn the synthetic
mechanissm. No (lata were obtained whliclh woul(l
enable clhoice betw-een any of the other proposed
mechanismiis.
The kinetic (lata her-e in(licate that Nor(lal and
Benlsoil (14) may have been prophetic in their proposal that mialnnolheptulose was formed from normiial
interme(liates alnd miight accumiiulate in avocados due
to an active mlanlnolleptulose phosphate plhosplhatase
and/or a sluggislh kinase. MVannoheptulose phosphate appears after only a few second(s of photosyinthesis alonig with the normiial phosphate esters.
The ketoses. fructose. se(lolheptulose. mlannlolheptulose.
and probably ribulose. appear almost simiiultaneously
with their correspon(ling phosphate esters. A number of factors suggest that this early labeling of the
unphosplhorylate(d ketoses iay be normal within the
avocado leaf andl associate(d with the accumliulation of
mannoheptulose. It does not seenm likely that the
rapid hy(lrolysis of the plhosplhate esters could be
sinmply an artifact of procedure. No simlilar hydrolysis of photosynthetically labeled phosplhate esters was
detectedl in leav-es frollm a number of other plants
(pinto Ibean. soy bean, sugar beet, barley) subjected
to idcentical proce(lures of exposure. killing, extraction, andl chromatography. Other experimiients show
(unpublishedl) the phosplhate esters formiied in avocado leaf disks frolmi labele(d sugars have little tendency to hydrolyze unider simiiilar conditioils of processing mlaking it unlikely that an unique condition
of the avocado leaf is responsible for the ester hydrolysis. Instead, it -ould seeml mlore likelv that
the rapid appearanice of the unphosphorylated sugars
may be due to the action of a phosphatase during
photosynthesis. The higlh activity of the ketoses,
relative to the activity in glucose, even wvhere glucose phosphates may pre(lominate, suggests that the
phosphatase might be specific for ketoses.
The pools for sedoheptulose andl for fructose appear to saturate rapidly (fig 4) wlhile that for
mannoheptulose continues to accumiiulate activity on
a linear basis over a long period (3). This suggests
that there may be a rather close equilibriuml between
the reactions for producing and re-utilizing the fructose andl sedoheptulose wlhile mlannoheptulose is poorly re-utilized. A kinase. specific for the C-3. C-4
configuration of fructose and of sedoheptulose, lo-
284
PLANT PHYSIOLOGY
cated in close proximity to the phosphatase coul(d satisfactorily account for the observedl behavior. Thus,
the basis for mannoheptulose accumulation in avocado
leaves might be the conlibine(d action of a phosphatase,
giving rise to all ketoses rapidly, anid a specific
kinase which would return fructose and sedloheptulose, but not mannoheptulose, to active metabolic
pool S.
Previous studies (3) shoxwe(l that fructose coIntinue(l to accumulate activity at a slow rate (lurinig
photosynthesis but (lark experiments made it apparent that the fructose wvas being formiied at the
expense of labeled sucrose rather than fromn hydrolysis
of photosynthetically forlmle(d fructose plhosplhate.
Thts, it is clear that two (listiiuct fructose pools are
involve(l in the metabolismii of the avocado leaf. One.
a very small pool, rapidly labeled (lIuing plhotosy-snthesis, lalV be located -within thle clhlor-oplast. The
secondI pool, (lerived fromii the splittinlg of sucrose.
Vouldl probably be in the cytoplasmi or vacuole. The
dark accutmiulation of activity in fructose xx-as not
observedl in somiie other plants (3). Thuus fructose
in the latter pool miay be subject to the same sluggislh metabolismimiaui fest for manaohlleptulose. This
is in shliarp contrast x-itlh the al)parent rapi(l re-uitilizationi of fructose produced in the h.9hrt perio(ds of
photosynthesis.
Summary
The assimiiilation of C"'O. inlto sugars and plbosphate esters in avoca(lo leaf (lisks dutrinlg photosnllthetic periods of 10 secoind(s to 16 mliinutes has beeni
studied. The labeling of mannolheptulose phosphate
after a slholt photosynthetic perio(d was dem]onstrated.
No evidence for perseitol phosphate as foulnd(l.
IFructose, sedlolheptulose, ilmannohleptulose, and
possibly ribulose in their- unphosphorylated formli becomiie labeled in avocado leav-es verv early but the
pools of fructose and se(lohel)tulose appealr to satutrate
rapidlyvrwhile miiannolheptulose alnd sucrose )ools conItinue to accumilulate activity at a linear rate over a
long, periodl of time.
A imiechlaniislml for the accumutilation of miannloheptulose based oln a phosphatase specific foI- ketose
esters ;and a kinase specific for fruictose and( sedo-
lheptulose coinfigulrationi
is
suiggested.
Literature Cited
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I).xAVIwS D. A.
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