-
J. Cell Sci. ia, 725-739 (i973) 725Printed in Great Britain
EFFECT OF ANAEROBIOSIS ON RESPIRATORY
RATE, CYTOCHROME OXIDASE ACTIVITY AND
MITOCHONDRIAL STRUCTURES IN
COLEOPTILES OF RICE (ORYZA SATIVA L.)
HELGIOPIK
Department of Botany and Microbiology,University College of
Swansea, Singleton Park, Swansea SA2 8PP, Wales, U.K.
SUMMARY
An attempt has been made to correlate respiration rate,
cytochrome oxidase activity andmitochondrial structure in
coleoptiles of rice, Oryza sativa L., germinated under aerobicand
anaerobic conditions. The rice coleoptiles emerge from the grain
and elongate considerablyeven under complete anaerobiosis, which
totally suppresses root and leaf growth. Cell number,dry weight and
nitrogen content per coleoptile are all lowered, although some cell
divisionand translocation of reserves into the coleoptile does take
place under anaerobiosis. Comparedwith coleoptiles from air-grown
seedlings, anaerobically grown coleoptiles have a much
lowercapacity for respiratory oxygen uptake and their cytochrome
oxidase activity is depressed evenmore. Mitochondria, however, are
still abundant in 4-day-old anaerobic coleoptiles, with acrista
density only slightly lower than in cells of aerobically grown
coleoptiles. Since, in theembryonic coleoptile of the ungerminated
grain, mitochondria show very little internal structure,a
considerable amount of elaboration of mitochondrial structure must
occur in the rice coleop-tile under anaerobiosis, contrasting with
the situation in yeast, where mitochondria of normalstructure are
formed only in aerobic conditions. Since a high crista density
develops in ricecoleoptile mitochondria with a very much depressed
cytochrome oxidase activity, there is noobligate correlation
between crista density and cytochrome oxidase activity in this
tissue.
INTRODUCTION
Higher plants are obligate aerobes and although many tissues can
survive limitedperiods of anaerobiosis, growth is generally
inhibited. Some species, however, areable to germinate in the
absence of oxygen, and rice, Oryza sativa, is one suchexample,
germinating normally in mud with very poor aeration, and its
coleoptile cangrow to a length of several centimetres even under
complete anaerobiosis.
In 1962-4 it was reported that in the facultatively anaerobic
yeasts Torula utilisand Saccharomyces cerevisiae, no mitochondria
were formed under anaerobic condi-tions but appeared rapidly on
aeration (Linnane, Vitols & Nowland, 1962; Wallace&
Linnane, 1964). Continued research on yeast has revealed a more
complex situa-tion: even under anaerobiosis, there is a formation
of at least morphologically verysimple mitochondria (Criddle &
Schatz, 1969; Damsky, Nelson & Claude, 1969;Watson, Haslam
& Linnane, 1970), detectable when special precautions are
takenduring fixing and isolation. But the anaerobically formed
yeast organelles are unableto carry out normal mitochondrial
oxidations, lacking cytochrome oxidase and
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726 H. Opik
possessing other mitochondrial enzymes in abnormally low
concentrations. Clearlyoxygen tension is a very important
controlling factor for the development of mito-chondrial enzymes
and normal mitochondrial structure in yeasts. It seemed thereforeof
interest to investigate how far this is also true of the rice
coleoptile, a higher plantorgan which can be considered to be a
'facultative anaerobe'. Accordingly growth,capacity for aerobic
respiration, cytochrome oxidase activity and mitochondrialstructure
have been compared in coleoptiles of rice germinated in air or in a
hydrogenatmosphere. The results indicate that although anaerobiosis
strongly suppresses thedevelopment of a capacity for aerobic
respiration and cytochrome oxidase activity,the structure of
mitochondria in rice coleoptiles maintained under anaerobiosis
ismodified only slightly.
MATERIALS AND METHODS
Plant material
Two rice varieties, Italpatna (from Italy) and Kakai (from
Hungary) have been used; theirphysiological behaviour and cell fine
structure showed no significant differences, but in anyone
experimental series one variety only was utilized.
Growth conditions
Plantings were carried out in a sterile cabinet. The rice grains
were surface-sterilized fori min in 70% methylated spirits,
followed by 25 min in sodium hypochlorite (3-5 %
availablechlorine), containing 05 % of Tween 20 as wetting agent.
The grains were then rinsed with12 to 15 portions of sterile water
and planted in batches of 50 to 60 in sterile glass dishes,9 cm in
diameter and 4-5 cm deep, containing a double circle of filter
paper and 10 ml sterilewater. The dishes were covered with Petri
dish lids and incubated at 27 °C in the dark usinga green safelight
(Ilford bright green 909) for handling. Anaerobic conditions were
achievedin a hydrogen atmosphere in microbial anaerobic culture
jars (Gallenkamp, modified Mclntoshand Fildes pattern),
accommodating up to 3 culture dishes. Each jar contained a
test-tubeof 10% KOH to prevent accumulation of carbon dioxide, and
a redox indicator (Cruikshank,1965) to check against air leaks.
Sampling
At each age, plants within a chosen length limit were sampled.
For instance, after 3 days ofgrowth in air, when most of the
coleoptiles measured about 8 mm, only those with lengthsbetween 7-5
and 85 mm were taken; these are referred to as '8 mm long', while
'14 mm'samples from 5 days under anaerobiosis included a length
range of 12-16 mm. For physio-logical experiments the leaf or leaf
primordium was removed after slitting the coleoptile witha sharp
blade.
Dry weight and nitrogen content
The coleoptiles were dried at 95-97 °C for dry weights, and the
dried samples (80-200coleoptiles) were then subjected to
micro-Kjeldahl digestion and steam distillation of theammonia,
which was collected in 2% boric acid and titrated against standard
001 N acid.
Cell number
Coleoptiles were frozen at — 20 °C and if necessary stored at
that temperature. Afterthawing they were vacuum infiltrated with
the macerating fluid, mixed from equal volumes of10% chromium
trioxide and 10% nitric acid (Purvis, Collier & Walls, 1966),
and incubatedat 27 °C. The cells were dispersed by shaking on a
microid shaker at top speed. Aerobically
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Anaerobiosis in rice 727
grown coleoptiles needed 16-18 h of digestion followed by 1-5
min shaking, while the morefragile anaerobic coleoptiles were
digested for 6-15 h and shaken for 20 s to 1 min. The cellswere
counted in a haemocytometer; from each suspension at least 5
aliquots were taken anda minimum of 600 cells scored.
Respiration rate
Standard Warburg manometric technique was employed, using 15-ml
capacity flasks, eachcontaining 25 coleoptiles laid on 'Kleenex'
paper tissue moistened with 0-25 ml distilledwater. Four flasks
were set up per experiment, 2 with 10% KOH in the centre wells and
2without, to obtain both oxygen uptake and carbon dioxide output.
The temperature wasmaintained at 30 °C. After 15 min equilibration,
readings were taken at 15-min intervals for1 h. At the end, the
coleoptiles were removed from the flasks for dry weight and
nitrogendeterminations.
Cytochrome oxidase
Samples of 25 coleoptiles were tied in moistened muslin sacs for
ease of handling and frozenat — 20 °C in tightly stoppered 2x3-5 cm
specimen tubes. Unfrozen coleoptiles gave verylittle reaction
except at cut edges, presumably due to failure of the reagent to
penetrate, butvariation of the period of freezing from 1 h to 16
days made no difference to activity. Thesamples were allowed to
thaw for 30 min at 27 °C. The reagent (Moraczewski &
Anderson,1966) was freshly prepared during this period, by
dissolving 10 mg of p-amino-diphenylamineand one small drop of
8-amino-i,2,3,4-tetrahydroquinoline (Sigma, practical, grade II)
in05 ml absolute ethanol, adding 35 ml distilled water and finally
15 ml of 02 M Tris buffer,pH 7-4. Filtered 10-ml portions were
added to the coleoptile samples in the specimen tubeswhich were
incubated unstoppered for 1 h at 27 °C in the dark. The coleoptiles
were trans-ferred to 10% ammonium molybdate for 30 min, rinsed
quickly with distilled water andblotted dry, and the blue reaction
product was extracted into 6 ml absolute ethanol, in a ground-glass
homogenizer with a power-driven pestle. The extract was cleared by
centrifugation atca. 2500 g for 6 min, the supernatant made up to 7
ml, and the optical density at 560 nm wasread immediately in a
Unicam SP 500 Series III spectrophotometer. Exposure of the
reagentor extract to bright light was avoided. Since the extracts
still showed a faint opalescence dueto very fine tissue debris,
tissue blanks were prepared with the coleoptiles incubated in
Trisbuffer instead of reagent, and the optical density reading of
these blanks (never exceeding10% of the experimental readings) was
subtracted from readings. The blank correction wassometimes omitted
from experiments involving replicate samples of identical
coleoptileswhere the corrections also would have been identical for
all.
Peroxidase and polyphenol oxidase can also react with the
reagent. Cytochrome oxidase is,however, much more heat-sensitive
than the other enzymes (Burstone, i960; Perner, 1952).The reaction
of the rice coleoptiles is very heat-sensitive, indicating it is
mediated by cyto-chrome oxidase.
Electron microscopy
Fixing and dehydration were carried out at 4°C; all plant
material was chilled beforeexcision, anaerobiosis jars being kept
in the cold for at least 30 min before being opened
forsampling.
Ungerminated or imbibed embryos, and coleoptiles up to 15 mm
long were fixed in glutar-aldehyde, 3-6 % in phosphate or
cacodylate buffer, 005 M, pH 7-2-7-4, for about 12 h, washedfor
2-12 h with several changes of the buffer containing 015 M sucrose,
sometimes storedovernight in the washing solution, and postfixed in
2 % osmium tetroxide in the buffer withsucrose for 3 h. After
dehydration in an ethanol series (occasionally acetone), the
specimenswere embedded in a mixture of n-butyl methacrylate 7 parts
and styrene 3 parts by volume,with 2% benzoyl peroxide as catalyst
(Mohr & Cocking, 1968). Some specimens were alsoembedded in
Araldite or TAAB Laboratories Epoxy resin; for this the specimens
were trans-ferred to epoxypropane after dehydration and resin was
added gradually over 5 days, before
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728 H. Opik
embedding in capsules. The methacrylate blocks, being easier to
section, were used for mostof the routine examinations, but the
more stable Araldite and TAAB resin blocks were usedfor some
high-power observations.
Once the coleoptiles had elongated beyond a few millimetres,
however, they became ex-tremely difficult to prepare for electron
microscopy. The final procedure adopted for oldermaterial involved
fixation for 3 h in a mixture of glutaraldehyde (final
concentration 2-5 %)and osmium tetroxide (final concentration 1 %)
in collidine buffer, 005 M, pH 72, with015 M sucrose. After a quick
buffer rinse, dehydration and embedding followed as beforeexcept
that the absolute ethanol contained 1 % uranyl acetate; treatment
with this lasted 2 h.Even with this procedure many cells from
coleoptile regions beyond the apical 1 mm weredamaged.
Sections with silver interference colours were cut on an LKB
Ultrotome, and post-stainedeither in 1 % aqueous uranyl acetate for
90 min followed by 8 min in 02 % alkaline leadcitrate (Venable
& CoggeshaJl, 1965); or if uranyl acetate had been applied
during dehydration,in lead citrate only. Either before the
collection of sections, or after post-staining, the gridswere
coated with a thin collodion film. Specimens were examined in an
AEI EM6 or EM6Gelectron microscope at 60-80 kV, or in an AEI
Corinth model at 60 kV.
Estimation of mitochondrial cross-sectional area and crista
density
For the first estimations on very young coleoptiles a planimeter
was used to measure theareas of mitochondrial profiles; each
mitochondrion was measured 3 times and the measure-ments averaged.
The cristae were counted on the same micrographs. Planimetry being
aslow and laborious method, it was later discarded in favour of one
based on stereologicalprinciples (Weibel, 1969). A transparent grid
with 36x36 points spaced at 05-cm intervalswas superimposed on
micrographs printed at x 45 000, and the number of points falling
oneach mitochondrion was counted 3 times, each time shifting the
grid slightly in relation to acorner of the micrograph; the counts
were averaged. The spacing interval of the grid pointsin relation
to the dimensions of mitochondrial profiles was such that no
mitochondria couldbe missed between 2 points. Then to calculate the
mitochondrial cross-sectional area:
Number of points per mitochondrion Area of mitochondrial
sectionTotal number of points on grid Total area under grid
Cristae per mitochondrion were still counted visually, twice,
and counts averaged. The sizeand shape of mitochondrial
cross-sections being highly variable, each sample consisted ofabout
250-275 mitochondria, and for the final calculations all the
cross-sectional areas for asample, and all the crista counts, were
pooled for calculation of average parameters. Whilethe
cross-sectional area is roughly proportional to volume, the
complexity of shape precludesthe calculation of precise
mitochondrial volumes.
RESULTS
Coleoptile growth under aerobic and anaerobic conditions
The coleoptile is the only organ which grows under anaerobic
conditions, root andleaf growth being totally suppressed. The
emergence of the coleoptile, and its initialrate of elongation, are
slowed by anaerobiosis (Table 1), but whereas in air the
coleop-tile ceases to grow and splits on the fourth or fifth day,
having attained a length of10-16 mm, under anaerobiosis it
continues to elongate further; the seedlings survivefor at least 11
days, with coleoptiles up to 45 mm long. Because of the different
ratesof elongation in air and in hydrogen, the data in Table 1 have
been expressed so thatcomparisons can be made for coleoptiles of
the same length, or, at 4 days, for thesame chronological age. The
anaerobically grown coleoptiles are narrow and delicate;their cell
walls are thinner and there is practically no xylem differentiation
at least
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Anaerobiosis in rice 729
Table 1. Growth, respiration rate and cytochrome oxidase
activityof rice coleoptiles at 27 °C in air and in hydrogen
The comparisons are made for coleoptiles of the same length; or,
at 4 days, plants of thesame age can be compared. Each value for
weight and nitrogen content is based on a total ofat least 100
coleoptiles; the number of coleoptiles used for cell counts is in
parentheses aftereach value. Respiration rates are averages of 2
experiments each using 100 coleoptiles asdescribed in Methods;
cytochrome oxidase values are averages of at least 3 samples of
25coleoptiles. Variety: Italpatna.
Growth conditions . . .
Coleoptile length, mm . . .
Days to reach this length . . .
Per coleoptileFresh wt, mgDry wt, mgCell no. x 10-4
Nitrogen content, mg x io3
Per cellNitrogen content, mg x io8
Dry wt, mg x 10'
»UO,/h/coleoptile/g dry wt./mg nitrogen/cell x io5
RQ
Cytochrome oxidase activity,O.D. units
/25 coleoptiles/cell x io7
/mg dry wt. x 10/mg nitrogen
AerobicA
1
8
3
2-230-273
10-2(49)
9-20
8892-57
2-579890
2 7 02 3 9
0-97
O-333
1-3°0-487i-45
13
4
5 2 00-420
13-0(40)
I3-4
1 0 2
3 2 1
3 9 27I3O
3O43 0 0
0 9 9
0363n o
O-3451 08
AnaerobicA
8
4
1 6 20-122
4-48(32)5-57
1 2 4
2 7 3
0-817
5630144
182
i-5
0029025900950208
14
5
2 6 1
01866-37(46)977
1 5 42 8 2
0-747
379O1 0 5
1-17
2-O
OO52O 3 1 9OI 120-215
over the 5 days investigated (although mature sieve tubes are
present already ini-mm-long anaerobic coleoptiles). Lignified xylem
is formed in aerobically growncoleoptiles. Even in hydrogen,
however, dry weight and nitrogen content of thecoleoptiles increase
(Table 1), indicating a translocation of reserve materials from
theendosperm to the coleoptile, and iodine solutions stain the
anaerobically growncoleoptiles deep blue-black all over, showing a
high starch content. The elongationof the coleoptiles involves both
cell division and cell elongation throughout the periodstudied
(Fig. 1). During the time that the aerobically grown coleoptiles
survive, theirweight, nitrogen content and cell number are always
higher than in anaerobicallygrown material of the same length or
age.
When seedlings are transferred after 5 days in hydrogen to air,
root and leafgrowth commence, with no sign of the plants having
sustained any damage due toanaerobiosis.
47 CE L 12
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730
13 -
H. Opik
7 8 9 10 11
Coleoptile length, mm
12 13 14
Fig. i. Changes in cell number in aerobically (#) and
anaerobically (O) germinatedrice coleoptiles during elongation of
the organ from 5 to 13-14 mm. Each point is basedon at least 22
coleoptiles.
Effect of anaerobiosis on respiration rate
Per organ, the oxygen uptake is appreciably diminished by
anaerobiosis (Table 1),and whereas in the aerobically grown
material, the oxygen uptake per coleoptileincreased as the organ
elongated from 8 to 13 mm, during the equivalent amount
ofelongation in hydrogen, there was actually a slight fall in
oxygen uptake, though therate of CO2 output increased. When the
results are expressed per cell, the differencesbetween the rates in
aerobically and anaerobically grown coleoptiles become muchless
(Table 1). The RQ (respiratory quotient) of the aerobically grown
coleoptiles isclose to unity, but is well above one in the
coleoptiles grown anaerobically and risesbetween days 4 and 5.
Effect of anaerobiosis on cytochrome oxidase activity of the
coleoptiles
Cytochrome oxidase activity is also very much lower in
anaerobically grown plants(Table 1); the depression is again most
marked on a per organ basis. In the aerobicallygrown plants, there
is a small rise in total activity between days 3 and 4 as the
coleop-tiles elongate from 8 to 13 mm, while expressed per cell,
unit weight or unit nitrogen,the activity falls. The same degree of
elongation under anaerobic conditions isaccompanied by almost a
doubling of the feeble cytochrome oxidase activity per
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Anaerobiosis in rice 731
Table 2. The effect of anaerobiosis on respiration rate and
cytochrome oxidase activity
Data of Table 1 expressed to show the percentage reductions
caused by anaerobiosis. In A,comparison is made for coleoptiles of
the same length, 13 mm; in B, for coleoptiles of thesame age, 4
days. Variety: Italpatna.
Dry UnitBasis of measurement . . . Coleoptile Cell wt.
nitrogen
. j % reduction of respiration rate 81 61 47 65\ % reduction of
cytochrome oxidase activity 86 71 68 80
•af % reduction of respiration rate 79 39 21 53\ % reduction of
cytochrome oxidase activity 92 76 72 81
coleoptile; per cell, unit weight or unit weight of nitrogen,
there is a slight rise inactivity. Table 2 shows that cytochrome
oxidase activity is more strongly inhibitedby anaerobiosis than the
rate of oxygen uptake.
When seedlings were transferred from aerobic to anaerobic
conditions after 3 daysof growth, the effect on respiration rate
and cytochrome oxidase activity was some-what variable. Over a
further 1-3 day period under hydrogen, there was sometimesno
change, sometimes a slight increase, sometimes a slight decrease.
During theseperiods the coleoptiles elongated considerably. When
the reverse transfer was made,of seedlings germinated for 3 days
anaerobically to air, the cytochrome oxidaseactivity of the
coleoptiles rose rapidly, reaching after 1 day in air a value of
approxi-mately 70 % of the normal aerobic 4-day value.
Mitochondrial structure
In coleoptile mitochondria of the ungerminated grain, only few,
narrow, faintlystaining cristae are visible (Fig. 2); the matrix is
electron-transparent, except fordarker granules which are often
centrally grouped in a mitochondrial profile and mayrepresent
mitoribosomes. On imbibition, a gradual increase in cristae and
enhance-ment of mitochondrial membrane contrast become apparent,
till by 36 h wellformed mitochondria are observable. This process
of mitochondrial elaborationproceeds identically in air and under
hydrogen.
A thorough examination of mitochondrial structure was carried
out when thecoleoptiles were not more than 1-5 mm long. With the
fixative used, the mitochondriawere in the condensed configuration
(i.e. with an electron-dense matrix and dilatedcristae), and looked
very similar in the aerobically and anaerobically grown
material(Figs. 3, 4); possibly mitochondria from anaerobically
grown coleoptiles had largercentral crista-free areas.
Ribosome-like particles were numerous in mitochondriafrom both
treatments. A quantitative analysis (Table 3) confirmed that there
is verylittle difference in mitochondrial size and crista density;
the organelles from anaerobiccoleoptiles are a little larger and
are slightly more highly cristate. The fine structureof the cells
of aerobically and anaerobically grown coleoptiles was identical
also inother respects.
Attempts to make a similar analysis on mitochondria in
coleoptiles of ages at which47-2
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732 H. Opik
Table 3. Comparison of mitochondrial cross-sectional area
{obtained by planimetry),number of cristae per mitochondrion, and
aristae per unit mitochondrial cross-sectionalarea, in aerobically
and anaerobically grown rice coleoptiles, 0-5 to 1-5 mm long
{germi-nated 42 h in air, or 70 h in hydrogen)
Cells of ground parenchyma only have been included in the
measurements. Sequentialglutaraldehyde/osmium tetroxide fixation in
phosphate buffer; mitochondrial configurationcondensed. Variety:
Italpatna.
Growth conditions
No. coleoptiles examinedNo. mitochondria
measuredCristae/mitochondrionMitochondrial cross-sectional area,
fim%
No. cristae//im*
Aerobic
5271
9 1
0-14
63
Anaerobic
8284
1 1
0-16
68
the cytochrome oxidase assays were carried out, met with
considerable difficulty, thehighly vacuolated cells showing a great
tendency for rupture of tonoplasts, and adifferent fixative had to
be employed. With the older coleoptiles, a complication alsoarises
because in the tipmost 0-5-0-75 mm, the cells are much smaller,
less elongated,less highly vacuolated, than in regions further back
and mitochondria in the 2 regionsare not quite identical; they are
therefore illustrated and analysed separately as wellas
averaged.
Figs. 5-9 show mitochondria of 4-day-old aerobically and
anaerobically growncoleoptiles. The anaerobic mitochondria now have
more dilated cristae, and lesselectron-dense matrices. This is
particularly evident when the back region mito-chondria are
compared. Circular crista cross-sections, as illustrated in Fig. 7,
wereat this stage found only in mitochondria from anaerobic plants,
appearing in about8% of the organelles. (Circular cristae were seen
in younger aerobic coleoptiles.)Mitochondrial configuration is
orthodox (i.e. the matrix is electron-translucent andthe
intracristal spaces contracted), or intermediate, in contrast to
the condensed con-figuration observed in the younger coleoptiles,
but this is thought to reflect the effectof the different fixatives
used. When the older coleoptiles were treated identicallywith the
younger, such cells as remained intact had condensed mitochondria.
Ribo-some-like particles are now sparse in mitochondria, especially
in anaerobic coleoptiles.
Table 4 shows that the back cell mitochondria from the anaerobic
coleoptiles arelarger, and their crista density is lower than in
aerobic coleoptiles. In the tips, mito-chondrial sizes are more
similar, but the crista number per mitochondrion is higherin
aerobic material, giving them an appreciably higher crista density
per unit cross-sectional area.
With respect to cell fine structure in general, the cytoplasm of
anaerobic cellstends to be more electron-transparent, with fewer
ribosomes and perhaps withfewer elongate endoplasmic reticulum
profiles, but more small vesicles. Certainirregularly shaped,
moderately electron-dense inclusions (Fig. 9) are very commonin the
anaerobic cells, but rare in the air-grown.
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Anaerobiosis in rice 733
Table 4. Comparison of mitochondrial cross-sectional area
{obtained by point counts),number of aristae per mitochondrion, and
aristae per unit mitochondrial cross-sectionalarea, in aerobically
and anaerobically grown rice coleoptiles 4 days old
Cells of ground parenchyma only have been included in the
measurements. 'Tip ' and' back' refer to regions between 0—0-75 a
nd 5-7 mm from the apex, respectively. In the caseof the back
regions, most of the well preserved cells which could be used in
the analysis werein 2-3 rows immediately beneath the epidermis, and
another 2-3 rows just external to thevascular bundles. In the
'Total' column, average values for tip and back cell
mitochondriaare given. Simultaneous glutaraldehyde/osmium tetroxide
fixation in collidine buffer; mito-chondrial configuration
orthodox. Variety: Italpatna.
Growth conditions
Region of coleoptile
No. coleoptiles examinedNo. mitochondria
measuredCristae/mitochondrionMitochondrial cross-sectional area,
/ira1
No. cristae//tm'
DISCUSSION
Tip
7245
1 1
0 1 573
Aerobic
Back
92397-100997i
Total
164849-20 1 3
72
Tip
6254970-17
57
AnaerobicA
Back
82517-10-15
48
Total
145058-40-16
52
Under anaerobiosis, vigorous fermentation has been observed in
rice seedlings bye.g. Taylor (1942) and Phillips (1947), the latter
showing it to be of the alcoholictype. This fermentation must
energetically suffice to support cell division, cellelongation,
nutrient hydrolysis and nutrient translocation, although these
processesdo occur more slowly in hydrogen than in air. The aim of
the present investigationwas to see how far the cellular apparatus
for aerobic respiration is developed in therice coleoptiles under
conditions in which they are entirely dependent on
fermentativemetabolism and aerobic respiration is impossible.
Per coleoptile, capacity for oxygen uptake and cytochrome
oxidase activity arevery much depressed by anaerobiosis. Per cell
or unit weight, however, the depressionis somewhat smaller, showing
that it does result to some extent from the smaller cellnumber in
the anaerobically grown coleoptiles. Even per cell, the activities
arenevertheless strongly decreased in spite of a higher cellular
nitrogen content. It maybe inferred that in anaerobiosis a smaller
proportion of the cellular nitrogen isincorporated into enzymes of
aerobic respiration. It is unlikely that shortage of sub-strate
could account for the lower oxygen uptake under anaerobiosis, for
carbondioxide output increases while oxygen uptake is falling, and
starch is abundant.The greater depression of cytochrome oxidase
activity compared with the capacityfor oxygen uptake may indicate a
relatively greater participation of alternative terminaloxidases in
the anaerobically grown plants. However, even the small amount
ofcytochrome oxidase in the anaerobically grown coleoptiles might
suffice for all theirrespiratory oxygen uptake, since the
cytochrome oxidase assay does not measure theactivity in terms of
oxygen equivalents.
Some of the observed cytochrome oxidase activity of anaerobic
coleoptiles may be
-
734 H. Opik
mediated by enzymes already present in the ungerminated grain.
However, cytochromeoxidase activity shows a rise under anaerobiosis
as the coleoptile elongates from 8 to13 mm, indicating that some
synthesis does proceed under anaerobiosis. Once formed,the
cytochrome oxidase of the rice coleoptile is fairly stable under
anaerobicconditions.
The most unexpected finding was the lack of a really drastic
effect of anaerobiosison mitochondrial structure, at least at the
level observable by conventional fixing andthin-sectioning
techniques, for in yeasts, oxygen tension influences
mitochondrialstructure profoundly. In Saccharomyces cerevisiae,
mitochondria of anaerobicallygrown cells are much simpler in
structure than in cells from aerated cultures (Watsonet al. 1970);
in the obligately aerobic yeast Candida parapsilosis, the extent of
cristaformation is less, the lower the aeration (Kellerman, Biggs
& Linnane, 1969). It is acommon observation that the
development of physiological activities in micro-organisms is more
directly influenced by environmental conditions than it is in
higherorganisms. Nevertheless, the degree of internal control in
the rice coleoptile seemssurprisingly rigid. The cristae begin to
develop at the same rate during imbibitionunder both aerobic and
anaerobic conditions. By 4 days, some qualitative and quan-titative
differences are apparent in the mitochondria, but these still are
relativelyminor distinctions, and could be more apparent than real,
resulting from differentdegrees of swelling or contraction during
fixation: one cannot assume that mito-chondria from aerobic and
anaerobic material react identically even to identicalfixation. The
appearance of the mitochondria from anaerobic cells does not
suggestthat their cytochrome oxidase activity should be reduced to
the extent actuallyobserved. Admittedly enzyme activity has been
calculated per coleoptile or per cell(not per mitochondrion),
whereas accurate estimates of mitochondrial number per cellor
coleoptile are not available. But counts made on a limited number
of cells under theelectron microscope showed no decrease of
mitochondria in anaerobic coleoptiles, andsquashes of fresh
coleoptiles treated with iodonitrotetrazolium showed
apparentlyequally abundant staining of particles of mitochondrial
dimensions in aerobicand anaerobic coleoptiles. The inhibition of
cytochrome oxidase activity per cell isover 70 %; there can be
nothing like that degree of decrease of mitochondrial numbersper
cell (if indeed there is any decrease), and one must accept a
lowering of activityper mitochondrion.
One factor involved in the different reaction of rice and yeast
mitochondria toanaerobiosis may be lipid supply. In yeast, the
synthesis of ergosterol and un-saturated fatty acids is
oxygen-dependent, and anaerobiosis therefore induces asevere
shortage of unsaturated lipids in the mitochondria (Paltauf &
Schatz, 1969);in a lipid-supplemented growth medium there is some
improvement in crista develop-ment (Wallace, Huang & Linnane,
1968). The very low membrane contrast of theanaerobic yeast
mitochondria is probably due to their abnormal lipid
composition;freeze-etching may be a better way of revealing
mitochondria in anaerobic yeast(Plattner & Schatz, 1969). In
rice coleoptiles, the cells in the dry grain containreserve lipid;
the dry coleoptile primordium stains with Sudan III and lipid
inclusionscan be seen in the cells by electron microscopy (Opik,
1972). Thus rice is less likely
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Anaerobiosis in rice 735
to suffer from a lipid shortage. The membrane contrast of
anaerobic rice mito-chondria is normal. Lipid supply is of course
only one possible controlling factor.
The fact that mitochondria from anaerobically grown rice
coleoptiles still have ahigh density of cristae in spite of a very
low cytochrome oxidase activity implies thatmitochondrial membranes
can be formed even when some membrane component isalmost lacking,
cytochrome oxidase normally being an integral part of the
innermembrane and cristae. This further suggests that the normal
way of mitochondrialformation may be the synthesis of a skeleton
membrane to which components arethen added. Since cytochrome
oxidase activity increases very quickly when coleop-tiles are
transferred to air after 3 days of anaerobic growth, it would
appear that theenzyme can still be added to membranes which have
been formed some time pre-viously. In yeast also, radioactive
labelling has indicated that the promitochondria ofanaerobic cells
develop into normal mitochondria on aeration by the addition
ofcomponents to their undifferentiated membranes (Schatz &
Criddle, 1969). Anotherimplication of the present findings is that
in the rice coleoptile, crista density is apoor guide to the
oxidative activity of the mitochondria. It is generally
consideredthat there is a correlation between the intensity of
oxidative metabolism of a tissueand the density of cristae in its
mitochondria. In the spadix of Arum maculatumduring a certain
developmental period some oxidative activities are almost
directlyproportional to crista density (Simon & Chapman, 1961).
In the rice coleoptilethere appears to be no obligatory
relationship between crista density and cytochromeoxidase activity.
Cytochrome oxidase is, however, only one enzymic component ofthe
mitochondrial membranes, and it is hoped to continue this study
with an examina-tion of the relationship between crista density and
the activity of succinic dehydro-genase, another component of the
inner membrane and cristae.
Part of this work was carried out while the author was in
receipt of a research grant from theScience Research Council.
Grateful acknowledgement is made to Mrs M. Fletcher and to Mrs
M. Metcalfe for technicalassistance, and to Mr K. Jones for help
with photography. I am also grateful to the Food andAgriculture
Organization of the United Nations, and to the Orszagos
Agrobotanikai Intezet,Tapioszele, Hungary, for gifts of rice.
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{Received 2 August 1972)
Fig. 2. Mitochondria in coleoptile of ungerminated rice grain.
Cristae (c) are sparse;the electron-dense granules (r) may
represent mitoribosomes. Sequential glutar-aldehyde-osmium
tetroxide fixation in phosphate buffer; embedded in TAABepoxy
resin, x 45000.Fig. 3. Mitochondria in rice coleoptile grown in air
to 0-5—15 mm. Numerous dilatedcristae are present; the matrix is
electron-dense; some ribosome-like granules arepresent, v, vacuole;
w, cell wall. Fixation as for Fig. 2; embedded in methacrylate.x 45
000.
Fig. 4. Mitochondria in rice coleoptile grown anaerobically to
0-5-1 5 mm. Structureis very similar to mitochondria in coleoptiles
grown aerobically to the same size, butcentral areas (a) free of
cristae are more conspicuous. Preparation as for Fig. 3.x
45000.
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Anaerobiosis in rice 737
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738 H. Opik
Fig. 5. Mitochondria (m) in 4-day-old aerobically germinated
rice coleoptile, fromcells 5-7 mm behind the tip. g, Golgi
apparatus; n, nucleus. Simultaneous glutaralde-hyde-osmium
tetroxide fixation in collidine buffer, embedded in
methacrylate.X45OOO.
Fig. 6. Mitochondria in 4-day-old aerobically germinated rice
coleoptile, from cellswithin ca. 0-5 mm of the apex. The dense body
b is probably a microbody; v, vacuole.Preparation as for Fig. 5. x
45 000.Fig. 7. Mitochondrion in 4-day-old anaerobically germinated
rice coleoptile, fromcell within ca. o'5 mm of the apex, selected
to show a circular crista profile. Prepara-tion as for Fig. 5. x 45
coo.Fig. 8. Mitochondria in 4-day-old anaerobically grown rice
coleoptile, from cellwithin o'S mm of the apex, showing the typical
form. The irregularly shaped inclusion(»') is a very common feature
in anaerobically grown coleoptiles at this age. n,
nucleus.Preparation as for Fig. 5. x 45000.Fig. 9. Mitochondria in
4-day-old anaerobically germinated rice coleoptile, fromcells 5-7
mm behind tip. Cytoplasmic inclusions (1) are again present; v,
vacuole;K, cell wall. Preparation as for Fig. 5. x 45000.
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Anaerobiosis in rice 739
w