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

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

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

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

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

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

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

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.

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

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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and biochemical properties. Biochemistry, N. Y. 8, 322-324.CRUIKSHANK, R. (1965). Medical Microbiology: a Guide to the Laboratory Diagnosis and Control

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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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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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w