Quinones and α-Tocopherol in Greening Callus Cultures of Kalanchoe crenata

Quinones and α-Tocopherol in Greening Callus Cultures of Kalanchoe crenataAuthor(s): D. R. Thomas and A. K. StobartSource: New Phytologist, Vol. 70, No. 1 (Jan., 1971), pp. 163-171Published by: Wiley on behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/2431064 .

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New Phytol. (I97I) 70, I63-I7I

QUINONES AND a-TOCOPHEROL IN GREENING CALLUS CULTURES OF KALANCHOF CRENATA

BY D. R. THOMAS AND A. K. STOBART

Department of Botany, University of Newcastle on Tyne, and Department of Botany, University of Bristol

(Received 2 May 1970)

SUMMARY

Dark-grown Kalanchoi callus tissue subcultured into i6-hour days produce chlorophyll only after several generations. Plastoquinone:chlorophyll molar ratios in extracts from green callus indicate that the level of plastoquinone is somewhat lower than in leaf tissue. This was confirmed in purified chloroplast preparations from callus and leaves. Hill reaction activity was lower in green callus chloroplasts than in those from mature leaves.

oc-Tocopherol, although present in non-green callus, increased with chlorophyll synthesis. D, L[2z4C]-mevalonate feeding experiments seem to indicate active sites of oc-tocopherol synthesis other than the mature chloroplast.

Changes in ubiquinone and oc-tocopherol content were observed during the growth of dark- grown callus. Such changes may be due to an increase in mitochondrial populations and to the onset of senescence.

INTRODUCTION

Kalanchoe crenata callus cultures, because of their ability to produce chlorophyll in the light (Stobart, McLaren and Thomas, I967) have been used extensively in the study of the biochemical morphogenesis of the chloroplast (Stobart and Thomas, I968a, b; Stobart, Weir and Thomas, I969; Thomas and Stobart, 197oa).

The failure of green callus cultures to photosynthesize at rates sufficient to support growth (McLaren and Thomas, I967) might be correlated with low levels of quinones in the callus chloroplasts so that photosynthetic phosphorylation might be impaired. Therefore plastoquinone, vitamin K and ubiquinone levels in the callus and mature Kalanchoe leaves were determined. oc-Tocopherol was assayed although its role in plant tissues has yet to be clearly defined.

MATERIALS AND METHODS

The methods of culture for Kalanchoe callus have been reported elsewhere (McLaren and Thomas, I967; Stobart et al., I967). A summary of the types of callus used in the experiments is given in Table i. Whole plants of K. crenata were obtained from the experimental grounds of the Botany Department at Newcastle. Leaves for experimental purposes were removed from the third and fourth nodes and stripped of their midribs before analysis or fractionation.

Leaves and callus were extracted in peroxide-free acetone in an M.S.E. overhead blender at 20 C. The lipid soluble components were passed into petroleum ether (bp

i63



I64 D. R. THOMAS AND A. K. STOBART

40-60? C) by phase separation and traces of acetone removed by washing with distilled water. The ether phase was dried over anhydrous sodium sulphate and concentrated to a small volume under a stream of nitrogen before analysis by paper chromatography.

Fractions for oc-tocopherol analysis were obtained using NaCl-buffer, pH 7.2 (Stobart and Thomas, i968b). Purified chloroplasts for chlorophyll and plastoquinone estimation were obtained using the density-gradient technique of James and Das (I957). Chloro- plasts for Hill reaction assays were prepared in sucrose-phosphate buffer medium, pH 6.5.

For the initial chromatography the technique modified by Booth (I963a) for plant tissue was used. Further purification of the lipids after elution was carried out on Schleicher and Schiill filter papers, No. 288 (Lichtenthaler, I964) using cyclohexane- benzene (7: 3 v/v). For good separation the papers were activated at i i0? C for 20 minutes prior to use. The reverse phase TLC technique of Wagner and Dengler (I962) was used to identify the purified isoprenologues of ubiquinone, plastoquinone and vitamin K.

Table i. Terminology used to describe Kalanchoe callus grown for various periods in i6-hour days

Terminology Description Dark-grown Callus cultures derived from stem cultures and which

have been grown for 3 years in the dark with monthly subculturing

First generation Callus subcultured from dark-grown callus and then grown in i6-hour days for 4 weeks

Second generation Callus subcultured from first generation callus and then grown in i6-hour days for 4 weeks

Third generation Callus subcultured from second generation callus and then grown in i6-hour days for 4 weeks

Subsequently six generations produced Green Green grown in i6-hour days for 3 years with sub-

culturing at monthly intervals

ox-Tocopherol was detected by the Emmerie-Engel reaction (Emmerie and Engel, 1938). Quantitative measurements were made colorimetrically using the method of Booth (I963a). Quinones were detected by the neotetrazolium reaction (Lester and Ramasarma, 1959). Purified plastoquinone, ubiquinone and vitamin K were quantitatively estimated from their oxidized and reduced spectra in alcohol. Reduction was achieved by adding sodium borohydride. The amounts of quinone were calculated using published extinction values: plastoquinone, oxidized maximum in ethanol 255 nm, E lcxn - 210

(Crane, 1959); ubiquinone, 276 nm, E i = I65 (Lester et al., I9); vitamin K1, 249 nm, E Lc = 540 (Ewing, Vanderbelt and Kamm, 1939). Chlorophyll was determined in diethyl ether (Comar and Zscheile, 1942).

Quinones and ox-tocopherol were identified by co-chromatography with authentic material and from their oxidation/reduction spectra (Morton, I965). The isoprenologues of ubiquinone, plastoquinone and vitamin K were identified as UQ10, PQ9 and K, respectively.

The Hill reaction was measured manometrically with the oxidant solution of Holt and French (1946). The results are expressed as Q och values (vol. 02 evolved/hour/mg chlorophyll).

DL-[2-14C]-mevalonic acid lactone was purchased from the Radiochemical Centre, Amersham, the acid being prepared from the lactone by the method of Rogers, Shah



Quinones and a-tocopherol in callus tissue I65 and Goodwin (I966). Eluted tocopherol was made up to I5 ml with liquid scintillator fluid consisting of 5 g 2,5 diphenyloxazole and 0.3 g I,4-bis-2-(5-phenyloxazolyl)- benzene in i 1 toluene. Samples were counted on a Packard Tri-Carb Model 2003.

Organic solvents were dried over sodium wire and distilled before use.

RESULTS

Plastoquinone was identified in all chlorophyllous callus and in Kalanchoe leaves. Ubiquinone and a-tocopherol were found in all callus and leaves, but vitamin K was positively identified only in the green callus tissues and not in the leaves of Kalanchoe. No evidence was obtained for the presence of plastoquinones B, C and D, the tocopherol quinones or other tocopherols in leaves or callus.

Dark-grown callus was subcultured into the light and first, second generations, etc. obtained in the usual way (Table I). Quinones, x-tocopherol and chlorophyll were

Table 2. Chlorophyll, plastoquinone, ubiquinone and vitamin K content of Kalanchoe leaves and the various callus cultures (results are expressed as pu-moles/g dry weight; each result is the mean of three replicates)

Material Chlorophyll Plastoquinone Ubiquinone Vitamin K Dark grown 0 0 0.038 0 First generation 0 0 0.03I 0 Second generation 0 0 0.035 0 Third generation o 0 0.034 0 Fourth generation 0.36 0.005 0.043 o.ooi8 Fifth generation 0.50 0.007 0.04I 0.0035 Sixth generation 0.74 O.OI 0.042 o.oo88 Green (a) I-35 o.oi6 o.o62 0.015

(b) i.6o 0.03 I o.o62 O.OI4 (C) 1 .89 0.029 0.047 o.oi8

Leaf (a) 33-4 I.84 o.i8i a (b) 37.1 2.i6 0.I73 0 (C) 34.0 I .93 0.I9I 0

determined for each kind of callus and the leaves of Kalanchoe? (Tables 2 and 4). Plasto- quinone and vitamin K were not detected in extracts of dark-grown callus and in light- grown callus lacking chlorophyll. However, with the onset of greening in the fourth generation in the light small amounts of plastoquinone and vitamin K were detectable. The plastoquinone content increased parallel to chlorophyll synthesis in the fifth and sixth generations in the light. The green callus clone contained over twice as much plastoquinone as the sixth generation callus but as the molar ratios plastoquinone- chlorophyll (P/C, Table 3) for all chlorophyllous callus were similar, this difference in plastoquinone content was correlated with a higher chlorophyll level and possibly the greater development' of plastids in the green clone. The P/C molar ratios for leaves were three times higher than those of green callus (Table 3). Vitamin K, although present in the callus in much smaller amounts than plastoquinone, showed a similar increase with greening (Table 2). However, there were wide fluctuations in the vitamin K: chlorophyll molar ratios (Table 3) than in the P/C ratios. This may have been due to errors introduced by analysis of the small amounts of vitamin K in callus or a greater lability during extraction. Vitamin K was not conclusively identified in leaf extracts and it is thought that this may be due to its 'adsorption' onto large quantities of fl-carotene.

Ubiquinone levels were similar in all types of callus analysed (Table 2). Greater



i66 D. R. THOMAS AND A. K. STOBART

amounts were present in leaves. The ratio plastoquinone: ubiquinone (Table 3) was high for leaves but low for chlorophyllous callus. Even in the green clone ubiquinone was always present in excess of plastoquinone.

ox-Tocopherol concentrations were similar for all callus not containing chlorophyll. However, as soon as chlorophyll synthesis commenced (Table 4) x-tocopherol content increased. This increase did not keep pace with chlorophyll, the chlorophyll: tocopherol ratios (Table 4) falling from 0.I7 in the fourth generation callus to o.o6 in the sixth generation and green callus. The ratios for leaves were always lower than those obtained for green callus.

Table 3. Molar ratios in chlorophyll-containing tissues

Material P: C P:U K:C Fourth generation O.OI 3 O. II 0.005 Fifth generation 0.0O7 0.I7 0.007 Sixth generation 0.02I 0.23 O.OI2 Green (a) O.Oi I 0.25 O.0I I

(b) 0.0Ig 0.50 0.009 (c) O.OI5 o.6i O.OI

Leaf (a) o.os6 10.2 (b) o.o6 I2.5 (c) o.os6 9.8

C, Chlorophyll; P, plastoquinone; U, ubiquinone; K, vitamin K.

Table 4. Chlorophyll and cx-tocopherol content of Kalanchoe leaves and the various callus cultures

Chlorophyll a-tocopherol Ratio Material (mg/g dry weight) p-g/g dry weight) a-T: C

Dark-grown o 20 First generation o 25 Second generation o 27 Third generation 0 23 Fourth generation 0.31 50 O.I7 Fifth generation 0.41 50 O.I2 Sixth generation o.96 58 o.o6 Green 1.41 72 o.o6 Leaf 27.1 625 0.02

cx-T, aX-Tocopherol; C, chlorophyll.

Intracellular location of plastoquinone and a-tocopherol Chloroplasts from callus tissue and leaves were purified on density gradients. The

plastids were extracted and plastoquinone and chlorophyll content determined. Table 5 shows the results of such an analysis. The P/C ratios were similar to those obtained for whole tissue (Table 3). The green callus chloroplasts seemed deficient in plastoquinone as compared to leaf chloroplasts. The similarity of the ratios for whole tissue and isolated chloroplasts suggests that the plastoquinone was located in the chloroplast.

In determining the intracellular distribution of ac-tocopherol a different experimental approach was used. Dark-brown callus and green callus were grown on complete medium for 2 weeks before being transferred to IO pCi DL-[2-14C]-mevalonate in I5 ml complete medium for a further 2 weeks. Dark-grown callus was kept in the dark at 250 C and the green callus kept under daylight conditions. oc-Tocopherol was extracted and after purification assayed for radioactivity. The specific activities (Table 6) of



Quinones and a-tocopherol in callus tissue I67

oc-tocopherol from dark-grown callus were higher than green callus although a similar total number of counts were incorporated into it (Table 6).

In a similar experiment dark-grown and green callus were fractionated in NaCl- buffer, pH 7.2. oc-Tocopherol in the 3000 g and 20,000 g fractions was extracted and purified and its radioactivity determined (see Table 7). No oc-tocopherol was detected

Table 5. Plastoquinone and chlorophyll recovered from purified chloroplasts of Kalanchoe leaves and

green callus

Chlorophyll Plastoquinone Molar ratio Source (pmoles) (pmoles) P: C

Leaf 3.2 o.2o8 o.o65 Green callus I.3 O.OI4 O.OI I

P, Plastoquinone; C, chlorophyll.

Table 6. Radioactivity incorporated into ox-tocopherol from D, L-2[14C]-mevalonate by dark-grown and

green callus

Total counts/minute Callus oa-Tocopherol in SA

(pg/g dry weight) oa-tocopherol Dark-grown 45 I 596 3 5 Green 90 I338 I5

SA, Specific activity; counts/minute/,ug, oa-tocopherol.

in the 3000 g fraction from dark-grown callus. However, oc-tocopherol was present in the 20,000 g fraction from dark-grown callus and was radioactive, its specific activity being 41 (counts/minute/,ug tocopherol). The 3000 g fraction from green callus contained nearly 78% of the total oc-tocopherol with a specific activity of i2. The specific activity of the 20,000 g fraction from the green callus was nearly treble this.

Ubiquinone and o-tocopherol in dark-grown callus It was observed that levels of ubiquinone and oc-tocopherol in dark-grown callus

varied according to the age of the callus. A series of experiments were set up to investigate these differences.

Dark-grown callus was subcultured onto fresh medium and ubiquinone and oc-tocopherol determined at intervals over 55 days growth. The results are given in Fig. i.

Three days after subculture the ubiquinone content was similar to that of 4-week-old dark-grown callus. At the seventh day, the ubiquinone content had increased and finally reached a maximum around the twentieth day. Thereafter, the ubiquinone content decreased to reach a low level at the termination of the experiment. oc-Tocopherol remained steady until the fortieth day of growth when the level increased and attained a maximum after 55 days.

Hill reaction activity Chloroplasts from green callus and Kalanchoe leaves evolved oxygen and were

capable of the Hill reaction. The Qc2 values (Table 8) for green callus chloroplasts were lower than those for leaf chloroplasts.



i68 D. R. THOMAS AND A. K. STOBART

Table 7. o-Tocopherol content and radioactivity in fractions prepared from dark-grown and green callus (the callus was grown on D, L-2['4C]- mevalonate in complete medium for the last 2

weeks of the 4 week growth period)

Counts/minute Fraction assayed ax-Tocopherol in SA

(pg) a-tocopherol Dark-green callus

3000 g 0 0 0 20000 g I79 7339 4'

Green callus 3000? 406 4872 I2

20000 g 39 I0527 33

SA, Specific activity; counts/minute pg, oa-tocopherol

Table 8. Hill reaction activity of chloroplasts fromgreen callus and Kalanchoe leaves

Source of chloroplasts Chlorophyll (mg) Qch Green callus 0.I7 446

0.I3 48I 0.I0 490

Leaf 0.30 685 0.2I 577 0.20 6oo

QCo2, Volume of oxygen evolved/hour/mg chlorophyll.

4

3 /120

;D

r i Lf

EL~~~~~~~~~~~~~~~~~~~~~) a, D

ioo

20

0 5 ' 10 15 20 25 30 35 40 45 50 55 Days

Fig. I. Changes in oc-tocopherol and ubiquinone during the growth of dark-grown callus.



Quinones and a-tocopherol in callus tissue I69

DISCUSSION

Hill reaction activity in the green callus chloroplasts was to be expected as McLaren and Thomas (I967) concluded that the green callus was capable of some photosynthetic fixation of carbon dioxide and hence contained functional chloroplasts presumably possess- ing the mechanisms for the production of ATP and NADPH. The plastoquinone content of green callus chloroplasts was less than that of KalanchoeF leaf chloroplasts. Thus, a deficiency in plastoquinone may account, in part, for the lower Hill reaction activity observed in green callus chloroplasts. Bishop (I959) originally reported that at least 7000 of the plastoquinone present in chloroplasts could be removed before any reduction in Hill reaction activity was evident. He later (I96I), produced new evidence demonstrating that highly active chloroplasts were extremely sensitive to plastoquinone extraction and when extracted chloroplasts were reconstituted with added plastoquinone, there was a reactivation of Hill reaction activity. Friend and Redfearn (I962), using polarographic techniques, also showed there was an immediate loss of Hill reaction activity following the removal of small amounts of plastoquinone. Hence, it would seem that the lower Hill reaction activity in green callus chloroplasts could be attributed to lower plastoquinone levels, and so may, in part, account for their poor photosynthetic ability. McLaren and Thomas (I967) demonstrated that the green callus in bright light did not photosynthesize at rates sufficient to supply the total carbon and energy needs of the callus.

The levels and molar ratios of plastoquinone and chlorophyll clearly demonstrate that the plastoquinone is wholly present in the chloroplasts of green callus. However, the results obtained for oc-tocopherol indicate that some oc-tocopherol may be located at sites other than the chloroplast. The chloroplast membrane is known to be impermeable to mevalonate (Rogers et al., I966) and so the isoprenoid side chain of oc-tocopherol seems unlikely to have been derived from the radioactive mevalonate supplied to the callus. Thus, the specific activity of oc-tocopherol located and presumably synthesized in the chloroplast would be relatively low when compared to any oc-tocopherol synthesized extraplastidically. The difference in specific activity of oc-tocopherol in the 3000 g and 20,000 g fractions of green callus possibly indicates that there is some oc-tocopherol synthesized elsewhere, perhaps in mitochondria, even though the 20,000 g fraction is undoubtedly contaminated with some oc-tocopherol from mature plastids as this fraction contained chlorophyll. It is also possible that more immature, smaller plastids, may be recovered in the 20,000 g fraction and this may account for some oc-tocopherol recovered in this fraction. Similar differences in the specific activities of phytol after feeding radioactive mevalonate to callus tissues have been observed (Stobart et al., I969). It was suggested that plastids sedimenting above 3000 g may be at a juvenile stage of development in which the plastid membranes are more permeable to mevalonate than are the membranes of more mature chloroplasts or etioplasts. It is known that the activity of mevalonate-activating enzymes in greening callus increases and this is associated with chloroplast development (Thomas and Stobart, 197ob). This would, in part, account for a high terpenoid component in the chloroplast having a low specific activity after feeding radioactive mevalonate. Dilley and Crane (I963) concluded that oc-tocopherol present in spinach and lilac leaves was present in the chloroplast. Similarly Booth (I963b) con- siders all oc-tocopherol in higher plants to be present in the chloroplasts. If in callus there exists extraplastid sites of oc-tocopherol synthesis then there is a discrepancy between this suggestion and those given by other workers.



I70 D. R. THOMAS AND A. K. STOBART

The significance of such changes in ubiquinone content with age of cultures is not immediately apparent. If these results are superimposed over a growth curve for dark- grown callus (Fig. i) it can be seen that the increase in ubiquinone level was associated with the rapid growth phase which occurred to about the thirtieth day of growth. It is possible that the increase in ubiquinone during the rapid growth phase was associated with increased respiratory metabolism, perhaps a rapid increase in number and activity of mitochondria was taking place.

Booth (i 964) reported that an increase in oc-tocopherol can take place in leaves removed from plants and in senescent leaves (Booth and Hobson-Frohock, I96I). As previously stated, oc-tocopherol increased after the fortieth day of callus growth. After 4 weeks growth the callus became dehydrated, often becoming pigmented with anthocyanins as the growth rate declined. Thus, the increase in a-tocopherol after 4 weeks growth might be associated with the onset of senescence.

ACKNOWLEDGMENTS

The authors are grateful to Dr Pennock, Department of Biochemistry, University of Liverpool, for providing purified samples of ubiquinone10, plastoquinoneg, ac-tocopherol and vitamin K1.

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