BOOTH-CAROTENE IN CARROT LEAVES 37 1

CAROTENE IN THE LEAVES OF THE CARROT

.By V. H. BOOTH*

Leaves of carrot plants of cultivated varieties contained an average of I 19 mg. of total carotene per kg. fresh weight, or 546 mg. per kg. dry weight. The concentration of total carotene bused on uvevuges of many batches was approximately constant through the season so long its the leaves remained healthy in appearance, and except for small seedlings, was independent of the age of the plant. The average concentration was the same during three successive years. There was, however, a variation between individual batches from different varieties, seasons and growing con- ditions. A measure of this overall variation is given by the standard deviation, 19 mg. per kg. fresh weight. Part of this variation was due t o experimental error of the analytical method, and part t o sampling. Beyond these there was a residual variation between different batches which has not been explained.

The concentration of leaf total carotene was independent of variety among cultivated types of carrot. There was no correlation between carotene concentrations in leaf and in root althhough the latter varied several hundredfold in different varieties, which included deep red, orange and white types.

In these respects leaves contrasted with roots.

Introduction Among commonly-grown commercial varieties of carrot (Daucus carota L.) used for human

food the concentration of carotene in the roots varies little at maturity (Booth & Dark1). When the survey is extended, however, to embrace the less common types, which include on the one hand fodder types, wild carrots and albinos (all of which have carotene contents, based on fresh weights, of less than 5 p.p.m.), and on the other hand, the long deep red varieties described by Dark & Booth2 (having up to 1000 p.p.m.), the range is considerable.

Although carotenoids are synthesized in roots (Rygg3 Booth*) it seemed possible that factors controlling the concentration in roots might have some effect on the concentration in leaves, and that there might be some correlation between these concentrations. It is shown below, however, that the concentration of total carotene in the leaves was almost independent both of variety and of growing conditions, factors that produce wide variation in the roots.

Experimental Methods and materials

The carrot plants were of four types, grouped according to the concentration of carotene in the roots : (I) ' high-carotene ' varieties having exceptionally high root concentrations, bred by Mr. S. 0. S. Dark at the Horticultural Research Station, Cambridge ; (2 ) ' ordinary ' or commonly- grown varieties having carotene contents near 125 p.p.m. at maturity ; (3) ' fodder ' carrots with no carotene in their roots ; (4) other ' white ' rooted carrots. All these carrots are described in greater detail elsewhere.2,

The term ' batch ' means a quantity of leaves taken from one plot at one time. Batches differed from one another either as to origin of seed, location (soil and climate) or year of growing. Thus, leaves from the same stock of seed grown under different conditions, or gathered when the roots were at different stages of maturity, constituted different batches. For each batch, leaves were gathered by snipping off a secondary axis with its leaflets from at least 10, and usually 25, growing plants. With one exception, which is described, the leaves were all from first-year foliage. The leaves were assayed immediately after collection. Each batch was analysed by weighing and extracting duplicate or triplicate sub-samples. The mean of the replicate analyses (the batch mean) was used for calculating statistics, except that of experimental variation.

Carotene determinations were made by extracting small samples of leaves from each batch with a mixture of cold light petroleum, acetone and quinol, and chromatographing on a mixture of alumina and Na,SO, (Booth6). Total carotene is expressed as p.p.m. of the fresh weight.

The cause of these varietal differences is unknown.

Only healthy green leaves were used, and main axes were excluded.

* Member of the scientific staff of the Agricultural Research Council

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372 BOOTH-CAROTENE I N CARROT LEAVES

The carotenc is mostly p-, but leaves of all carrots except ' white ' varieties also contain some cc-carotene.

Results Experimental variation

An estimate of the precision of the carotene determinations was made from the within-batch standard deviation. This, calculated from 104 duplicates, 76 triplicates and 8 higher replicates was 7.0 p.p.m. Hence the coefficient of variation for a single determination was 5.9%. This value, which is reasonably small for a biochemical determination, is of the same order as that found for grass and other small leaves.' The major contribution to this experimental variation was presumed to be sampling error and this included variations (I) in the ratio of leaf to axis, ( 2 ) between individual leaflets on one plant, and (3) between individual plants in a batch.

Variations in di ferent parts of the leaf

visibly lower than in leaves. roo g. of carrot ' tops ' were dissected into three categories, weighed and analysed. are shown in Table I. of the batch, and had a carotene concentration only one-tenth that of the leaves.

The mean carotene concentration in the 188 samples was 118 p.p.m.

The concentration of pigment in axes and petioles (together popularly termed ' stem ') is In an experiment designed to find how carotene is distributed,

The results The petioles and main axes (large stems) represented 60% of the weight

The secondary

Table I Carotene content of entire leaf and its components

Weight, Carotene g. total, pg. p.p.m.

Petiole + main axes or rachis 15 210 14

Laminae or leaflets 9 1278 142 Rachillae or secondary axes I 40 40)1 32*

__ ~

Entire leaf system, or crown 25 1528 61 * Calc. on total weight of rachillae and laminae

axes (side stems) also had considerably less carotene than the leaves, but represented only 4% of the total weight. In this experiment, taking all the secondary axes with the leaves would have lowered the carotene value from 142 to 132 p.p.m., a reduction of only 7%. To save time in the sampling technique normally practised during this investigation, parts of the secondary axes were taken together with the leaves, but the stems were discarded.

Also the carotene content of similar plants similarly cultivated in one plot varied. The results of 130 determinations are summarized in Table 11. The variation from leaf to leaf on one plant was a little greater than from plant to plant and both are greater than experimental error, but not

The carotene content of different, but similar, leaflets on the same plant varied.

Table I1 Variation within sets of batches of carrot leaves taken in different mays

Scatter between Batches Determinations Mean carotene, Standard in each batch p.p.m. deviation

Leaflets on a plant 8* 5-8 127 16.0 Similar plants in one row 7 t 10-1 2 113 I 1.4

* Eight plants from each of which some 5 to 8 separate leaflets were examined 7 Seven rows or plots of different varieties, and 10 to 12 plants from each examined separately

so great as the variation from batch to batch throughout the investigation (standard deviation, cultivated varieties, 19 p.p.m. in Table 111).

The highest carotene value found for a batch of apparently normal plants was 170 p.p.m. and the lowest was 60. Occasionally

J. Sci. Food Agric., 8, June, 1957

A normal carrot ' bolts to seed ' in its second summer.

BOOTH-CAROTENE I N CARROT LEAVES 373

i t fails to do this and the leaves have a peculiar, dark appearance. were extracted and they yielded 190 p.p.m. of carotene. abnormal appearance are not included in the tables.

Variation in water content Moisture determinations were made on batches of carrot leaves by weighing before and after

heating in a ventilated air oven at rooo. In order to cover extremes, the batches separately determined included young and old leaves, during dry summer and wet autumn weather, and also after frost. The values ranged from 18.8 to 23.5y0, and had a coefficient of variation of 6.0. An estimate of the ' within batch ' experimental precision is given by the standard deviation of a single determination, 0.61Y0 dry matter : hence the standard error of the mean of quadruplicate determinations was 0.31, and the coefficient of variation attributable to technique was 1.4.

Comparison of varieties of carrot The results appear in Table 111.

The third column shows the average concentration in the roots of the varieties at maturity. These are estimates for each variety, and are not necessarily for the same plants whose leaves were examined.

The leaves of one such plant This and other specimens of obviously

The average dry matter content of 11 batches was 21.8y0.

Carotene was determined in leaves of many varieties.

Table I11 Carotene in leaves of diffevent vavietzes of carrot

Variety Batches Carotene Carotene in leaves ~- ~ examined in root, p p m. s.d * P PJn.

Group I : High root-carotene varieties 31 290-jjo rrg 20'0

Group z : ' Ordinary ' varieties Red Cored Chantenay . 19 1.50 '77 21.3

Autumn King 9 I I0 128 13.8 Other ' ordinary ' varieties 65 100-160 119 19.6

Chantenay 18 114 I1 j 18.5

Group z pooled I11 118 19'3

Group 3 : Fodder varieties 10 0 120 12.9

' Albinos ' ex coloured varieties 5 3'0 108

Groups I , z and 3 pooled? 1.52 0-550 1 I9 19.0

-411 groups I 64 0-5 j o I18 19'2

Group 4 : Other ' white root ' varieties

Wild carrot 7 1'0 99 11.4

* Standard deviation of one batch t That is, all cultivated varieties

The mean leaf concentration for the 31 batches of high-carotene carrots was 119 p.p.m., and for the III batches of ordinary carrots was 118 p.p.m. Therefore it was concluded that there was no correlation between the concentration of carotene in the leaves and in the roots of pigmented carrots. This conclusion is supported by the results of experiments on age of plant below ; for young plants with low contents of root carotene had normal contents of leaf carotene. Since ( a ) the difference between groups I and z is insignificant, and ( b ) the estimated standard deviation of a single batch in group 2, namely 10.3, is only of the same order as that of the average standard deviation of a single batch within a variety, there is no evidence that there are varietal differences in carotene content of leaves within groups. Hence the apparently high value for Autumn King is probably not significant, and indeed the difference between Autumn King and the variety with the lowest value, namely Chantenay, was not significant (P > 0.05).

An early version of Table 111, made when fewer results were available, gave a hint of a difference between total carotene contents of the leaves of white rooted and pigmented carrots.

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374 BOOTH-CAROTENE I N CARROT LEAVES

Therefore, in order to test whether white fodder carrots resemble pigmented or wild types as to total carotene concentration in their leaves, a statistical trial was laid down. Four varieties of ordinary red, and four varieties of fodder, carrots were sown in randomized rows in each of four blocks. In August the 32 batches were analysed in duplicate. The average carotene contents were 136 and 131 p.p.m. for the red and the white varieties respectively. Analysis of variance showed no significant difference.

The overall mean carotene concentration for all the batches in Table 111, except wild and albino types, is 119 p.p.m. The mean concentration based on a dry weight of 21.8% is 546 p.p.m.

Does the carotene concentration in carrot leaves vary f ~ o n z year to year? Booth & Dark1 showed that the concentration of total carotenoids in the roots of carrots

varied from year to year. The average values for instance in the autumn of 1947-a year with a hot dry summer-werc 1.27 times the average values in the autumn of 1948-a cool wet year. On the other hand, the concentration of carotene in the leaves was not significantly different in the years 1946 to 1951 (Table IV). The harvesting period extended from summer to winter, the bulk of the batches being gathered in autumn.

Table I V Mean carotene concentration in carrot leaves in differezt years

Year Batches Carotene, p.p.m. mean %d.*

1946-47 7 117 22'3 '947-48 I9 I 2 2 77'3 1948-49 59 1 I9 19'3 1949-50 I1 117 14.6 1950-51 9 120 22.7

* Standard deviation of one batch

T h e efect of age Carotene concentration in the root of the carrot increases with age (see Booth & Dark1).

That this is so only in the early stages in leaves was shown by the following experiments in which carotene was estimated at different stages of leaf development. Leaves from stands of plants whose seed had been sown in spring (March, April or early May) were compared with leaves from stands of similar plants whose seed had been sown in summer (June, July or early August), by analysing them together in paired sets of several plants. The comparisons in Table V show that the ratio of carotene contents of leaves of spring-sown plants to that in summer-sown plants

Table V Effect of date of sowing on carotene concentration an carvot leaves

Harvested Carotene, p.p.m. Ratio

August 114 116 0.98

October 120 142 0.85 h'ovember 130 124 I '05

January 124 114 1.09

Mean 0.99

spring sown summer sown spring : Summer

September 124 124 1'00

December 119 125 0'95

~

is near unity. In other words carotene concentration in leaves on a given date is not related to time of sowing the seed. The carotene in the roots of the younger plants had only reached one-third to two-thirds the concentration found in the roots of the older plants of the same variety at the time the leaves were analysed.

Not only is the carotene concentration in roots on a given date higher the earlier the seed is sown, but it is also higher the later the roots are harvested. The results in Table V suggest that this is not so in leaves, but in order to increase the scope of the tests, the results of all the

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BOOTH-CAROTENE I N CARROT LEAVES 375

appropriate determinations in this investigation have been grouped according to the date when batches were examined. The batches included several varieties (but no ' whites I ) , were sown on various dates, and were taken over 7 years ; but since these factors have been shown not to influence carotene concentration in leaves it is permissible to pool the results.

Seven batches (differing as to variety or year) of cotyledons taken in summer had a mean value of 55 p.p.m. of carotene. There were no leaves at this stage of development, and the roots were threadlike, semi-transparent, almost colourless, weighed about 7 mg. each, and their carotene content was only 0.75 p.p.m. Four batches of leaves, taken in June from very young seedlings, whose tops were less than 10 cm. high, had a mean carotene value of only 60 p.p.m. Other batches of larger leaves have been grouped in Table VI.

Table VI Carotene content of carrot leaves in di-ffevent months

Month

May June July August September October November December January February March

Samples ( examined

5 I 0 I 2

15 18 I8 8

23 9 0 3

Standard deviation*

I 7.6 19.6 18.8 24'4

24'5 73'7 23 .6

22'1

11.5

* Of one batch

From July until after midwinter the concentration of carotene appears to be broadly independent of date. Moreover the scatter between the batches within any month (standard deviation of one batch) was about the same as for other groupings of the batches. In winter the larger leaves wither and lose their carotene. Only results from obviously healthy leaves appear in the tables. Had no discrimination been made in selecting leaves for analysis a decline would have been recorded from October. Moreover the earlier the carrots are sown the earlier do the leaves wither ; hence most of the later batches are from summer sowings. The amount of stem relative to leaf increases during growth ; hence had whole crowns been analysed instead of leaflets, a slight fall in concentration would haw: been observed during summer and autumn.

In contrast with the present findings, Shivrina8 observed that as the carotene in St. Valery carrots increased in the roots it decreased in the leaves, although no corresponding decrease was observed in ' white ' carrots.

Discussion No correlation (between carotene contents of roots and leaves

Although the concentration of carotene in the leaves of the carrot plant varies, i t does not vary nearly so much as in the roots. The range for roots of the carrots whose leaves were analysed was about 0-550 p.p.m., but carrot roots have been examined that had carotene contents of nearly 1000 p.p.m. On the other hand the range for fresh leaves, excluding obviously abnormal specimens, was 60-170, two-thirds of the observed values lying between 99 and 137. The estimated standard deviation of one batch in the whole investigation was 19.2 (Table 111) : this included experimental error, a measure of which is given by the standard deviation of one replicate within a batch, namely 7.0. Such variation as does occur in the concentration of total carotene in different leaves other than seedlings, albinos and wild varieties is not correlated with the concentration in the roots. Hence a knowledge of the total carotene concentration in leaves is of no value for predicting the concentration in the root of a growing carrot.

Since it has been shown (Booth5) that leaves of pigmented carrots contain u-carotene while those of white carrots do not, it will be seen that total carotene rather than p-carotene is the near-constant quantity in leaves of cultivated types of carrot.

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376 B O O T H - - C A R O T E N E I N C A R R O T LEAVES

Variation of carotene concentration f r o m batch to batch Much is known about conditions (variety, maturity, season, etc.) that affect carotene

concentration in the root of the carrot. On the other hand, knowledge of the nature of the conditions causing the much smaller variation in the concentration in the leaf is elusive. The variation cannot, for instance, be accounted for by the small variation in water content. Zafren9 found that the carotene content of clover leaves gathered in daylight was lower than in leaves gathered in darkness, and this observation has since been qualitatively supported by authors using other leaves, but Franklo stated that seedlings had no more carotene in dark than in light. Therefore variation in intensity of illumination m a y have affected the carotene content.

If the very young plants are omitted, and the batches of cultivated carrots are arranged in groups-according to year, or age, or season, or variety-the variation between groups is negligible, provided each group comprises many batches. For instance, when batches are arranged in monthly groups the concentrations of carotene in healthy leaves is seen to be nearly constant from July until winter. Further, not only the means, but also the within-group splays or variances are similar for the several groups.

The data presented provide support for the view that ( a ) a species of plant may have a characteristic carotene content, (b) that carotene content is not representable by a point value but by a frequency curve, whose characteristics would depend upon the times at which samples were taken and (c) the scatter is not merely a variation between individuals but is also due to a fluctuation with time within an individual. While the carotene content of carrot leaves does not vary much, there is indeed a fluctuation which has not yet been correlated with any condition.

Although the function of carotene in leaves is not certainly known it seems highly probable that it and luteol take a part in photosynthesis and/or other processes. If carotene does take a part in chemical reactions-if its function is chemically dynamic rather than static-then the concentration would be expected to fluctuate. Glover & Redfearnll found evidence that @-carotene in tomato leaves is oxidized to epoxide and furanoid compounds, and then further oxidized, not reduced back to carotene. If synthesis and catabolism follow different routes, as these authors suggest, and if the mechanisms regulating each were not coordinated, the concen- tration of carotene would fluctuate.

In the present work leaves were always gathered in daylight.

Acknowledgments

for some of the specimens. I thank Drs. L. J. Harris and T. Moore for their interest in this work and Mr. S. 0. S. Dark

Dunn Nutritional Laboratory Cambridge

Received 15 October, 1956

References 1 Booth, V. H., & Dark, S. 0. S., J . agric. Sci., 1949, ' Analytical Methods Committee, Society of Public

39, 226 Analysts and other Analytical Chemists, Analyst,

36. 142 Shivrina, A. N., Bull. appl . Bot. Gen. PI.-Breed. 2 Dark, S. 0. S., & Booth, V. H., J . ugric. Sci., 1946, 1950. 75, 568

.. I _

3 Rygg, G. L., Amer. SOC. hort. Sci., 1949, 54, 307 Booth, V. H., J . Sci. Fd pgr ic . , 1951. 2 , 353

1937. No. 235 Zafren, S. Ya., Dokl. Akad. N a u k SSSR, 1946, 52,

q r - 1 - 1 Booth, V. H., 1. sci. Fd Agric., 1956, 7, 386 10 Frank, S., Sci. Amer., 1956, 194, 80

6 Booth, V. H., ' Carotene, its determination in l1 Glover, J.. & Redfearn, E. R., Biochem. J., 1953, biological materials ', 1957 (Cambridge : Heffer) 54, viii

J. Sci. Food Agric., 8, June, 1957