carotenoids of and mutant strains of the green ... · krinsky and levine-carotenoids of...

PLANT PHYSIOLOGY

23. PALEG, L. 1963. Cellular localizatioIn of the gib-berellin-induced response of barley endosperm.5th Intern. Conf. on Natural Plant Growth Regula-tors, J. P. Nitsch, ed. (in press).

24. PEVELING, E. 1962. Der elektronenimikroskopischeNachweis der Spharosomeii in den Epidermiszellender Zwiebelschuppen von A/llhiumi ccpa. Proto-plasma 55: 429-35.

25. PFEFFER, W. 1872. Untersuchungen uiber die Pro-teinkdrner und die Bedentung des Asparagins beimKeimen der Sameni. Jahrb. f. Wiss. Bot. 8:429-571.

26. PORTER, K. 1961. The ground substanice; observa-tions from electroni microscopy. In: The Cell, vol.II. J. Brachet anid A. Mirsky, ed. Academic Press,N.Y.

27. SACHS, J. VoN. 1887. Lectures on the Physiologyof Plants. p 77. Clarendon Press, Oxford.

28. SALMON, J. 1940. Quelques remarques sur letatphysique et le comportement histochimique desgloboides. Compt. Rend. Acad. Sci., Paris. 211:510-11.

29. STEVENS, J. E., F. E. McDERMOTT, AND J. PACE.1963. Isolation of enidosperm protein and aleuronlecell contents from wheat, and determiniation of theiramino-acid composition. J. Sci. Food Agr. 14:284-87.

30. STOECKENIUS, W. 1959. Anl electroni microscopestudy of myelin figures. J. Biophys. Biochem.Cytol. 5: 491-500.

31. WIELER, A. 1943. Der feinere Bau der Aleuroni-klrner und ihre Entstehung. Protoplasma. 38:21 -63.

Carotenoids of Wild Type and Mutant Strains of the GreenAlga, Chlamydomonas reinhardi 1, 2

N. I. Krinsky and R. P. LevineDepartment of Pharmacology, Tufts University School of Medicine, Boston 11, and The Biological

Laboratories, Harvard University, Cambridge 38, Massachusetts

Clilainivdomionas reinilimr(di is an excellent organ-isImi for research in photosyinthesis for there are sev-eral mutant strains with impaired plhotosynthesis(21), as well as a nunmber of nmutant strains in whichthe pigments are visibly altered (25). In view of therecent investigations on the photosynthetic compe-tence of these mutant strains (17, 19, 20) we felt itwas desirable to obtain a comlplete (lescription of theircarotenoid pignments.

In a preliminary study of the carotenoid pigmentsof C. rein/lardi, Sager and Zalokar (26) separated 15carotenoi(d fractions fronm their light-grown wild typestrain. They were able to identify a-carotene, ,-caro-tene ancd lutein. When grown in the dark, the wildtype strain produced only 6 fractions, including the3 identified in the light. In contrast, a pale greenmutant strain (no. 95) which dies when grown eitherphotosynthetically or heterotrophically in the light,produced only a-carotene and 8-carotene in the dark.

In the present study. 9 (lifferent carotenoid pig-1 Received Nov. 22, 1963.2 This investigation was supported in part by Public

Health Service Research Grants (GM-07984) from theNational Institute of General Medical Sciences, (AI-01421) from the National Institute of Allergy and In-fectious Disease and by the National Science Founda-tion (GB-329).

mlenits could be i(lentifiedl in light-grown cultures ofthe wvild type strain of C. reinhardli. Dark-growniwil(d type cultures contain 7 of these pigmiients, haveless total pigment. and have a markeclly decreasedratio of 18-carotene/a-carotene. The latter 2 proper-ties are also characteristic of the mllutanlt strains (le-scribed here.

Materials and Methods

Organisms. Six strains of C. reinhar(di were usedin the experinments reported; wild type (strain 137c6a mlutant strain of spontaneous origin, 1-2, an(d 4UV-inducecl mutant strains, ac-16, ac-21, ac-115 and(lac-141 ( 18).

Wild type C. reinhardi is green when cultured inthe light or (lark, whereas v-2 is green when culturedin the light but yellow when cultured in the (lark.The nmutant strain is, therefore, phenotypically similarto the yellow in the (lark or yellow strain described bySager (24). This yellow phenotype results fronm theinability of the nmutant strain to synthesize chloro-phyll in the dark. During the course of this investi-gation, it became apparent that we were working withmixed cultures of wvild type and y-2. Cell populationstuclies were therefore carried out on cultures grown

680

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KRINSKY AND LEVINE-CAROTENOIDS OF CHLAMYDOMONAS REINHARDI

under identical conditions to those used for the pig-ment analyses. Several wild type and y-2 liquid cul-tures were grown in the light and in the dark andplated out. These plates yielded several hundredcolonies from each original culture. Wild type cul-tures were found to contain 12 % y-2 whereas thev-2 cultures contained 3 % wild type. These re-sults were the same whether the plates were cul-tured in the light or in the dark. Pigment analyseswere carried out on similar liquid cultures and thefollowing corrections applied: let w = pigment con-centration in pure wild type cultures, x = pigmentconcentration found in wild type cultures, y = pig-ment concentration in pure y-2 cultures and z = pig-ment concentration found in y-2 cultures: then x =(0.88) (w) + (0.12) (y) and z = (0.03) (w) +(0.97) (y): therefore w = (1.141) (x) - (0.141)(z) and y = (1.035)(z) - (0.035)(x).

Growth Conditions. Cultures were grown on ashaking apparatus at 250 in high salt minimal mediumsupplemented with 2 % sodium acetate (30). Theywere either illuminated continuously at a light in-tensity of 4500 lux from daylight fluorescent lampsor cultured in the dark in flasks made light-tight withblack masking tape. Pigment analyses of wild typeandl v-2 were carriel out on both light- and dark-grown cultures whereas analyses of the remainingstrains were made on light-grown cultures.

Pigmnent Analysis. Cells were harvested duringthe logarithmic phase of growth by centrifugation at4000 X g for 5 minutes, and were examined directlyor after they had been stored at - 200 for up to 48hours. Packed cells (1.2-6.0 g wet wt) were sus-pended in 6 % KOH in methanol (5 ml/g wet wt)and saponified in the dark for 5 minutes at 40°. Thismixture was rapidly chilled and the methanol de-canted after a brief centrifugation. The cell residuewas reextracted several times with methanol (5.0 ml/gwet wt) until it was colorless. All of the carotenoidpigments were transferred to diethyl ether (peroxide-fre2) following the addition of an equal volume of a5 % sodium chloride solution to the pooled methanolextracts. Alkali was removed by washing the etherlayer with water, and this extract was taken to dry-

ness in vacuo at 40°. The pigments were redissolvedin 10 % acetone in petroleum ether and were chro-matographed on columns of weakened aluminumoxide, as described earlier (15). Four fractions (A,B, C, D) were eluted from these columns with 20 %acetone in petroleum ether, 40 % acetone in petro-leum ether, acetone and absolute ethanol respectively,and these fractions were resolved into their individualcarotenoid components by chromatography on mag-nesium oxide (Sea Sorb 43): Celite (1: 1).

Fraction A. This fraction contained the hydro-carbon pigments and could be separated on a mag-nesia column by development with petroleum ether.

Fraction B. Using 2 % ethanol in petroleumether on a magnesia column this fraction resolvedinto cryptoxanthin (when present), lutein, zeaxan-thin, and luteoxanthin (zeaxanthin monoepoxidemonofuranoid).

Fraction C. Development with 6 % ethanol ona magnesia column resulted in a separation of 2 pig-ments, violaxanthin and trollein.

Fraction D. This fraction contained only a singlepigment, neoxanthin, when developed with 10 % etha-nol on a magnesia column.

Each pigment was identified by a number of pro-cedures, which included spectrophotometry, mixedchromatography with authentic samples and color re-actions with concentrated hydrochloric acid. In ad-dition, quantitative partition coefficients were ob-tained, as described by Petracek and Zechmeister(23), and from these, M50 values were obtained, asdescribed by Krinsky (14). M., values representthe percentage methanol, in a solvent system petro-leum ether-aqueous methanol which are required togive a partition coefficient (concentration in petro-leum ether/concentration in methanol) of 50: 50 andare directly proportional to the number and types ofpolar substitutes in carotenoid molecules. Epoxideswere detected by their characteristic hypsochromicspectral shift in the presence of dilute acid (12).

The results are expressed as micrograms caroten-oid per gram wet weight of packed cells. The

E1 cm values are listed in table I. along with theXmax values of the isolated pigments.

Table I. Spectral Characteristics of the Carotentoids of Chlantzydomonias reinhardi

Solvent Xmax(mA) E1 cm at Xmax

a-Carotene,8-CaroteneP-460CryptoxanthinLuteinLuteoxanthinZeaxanthinViolaxanthinTrolleinNeoxanthin* Estimated value.

Petroleum ether

EthanolPt

Carotenoid Reference

445.5451.0460.0451.0445.5423.0452.5442.5445.5437.5

273525802720*247025502280249025502350*2270

3232

2727272713

27*Estimatedvalue.

610



PLANT PHYSIOLOGY

Table II. The Carotenoids of Wild Type Chlanydornonas reinhardi

Light-grownPigment

,Ag/g wet wt

(t-Carotene3-CaroteneP-460LuteinLuteoxanthinZeaxanthinViolaxanthinTrolleinNeoxanthinUnknowns

Total(range of 4 cultures)

,8-carotenea-carotene

xanthophyllcarotene

11.6374.151.6

210.942.826.8

274.2120.3148.518.2

1279.0(996-1424)

0.929.24.0

16.53.32.1

21.49.411.61.4

32.3

1.93

Results

[Wild Typpe. Results of the pigment analyses car-

rie(l out on both light-grown and dark-grown culturesof wild type C. reinhardi are given in table II. Mostof the carotenoid pigments foun(d have been reportedpreviously in the Chlorophyceae (10, 22, 29). Theidentifications of a-carotene, 8-carotene, lutein, ze-

axanthin, violaxanthin, and neoxanthin were made on

the basis of their spectral properties, M5;, values (14),an(d cochromatography with authentic samples from

*P-460

.960 - PRO-Y-CARO-U TENE ISO-

< .800 MERATE

< 8000 640-

<.480~

.320-

.160

320 360 400 440 480 520

FIG. 1. The absorption spectra of P-460 (0) and ofthe iodine isomerate of pro-y-carotene (0) (31) in pe-

troleum ether.

natural sources. In addition, a number of pigmentswere foundl which had not been observe previouslyin the Chlorophyceae.

z

co0(I)

co

350 400 450m,u

500

FIG. 2. The absorption spectrum of luteoxanthin(-) in absolute ethanol. Also plotted is the spectrumof the auoxanthin (---) formed from luteoxanthin by theadditioin of hydrochloric acid at a final concentration of0.006 N.

682

Dark-grown

,ug/g wet wt

28.6387.719.5

128.6. . .

150.8115.695.3

926.1(908-943)

3.141.82.1

13.9

16.312.510.3

13.6

1.1




z

im0 0.5nm

a

. .

M)J'A

FIG. 3. The absorption spectrum of trollein in ab-sclute ethanol.

Pigment P-460 was observed as a pinkish-orangeband moving behind ,8-carotene on magnesium oxide-celite columns when developed with petroleum ether.It has a partition coefficient of 100: 0 in a 95 %methanol: petroleum ether system, which remainedunchanged after saponification. The spectrum of thispigment, plotted in figure 1, was virtually unchangedwhen exposed to light in the presence of iodine.

Figure 2 represents the absorption spectra, inabsolute ethanol, of luteoxanthin and the productformed by the addition of a trace of hydrochloric acid.

Luteoxanthin can be characterized by its M50 value of64.3, characteristic of a compound containing 2 hy-droxyl groups and 2 oxide groups (14), by the hypso-chromic displacement of 19 m,u when it is convertedto auroxanthin following the addition of a trace ofhydrochloric acid, and by cochromatography with anauthentic sample of luteoxanthin isolated from aceticacid-treated violaxanthin (28). This pigment isfound in the light-grown cultures, but not in dark-grown cultures of the wild type strain (table II).

The absorption spectrum of trollein, present inboth light-grown and dark-grown cultures of wildtype C. reinhardi, is shown in figure 3. Epoxidegroups are absent, for when the pigment is treatedwith dilute hydrochloric acid, there is a hypsochromicdisplacement of only 1 to 2 m,u, apparently due to theformation of cis-isomers. When an ethereal solutionof the pigment is treated with concentrated hydro-chloric acid, there is no color development, whichindicates a lack of a furanoid group. Quantitativepartition coefficients yield an M50 value of 53.3, in-dicative of 3 hydroxyl groups.

On occasions, small quantities of pigments withproperties similar to mutatochrome, flavoxanthin, andauroxanthin were observed, but they were not presentin sufficient quantities for positive identification. AnE ' es value of 2250 at 440 mu was used to deter-mine the concentration of the unknown pigments.

Dark-grown wild type cultures contain about 70 %of the carotenoid pigment present in the light-growncultures. This decrease is due to a lower concentra-tion of xanthophylls in the dark-grown cultures, forthe total carotene level does not differ when wild typecultures are grown in the light or in the dark. Thereis, however, a change in the 18-carotene/a-caroteneratio, which drops from a value of 32.3 in the light-grown cultures to 13.6 in the dark-grown cultures.In addition, we were unable to detect zeaxanthin or

luteoxanthin in these dark-grown cultures.

Table III. The Carotenoids of Mutant Strain y-2

Light-grown Dark-grownPigmentLih-rwPtng/g wet wt % Ag/g wet wt go

a-Carotene 18.8 2.3 15.6 5.2,8-Carotene 193.8 23.9 140.7 47.3P-460 5.9 0.7 3.7 1.2Cryptoxanthin 7.2 0.9 4.5 1.5Lutein 202.1 24.9 71.8 24.2Zeaxanthin 2.5 0.3Violaxanthin 149.6 18.4 31 10.5Trollein 151.3 18.6 22.9 7.7Neoxanthin 79.8 9.8 6.8 2.3

Total 811.0 297.1(range of 2 cultures) (762-860) (290-304)

1-carotenea-carotene 10.3 9.0

2.710.86~~~~~~~~

xanthophyllcarotene

6083

2.71 0.86



Table IV. T'he Carotentoids of Photosyntthetic Muttants of Chlaomdomomos reinhardi

ac-16 ac-21 ac-lAlPigment

a-Carotenie(-CaroteineP-460CryptoxanithinLuteinLuteoxanthinZeaxanthinViolaxanithitiTrolleinNeoxanthinUnkniowns

Total

Mg/gwet wt

6.7109.9

112.542.3

58.892.480.014.0

516.6

/g,/g<70 wet wt

1.321.3

21.88.2

11.417.915.52.7

% of light-grownwild type 40.4

(-carotene 16.4a-carotene

xanthophyllcaroteine

3.43

22.966.33.43.3

147.95.2

25.889.251.561.6

. .1477.1

37.3

2.9

4.15

Lg/g%7 wet wvt

4.813.90.70.7

31.01.15.4

18.710.812.9

35.9186.2

5.76.6

258.111.421.7154.2127.6125.711.3

944.4

73.8

5.2

3.15

'v-2. The carotenoid pignments foundl in botlh light-grown an(ldark-grown cultures of the y-2 strain are

listed in table III. In general, the pigmlent distribu-tion was similar to that observed with the wild typestraiin (table II), but 1-2 was founcl to contain smallamiiounts of cryptoxanthin and to lack luteoxanthin.

Light-grown cultures of y-2 contain about 63 %of the total pigment found in conmparable wild typeclultures. However, the dlark-grow n cultures of y-2contain only 32 % of the carotenoid pigment found indark-grown cultures of wil(d type. Botlh light- andt1ark-groxvn cultures of y-2 halve /3-carotene/a-caro-tene ratios close to the value found( for the dlark-growncultures of wild type.

ac-16. This mutant strain (table IV) containsonly 40 C%i of the carotenoids found( in the wild type.Tt lacks P-460 and zeaxanithin, and(l contains rela-tively less /3-carotene and violaxanthini and relativelymore trollein, neoxanthin, and luteoxanthin. The/3-carotene/a-carotene ratio is sinmilar to the valuefoundl for wild type cultures grown in the dark.

ac-21. While this mutant strain contains approxi-mlatelyx the same amount of carotenoid pigment as doesac-16 (37 °S, of the light-grown wildl type value,table IV), there is a very marked alteration in thecarotene fraction. The nmutant contains only 17 to18 % of the /8-carotene found in both light- and dark-grown cultures of wild type strain, ancd the,8-carotene/a-carotene ratio is 2.9, the lowest value recorded forthese strains of C. reinhardi.

ac-115. With respect to its total carotenoid con-

tent, this mutant approaches the level found in light-grown wild type (74 %) and actually contains more

than the dark-grown wild type (102 %). However,clark-grown wild type contains twice as much caro-

tene as dloes ac-115. The distribution of pigments in

ac-115 is very similar to light-grown cultures of 1-2,wvith the single exception that ac-15 contains lute-oxanthin.

ac-141. Although this mutant contains only 41 cof the total caroten-oid found in light-grown wilc typecultures (table TV), there is essentially no decrease

in the anmount of lutein. Thus, the mutant strain ishighly enrichedl with respect to this pigment. Thereis also an enrichimlent in the relative amlount of a-

carotenie, resultinig in a /8-carotene /a-carotene ratioof 5.t6.

Discussion

The distribution of carotenoi(d pigmlenits amilonig thevarious classes of algae has been reviewe(l recently(10, 22, 29). In general, it appears that the Chloro-phyceae are very similar to green plaints, for bothgroups contain a-carotene, /3-carotene, lutein, vio-laxanthin, and neoxanthin. This pattern can be al-tered by mutationi. Claes describedl 4 nmutant strainsof Chlorella, zuitlgaris in which the synthesis of thecarotenoid pigmlents was altered (2-6). Allen, Good-win, andl Phagpolngarm described 3 mutant strains ofChliorella pyretioidosa which also ha(l altered carotenl-oicl i)atterns (1).

The carotenoids of C. reinliardi have been studiedlby Sager ancl Zaloker (26), ancl they idlentified a-

carotene, /3-carotene, and lutein in their pignment frac-tions froml light-grown wild type cultures. Our re-

sults confirnm the presence of these 3 well-knownChlorophyceae pignments, and we have also identifiedthe epoxide carotenoids, violaxanthin and neoxan-

thin, in similar cultures. In addition, we have isolatedand identified 3 pigments not previously reportedl inC. reiniiar(di.

ac-141

wgtg% wet wt

3.819.70.60.7

27.31.22.3

16.313.513.31.7

23.2129.0

8.4

194.93.27.4

68.532.659.0

. .

526.2

4.424.51.6

37.00.61.4

13.06.2

11.2

41.1

5.6

2.28

684 PLANT PHYSIOLOGY




One of these has been designated P-460 from thewave length of the major absorption peak in petro-leum ether. From its position on magnesium oxide-celite columns and its partition coefficient, which re-mains constant with saponification, we conclude thatP-460 is a hydrocarbon. As seen in figure 1, the spec-trum of P-460 is almost identical to the spectrum ob-tained by isomerizing pro-y-carotene (31), and itwould therefore appear that P-460 is a naturally oc-curring cis-isomer of y-carotene. Haxo and Clen-denning have reported large amounts of y-carotene inthe gametes of the green alga, Ulva (11).

An additional pigment has been identified as trol-lein. This was first described by Curl and Bailey (8)as a polyhydroxy xanthophyll present in orange juice.This pigment appears identical to a xanthophyll iso-lated from green algae by Strain (29). When chro-matographed on sugar columns, and developed with0.5 % n-propanol in petroleum ether, the unidentifiedxanthophyll described by Strain adsorbed betweenviolaxanthin and neoxanthin. We find that trolleinbehaves similarly when chromatographed with vio-laxanthin and neoxanthin on a sugar column, formingthe middle zone. Krinsky (14) has suggested thatthis compound is a tri-hydroxy xanthophyll, andKrinsky et al. (16) have also isolated this pigmentfrom another alga, Euglena gracilis.

The third new pigment which we have character-ized from C. reinhardi does not appear to have beenobserved previously in any algal species. This pig-ment is luteoxanthin, a monoepoxide monofuranoidxanthophyll first isolated by Curl and Bailey (7) froma number of fruits. Luteoxanthin can be formed invitro during the acid-catalyzed conversion of vio-laxanthin to auroxanthin (28). Inasmuch as tracesof an auorxanthin-like pigment were occasionallyfound in the light-grown cultures along with lute-oxanthin, there was the possibility that both of thesepigments arose from violaxanthin during the extrac-tion process. This seems unlikely, in view of the factthat we found luteoxanthin in all of the light-grownwild type cultures that we examined (both fresh andfrozen material), but found none of this pigment indark-grown cultures of the wild type strain whichreceived identical treatments (table II). In addition,if conditions during the extraction permitted the con-version of violaxanthin to luteoxanthin, one wouldexpect to find neoxanthin converted to its correspond-ing furanoid, neochrome (9), but this latter pigmentwas never observed in these extracts. Thus, it ap-pears that the presence of luteoxanthin specificallydifferentiates light-grown cultures of the wild typefrom dark-grown cultures.

Zeaxanthin, found frequently in higher plants, hasnot been previously reported in the Chlorophyceae.This pigment is present in all of the light-grown cul-tures of C. reinhardi reported here except ac-16.

There are, therefore, 3 major differences betweenthe light-grown and dark-grown cultures of the wildtype strain of C. reinhardi with respect to theircarotenoid content. (A) Dark-grown cultures con-

tain 28 % less carotenoids than do the light-growncultures. This decrease in total carotenoid occurscompletely at the expense of the xanthophyll fraction,for the total carotenes remain constant in both thelight and dark. A consequence of this is the fact thatthe xanthophyll/carotene ratio falls from a value of1.9 in the light-grown cultures to 1.1 in the dark-grown cultures. (B) The ,8-carotene/a-carotene ra-tio also decreases, from a value of 32.3 in the light-grown cultures to a value of 13.6 in the dark-growncultures. (C) Zeaxanthin and luteoxanthin are absentfrom dark-grown cells. Although these 2 pigmentsaccount for only about 5 % of the total carotenoid inthe light-grown cultures, they should have been read-ily detectable in the dark-grown cultures, if present.

The full significance of these observations is asyet unknown. It has been shown, however, that dark-grown cultures of the wild type strain cannot carryout a Hill reaction using either p-benzoquinone orferricyanide as an electron acceptor until they havebeen exposed to light for 15 minutes (R. P. Levineand D. Graham, unpublished observations).

Our results differ from those presented by Sagerand Zalokar (26), particularly with respect to theamount of xanthophylls present. We find 12 times asmuch xanthophyll present in light-grown wild typecultures and twice as much in dark-grown wild typecultures. One factor which might account for someof these differences is the age of the cultures at har-vesting. Sager and Zalokar reported using cells har-vested at the end of the logarithmic phase of growth,whereas we harvested cells during the logarithmicphase of growth. In addition, they did not report thelight intensity at which their cultures were grown.

The mutant strain, v-2, appears very similar towild type when grown in the light, with the exceptionsthat it contains a small amount of cryptoxanthin, andluteoxanthin is absent. In the dark, when v-2 is nolonger able to synthesize chlorophyll, there is a verymarked decrease in the total carotenoids, and this de-crease is seen in both the carotene and in the xantho-phyll fractions.

In comparison, the pigment mutant described bySager and Zalokar (26) as a pale green mutant (no.95) lacks all xanthophylls and contains only a- and,8-car3tene. The 18-carotene/a-carotene ratio is 2.9.This mutant strain was grown in the dark, for it dieswhen grown aerobically in the light. In this respect,it resembles some of the mutant strains of Chlorellavulgaris (2-6) and Chlorella pyrenoidosa (1) whichwill also be killed when exposed to light and to oxy-gen. The Chlorella mutants lack xanthophylls, butcontain colorless polyenes, such as phytofluene andphytoene, which are absent in the wild type strains.These colorless polyenes were not reported in thepale green mutant (26), and they are also absent fromstrain y-2.

The 4 remaining mutant strains of C. reinhardiunder consideration have impaired photosynthesis.They are unable to fix CO2 in the light at a rate com-parable to the wild type strain (21) and each is pre-

6805



PLANT PHYSIOLOGY

sumed, therefore, to be blocked in some step of photo-synthesis. These steps have been partially elucidated.Whole cells or isolated chloroplast fragments of ac-16an(l ac-21 give a Hill reaction whereas similar prepa-rations of ac-115 and ac-141 (lo not (20, 21). It hasalso been shown that both ac-115 an (lac-141 are de-ficient in plastoquinone (20).

The analysis of the carotenoidl pigments in light-grown cells of these 4 mutant strains reveals sinmilar-ities aimongst the mutant types which ten'l to (listin-guish them from the wild type strain (table IV). All4 of these mutant strains have a lower carotenoidcontent antI a lower P-carotene/a-carotene ratio thanthe light-grown wild type strain. In this respect,these mutants resemble the dlark-grown wild typestrain, which has similar characteristics. They dif-fer from dark-grown wild type, however, for theyhave a higher xanthophyll/carotene ratio.

In spite of the similarities between the mutantstrains an(l the dark-grown wildl type strain, eachstriain has a characteristic pattern of types andamounts of carotenoids which (listinguishes it fromthe other nmutant strains an(d fromii both the (lark- andlight-grown wild type.

It must be concluded, therefore, that the carotenoidpignments founid in these 4 mutants,, while character-istic for each strain, (lo not appear to show sufficient(lifferences fronm those present in the wild type strainto account for their inability to carry out normalphotosynthesis.

Summary

Light-grown cultures of the wild type strain ofClhlaniividoiiionas reinhardi have been shown to con-tain the usual pigments of the Chlorophyceae: a- and,8-carotene, lutein, violaxaintlhin, an(l neoxanthin. Inaddition, zeaxanthin, luteoxanthin, trollein, an(d a newcarotene, P-460, which appears to be a cis-isomler ofy-carotene have been identifiedl.

Dark-grown cultures of the wil(d type straiin con-tain less total carotenoil, andl this (lecrease occursexclusively in the xanthophyll fraction, resulting in alower xanthophyll/carotene ratio than that found inthe light-grown cultures. The (lark-grown culturesare also characterized by a lowver 18-carotene/a-caro-tene ratio, and 2 xanthophylls, zeaxanthin an(d lute-oxanthin, are absent.

The mlutant, v-2, when culture(d in the light, issimilar to the wild type strain, except that it lacksluteoxanthin and contains a smlall amnount of cryp-toxanthin. In the dark it shows a very mlarked de-crease in total carotenoidls.

Light-grown cultures of 4 mutant strains with im-pairedl photosynthetic ability, ac-16, ac-21, ac-l15, andac-141, all have characteristic carotenoid patternswhich distinguish them from the light-grown wildtype strain. They share the properties of havinghigher xanthophyll-carotene ratios, lower total caro-tenoitls, and lower 13-carotene/a-carotene ratios thanthe corresponding wild type culture. The latter 2

properties are also characteristic of the wild ty)pestrain culturedl in the (lark.

Acknowledgment

The authors acknowledge the expert technical assist-ance oI Adrian Gordon and Joanne Brunigard during thecourse of this work. VTiolaxanthin was a gift of Dr.J. C. Bauernfeind of Hoffman-LaRoche.

Literature Cited

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Determinations of the Heat Transfer Coefficient of a Leaf'Edward Thornton Linacre

Commonwealth Scientific and Industrial Research Organization, Irrigation Research LaboratoryGriffith, N.S.W., Australia

In equilibrium conditions the net radiant energyflux R falling onto a leaf is divided mainly betweenthe flow of latent heat for the evaporation of trans-pired moisture LE, and sensible heat passing into theambient air H. The latter defines a heat-transfercoefficient h in terms of a temperature difference, thus

H = h(TleafTair)* IWhere the heat transfer is the result of convection,the coefficient may be replaced (10, 14, 15) by anequivalent diffusion resistance r

r = cp/lh = 0.018 sec/cm, IIh

approximately, where cp is the specific heat of air atconstant pressure (cal/g°C), p is the density of air

1 Received Nov. 22, 1963.

(g/cm3), and h is the heat-transfer coefficient (cal/cm2min °C). However, it is convenient in the pres-ent note to retain the concept of the transfer coeffi-cient. The aim is to review previous experimentalmethods of deriving the coefficient, and then to de-scribe a modification.

Methods

Steady-State Methods. Brown and Escombe (2)used equation I to derive the coefficient, which wascalled the 'emissivity' in their paper. This termimplies radiative transfer, which certainly occurs, butit accounts for only 0.008 cal/cm2min °C of the valueof the heat-transfer coefficient (18). The flux H wasreckoned by Brown and Escombe as the difference be-tween the radiation flux R and the latent-heat flux

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carotenoids of and mutant strains of the green ... · krinsky and levine-carotenoids of...

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