Molec. gen. Genet. 179, 259 263 (1980) © by Springer-Verlag 1980

Three New

yellow Loci in Chlamydomonas reinhardtii

Clark F o r d and Wei-yeh W a n g Department of Botany and Genetics Ph.D. Program, University of Iowa, Iowa City, Iowa, 52242, USA

Summary. Three phenotypically ' y e l l o w ' , mendelian mutants of Chlamydomonas reinhardtii have been isolated and tested for allelism with the yellow m u t a n t y-1 a 1 and with each other. The three m u t a n t s represent three new yellow loci, two of which are located on linkage g r o u p I. Like y-1 a, the mutants accumulate protochlorophyllide when grown under dim light, but have a wildtype phenotype when grown in the light. W e conclude that the control of light-independent protochlorophyllide reduction is m o r e complex than has been t h o u g h t previously.

Introduction The reduction o f protochlorophyllide (Pchlide) to chlorophyllide plays a key role in the biosynthesis of chlorophyll and in the biogenesis of functional chloroplasts. All plants are capable of Pchlide reduction via a light-dependent reaction involving the Pchlideh o l o c h r o m e complex (Harel, 1978). In some lower plants and algae, including the green alga Chlamydomonas reinhardtii, a second means of Pchlide reduction exists which allows chlorophyll synthesis to proceed in the absence of light (Bogorad, 1976). Very little is k n o w n a b o u t this light-independent reaction. M u t a n t s apparently defective in light-independent Pchlide reduction have been described in C. reinhardtii and in the related green alga ChIorella vulgaris. These mutants, called yellow mutants, fail to synthesize chlorophyll when grown in the dark, but instead accumulate small a m o u n t s of Pchlide. Light-dependent, h o l o c h r o m e - m e d i a t e d Pchlide reduction is unaffected in these m u t a n t s since chlorophyll synthesis is n o r m a l when they are grown in the light (Sager, 1961 ; Granick, 1950).

Offprint request to." Wei-yeh Wang 1 y-la was isolated by Wang et al. (1977) and was shown to be allelic to the y-1 mutant isolated by Sager (1955)

Several independently isolated yellow m u t a n t s of C. reinhardtii have been analyzed genetically (Sager and Tsubo, 1962; Winig, 1965 ; Stolbova, 1971 ; W a n g et al., 1977) and are all apparently allelic to the yellow m u t a n t y-1 isolated by Sager (1955). y-1 is a nuclear, centromeric m u t a t i o n as shown by 2 : 2 segregation in crosses with wildtype, and by the absence of second division segregation in meiosis (Sager, 1955). Several attempts to m a p y-1 to the 16 k n o w n linkage groups of C. reinhardtii have failed (Hastings et al., 1965; Stolbova, 1971 ; Smyth et al., 1975) p r o m p t i n g Hastings et al. (1965) and Smyth et al. (1975) to propose its location on a 17th linkage group. We have isolated three new yellow mutants of C. reinhardtii which are non-allelic either to y-1 or to each other. All three accumulate Pchlide when grown under dim light and synthesize chlorophyll normally when grown in the light. Since mutations at four different loci in C. reinhardtii apparently cause defective light-independent Pchlide reduction, we think the reaction or its control is m u c h m o r e complex than has been t h o u g h t previously.

Materials and Methods Strains. Wildtype and mutant stocks of C. reinhardtii used in this study were derived from strain 137 c. The y-1 a mutant was isolated by Wang et al. (1977) and was shown to be allelic to the y-1 mutant originally isolated by Sager (1955). The argenine-requiring mutants arg-2 and arg-7 were also used. Stocks were monitored microscopically for the presence of pigment mutants or revertants and were cloned to purity when necessary. Culture Conditions. Cells were grown either on high salt medium (Sueoka, 1960) supplemented with 1.2 g/1 sodium acetate (HSA) or on Tris-acetate phosphate medium (TAP) (Gorman and Levine, 1965). Media were solidified by the addition of 15 g/1 Difco agar. 100-200 /ag/ml L-arginine was added to support the growth of the arginine auxotrophs. TAP of HSA medium made without NH4C1 ( - N ) were used for gametogenesis. The minimal medium of Ebersold (1956) solidified with 4% agar (40 g/L) was used for zygote maturation.

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260

C. Ford and W. W a n g : Three New yellow Loci in Chlamydomonas reinhardtii

Cells were grown at 2 5 ° C in the light (1500-6500 lux from fluorescent tubes) or under dim light (about 0.5 lux from fluorescent tubes mostly covered by black electrical tape). Light intensities were measured with an LI-185 photometer ( L a m b d a Instrument Corp.). D i m light instead of total darkness was used routinely because the y-1 a m u t a n t dies or reverts to wildtype after prolonged growth in the dark (Wang, 1978). After growth for several generations under dim light, yellow m u t a n t s can easily be distinguished from wildtype by their yellow color. Occasionally cells were grown under dim light at 35 ° C to shorten their generation time.

Mutagenesis. The produre used for UV mutagenesis was that of W a n g et al. (1974) with some minor alterations. Five ml of lightgrown wildtype cells at a concentration of about 5 x 106 cells/ml were irradiated 3 ~ 1 2 0 s while stirred by a spin bar in a small (5-cm diameter) Petri dish. The short-wave ultra-violet light source (mineralight UVSL25, Ultra-Violet Products, Inc.) was m o u n t e d in a box about 12 cm from the surface of the cell suspension. After irradiation the cells were held in darkness for 24 h to prevent photoreactivation. Cells were plated under room light and incubated under dim light conditions. Genetic Analysis. Genetic crosses were made following a procedure similar to that of W a n g et al. (1974). Cells were pregrown on agar plates in the light, transfered to -N plates and incubated in the light for 1-2 days to form gametes. Gametes of opposite mating types were suspended in distilled water or in liquid -N m e d i u m for mating. Zygotes, plated on 4% agar plates, were incubated for 1-2 days in light then transferred to darkness for maturation. F o r tetrad analysis routine procedures established by Ebersold and Levine (1959) were used. Zygotes were germinated in the light or under low light (about 200 lux from fluorescent tubes). Markers were considered unlinked if the ratio of parental ditype tetrads (PD): non-parental ditype tetrads (NPD) was 1 : i or if the ratio of non-parental ditype tetrads: tetratype tetrads (T) was greater than 1:4 based on X2 tests (p > 0.05) (Gowans, 1965). M a p distances between linked markers were determined from the formula: m a p units ( m . u . ) = ( 1 / z T + N P D / P D + N P D + T ) x l 0 0 (Smyth et al. 1975). Centromeric distances were calculated from the frequency of tetratype tetrads in crosses with the centromeric marker yla:m.u.=(1/z/PD+NPD+T) x 100 (Hastings et al., 1965). For zygote clone recombination tests, mature zygotes and unmated vegetative cells from 4% plates were collected and plated on T A P or H S A plates and then exposed to ehlorofrom vapor for 30 s to kill the vegetative cells. The zygotes were germinated and allowed to grow for 1-3 days in the light, then incubated under dim light for several generations before scoring. Zygote clones containing green sectors were scored as recombinant clones; those without green sectors were scored as non-recombinant. Complementation tests were done by the m e t h o d of Gillham (1963). yellow m u t a n t s combined with the auxotrophic marker arg2 were crossed to yellow m u t a n t s carrying the complementing marker arg-7. Diploid cells formed by mating as well as u n m a t e d gametes from the mating mixture were plated on minimal medium and incubated in the light for 1-2 days before transfer to dim light. Only the diploid cells which carried both complementing auxotrophic markers survived on minimal medium. After several generations of growth under dim light, diploid clones were scored on the basis of their coloration. Yellow clones indicated lack of complementation between two yellow m u t a n t s ; green diploid clones indicated that complementation had occurred. Pigment Analysis. Pigment determinations were performed both on cells grown in the light (3000 4000 lux) and under dim light conditions based on the method of W a n g (1978). Cells were pregrown on H S A plates, transferred to liquid H S A m e d i u m in Erlen-

meyer flasks, and grown for several generations on a shaker. Cells were kept in log phase by transfer and dilution. Cell concentrations were determined by hemacytometer counts. Two ml light-grown or 35 ml dim light-grown cell cultures were harvested by centrifugation, and the pellets were frozen. A n equal volume of 0.1 N N H 4 O H was added to the light-grown cell suspension before centrifugation to kill the cells. Dim light-grown cell cultures were transferred to dark flasks for 24 h (~1 generation) before harvesting to dilute chlorophyll produced by photoconversion. Frozen pellets were thawed on ice and extracted with 5 ml cold alkaline acetone (acetone: 0.1 N N H , O H ; 9:1; v/v) for 30 min. Extraction procedures were carried out under a green safelight or in the dark. Pigment extracts were centrifuged, and the supern a t a n t was brought back up to a volume of 5 ml with acetone. Extracts were scanned from 400 to 700 n m with an Aminco DW-2 spectrophotometer. After scanning, the acetone extracts were then reextracted with equal volumes of petroleum ether. The partitioned petroleum ether fractions (containing phytolated pigments) were discarded and the remaining acetone fractions were adjusted to their original volume with the 90% alkaline acetone and rescanned in the spectrophotometer. Chlorophyll content of the samples was determined by analysis of the first scan while Pchlide concentrations were determined from the second scan. Total chlorophyll was calculated by A r n o n ' s equation (1949), with chlorophyll a and be measured at 663 n m and 646 rim, respectively. In samples with more Pchlide than chlorophyll, only chlorophyll a was measured using the extinction coefficient 90.8 (mM -1) (Falk, 1964). Pchlide was recognized by its characteristic absorption spectrum (Gough, 1972). Pchlide concentrations were calculated using the extinction coefficient 34.5 (riM-~) at 628 n m (Gough, 1972).

Results

Description of Mutants. We have isolated three phenotypically 'yellow' mutants from wildtype C. reinhardtii. The mutants appear yellow when grown under dim light, and are indistinguishable from wildtype when grown in the light. The three mutants are designated y-5, y-6, and y-7. y-5 was isolated by UV mutagenesis following the procedure of Wang et al. (1974). y-6 was isolated as a spontaneous mutant, y-7 was isolated following the mutagenesis procedure given in the Materials and Methods. Crosses made between the mutants and wildtype were scored using tetrad analysis (Table 1). All of the crosses showed 2: 2 segregation, indicating nuclear location of the mutations. In all of the crosses, only parental ditype tetrads were found, indicating that the mutant phenotypes result from single gene mutations. When grown under dim light and then in darkness for 24h, all three mutants accumulated small amounts of Pchlide (Table 2). The total amount of chlorophyll synthesized under these conditions was about 102-103 times less than that made by the wildtype. Compared with the other yellow mutants, y-5 accumulated about 90% less Pchfide and about 10 times more chlorophyll, y-5 clearly accumulated more chlorophyll than Pchlide, although the opposite was true for the other yellow mutants. To determine whether

C. Ford and W. Wang: Three New yellow Loci in Chlamydomonas reinhardtii Table 1. Tetrad analysis of crosses between yellow mutants and wildtype

261

Table 5. Tetrad analysis of pairwise crosses between yellow mutants

y-5 y - l a x y - l a + 91:0:0 y-5 x y - 5 + 99:0:0 y-6 x y - 6 ÷ 54:0:0 y-7 x y - 7 + 87:0:0

y-Ia

y-5

Ratios represent parental ditypes (PD): non-parental ditypes (NPD): tetratypes (T)

y-la

y-5 y-6 y-7

Wildtype

Dim light gmoles/1011 cells Pchlide

Chl

Light gmoles/109 cells Chl

2.1 _ + 1 . 8 0.25_+0.03 1.7 + _ 0 . 4 1.9 _ + 0 . 0 ND

0.14_+0.07 4.3 ±0.2 0.14_+0.07 0.41_+0.02 360 -+ 10

7.7_+2.1 3.5_+0.9 4.4+_0.9 4.1_+0.1 9.9 _+1.2

Table 3. Zygote clone recombination tests of pairwise crosses between yellow mutants y-1 a

y-5

y-6

y- 7

-

+ -

+ + -

+ + +

y-5 y-6 y-7

Zygote clones containing green sectors were scored as recombinant clones; those without green sectors were scored as non-recombinant. Minus ( - ) indicates no recombinant clones observed in a sample of over 2000. Plus ( + ) indicates greater than 10% recombinant clones observed in a sample of over 100

Table 4. Complementation tests of pairwise crosses between yellow mutants

y-la

y-5 y-6

y-1 a

y-5

y-6

y-7

-

+

+ + --

+ + +

-

-

112:109:0

82:81:1

68:0:31 (t6 m.u.)

15:23:17 20:22:0

low mutant, y-6 and y-7 are much more stringent than y-5, but no definitive tests of their leakiness were made. When grown in the light all three mutants make roughly wildtype levels of chlorophyll.

N D = n o t detectable; Pchlide=protochlorophyllide; Chl=chlorophyll

y-la

y-7

Ratios represent PD:NPD:T. The underlined ratio indicates linkage. The number in parentheses indicates the approximate recombination distance in map units (m.u.) between the linked markers

Table 2. Pigment analysis of yellow mutants and wildtype Genotypes

y-6

22:30:21

y-6

y-7

Minus ( - ) indicates lack of complementation (yellow diploids). Plus (+) indicates complementation (green diploids)

the high level of chlorophyll accumulated by y-5 was due to photoconversion by dim light or to leakiness, y-5 was grown for several additional generations in the dark after transfer from dim light. N o significant dilution of the chlorophyll accumulated by the mutant was found. We therefore interpret y-5 as a leaky yel-

Genetic Analysis. Pairwise zygote clone recombination tests between the new yellow mutants and with y-1 a showed that the four mutations recombine freely, indicating that they are non-allelic to one another (Table 3). Complementation tests between the foulr mutants showed that y-1 a, y-5, y-6, and y-7 occupy separate complementation groups, confirming that the mutations are from four different genetic loci (Table 4). The presence of wildtype pigmentation in the diploids formed between the different yellow mutants indicated that all four of the yellow mutations are recessive to their respective wildtype alleles. Tetrad analysis of pairwise crosses between the four yellow mutants indicated that the y-5 and y-6 loci are linked, separated by a distance of about 16 map units (m.u.) Table 5). The y-7 and -1 loci are unlinked both to each other and to the linked y-5 and y-6 loci. Map distances between the new yellow mutations and their respective centromeres were determined from the crosses with y-1 a, since the y-1 locus is centromeric (Table 5). N o second division segregation in over 200 tetrads scored was found in the cross between y-6 and y-la, indicating that the y-6 locus is very tightly linked to its centromere. The y-5 locus is located about 14 map units from its centromere. Since the y-6 locus is centromeric, the distance between the y-5 locus and its centromere can be independently determined from the map distance between the y-5 and y-6 loci (16 m.u.). The two centromeric distances are in fair agreement. Like the y-1 and y-6 loci, the y-7 locus is also closely linked to its centromere ( < 1 m.u.). In mapping crosses, both the y-5 and y-6 loci were found to show linkage with the marker arg-7 located on linkage group 1 (Hastings et al., 1965) (Table 6). The y-5 locus is located about 8 map units

C. Ford and W. Wang: Three New yellow Loci in Chlamydomonas reinhardtii

262

Table 6. Tetrad analysis of pairwise crosses between the linkage

group 1 marker arg-7 and yellow mutants Genotypes

y-1 a

y-5

y-6

y-7

arg-7

6:8:1

39:0:7 (8 m.u.)

45:0:40 (21 m.u.)

4:9:5

Ratios represent P:NPD:T. Underlined ratios indicate linkage. Numbers in parentheses indicate approximate recombination distances in map units (m.u.) between linked markers

y-6

y-5

•

arg-7

o

o

C

~

1

6

~

8 21

y-5 and y-6 in relation to arg-7 and the centromere (c) of linkage group 1. Numbers indicate approximate recombination distances in map units according to the data in Tables 5 and 6

Fig. 1. Map locations of

to the left o f arg-7 on the right a r m o f l i n k a g e g r o u p 1 (Fig. 1).

Discussion

W e have i s o l a t e d three new yellow m u t a n t s o f Chlamydomonas reinhardtii, d e s i g n a t e d y-5, y-6, a n d y-7. The p h e n o t y p e s o f the three m u t a n t s are very similar to t h a t o f the y-1 m u t a n t . A l l a c c u m u l a t e Pchlide a n d h a v e c h l o r o p h y l l levels m u c h r e d u c e d f r o m wildtype w h e n g r o w n u n d e r d i m light. T h u s we a s s u m e t h a t they are defective in the m e c h a n i s m for lighti n d e p e n d e n t Pchlide r e d u c t i o n . W h e n g r o w n in the light, the new m u t a n t s , like y-l, p r o d u c e c h l o r o p h y l l at a b o u t the w i l d t y p e level. T h u s the m u t a n t s h a v e a functional light-dependent (holochrome-mediated) Pchlide reduction mechanism. T h e defects in the three m u t a n t s we have i s o l a t e d are all caused b y single, recessive n u c l e a r m u t a t i o n s . y-6 is a s p o n t a n e o u s m u t a t i o n while y-5 a n d y-7 are U V - i n d u c e d . T h e three n e w yellow m u t a t i o n s t o g e t h e r with y-1 define f o u r r e c o m b i n a t i o n a l l y d i s t i n c t loci as s h o w n b y z y g o t e clone r e c o m b i n a t i o n tests a n d t e t r a d analysis. H e t e r o z y g o u s d i p l o i d s f o r m e d b y c o m p l e m e n t a t i o n tests h a v e a w i l d t y p e p h e n o t y p e which shows t h a t the f o u r yellow m u t a t i o n s are recessive a n d t h a t w i l d t y p e gene p r o d u c t s f r o m the yellow loci are able to c o m p l e m e n t each o t h e r in trans. This indicates t h a t at least f o u r p r o t e i n s are i n v o l v e d in the genetic c o n t r o l o r the b i o c h e m i c a l m e c h a n i s m o f Pchlide reduction. T w o o f the yellow loci, y-5 a n d y-6, are linked,

a n d these have been m a p p e d to the first linkage g r o u p o f C. reinhardiii. T h e c e n t r o m e r i c l o c a t i o n o f the y-6 locus m a k e s it an ideal m a r k e r f o r genetic m a p p i n g . P r e l i m i n a r y tests indicate t h a t the y-7 locus is l o c a t e d on linkage g r o u p III. F r o m their d i s t r i b u t i o n in the g e n o m e , we can c o n c l u d e t h a t the f o u r yellow loci d o n o t constitute a s p a t i a l l y related f u n c t i o n a l g r o u p such as an o p e r o n . It has been p r e v i o u s l y a s s u m e d t h a t y-1 m u t a t i o n s d e f i n e d the o n l y yellow locus in C. reinhardtii. W e believe the r e a s o n t h a t o t h e r yellow loci have n o t been described is due to the highly m u t a b l e n a t u r e o f the y-1 locus (Sager a n d T s u b o , 1962; Winig, 1965), a n d conseq u e n t l y the ease with w h i c h y-1 alleles can be isolated. Since there was only one k n o w n genetic lesion o f the reaction, the l i g h t - i n d e p e n d e n t Pchlide r e d u c t i o n m e c h a n i s m was n o t t h o u g h t to be c o m p l e x ( G r a n i c k , 1967). W e have n o w s h o w n t h a t there are at least f o u r yellow loci in C. reinhardtii. T h e r e f o r e the c o n t r o l o f l i g h t - i n d e p e n d e n t Pchlide r e d u c t i o n m u s t be m o r e c o m p l e x t h a n c o u l d be a s s u m e d with k n o w l e d g e o f o n l y the y-1 m u t a t i o n a n d its alleles. This c o n c l u s i o n is s t r e n g t h e n e d b y p r e l i m i n a r y d a t a i n d i c a t i n g the existance o f several m o r e yellow loci in a d d i t i o n to the f o u r r e p o r t e d here. A l t h o u g h we have identified several genetic loci w h o s e p r o p e r f u n c t i o n s are a p p a r ently necessary for a l i g h t - i n d e p e n d e n t Pchlide reduction, it is n o t k n o w n h o w m a n y o f the yellow loci p r o d u c e p r o t e i n s directly involved in the b i o c h e m i c a l r e a c t i o n p e r se. O n e or m o r e o f the loci m a y p r o d u c e positive r e g u l a t o r y p r o d u c t s necessary for p r o d u c t i o n o f the e n z y m a t i c proteins.

Acknowledgments. We would like to thank Lou Marino for his technical assistance. C.F. is an N.I.H. pre-doctorial trainee. References

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Note Added in Proof

A correction factor was used in the determination of Pchlide concentrations to compensate for chlorophyllide absorbance at 628 nm. Dim light spectra of wildtype were used to establish the level of chlorophyllide absorbance at 628 nm. A mean ratio of about 0.223 was found between the chlorophyllide peak heights at 628 nm and at 663 nm. Thus a factor of 0.223 times the peak height at 663 nm was subtracted from the peak heigt at 628 nm in samples with both chlorophyllide and Pchlide. The remainder of the peak height at 628 nm was attributed to Pchlide absorbance.

263

Stolbova, A.V. : Genetic analysis of pigment mutations of Chlamydomonas reinhardi II. Analysis of the inheritance of mutation of chlorophyll deficiency and light sensitivity in crosses with the wild type. Genetika 7, 124-129 (1971) Sueoka, N. : Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardi. Proc. Natl. Acad. Sci. USA 46, 83-91 (1960) Winig, H.R. : Genetic investigations of the Y mutant in Chlamydomonas reinhardi. Bachelor Thesis, Harvard University (1965) Wang, W.Y., Wang, W.L., Boynton, J.E., Gillham, N.W. : Genetic control of chlorophyll biosynthesis in Chlamydomonas: analysis of mutants at two loci mediating the conversion of protoporphyrin IX to magnesium protoporphyrin. J. Cell Biol. 63, 806 823 (1974) Wang, W.Y., Boynton, J.E., Gillham, N.W.: Genetic control of chlorophyll biosynthesis: effect of increased b-aminolevulinic acid synthesis on the phenotype of the y-1 mutant of Chlamydomonas. Mol. Gen. Genet. 152, 7 12 (1977) Wang, W.Y. : Effect of dim light on the y-1 mutant of Chlamydomonas reinhardtii. Plant Physiol. 61, 84~846 (1978) Communicated

by H. B6hme

Received March 24, 1980