Photosynthesis-deficient Mutants of Chiamydomonas reinhardii with Associated Light-sensitive Phenotypes'. Received for publication July 15, 1980 and in ...
Plant Physiol. (1981) 67, 565-569 0032-0889/8 1/67/0565/05/$00.50/0
Photosynthesis-deficient Mutants of Chiamydomonas reinhardii with Associated Light-sensitive Phenotypes' Received for publication July 15, 1980 and in revised form October 14, 1980
ROBERT J. SPREITZER AND LAURENS METS Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106 ABSTRACT A series of non-photoautotrophic mutants of Chlamydomonas reinhardii was isolated by replica-plating mutagenized cells which had been grown in the dark. Many of these acetate-requiring mutants are photosensitive, showing poor growth on acetate medium in the light, but normal growth in the dark. Biochemical characterization showed that the photosensitive mutants all had specific lesions in photosynthesis or photosynthetic pigment accumulation. The acetate-requiring mutants which were not photosensitive were all able to fix CO2. Among the light-sensitive mutants are 15 which show uniparental inheritance. These include six with specific lesions in photosystem II and one with an altered large subunit of ribulose1,5-bisphosphate carboxylase. Since these two classes of uniparental mutants have been rare or not previously reported, it seems likely that photosensitivity is an important factor which limited their detection in previous mutant isolation experiments.
The unicellular, green alga Chlamydomonas reinhardii can grow heterotrophically when supplied with acetate. This characteristic allows strains incapable of photoautotrophic growth to be selected and maintained as acetate-requiring mutants. Although some acetate-requiring mutants have been described which fix CO2 normally (19), most have been shown to possess specific defects of photosynthesis. These nonphotosynthetic mutants have been useful for investigating photosynthetic electron transport (17), chloroplast structure (11, 12), Chl synthesis (34, 35), thylakoid membrane polypeptide associations (4, 7), and chloroplast ribosomes (6, 13). Because C. reinhardii is a sexual alga, it has been possible to classify mutants as mendelian or nonmendelian and to map both types (9, 19, 24). The nonmendelian mutants are inherited in a uniparental (maternal) pattern which is a characteristic of chloroplast gene transmission in Chlamydomonas (10, 22). Uniparental acetate-requiring mutants, with defects in photosynthesis, have allowed the assessment of chloroplast gene contributions to the structure and photosynthetic function of the chloroplast (4, 5, 16,
27, 29). The analysis of acetate-requiring mutants has been very useful for understanding certain aspects of the biochemistry and genetics of photosynthesis and the chloroplast. It has not lived up to its full potential, however, since certain types of photosynthesis-deficient mutants have not been recovered easily. At the time the work reported here was begun, only one mutant had been described with a defect in the dark reactions of photosynthesis. This
mendelian mutant lacks Calvin cycle phosphoribulokinase activity (23). Furthermore, no uniparental acetate-requiring mutants had been described which lacked PSII activity. During attempts at selecting mutants of RubPCase,3 we reasoned that Calvin cycle mutants, in general, may have lightsensitive phenotypes. The absence of Calvin cycle activity might lead to an accumulation of unused reductant, generated by an intact photosynthetic electron transport system. Side reactions of the excess reductant could potentially generate toxic compounds. A light-sensitive phenotype would account for the difficulty of recovering Calvin cycle mutants as those requiring acetate because previously described selection experiments have always been carried out in the light. Therefore, we developed a selection protocol that would allow the recovery of light-sensitive, acetate-requiring mutants. We present a preliminary biochemical and genetic description of a collection of non-photoautotrophic mutants which we have obtained by this method. One of these mutants, rcl-u- 1-10-6C, has RubPCase with no activity and an altered large subunit and has recently been described in detail (29). It is photosensitive as postulated. However, all of the photosynthesis-deficient mutants in the collection are light-sensitive. These include both mendelian and uniparental strains with specific defects in PSI or PSII activity. The only acetate-requiring mutants which are not light-sensitive have normal CO2 fixation. Photosensitivity appears to be a prevalent and unique property of acetate-requiring mutants with defects in a wide range of photosynthetic functions. MATERIALS AND METHODS
Strains and Culture Conditions. C. reinhardii WT strain 2137 mt+ was selected for vigorous growth in the dark from a cross between WT strains 21gr mt+ (R. Sager) and 137c mt- (R. P. Levine). It maintains full photosynthetic competence during dark growth. Centromere marker strains y-1 mt- and pf-2 mt- were obtained originally from R. P. Levine. Photoautotrophic strains were maintained on the Tris-minimal medium of Surzycki (32), except that phosphate buffer I was used at 10 ml/l and no MgSO4 was added in addition to that present in the salt solution. All nonphotoautotrophic mutants were grown in the dark on 10 mm sodium acetate in minimal medium. Cultures were grown in flasks on a rotary shaker platform or on medium solidified with 15 g/l Bacto agar in Petri dishes. All cultures were kept at 25 C. For biochemical analysis, cells were grown in 50 to 250 ml acetate medium cultures in the dark until they reached early stationary phase (2 x 106 cells/ml). Cells were counted with a hemacytometer.
'This work was supported by a grant PCM 78-20684 from the National Science Foundation. 2 Present address: Agronomy Department, Illinois, Urbana, IL 61801.
Turner Hall, University of 565
3Abbreviations: RuBPCase, ribulose-1,5-bisphosphate carboxylase; WT, wild-type; PD, parental ditype; NPD, nonparental ditype; T, tetratype; DCPIP, dichlorophenolindophenol.
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Mutagenesis and Selection. C. reinhardii WT strain 2137 mt+ 21gr mt+ was grown in minimal medium at 4,000 lux. Using these cells, independent cultures within a single experiment were set up containing 3 x 104 cells/ml in 1 mm 5-fluorodeoxyuridine in acetate medium and grown in the dark. 5-Fluorodeoxyuridine was included to enhance the recovery of uniparental mutants (36). When the cultures reached stationary phase, each was subjected to ethyl methanesulfonate mutagenesis (21). After 2 h, the cells were washed and resuspended in acetate medium at 3 x 104 viable cells/ml and grown to stationary phase in the dark. Survival after ethyl methanesulfonate mutagenesis was approximately 15%. Cells from each independent culture were plated at 100 to 500 cells/ plate on acetate medium and grown in the dark. When colonies appeared after 1 to 2 weeks, the plates were replica plated to minimal medium at 4,000 lux. Nonphotoautotrophic colonies were saved for further characterization. Of the mutants recovered, only those of independent origin were characterized in detail. Genetic Analysis. Each mutant was crossed to y-l mt- or pf-2 mt- to determine if it was uniparental or mendelian, and, if mendelian, its centromere distance was determined. From these initial crosses, mt+ and mt- mutant strains were obtained. Gametes were induced in nitrogen-free acetate medium at 2,000 lux and mated for 1 h at 4,000 lux. Mated cells were plated onto minimal medium containing 40 g/l Bacto agar. After 23 h at 4,000 lux, the plates were placed in the dark. After 2 weeks, zygotes were germinated at 500 to 1,000 lux on acetate medium and tetrads of progeny cells were manipulated into rows, following standard procedure (18). These plates were incubated in the dark until colonies were large enough for replica plating. Phenotypes of the cells in tetrads were determined directly for y-l in crosses. The tetrads were replica-plated to minimal medium at 4,000 lux and acetate medium at 2,000 and 4,000 lux to determine acetate-requiring and light-sensitive phenotypes. The pf-2, paralyzed flagellae, phenotype was scored by checking for motility of cells in a colony in a drop of water under the microscope. Mating type was determined by checking under the microscope for flagellar agglutination with gametes of known mating or
type.
Uniparental inheritance was identified as a 4:0 ratio of progeny phenotypes in a tetrad, corresponding to the mt+:mt- phenotypes donated in a cross. Mendelian inheritance was identified as a 2:2 ratio of progeny phenotypes in a tetrad. Since a centromere marker (y-l or pf-2) was present in every cross, mapping information was obtained by scoring each individual tetrad as a PD, NPD, or T (18). This information was used to calculate genecentromere distance for the mutants based on the frequency of second division segregation from the formula 100 x ½h T/(PD + NPD + T). When markers appeared linked (PD:NPD > 1), map distance between them was calculated as 100 x (NPD + 1/2 T)/ (PD + NPD + T). Spot Tests. All original mutant isolates, and mt+ and mtstrains derived from them, were suspended in distilled H20 at 2 x 10r cells/ml. From suspension, 1 drop from a Pasteur pipette was placed on solid minimal medium at 2,000 and 4,000 lux and acetate medium at 2,000 and 4,000 lux and in the dark. Growth was assessed with respect to both WT growth and growth in the dark after I week. Biochemical Analysis. PSII activity (Hill reaction) of chloroplast membrane fragments, prepared as described by Levine and Gorman (20), was measured as the rate of reduction of DCPIP, as described previously (29). All values were recorded as the percentage of the WT rate per unit Chl measured during the same experiment. CO2 fixation was measured in whole cells as the rate of incorporation of 14C from NaH"4C03 into acid-precipitable material as described previously (29). All values were recorded as the percentage
Plant Physiol. Vol. 67, 1981
SPREITZER AND METS
of the
WT
rate.
Chl was measured spectrophotometrically from the clarified
supernatant of an 80% acetone extract of whole cells (1). RubPCase protein was estimated from its peak height from a scan of the absorbance of a sucrose gradient of total soluble cell protein as previously described (29). Values were recorded as the percentage of WT RubPCase per cell. Fluorescence Induction Kinetics. Fluorescence transients were measured on dark-adapted colonies grown on acetate medium plates. The apparatus used was a fluorescence microscope adapted as described by Bennoun (2) except that excitation was delivered by epi illumination. The patterns of fluorescence were used to describe a mutant as PSI-deficient, PSII-deficient, or neither (WT fluorescence), using criteria outlined by Bennoun and Chua (3).
RESULTS Recovery of Mutants. Table I shows that mutants were recovered at a frequency of approximately 1/103 cells plated in the dark. These mutants consisted of three types: (a) pigment-deficient mutants, which were light-sensitive and acetate-requiring, (b) light-sensitive, acetate-requiring mutants, which had normal green pigmentation, and (c) non-light-sensitive, acetate-requiring mutants. Strains were described as light-sensitive if they grew better in the dark with acetate than in the light (4,000 lux) with acetate, when spot tests were scored after 1 week. All strains were indistinguishable from WT when the amount of growth in the dark was compared. Pigment-deficient Mutants. These mutants are listed in Table II. Unlike WT, they were not green in the dark, nor did they become green in the light like y- 1. Althoughy- l-like mutants were observed in the present investigation, they were not categorized due to their capacity for photoautotrophic growth. Two pigment-deficient mutants, 1 -3B and 1 1-7C, were obligate heterotrophs, unable to grow at the lowest light intensity tested (2,000 lux). They are similar to previously described pigmentdeficient obligate heterotrophs (25, 30, 34). The other mutants survived on acetate medium at 2,000 lux but grew better in the dark on acetate than on acetate medium at 2,000 or 4,000 lux. They did not grow, or they displayed leaky growth, on minimal medium. Therefore, most of the pigment-deficient mutants also required acetate. They seem to be different from other pigmentTable I. Selection of Non-photoautotrophic Mutants ExperiWT Strain
ment
Cells Plated
Number
Mutants Mutants
Mutants/
Recovered
Plated
8 17 25 25
2.0 1.7 0.9 2.3
IO' Cells
xlO-3 21gr 2137 2137 2137
8 10 11 12
4.1 9.9 28.9 11.0
Table II. Pigment Deficient Mutants Cross to pf-2 Color in Dark Strain T PD NPD 7 23 5 10-6A Yellow-green 6 12 16 Pale green 10-8B 4 10 12 10-8C Yellow-green 7 9 5 Yellow 11-2B 8 8 8 Brown 11-3B 9 10 11 Tan 11-6A 7 19 2 Brown 11-7A 8 1 44 White 11-7C (w-7) ' Map distance between w-7 and pf-2.
Centromere
Distance 33 9 8 17 17 15 34 8a
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deficient mutants which require acetate but do not have an associated light sensitivity (19). Pigment-deficient, acetate-requiring, and light-sensitive phenotypes did not segregate in crosses. None of the pigment-deficient mutants were inherited in a nonmendelian pattern. One pigment-deficient mutant, 11-7C, was found to be linked
sensitive (see ref. 29 and below). All of the uniparental mutants recovered in this investigation were light-sensitive (Table IV), growing better in the dark on acetate than in the light (2,000 lux) on acetate. None survived on minimal medium, but all grew on acetate at 2,000 lux. Some could not be maintained at 4,000 lux. to pf-2 (Table II). This marker was assigned the locus name w-7. The uniparental mutants all lacked the ability to fix CO2 but Preliminary mapping data (28) indicates that w-7 is to the left of had normal levels of Chl. Fluorescence induction kinetics indithe centromere in linkage group XI of the C. reinhardii genetic cated that two mutants lacked PSI. Seven others had WT patterns map (19). of fluorescence. One of these, 10-6C, was found to lack RubPCase Light-sensitive, Acetate-requiring Mutants. All of the mutants activity and possessed an altered isoelectric point for the large in this class grew better in the dark on acetate medium than in the subunit of the enzyme. This is the first uniparental Calvin cycle light on acetate medium. All had normal green pigmentation in mutant reported in Chlamydomonas and has been described in the dark and apparently WT levels of Chl. Both mendelian and detail recently (29). Seven mutants lacked Hill reaction activity. uniparental mutants were in this group. All of these, except 10-7C, had patterns of fluorescence indicating The mendelian mutants (Table III) include two strains, 11-8B PSII deficiency. Strain 10-7C shows normal in vivo fluorescence and 12-6A, which are unable to grow on acetate medium at 2,000 induction kinetics. It is possible that some component of the PSII lux and can be described as obligate heterotrophs. Stolbova (31) reaction can function relatively normally in vivo but does not has described similar mutants, but without characterizing photo- survive cell fragmentation. Because of this discrepancy, it is diffisynthesis. Although fluorescence induction kinetics indicated that cult to classify the primary defect in this mutant. In five of these our two mutants were deficient in PSI activity, other PSI mutants mutants, the amount of RubPCase protein was measured and in this group were capable of some growth on acetate medium at found to be at WT levels. Some mendelian (6, 13) and uniparental 2,000 lux. One mutant in particular, 11-6F, with fluorescence (27) mutants of C. reinhardii have been described which lack both indicating defective PSI, showed leaky growth on minimal me- Hill reaction and RubPCase activity and which are also defective dium at 2,000 lux. in chloroplast ribosomes. The PSII-deficient mutants recovered The other mendelian mutants in this group were all capable of here are clearly not of this type. growth on acetate at 2,000 lux. Some could not be maintained on Non-light-sensitive, Acetate-requiring Mutants. This group (Taacetate medium at 4,000 lux and none survived on minimal ble V) consisted of mendelian strains that required acetate for medium. Genetic analysis did not show any instance of segregation growth but were not photosensitive. Genetic analysis did not of the light-sensitive and acetate-requiring phenotypes. reveal segregation of photosensitive phenotypes. They displayed All of the mendelian mutants, except 12-8A, had apparently growth on acetate at 4,000 lux better than, or equal to, their normal Hill reaction. Fluorescence induction kinetics also indi- growth or WT growth in the dark. All were stringent acetatecated that 12-8A was defective in PSII. The fluorescence pattern requiring mutants, except for 8-10, which could grow on minimal of 8-34B was also consistent with a PSII defect, although it had medium for about 1 week before bleaching. Preliminary studies measurable Hill reaction activity. The reason for this discrepancy have shown that this mutant dies even more rapidly when prois not yet known. Several strains had WT patterns of fluorescence. vided with 5% CO2 in air. All of the non-light-sensitive, acetateThese may include mutants with defects in photophosphorylation requiring mutants had normal amounts of Chl and PSII activity, or photosynthetic carbon metabolism. We know that a mutant as well as WT fluorescence. Furthermore, all had rates of CO2 deficient in RubPCase has normal fluorescence and is also light- fixation that were not conclusively different from WT. Table III. Light-sensitive and Acetate-requiring Mendelian Mutants Cross to y- I CenFluo- PSII Ac- CO2 troStrain res- tvy' Fixa-2 Chl8 mere PD NPD T Discence tiona
DISCUSSION
An important feature of the mutant isolation protocol used here is that the cells were not exposed to light, either during the recovery from mutagenesis or during colony growth prior to replica plating. This was done for two reasons. First, WT cells, tance which are capable of photoautotrophic growth, would have no selective growth advantage over non-photoautotrophic mutants % control under these conditions. Second, we anticipated that at least some 8-32 WT 2 4 6 25 of types nonphotoautotrophic mutants would be light-sensitive. 0 8-33 PSI 4 6 30 cells which are either killed by light or whose growth is Any 8-34Bb PSII 28
