Greening in a virescent mutant of maize

Department of Botany, University of Illinois, Urbana, USA

Greening in a Virescent Mutant of Maize I. Pigment, Ultrastructural, and Gas Exchange Studies 1) RAYMOND CHOLLET and DOMINICK

J. PAOLILLO, Jr.

With 12 figures Received March 17, 1972

Summary The primary leaves of virescent seedlings (v 18) of Zea mays L. were studied during greening. In several regards v 18 resembled lethal rather than viable chloroplast mutants. Before greening, mesophyll and bundle sheath plastids were morphologically indistinguishable, both having only rudimentary vesicular and lamellar components. Chlorophyll and carotenoid content was low, but the carotenoid to chlorophyll ratio was above normal. In contrast to other viable mutants, the chlorophyll alb ratio was lower than normal. In addition, the rate of CO 2 fixation (per unit chlorophyll) and the light intensity required for photOsaturation were also reduced. The results suggest a block in the dark enzymatic reactions of photOsynthesis. Pigment content, plastid structure, and photosynthetic capacity normalized with greening.

Introduction The structure of developing chloroplasts has been studied in meristematic tissues (LAETSCH and PRICE, 1969; STETLER and LAETSCH, 1969), etiolated tissues that have been induced to green (KIRK and TILNEY-BASSETT, 1967) and greening tissue cultures (BLACKWELL et a!., 1969). Mutant plants have been investigated to elucidate the role of plastid pigments and ribosomes in deriving and maintaining structure (BACH1fANN et a!., 1967; LEVINE, 1969 ; SHUMWAY and WEIER, 1967), and the genetic control of chloroplast development (KIRK and TILNEy-BASSETT, 1967). The majority of mutants that have been studied are "static" in that the defective plastid structure caused by the genetic lesion remains uncorrected as the tissue ages or can be corrected only under special conditions (WALLES, 1963). Recently, virescent mutants have been investigated (BENEDICT and KOHEL, 1968, 1970; DALE and HEYES, 1970). These mutants are especially good tools for correlative studies of greening because they are characterized by a temperature-dependent chlorophyll deficiency that is spontaneously corrected as the seedling ages (PHINNEY and KAY, 1954). Grass leaves provide a distinct advantage in developmental studies because a gradient of decreasing maturity can be traced from the tip of the leaf to the intercalary 1) This work was supported in part by Grant GB-6S20 from the National Science Foundation. Z. Pflanzenphysiol. Bd. 68. S. 30-44. 1972.

Greening in a Virescent Mutant of Maize

31

meristem at the leaf base (FAHN, 1967). In the present investigation we sought to combine this advantage with others by utilizing a virescent mutant of maize (Zea mays L.), where the progress of greening can be followed along a basipetal gradient in the individual leaves of seedlings. An earlier report (PAOLILLO and REIGHARD, 1968) indicated that greening in several virescent mutants of maize was correlated with a structural normalization of aberrant mesophyll and bundle sheath plastids. This paper describes the changes in pigment content, plastid structure, and photosynthetic capacity during greening in a virescent mutant of maize. A companion paper (CHOLLET and OGREN, 1972) deals with the changes in the in vitro activity of several cytoplasmic, photorespiratory, and photosynthetic enzymes and in the in vivo labeling patterns of photosynthetic intermediates following short-term photosynthesis in 14CO z. These studies complement the limited literature on chloroplast structure, function, and ontogeny in plants possessing the C 4 -pathway of photosynthetic CO 2 fixation (HATCH et aI., 1971), and allow some interesting comparisons with the results reported with other higher plant chloroplast mutants and greening etioplasts.

Materials and Methods Plant Material The virescent mutant v 18 of maize differs from its normal sibling by a recessive allele at a single, undetermined locus on chromosome 10 (NEUFFER et a!., 1968). Seeds homozygous for the virescent condition and those of a normal, unrelated hybrid stock (W23/L317) were furnished by Dr. R. J. LAMRERT, Department of Agronomy and Maize Genetics Cooperative, University of Illinois (Urbana, Illinois). Mutant and normal plants were grown from seed in sterilized soil in a growth chamber with a 12-h day at 21 ° C and a 12-h night at 16° C. Light of 13.5 klux at the soil surface was provided by a bank of cool-white fluorescent tubes. Light intensities were measured with a Weston Model 756 illumination meter. The seedlings were watered daily with tap water and onl y the primary leaves were used for analysis. Sampling Procedures Under these growth conditions the primary leaves of v 18 greened basipetally, becoming visually indistinguishable from wild-type after 3-4 wks. Comparable mutant and normal seedlings were sampled at three arbitrary stages in the development of the virescent leaf: when the primary leaf was pale-yellow except for the distal light-green tip (v18, ungreened); when the leaves had developed a pale-green color (v 18, greening); and finally when the primar y leaf was essentially indistinguishable from normal (v 18. greened). Pigment Analysis Preweighed leaf material was thoroughly ground in aqueous 80 % (v/v) acetone using a Ten Broeck homogenizer. Following centrifugation, the concentration of total chlorophyll in the acetone extracts was determined spectrophotometrically (ARNON, 1949), as were chlorophyll alb ratios (BRUINSMA, 1963) and an estimate of total carotenoids (mainly fJ-carotene and lutein) (KOUCHKOVSKY, 1963). Room temperature absorption spectra were determined between 400 and 700 nm with a Cary 14 recording spectrophotometer. Electron Microscopy Narrow tissue strips were dissected from distal, middle, and basal regions of the leaves, midway between the leaf margin and midrib, and fixed and dehydrated as previously described (PAOLILLO et a!., 1968) . The specimens were embedded in Epon 812 according to

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PJlanzenphysiol. Bd. 68. S. 30-44. 1972.

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R. CHOLLET and D.

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PAOLILLO

(1961), sectioned with a diamond knife on a Reichert OmU2 ultramicrotome, stained for 15 min in 25 ~/o (w/v) methanolic uranyl acetate (STEMPAK and WARD, 1964) followed by 5 min in undiluted basic lead citrate (REYNOLDS, 1963), and examined in an RCA EMU-4 electron microscope. L UFT

Gas Exchange Studies

The exchange of COt by mutant and normal leaves in the dark and at various light intensities was determined with an infrared gas analyzer (Grubb-Parsons Model SB-2). Primary leaves were detached from seedlings, recut under water, and positioned with their bases in water in a plexiglass and brass assimilation chamber that formed part of a 0.60 I closed system constructed as described by JAGELS (1970). Gas flow through the system was 2 l/min. Rates of CO 2 exchange were measured within the range of 295-315 Ill/I CO 2 in air. Temperature was controlled by immersing the brass bottom of the chamber in a water bath at 25° ± 10 C. Illumination was provided by 300 W / 120 V reflector-flood lamps immersed in an 18-cm water filter, and the light intensity was varied by a series of cheesecloth filters that did not alter the spectral energy distribution of the light source. At the completion of the measurements, the leaves were quickly blotted dry, weighed, and their pigment content determined as described above. The CO 2 exchange of detached leaves of v 18, ungreened was measured in air and in a 2% (v/v) oxygen gas mixture (2 0/ 0 O 2 , balance N2 in 360,u1J1 CO2) with an infrared gas analyzer (Beckman Model 215 A) in a 0.63 I closed system similar to the one described by BOWES et al. (1972) with the following modifications: a 62-ml stoppered test tube served as the assimilation chamber; the chamber was immersed in a 24 ° ± 1° C water bath; and a YSI Model 53 oxygen monitor was incorporated into the system. Rates of CO 2 exchange at both oxygen tensions were determined in the dark and at 12.9 klux within the range of 315-345 ,ulll COt. Results

Pigment Studies Table 1 shows the changes in pigment content during the development of comparable normal and virescent leaves. The immature mutant was extremely deficient Table 1 Changes in pigment content during development of the primary leaves of v 18 and W23!L317. Leaf Material v 18, ungrecned W23/L317 v 18, greening W23/ L317 v 18, greened W23/L317

Days Postplanting 14 20 27

mg total chlorophyll/g leaf fresh wt. 0.139 2.00 0.642 2.81 1.95 2.88

mg total total carotenoids/ chlorophyll carotenoids/g total chlorophyll alb leaf fresh wt. 0.052 0.395 0.166 0.481 0.361 0.510

0.374 0.198 0.259 0.171 0.185 0.177

2.64 4.03 3.42 3.74 3.70 3.79

in chlorophyll and carotenoids, both of which increased to near-normal levels with greening. The ratio of carotenoids to total chlorophyll was greater in the ungrcened and greening mutants compared to normal. The ungreened virescent was relatively

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Greening in a Virescent Mutant of Maize

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less deficient in chlorophyll b than in chlorophyll a, resulting in lower chlorophyll alb ratios than for normal leaves. With greening, the ratios of carotenoids/total chlorophyll and chlorophyll a/ b were essentially normalized. Examination of the room temperature absorption spectra of acetone extracts of virescent and normal leaves at various ages revealed no major qualitative difference in chloroplast pigments. Plastid Fine Structure

Plastids in the primary leaves of normal maize (Figs. 1, 2) showed the structural dimorphism characteristic of this C 4 -species (Woo et aI., 1971). The general morphology of the mesophyll and bundle sheath chloroplasts remained essentially constant throughout the entire leaf at all ages sampled. The ungreened virescent leaves contained a variety of aberrant bundle sheath and mesophyll chloroplasts. The plastid types ranged from those with little internal structure or loosely constructed prolamellar bodies (PLBs) to those with nearly normal morphology (Figs. 3-8). The distinctly abnormal plastids (Figs. 3, 4) predominated in the middle and basal regions of the leaf, although their occurrence in the distal region was not uncommon. Near-normal plastids (Figs. 6, 8) were restricted to the distal region of the ungreened leaf where some green color was always evident. The variations in plastid structure were shown to be correlated with greening in the older tissues, v 18, greening and v18, greened. Hence, the variations in plastid structure in the ungreened leaf were part of a developmental sequence, and the basipetal direction of greening was evident in our earliest materials. Highly aberrant mesophyll and bundle sheath plastids were morphologically indistinguishable. Both possessed disorganized, non-crystalline PLBs that appeared to be augmented by vesicular invaginations from the inner membrane of the plastid envelope (arrows, Fig. 3). In somewhat more developed plastids the PLBs were associated with irregular lamellae that possibly originated by fusion of vesicles in the stroma. Following this stage the plastids became dimorphic. When additional lamellae were added to mesophyll plastids they were commonly aggregated into large granal stacks (Fig. 4). No grana of comparable size were observed in bundle sheath plastids. The subsequent development of mesophyll plastids was characterized by a proliferation of large, isolated grana (Fig. 5). The nearly normal plastids in the mesophyll possessed numerous grana but the fretwork was poorly developed (Fig. 6). In the bundle sheath plastids there was a gradual proliferation of long, parallel lamellae with short regions where adjacent membranes were appressed (arrows, Fig. 7). Near-normal bundle sheath plastids contained starch grains and relatively long grana consisting of several appressed lamellae (Fig. 8), but their membrane systems were not as extensive as those of normal bundle sheath plastids. Even in the most abnormal mesophyll and bundle sheath chloroplasts, numerous ribosomes, DNA-like fibrils and osmiophilic granules were observed. However, in the highly aberrant plastids a clearly defined peripheral reticulum could not be distinguished from other membranous components in the peripheral stroma (Fig. 3), unlike

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PJlanzenphysiol. Bd. 68. S. 30-44. 1972.

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Fig. 1. Mesophyll chloroplast of normal maize showing a well-developed peripheral reticulum (P R) and an extensive grana-fretwork system. X 21,500. Fig. 2. Portion of a bundle sheath cell of normal maize. The chloroplasts contain mostly nonappressed, parallel lamellae with only rudimentary grana and possess starch grains and a well-developed peripheral reticulum. The inset shows several short regions of appressed lamellae in one of the bundle sheath plastids at greater magnification. X 16,500; inset: X 65,100.

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Fig, 3. Highly aberrant bundle sheath chloroplast in the basal region of v 18, ungreened, Vesicular invaginations from the inner plastid membrane (arrows) appear to contribute to the non-c rystalline prolamellar body, Unlike the plastids, mitochondrial fine structure (M) is not affected by the mutation, Portion of a highly aberrant mesophyll chloroplast ~ Me) is visible in an adjacent cell. X 26,800, Fig. 4. Highly aberrant mesophyll plastid in the basal region of v 18, ungreened. Vesicle fusion in the stroma appears to give rise to structurally irregular lamellae which are aggregated into a relatively large granal stack. Portion of another mesophyll chloroplast (MC) is visible in an adjacent cell. X 20,800.

36

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and D.

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Fig. 5. Abnormal mesophyll chloroplast in the middle region of the ungreened virescent leaf showing the proliferation of large, isolated grana. X 18,100. Fig. 6. Near-normal mesophyll chloroplast in the distal region of v 18, ungreened. The plastid contains numerous well-developed grana but lacks an extensive fretwork system. X 15,000.

37

Greening in a Virescent Mutant of Maize

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Fig. 7. Abnormal bundle sheath chloroplast in the middle region of v 18, ungreened. The plastid profile consists mainly of parallel lamellae with several short regions of appressed lamellae (arrows). X 29,000. Fig. 8. Portion of a bundle sheath cell in the distal region of v 18, ungreened. The near-normal chloroplasts contain starch grains and relatively long grana consisting of several appressed lamellae, but lack the extensive lamellar system observed in normal maize sheath plastids. X 17,600.

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Fig. 9. Normal mesophyll chloroplast in the distal region of v 18, greening. Note the extensive grana-fretwork system. X 32,900. Fig. 10. Normal bundle sheath chloroplast in the distal region of v 18, greening. The plastid possesses an extensive system of mostly non-appressed, parallel lamellae with only rudimentary grana and copious starch. X 21,500.

Z. Pjlanzenphysiol. Bd . 68. S. 30-44. 1972.

Greening in a Virescent Mutant of Maize

39

in the more developed plastids (Fig. 8). The fine structure of other subcellular organelles, such as microbodies and mitochondria (Fig. 3), and guard cell chloroplasts was not affected by the mutation. In the leaves of v 18, greening, the distal region had a high frequency of normal plastids in mesophyll cells and in the bundle sheaths (Figs. 9,10). In the middle and basal regions of greening leaves, most plastids resembled those at the more advanced stages in the ungreened leaf (Figs. 5- 8) . In the leaves of v 18, greened, normal plastids were predominant in all regions of the leaf. During greening some plastids appeared to senesce. In leaves of both v 18, greening and v 18, greened, senescent plastids occurred either as the only plastids in individual cells or mixed in the same cell with developing plastids. In any case the senescent plastids were always rare, and they were more disorganized than any of the aberrant plastids observed in v 18, ungreened. There was never any abundance of degenerating plastids and no new population of plastids was ever recognizable. Instead, mostly the intermediate plastid types were observed during greening, and it can be concluded that greening was accompanied by the normalization of structure in most of the aberrant plastids in the ungreened leaf. Gas Exchange Studies

Figs. 11 and 12 are representative light saturation curves for the exchange of COt by detached leaves of normal and virescent maize. At each stage in greening, visually comparable mutant material showed a marked variation in rates of CO 2 exchange,

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Z. Pjlanzenphysiol. Bd. 68. S. 30-44. 1972.

40

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and D.

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PAOLILLO

probably because small differences in pigment content are associated with other variables that affect photosynthesis. In Fig. 11, the maximum and minimum rites obtained with ungreened virescent leaves are given along with a representative normal curve. For v 18, ungreened, CO 2 exchange saturated at about 40 klux, whereas with normal leaves the rate of CO 2 fixation was still increasing at 118 klux. At all light intensities tested, the ungreened virescent showed lower rates of true photosynthesis than comparable normal leaves, on both a chlorophyll and leaf fresh weight basis. In addition, the mutant material often evolved CO 2 in the light, especially at the lower light intensities. The immature mutant leaves respired in the dark at about half the rate of wild-type per unit leaf fresh weight. However, on a soluble protein basis, normal and virescent leaves had nearly identical rates of mitochondrial respiration. The rate of true photosynthesis in the ungreened virescent was inhibited by 2 0 / 0 on going from a low ambient oxygen concentration to air. Increasing the ambient CO 2 concentration from 275 to 520 /NI CO 2 in air caused only a slight increase in the saturated rate of CO 2 exchange at 91.5 klux. A substantial increase in the photosynthetic capacity of the mutant accompanied the rise in pigment content and normalization of aberrant plastids during development. Greening virescent leaves (data not shown) showed a slightly higher rate of CO 2 uptake per chlorophyll than normal, irrespective of the incident light intensity, but on a leaf fresh weight basis the mutant still fixed considerably less CO 2 than W23jL317. Fig. 12 shows that the rate of CO ~ fixation in v 18, greened and W23 jL317 were

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Greening in a Virescent Mutant of Maize

41

nearly identical on both a leaf fresh weight and chlorophyll basis, and that both normal and virescent leaves responded similarly to increasing light intensities. Discussion The leaves of viable chloroplast mutants invariably contain reduced amounts of total chlorophyll, ranging from 20 % of the normal amount in an aurea mutant of Lespedeza procumbens (CLEWELL and SCHMID, 1969) to 70% in a barley mutant lacking chlorophyll b (BOARDMAN and HIGHKIN, 1966). The deficiency in carotenoids is often less than that in chlorophyll, yielding an enrichment in carotenoids per unit chlorophyll (CLEWELL and SCHMID, 1969). The deficiency in chlorophyll a is usually less than that in chlorophyll b, raising the chlorophyll alb ratio above normal (BOARDMAN and HIGHKIN, 1966; HIGHKIN et al., 1969; KECK et al., 1970 a). In ungreened leaves of v 18 of maize, the relatively greater deficiency in chlorophyll a reduces the chlorophyll alb ratio below normal. We regard this difference as a distinctive characteristic of this virescent mutant. The leaves of v 18, ungreened are considerably more deficient in total chlorophyll compared to uniformly pale-green mutants, but this is due to the fact that chlorophyll is predominantly located in the tip of the leaf. Carotenoids are detectable throughout the ungreened leaf, and as is often the case with other viable mutants, the carotenoid/chlorophyll ratio is higher than normal. If the carotenoids are functioning to protect chlorophyll from photo-oxidation (KRINSKY, 1966), then photodestruction is probably not the cause of chlorophyll deficiency in the viable chloroplast mutants. The structural differences between the plastids of v 18, ungreened and those of other viable mutants are more striking than the differences in pigments. Uniformly pale-green mutants invariably contain well-developed chloroplasts, but the grana have fewer compartments than normal and there is an increase in unpaired lamellae (BENEDICT and KOHEL, 1970; CLEWELL and SCHMID, 1969; DALE and HEYES, 1970; HIGHKIN et al., 1969; KECK et al., 1970 a; SCHMID, 1967). In contrast, v 18, un greened contains plastids that range from near-normal to those with only loosely constructed prolamellar bodies. The lack of an extensive fretwork in the best developed mesophyll chloroplasts in ungreened leaves is probably reflected in the lower than normal chlorophyll alb ratios (cf. GOODCHILD and PARK, 1971). The most distinguishing feature of v 18, ungreened is its ineffectiveness in fixing CO 2 • In other viable mutants, photosynthesis saturates at the same (BENEDICT and KOHEL, 1968; KECK et al., 1970 b) or higher light intensities (CLEWELL and SCHMID, 1969; HIGHKIN et al., 1969; SCHMID, 1967) than normal plants, with rates of photosynthesis 2 to 10 times greater than wild-type per unit chlorophyll, and at least equal to normal on a leaf area or weight basis. One expects low rates of photosynthesis per unit fresh weight in v 18, ungreened because chlorophyll and probably photosynthesis are essentially restricted to the tip of the leaf. However, the rate of true photosynthesis per unit chlorophyll is also low, and CO 2 exchange is saturated at moderate light intensities. Increasing the ambient CO 2 concentration does not markedly affect the

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light-saturated rate of CO 2 exchange. This observation suggests that the saturation at moderate light intensities is probably due to a limitation in the dark enzymatic reactions of CO 2 fixation, rather than to a stomatal malfunction. This interpretation is further supported by the observed normal fine structure of guard cell chloroplasts and the presence of chlorophyll in these plastids, which was demonstrated with fluorescence microscopy. The lack of a significant oxygen inhibition of photosynthesis suggests that photorespiration is not the cause of the low rates of CO 2 fixation (JACKSON and YOLK, 1970). Overall, v 18, ungreened bears little resemblance to the viable chloroplast mutants previously studied. In fact, in regard to its pigment content, plastid morphology, and photosynthetic capacity, the immature virescent more closely resembles the lethal chloroplast mutants of barley (WALLES, 1963) and corn (MILLERD et al., 1969), and the non-green areas in the leaves of iojap maize (SHUMWAY and WEIER, 1967) and variegated tobacco (SCHMID, 1967) and cotton (KoHEL and BENEDICT, 1971). Considering the structural and physiological similarity between v 18, ungreened and several lethal chloroplast mutants, the capacity of v 18 to green spontaneously seems somewhat remarkable. Studies on greening etioplasts reveal two developmental phases: (1) long, largely unpaired lamellae are formed and the chlorophyll alb ratio is high; (2) grana are formed and the chlorophyll alb ratio is reduced (PARK and SANE, 1971). In v 18 mesophyll plastids, the situation is reversed; grana form before an extensive fretwork is developed. Accordingly, the chlorophyll alb ratio begins at a value lower than normal and subsequently rises. The plastids of v 18 become dimorphic when the mesophyll plastids form grana and the bundle sheath plastids do not. This resembles the situation during the lightinduced greening of etiolated sugarcane leaves (LAETSCH and PRICE, 1969), but differs from the sequence in light-grown leaves of sorghum (DOWNTON and PYLIOTIS, 1971) and sugarcane (LAETSCH and PRICE, 1969), where the bundle sheath plastids pass through a grana-building phase in their ontogeny. On the other hand, the sequence of structural changes in v 18 mesophyll plastids resembles those reported for mesophyll plastids in light-grown maize (MILLERD et aI., 1969 ; SHUMWAY and WEIER, 1967), sugarcane (LAETSCH and PRICE, 1969), barley (RHODES and Y EMM, 1966), and elodea (MUHLETHALER and FREY-WYSSLING, 1959). PAOLILLO (1970) suggested that the threedimensional structure of mature chloroplasts is best explained if one postulates that an extensive fretwork comes after grana formation in plastid ontogeny. We concur with BLACKWELL et ai. (1969) that an all-embracing scheme of plastid development cannot be proposed when the various patterns and circumstances of chloroplast development are taken into account. Concomitant with the light-induced greening of etioplasts there is an increase in several phases of cell metabolism, notably the de novo synthesis of plastid proteins (GRAHAM et aI., 1970; IRELAND and BRADBEER, 1971; KIRK and TILNEy-BASSETT, 1967). It is evident that metabolic changes also occur during greening in v 18. An

Z. P/lanzenphysiol. Bd. 68. S. 30-44. 1972.

Greening in a Virescent Mutant of Maize

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increase in plastid pigments and photosynthetic capacity accompanies the structural normalization of aberrant plastids during development. Since photosynthesis in the un greened virescent is probably limited by the dark reactions of CO 2 fixation, it is likely that photosynthetic carbon metabolizing enzyme(s) also increase with greening. In the companion paper (CHOLLET and OGREN, 1972), an account of the changes in plastid and non-plastid enzyme activity during greening in v 18 is presented. References ARNON, D. 1.: Plant Physio!. 24, 1 (1949). BACHMANN, M. D., D . S. ROllERTSON, C. C. BOWEN, and I. C. ANDERSON: ]. Ultrastruct. Res . 21,41 (1967). BENEDICT, C. R., and R. ]. K OHEL: Pl ant Physio!. 43, 1611 (1968). - - Plant Physio!. 45, 519 (1970). BLACKWELL, S. J., W. M. LAETSCH, an d B. B. HYDE: Amer.]. Bot. 56, 457 (1969). BOARDMAN, N. K., and H. R. HIGHKIN: Biochim. Biophys. Acta 126, 189 (1966). BOWES, G., W. L. OGREN, and R. H. H AGEMAN: Crop Sci. 12,77 (1972). BRUINSMA, ].: Photochem. Photobio!' 2 . 241 (1963). CHOLLET, R ., and W. L. OGREN: Z . Pflan zenphysio!. 68, 45 (1972). CLEWELL, A. F., and G. H . SCHMID: Planta 84, 166 (1969). D ALE,]. E., and]. K. HEYES : New Ph ytologist 69, 733 (1970). DOWNTON, W. ]. S., and R . B. P YLIOTIS: Can. ]. Bot. 49, 179 (1971) . FAHN, A.: Plant Anatomy. Per gamon Press, Oxford (1967). GOODCHILD, D. J., and R. B. PARK: Biochim. Biophys. Acta 226, 393 (1971). GRAHAM, D., M. D. HATCH, C. R. SLACK, and R. M. SMILLIE: Phytochemistry 9,521 (1970). HATCH, M. D., C. B. OSMOND, and R. O. SLATYER (eds.): Photosynthesis and Photorespiration. Wiley-Interscience, New York (1971). HIGHKIN, H. R., N. K. BOARDMAN, and D.]. GOODCHILD: Plant Physio!. 44, 1310 (1969). IRELAND, H. M. M., and]. W. BRADBEER: Planta 96, 254 (1971). JA CKSON, W. A., and R.]. YOLK: Ann. Rev. Plant Physio!. 21, 385 (1970). JAGELS, R.: Can. ]. Bot. 48, 1843 (1970). KECK, R. W., R. A. DILLEY , C. F. ALLEN, and S. BIGGS: Plant Physio!. 46, 692 (1970 a). KE CK, R. W., R . A. DILLEY, and B. KE: Plant Physio!. 46, 699 (1970 b). KIRK, ]. T. 0., and R. A . E. TILNEY-BASSETT: The Plastids. Their chemistry, structure, growth, and inheritance. W. H . Freeman, London (1967). KOHEL, R . ]., and C. R . BENEDICT: C rop Sci. 11, 486 (1971). KOUCHKOVSKY, Y. DE: Ph ysio!. Veg 1, 15 (1963). KRINSKY, N. I: In: Biochemistry of Chloroplasts, vol. I, p. 423, T. W. Goodwin, ed., Academic Press, New York (1966). LAETSCH, W. M., and 1. PRICE: Amer. J. Bot. 56, 77 (1969). LEVINE, R. P.: Ann. Rev. Plant Physio!. 20,523 (1969). LU FT,]. H.: J. Biophys. Biochem. Cyto!. 9, 409 (1961). MILLER D, A., D. J. GOODCHILD, and D. SPENCER: Plant Physio!. 44,567 (1969). MUHLETHALER, K., and A. FREy-WYSSLING: J. Biophys. Biochem. Cyto!' 6, 507 (1959). N EUFFER, M. G., L. JONES, and M. S. ZUBER: The Mutants of Maize. Crop Science Soc. of Amer., Madison (1968). PAOLILLO, D. ]., Jr. : ]. Cell Sci. 6, 243 (1970). P AOLILLO, D . ]., Jr., and]. A. REIGHARD: Trans. Amer. Microsc. Soc. 87, 54 (1968). P AOLILLO, D.]., Jr., G. L. KREITNER, and J. A. REIGHARD : Planta 78, 226 (1968). PARK, R. B., and P. V. SANE: Ann. R ev. Plant Physio!. 22, 395 (1971). PHINNEY, B. 0., and R. E. KAY: Hilgardia 23,185 (1954).

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S. 30-44. 1972.

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PAOLILLO

REYNOLDS, E. S.: J. Cell Biol. 17, 208 (1963). RHODES, M. J. c., and E. W. YEMM: New Phytologist 65, 331 (1966). SCHMID, G. H.: J. Microscopie 6, 485 (1967). SHUMWAY, L. K., and T. E. WEIER: Amer. J. Bot. 54, 773 (1967). STEMPAK, J. G., and R. T. WARD: J. Cell Biol. 22, 697 (1964). STETLER, D. A., and W. M. LAETSCH: Amer. J. Bot. 56, 260 (1969). WALLES, B.: Hereditas 50, 317 (1963). Woo, K. c., N. A. PYLIOTIS, and W. J. S. DOWNTON: Z. Pflanzenphysiol. 64, 400 (1971). Dr. R. CHOLLET, Department of Agronomy, University of Illinois, Urbana, Illinois 61801, U.S.A. Dr. D. J. PAOLILLO, Jr., Section of Genetics, Development & Physiology, Cornell University, Ithaca, New York 14850, U.S.A.

Z. l'/lanzenphysiol. Bd. 68. S. 30-44. 1972.