Carotenoid-deficient maize seedlings fail to accumulate light-harvesting chlorophyll a/b binding protein (LHCP) mrna

Transcription

1 Eur. J. Biochem. 144, (1984) (( FEBS 1984 Carotenoid-deficient maize seedlings fail to accumulate light-harvesting chlorophyll a/b binding protein (LHCP) mrna Stephen P. MAYFTELD and William C. TAYLOR Department of Genetics, University of California, Berkeley, California (Received April 17/July 10, 1984) - EJB Yellow leaves of chlorophyll-deficient seedlings and white leaves of carotenoid-deficient seedlings contain no detectable light-harvesting chlorophyll u/b binding proteins (LHCP). Chlorophyll-deficient leaves contain plastids which are arrested in development prior to chloroplast formation [Mascia, P. N. and Robertson, D. S. (1978) Plunta (Berl.) 143, while carotenoid-deficient leaves contain plastids which are arrested in development at a rudimentary stage [Bachmann, M. D., Robertson, D. S., Bowen, C. C., and Anderson, I. C. (1967) J. Ultrustruc. Res. 21, Chlorophyll-deficient leaves have normal levels of nuclear-encoded LHCP mrna while carotenoiddeficient leaves contain only trace amounts of LHCP mrna. Similar results were obtained with carotenoid deficiences caused by nuclear gene mutations and by treatment with the herbicide norflurazon which blocks carotenoid biosynthesis. We conclude that events at early stages of plastid development influence the accumulation of a nuclear-encoded mrna. Interactions between nuclear and chloroplastic genomes play key roles in leaf development and function. Many chloroplastic proteins are nuclear encoded and synthesized on cytosolic ribosomes. Some of these nuclear-encoded proteins form functional complexes with chloroplast-encoded proteins. Polypeptides comprising a given complex generally accumulate in a coordinate fashion. The basis of this apparent coordinate gene expression is unknown. The light harvesting chlorophyll u/b binding protein of photosystem I1 (LHCP) is encoded by a nuclear [I] multigene family and is an integral component of the antenna chlorophyll complex [2]. It is synthesized in the cytosol as a soluble precursor polypeptide 4-5 kda larger then the mature protein [3]. The precursor is imported post-translationally into the chloroplast, where the apoprotein is cleaved to its mature size before combining with chlorophylls a and b. The amount of LHCP is proportional to the amount of chlorophyll u and b present in the leaf [4]. In the absence of both chlorophylls the mature polypeptide never accumulates in the thylakoid membrane [5]. Chloroplast development is also dependent on the synthesis of chlorophylls and carotenoids. Chlorophyll-deficient plastids contain many of the chloroplastic enzymes, such as ribulose- 1,5-bisphosphate carboxylase and the ATP-synthesizing complex (CF,), but are deficient in light-harvesting and chlorophyll-binding proteins [4]. Chlorophyll-deficient plastids contain internal membrane structures although they are not well organized [6]. Carotenoid-deficient plants contain only rudimentary plastids which lack internal membrane structure Abbreviations. SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis ; norflurazon, 4-chloro-5-(methylamino)-2- (a,a,x-trifluro-rn-tolyl)-3 (2H)-pyridazinone; LHCP, light-harvesting chlorophyll n/b binding protein. [7-91, are devoid of chlorophyll-binding proteins and CF,, and are greatly reduced in many other chloroplastic proteins [41. The expression of the LHCP gene family appears to be regulated by light. Plants grown in the dark contain very low levels of LHCP mrna. Exposure to light results in the rapid accumulation of LHCP mrna [lo However, when dark-grown plants are exposed to red light or intermittent white light, LHCP mrna accumulates to near normal levels. LHCP polypeptides fail to accumulate in these plants [I 4, 151. LHCP mrna in both dark-grown and light-treated plants is found in the polysome fraction [12, 15, 161. It has been postulated that LHCP is synthesized and transported into the chloroplast, but is rapidly degraded if not stabilized by chlorophyll [17, 181. Many pigment-deficient nuclear mutants of maize have been described which are blocked in their ability to produce chlorophyll or carotenoids. Chlorophyll-deficient mutants contain carotenoid pigments and appear as dark yellow seedlings. Carotenoid-deficient mutants produce white seedlings due to the photooxidation of chlorophyll in the absence of carotenoids [19]. It is also possible to make white seedlings by treating non-mutant seeds with the herbicide norflurazon (Sandos 9789). These seedlings are white due to the inhibition of carotenoid synthesis and the subsequent photooxidation of chlorophyll [20]. Both chlorophyll-deficient and carotenoid-deficient seedlings lack chlorophyll and mature LHCP [4]. In this study we examined the effect of deficiencies of chlorophyll and carotenoids on LHCP mrna levels in plants grown under normal diurnal conditions. We find that chlorophyll-deficient mutants of maize contain levels of LHCP mrna similar to those of normal seedlings. Carotenoid-deficient seedlings, however, have levels of LHCP mrna greatly reduced compared with normal seedlings.

2 80 MATERIALS AND METHODS Plant material Seeds were planted onto a 1 : 1 mixture of soil and sand, covered with 1 cm of vermiculite and grown in a green house with h of sunlight. Seedlings were harvested 7-10 days (d) later when the plants were 10-14cm in height. Carotenoidand chlorophyll-deficient plants were still growing at this stage and showed no evidence of necrosis. Seedling death did not occur until about 21 d after germination. Leaves were harvested into liquid nitrogen and then held at -70 'C until used. The mutants oil yellow (Oy), luteus 13 (113), and oil yellow 1040 (0~1040) were from Peter Mascia (Monsanto, St Louis, MO, USA). The mutants wi-l43(b), wlv-.587(a), and wlv- 66(A) were from M. G. Neuffer (University of Missouri, Columbia). Lemon white (lw) was from S. Hake and M. Freeling (University of California, Berkeley), viviparous 7 (vp7) was from E. Daniel (University of California at Berkeley), and white 3 (w3) was from the Maize Genetics Cooperative stock center (University of Illinois, Urbana). All plants were grown under the same conditions except for vp7. vp7 is a recessive mutant of maize which fails to become dormant (viviparous) and therefore germinates while still on the ear. These plants were propagated by removing the embryo from the seed while still on the ear and placing onto agar media [21]. White and normal green seedlings were harvested 7-10 d later and treated in the same way as the other seedlings. All of these mutants are nuclear and segregate as if they had lesions at single loci. Seeds of the inbred line B73 (Pioneer Hi-Bred, Johnston, la, USA) were germinated and watered with 10pM norflurazon (Sandoz 9789), resulting in white seedlings. Protein isolation and blotting Protein isolation, antibody production and protein blotting were as previously described [4]. Translation of poly(a)-rich RNA in vitro Poly(A)-rich RNA was purified from total RNA by chromatography on oligo(dt)-cellulose. Poly(A)-rich RNA (0.5 pg) was added to 10-pl aliquots of rabbit reticulocyte lysate cellfree translation system (Amersham) with pci [35S]methionine. The translation products were separated on a gradient polyacrylamide gel with 0.1 % SDS run at 10mA for 12-14h [24]. The gel was impregnated with Enhance scintillant (New England Nuclear), dried under vacuum onto filter paper and exposed to Kodak XAR film with an intensifying screen. IdentiJicatioiz of a maize LHCP cdna clone The maize LHCP cdna clone pm7 was isolated from a cdna clone bank made from leaf poly(a)-rich RNA isolated from seedlings grown for 7 d in the dark followed by 48 h of continuous light. Initial identification of pm7 was madee by differential colony filter hybridization [25] using RNA labeled in vitro [38] isolated from dark-grown or light-treated maize leaves as probes. Positive identification was made by translation in vitro of mrna selected by hybridization to pm7 DNA immobilized on Sephacryl S-100 [26] and immunoprecipitation of translation products with maize LHCP antiserum [27]. The translation products obtained in vitro were analyzed by SDS- PAGE (Fig. 1). Restriction mapping of pm7,shows that the cloned cdna represents about 1000 bases of the base LHCP mrna. RESULTS Pigment contents of mutant and herbicide-bleached plants Maize seedlings grown under daylight conditions contain large amounts of both chlorophylls a and b, and carotenoid pigments. Maize seedlings grown in complete darkness for 7 d Pigment nieasurementj Frozen leaves were ground into a fine power in a coffee grinder chilled with solid CO,. The frozen powder was thawed in 100 acetone in the dark. The solution was filtered through scintered glass and the spectra recorded on a Cary visible spectrophotometer using 100 % acetone as the blank. Chlorophyll content was determined by the method of Arnon [22] and carotenoid content (including xanthophylls) was estimated from the absorbance at 480 nm [23]. RNA isolation and blotting RNA was isolated, subjected to denaturing gel electrophoresis and blotted as previously described by Nelson et al. [13], with the exception that total RNA was used for the RNA blots and not poly(a)-rich RNA. Maize LHCP cdna clone pm7 was hybridized to the RNA blots in 50% formamide, 5 buffer A (buffer A = 0.15 M N~CI, 1 m~ EDTA, 0.01 M 'Odium phosphate, ph 7.0), % SDS, mg/ml poly(a) and o.l mg/ml sperm DNA at 42 OC for h. The blots were washed three times for 1 h each in 50 % formamide, o.l % SDS, and 5 x buffer A at 42 "C then exposed to Kodak XAR film with an intensifying screen (DuPont Cronex). Fig. 1. Maize cdna clone pm7 hybridizes to LHCP mrna. Translation products obtained in vitro were separated by SDS-PAGE. Translated RNAs were (A) IeafmRNA which had been hybridized to pm7 DNA, (B) no added RNA, (C) leaf poly(a)-rich RNA, followed by immunoprecipitation with LHCP antiserum. 32 indicates a band of molecular mass 32 kda. The dark band (I) is endogenous - to the translation system

3 81 Table 1. Chlorophyll, carotenoid, and LHCP content of mutant and normal seedlings Chlorophyll and carotenoids were determined as in Materials and Methods. LHCP content was estimated from autoradiographs of membrane protein blots reacted with LHCP antisera, and 251-labeled staphylococcal protein A. Protein quantity is designated as abundant (+ +), present (+), or not detectable (n.d.) Seedling Chlorophyll Carotenoids LHCP llslg fresh wt AA4,olg fresh wt oy1040 green" oy1040 yellow n.d. 113 green yellow n.d. Oy yellow-green Fig. 3. LHCP is not detectable in chlorophyll- or carotenoid-deficient w3 green seedlings. Membrane proteins were isolated from yellow leaves of w3 white 0 0 n.d. homozygous oy1040 seedlings (B), from green leaves of their phenolw white 0 0 n.d. typically normal sibling plants (A), white leaves of homozygous lw seedlings (D), and green leaves of their phenotypically normal sibling vp7 white 0 0 n.d. plants (C). Electrophoresis, blotting, and immunodetection were as B73 green described in Fig Indicates a band of molecular mass 27.5 kda B73 whiteb 0 0 n.d. B73 yellow' n.d. wl-l43(b) green wl-i43(b) white n.d. wlv-587(a) green wlv-587(a) white n.d. wlv-66(a) green wlv-66(a) white n.d. a Phenotypically normal sibling plant of either white or yellow mutant seedling. Norflurazon-bleached seedling. Dark-grown seedling. Fig. 2. Light-induced accumulation of LHCP. Membrane proteins were isolated from leaves of seedlings grown for 8 d in darkness (A), 7 d darkness followed by 48 h white light (B), or 8 d under normal daylight conditions (C). Proteins were separated by SDS-PAGE, blotted, and then reactted with LHCP antiserum and '251-labeled staphylococcal protein A before autoradiography Indicates a band of molecular mass 27.5 kda contain neither chlorophyll a nor b, but contain approximately 50% of the carotenoid pigments of light-grown plants (Table 1). The lethal recessive mutants oy1040 and 113 contain large amounts of carotenoid pigments, but no chlorophyll, producing dark yellow seedlings. These mutants are blocked at Fig. 4. LHCP mrna accumulates in chlorophyll-deficient seedlings. Total RNA was isolated from leaves of yellow homozygous oy1040 seedlings (B), green leaves of their phenotypically normal siblings (A), yellow leaves of homozygous 113 seedlings (D), green leaves of their phenotypically normal siblings (C), and yellow-green leaves of Oy seedlings (E). RNA samples were fractionated on denaturing formaldehyde gels, blotted and hybridized with nick-translated LHCP cdna clone pm7 as described in Materials and Methods. The numbers represent residues x the conversion of protoporphyrin IX to Mg-protoporphyrin and thus are unable to synthesize chlorophyll [28]. The dominant mutant Oy is a non-lethal allele of oyz040 and contains enough chlorophyll to allow the plant to reach maturity and set seed [29]. This mutant contains high levels of carotenoid pigments and produces a yellow-green seedling and plant. The recessive white lethal mutants Iw and w3 contain no measurable carotenoids, xanthophylls, or chlorophylls when grown under daylight conditions [30], (Table 1). The lethal recessive mutant vp7 is able to produce the carotenoid pigment

4 82 Fig. 5. LHCP rnrnafails to accumulate in carotenoid-deficient seedlings. Total RNA was isolated from leaves of white homozygous w3 seedlings (B). green leaves of their phenotypically normal siblings (A), white leaves of homozygous Iw seedlings (D), green leaves of their phenotypically normal siblings (C), white leaves of homozygous vp7seedlings (F), and green leaves of their phenotypically normal siblings (E). RNA was isolated from leaves of the inbred line B-73 grown in normal daylight for 8 d (G), or for 8 d in complete darkness (H), and from norflurazon-treated seedlings grown in daylight for 8 d (I). RNA was fractionated, blotted, and hybridized as in Fig. 4. The numbers represent residues x continuous white light. Concentrations of LHCP similar to those of plants grown under normal diurnal conditions are reached after 48 h of continuous white light exposure (Fig. 2). Similar patterns have been reported for other plant species [5, 11, 121. LHCP is not detectable in the carotenoid-deficient mutants lw or w3, nor in the leaves of herbicide-bleached seedlings (Fig. 3, Table 1). LHCP was not detectable in the chlorophyll-deficient mutants oy1040, or 113 (Fig. 3, Table 1). Fig. 6. LHCPmRNA fails to accumulate in white seedlings. Total RNA was isolated from leaves of white homozygous w1-143(b) seedlings (B), from their green phenotypically normal siblings (A), from leaves of white homozygous wiv-587(a) seedlings (D), from their green phenotypically normal siblings (C), from leaves of white homozygous wlv- 661 A) seedlings (F), and their green phenotypically normal siblings (E). The RNA was fractionated, blotted, and hybridized as described in Fig. 4. The numbers represent residues x lycopene, which accumulates in the embryo [31], but the pigment does not accumulate in seedling leaves under normal daylight conditions (Table 1). The recessive seedling lethals ~1-143( B), wdv-587(a), and wlv-66ja) are unmapped ethylmethylsulfonate-induced mutants which contain trace amounts of both carotenoids and chlorophylls (Table 1). Seedlings germinated and watered with 10 pm norflurazon contained neither chlorophylls nor carotenoids (Table 1). LHCP,fails to uccumulute in chlorophyll- or carotenoid-deficient seedlings Blots of membrane proteins from dark- and light-grown maize leaves show that LHCP is absent from dark-grown seedlings, but appears within several hours of exposure to LHCP mrna does not accumulate in car0 tenoid-dejkien t seedlings Maize seedlings germinated and grown in complete darkness contain only trace amounts of LHCP mrna, but upon exposure to light rapidly accumulate high levels of the mrna (Fig. 5). The increase in mrna preceeds the accumulation of the mature protein. Levels of LHCP mrna from the chlorophyll-deficient seedlings oyl040 and I13 are similar to those of their phenotypically normal siblings, although LHCP polypeptides were undetectable in the yellow leaves. The dominant mutant Oy produces greenish-yellow plants and has normal LHCP mrna levels (Fig. 4). In contrast, LHCP mrna levels from the carotenoiddeficient mutants lw and w3 are greatly reduced compared with their phenotypically normal siblings (Fig. 5). The carotenoiddeficient leaves of vp7 have LHCP mrna levels similar to those of lw and w3. Leaves of the herbicide-bleached seedlings, which contain neither chlorophyll nor carotenoids, have LHCP mrna levels equal to those of the white mutant seedlings (Fig. 5). The amount of LHCP mrna which accumulates in these white seedlings is similar to levels of dark-grown normal seedlings, and is only a small percentage of the LHCP mrna levels of normal seedlings grown in the light. The white seedling lethal mutants wi-l43(b), wlv-587(a), and wlv-66ja) which contain trace amounts of both chlorophyll and carotenoids, have LHCP mrna levels that are slightly higher than the other white lethal seedlings, but still greatly reduced compared with phenotypically normal seedlings (Fig. 6). We estimate that none of the white seedlings have LHCP mrna levels greater than 10 % of their phenotypically normal siblings.

5 83 Fig. 7. Carotenoid-deficient seedlings accumulate many mrnas. Translation products obtained in vitro were separated by SDS-PAGE. Translated RNAs were (A) leaf mrna isolated from white homozygous lw seedlings and (B) leaf mrna isolated from their green phenotypically normal siblings. Equal amounts of poly(a)-rich RNA were translated and complete reactions were loaded onto the gel. 32 Indicates a band of molecular mass 32 kda Many m RNAs are not reduced in carotenoid-deficient seedlings Although LHCP mrna from carotenoid-deficient seedlings is greatly reduced compared to normal seedlings, most other mrnas are unaffected by the pigment defect. Translation in vitro of leaf poly(a)-rich mrna from Iw, compared with its phenotypically normal sibling, shows that most of the translation products are present in identical quantity in both samples (Fig.7). The prominant LHCP precursor bands at 32 kda are reduced in the white plants, as are a few other proteins. Most of the translation products are in similar concentration in both samples. DISCUSSION The accumulation of LHCP mrna has been shown to be under the control of the photoreceptor phytochrome in barley [5], pea [32], and Lemna [33]. A 45-s exposure to red light causes a severalfold increase in the amount of LHCP mrna. This increase can be reversed if plants are exposed to far- red light immediately following the red light treatment. Under these conditions neither chlorophyll nor mature LHCP accumulate in the thylakoid membranes. Treatment of dark-grown seedlings with intermittent white light results in the accumulation of LHCP mrna to levels similar to those of plants grown under normal daylight conditions, but neither chlorophyll b nor LHCP accumulate [15]. Red light treatment induces transcription of the Lemna LHCP gene family [34]. The accumulation of LHCP within the thylakoid membranes depends upon the light-induced accumulation of LHCP mrna, and on the accumulation of chlorophylls a and b. Harpster et al. [4] have shown that LHCP accumulates in a coordinate fashion with chlorophyll. It has been proposed that the stable intergration of LHCP into the thylakoid membrane requires chlorophyll, and without chlorophyll the protein is rapidly degraded [16, 171. The apparent light regulation of LHCP accumulation is therefore due to the light regulation of chlorophyll accumulation. Chlorophyll is not a requirement for the accumulation of LHCP mrna, as etiolated barley seedlings exposed to red light accumulate LHCP mrna but do not accumulate chlorophyll [5]. Therefore, the accumulation of normal amounts of LHCP mrna in chlorophyll-deficient (yellow) mutants of maize is not extraordinary. However, that all carotenoiddeficient (white) seedlings contain greatly reduced levels of LHCP mrna was unexpected. The reduced levels of LHCP mrna in carotenoid-deficient seedlings might be explained in several ways. First, the active form of phytochrome might not be present in white seedlings. Gorton and Briggs [35, 361 measured several phytochrome effects, including hypocotyl elongation and anthocyanin accumulation, in maize plants which had been treated with the herbicide norflurazon (white seedlings). They found no difference between treated and untreated plants. Mustard seedlings treated with norflurazon have also been reported to have normal phytochrome action [37]. Thus we think that phytochrome is present and functional in carotenoid-deficient seedlings. A second possibility is that phytochrome is not the only factor responsible for LHCP mrna accumulation. Perhaps a carotenoid pigment is required for light-induced transcription of the LHCP gene family, or is required for the stabilization of LHCP mrna. A third possibility is that both the failure to accumulate carotenoids and the failure to accumulate LHCP mrna are effects of another agent, and are not related. However, that several different nuclear mutants and norflurazon treatment show the same effects suggests that carotenoid deficiencies and failure to accumulate LHCP mrna are directly related. Finally, plastids of carotenoid-deficient mutants are arrested early in development and contain no internal structure [30]. These plastids are also deficient in chloroplastic ribosomes, as they are photobleached in light along with chlorophyll [39]. It is possible that a certain degree of plastid development is required before LHCP mrna can accumulate within the cell. Whatever the case, our results demonstrate that events in the developing plastid affect the accumulation of nuclear-encoded mrna. We thank Peter Mascia, M. G. Neuffer, E. Daniel, S. Hake, and M. Freeling for providing us with mutant seed stock. We also thank Peter Mascia for his most helpful discussion at the beginning of this project and Timothy Nelson for his careful reading of the manuscript. S. M. was supported by a McKnight Foundation Grant. Research was supported by a grant from the Competitive Research Grants Office of the US Department of Agriculture (82-CRCC ) to W. T. REFERENCES 1. Kung, S. D., Thornber, J. P. & Wildman, S. G. (1972) FEBS Lett. 24, Thornber, J. P. (1975) Annu. Rev. Plant Physiol. 26, Schmidt, G. W., Bartlett, S. G., Grossman, A,, Cashmore, A. & Chua, N.-M. (1981) J. Cell Bid. 91, Harpster, M. H., Mayfield, S. P. &Taylor, W. C. (1984) Plant Mol. Biol., in the press. 5. Apel, K. (1979) Eur. J. Biochem. 97,

6 84 6. Mascia, P. N. &Robertson, D. S. (1978) Planta (Berl.) 143, Bachmann, M. D., Robertson, D. S., Bowen, C. C. &Anderson, 1. C. (1967) J. Ultrastruc. Res. 21, Bachmann, M. D., Robertson, D. S. & Bowen, C. C. (1969) J. Ultrastruc. Rex 28, Faludi-Daniel, A., Fridvalszky, L. & Gyurjan, I. (1968) Planta (Berl.) 78, Apel, K. & Kloppstech, K. (1978) Eur. J. Biochem. 85, Tobin, E. M. (2978) Proc. Nut1 Acad. Sci. USA 75, Cumming,A. C. & Bennett, J. (1981) Eur. J. Biochem. 118, Nelson, T., Harpster, M. H., Mayfield, S. P. & Taylor, W. C. (1984) J. Cell Biol. 98, Apel, K. & Kloppstech, K. (1980) Planta (Berl.) 150, Viro, M. & Kloppstech, K. (1982) Planta (Bed.) 154, Slovin, J. P. & Tobin, E. M. (1982) Planta (Berl.) 154, Bennet, J. (1981) Eur. J. Biochem. 118, Ryrie, I. J. (1983) Eur. J. Biochem. 131, Anderson, I. C. & Robertson, D. S. (1960) Plant Physiol. 35, Bartels, P. G. & McCullough, C. (1972) Biochem. Biophys. Res. Cornrnun. 48, Robichaud, C. S., Wang, J. & Sussex, I. M. (1980) Dev. Genet. I, Amon, D. I. (1949) Plant Physiol. 24, Davies, B. H. (1976) in Chemistry and Biochemistry of Plant Pigments (Goodwin, T. W., ed.) 2nd edn, vol 11, pp , Academic Press, London. 24. Chua, N.-H. (1980) Methods Enzymol. 69, Grunstein, M. & Hogness, D. (1975) Proc. Natl Acad. Sci. USA 72, Bunemann, H. P., Westhoff, A. & Hermann, R. G. (1983) Nucleic Acids Res. 10, Kessler, S. W. (1981) Methods Enzymol. 73, Mascia, P. (1978) Mol. Gen. Genet. 161, Mascia, P. N. & Robertson, D. S. (1980) J. Hered. 71, Bachmann, M. D., Robertson, D. S., Bowen, C. C. &Anderson, I. C. (1973) J. Ultrastruc. Res. 45, Robertson, D. S. (1975) J. Hered. 66, Thompson, W. F., Everett, M., Polans, N. 0. & Jorgensen, R. A. (1983) Planta (Berl.) 158, Tobin, E. M. (1981) Plant Mol. Biol. 1, Silverthorne, J. & Tobin, E. M. (1984) Proc. Natl had. Sci. USA 81, Gorton, H. C. & Briggs, W. R. (1980) Plant Physiol. 66, Gorton, H. C. & Briggs, W. R. (1981) Planf Cell Environ. 4, Frosch, S., Bergfeld, R. & Mohr, H. (1978) in Chloroplast Development (Argyroudi, G. & Argyroudi-Akoyunoglou, J. H., eds) pp , Elsevier/North-Holland, Amsterdam. 38. Maizels, N. (1976) Cell 9, Troxler, R. F., Lester, R., Craft, F. O., Albright, J. T. (1969) Plant Physiol. 44, S. P. Mayfield and W. C. Taylor, Department of Genetics, University of California, Berkeley, California, USA 84720