Planta
Planta (1984)161:481-486
9 Springer-Verlag 1984
The appearance of photosynthetic proteins in developing maize leaves Stephen P. Mayfield 1 and William C. Taylor 2 , 1 Division of Molecular Plant Biology and 2 Department of Genetics, University of California, Berkeley, CA 94720, USA
Abstract. The appearance of photosynthetic pro-
teins was directly measured in developing maize (Zea mays L.) leaves. Third leaves of 10-14-d-old seedlings were dissected into six successive sections from the basal meristem to the tip of the leaf. The membrane and soluble proteins were separated by polyacrylamide gel electrophoresis and then transferred onto cyanogen bromide paper. After transfer of membrane proteins the paper was reacted with antisera raised against the light-harvesting chlorophyll a/b protein of photosystem II, the chlorophyll a-binding protein of reaction center P-700 of photosystem I and the 0~-subunit of chloroplast-coupling factor 1. The blots of soluble proteins were reacted with antisera raised against the electron-transport proteins plastocyanin and Fe-NADP reductase (EC 1.6.7.1), the carbon-fixing enzymes ribuloseq,5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31), as well as pyruvate orthophosphate dikinase (EC 2.7.9.1). The schedule of appearance of proteins shows that the lightharvesting and ATP-generating proteins are present in the most immature segments at the leaf base and accumulate rapidly as the cells mature. The carbon-reducing enzymes, however, appear only in tissue that has differentiated into mesophyll and bundle-sheath cells. Key words: Chloroplast membrane protein - Leaf
development - Photosynthesis (proteins) - Zea (chloroplast proteins). * To whom correspondence should be addressed Abbreviations: CFl~c~-Subunit of chloroplast coupling factor 1; C h l : c h l o r o p h y l l ; CPI=chlorophyll a-binding protein of reaction center P700 of PSI; LHCP - light-harvesting chlorophyll a/b protein of PSII; LSu=large subunit of RuBPCase; PEPCase = phosphoenolpyruvate carboxylase; P P D K = pyruvate orthophosphate dikinase; PS I, I I - p h o t o s y s t e m I and II, respectively; RuBPCase=ribulose-l,5-bisphosphate carboxylase; SSu = small subunit of RuBPCase
Introduction
Photosynthetic competence requires the light-capturing and energy-transformation mechanism of the chloroplastic photosynthetic membranes (the light reaction), as well as the CO2-fixing ability of the stromal enzymes (dark reaction). To help understand the appearance of these two systems within the developing leaf of maize we monitored the synthesis and assembly of the photosynthetic apparatus in relationship to cell age and stage of differentiation. The leaves of maize develop basipetally with cell division taking place at the basal meristem (Sharman 1942). Cells are diplaced by new divisions, resulting in the youngest cells always being present at the base of the leaf, and successively older cells towards the tip of the leaf. Because of this pattern of development, which results in a gradient of cellular development and differentiation, successive sections of maize leaves provide relatively homogeneous populations of cells (Leese and Leech 1976; Baker and Leech 1977). Plastid differentiation follows a similar pattern, with the base of the immature leaf containing only proplastids while the more mature cells contain fully differentiated chloroplasts (Leech et al. 1973). The base of the immature maize leaf contains both chlorophylls a and b and has photosystem I (PS I) and photosystem II (PS II) activity (Baker and Leech 1977). The basal sections also contain stomata, although they are neither mature nor functional, and CO 2 diffusion may be very limited in this region (Miranda et al. 1981). The ability to evolve 02, implying an active PS II, is present in the basal sections of young maize leaves, although not to the same degree as in the leaf tip (Baker and Leech 1977). Maize leaves, like those of most C 4 plants, have a typical Kranz anatomy in which mesophyll and
482
S.P. Mayfield and W.C. Taylor: Photosynthetic proteins in developing maize leaves
bundle-sheath cells are structurally and functionally differentiated. The initial step of carbon fixation occurs in mesophyll cells where phosphoenolpyruvate carboxylase (PEPCase) fixes CO 2 to form C 4 acids. These are transported to the bundlesheath cells where decarboxylation and refixation of CO 2, via ribulose-l,5-bisphosphate carboxylase (RuBPCase), occurs (for reviews see Bj6rkman et al. 1974; Edwards and Huber 1981). In maize, Kranz anatomy does not appear until after cell expansion and elongation are complete, which in young second leaves occurs 3-5 cm above the basal node (Miranda et al. 1981). Biochemical function within the maize leaf is dependent upon the cell type and thus upon cellular differentiation. To assess the effect of cell maturity and differentiation on the accumulation of photosynthetic proteins, we dissected developing maize leaves into successive sections from the basal node to the tip of the leaf. The appearance of specific proteins was measured immunologically on blots of protein separated by gel electrophoresis. Specific proteins were detected in crude leaf homogenates by their reaction with antisera raised against each protein. The use of crude homogenares allowed for the complete extraction of all protein and avoided the likelihood of differential protein recovery after subcellular fractionation. Proteins representative of both the light and dark reactions of photosynthesis were monitored. For the light reaction we followed the appearance of the light-harvesting chlorophyll a/b protein (LHCP) of PS II, the chlorophyll a-binding protein of reaction center P700 of PSI (CPI), the e-subunit of chloroplast coupling factor 1 (CF 0 and the electron-transport proteins plastocyanin and FeNADP reductase (Fe-NADP). For the dark reaction we followed the carbon-fixation enzymes RuBPCase and PEPCase, and pyruvate orthophosphate dikinase (PPDK).
Material and methods Plant material. Seeds (kernels) of Zea mays L. (inbred B73; Pioneer Hi-bred International, Johnston, IR., USA) were planted onto a 1:1 (v/v) mixture of soil and sand, covered with 1 cm of vermiculite, and placed into a growth chamber under 16 h light (65 W m -z from fluorescent [cool white; Westinghouse, Danvers, Mass., USA] and incandescent lamps) and 8 h dark. Plants were harvested 7-10 d after planting when the third leaves (after the coleopfile) were 12-14 cm in length. The first and second leaves were peeled away and the third and younger leaves were cut into six successive sections from the base of the leaf to the tip. The first and second sections were 1 cm in length while all other sections were 2 cm except for the last section, which was 4-6 cm in length. The tissue
was harvested, sectioned, frozen in liquid nitrogen, and stored at - 7 0 ~ C until needed.
Chlorophyll determination. Leaf sections were ground to a fine powder with dry ice in a mortar and pestle, thawed in 80% acetone, and filtered through Whatman (Clifton, N.J., USA) No. 1 filter paper. Chlorophyll content was determined by the method of Arnon (1949). Protein preparation. The leaf sections were homogenized in 3 : 1 (v/w) homogenization buffer with a Polytron homogenizer (Brinkman Instruments Co., Westbury, N.Y., USA) for 5-10 s. The homogenization buffer contained 0.4 M sucrose, 10 mM MgClz, 100ram 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris), pH 8.0, and 40 mM 2-mercaptoethanol (BME). The homogenate was filtered through two layers of cheesecloth and centrifuged at 10,000 g for 15 rain. The pellet, containing the membrane proteins, was resuspended in a solution containing 2% sodium dodecyl sulfate (SDS), 12% sucrose, 100 mM Tris (pH 8.0) and 40 mM BME, then frozen and stored at - 2 0 ~ C for later use. The supernatant was centrifuged at 15,600 g for 15 rain to remove any remaining membrane proteins and then brought to 2% SDS, 12% sucrose before being frozen and stored at - 20 ~ C. Concentrations of both membrane and soluble proteins were determined by the method of Lowry et at. (1951).
Polyacrylamide gel electrophoresis (PAGE). Protein samples were separated by electrophoresis through 7.5-15% polyacrylamide gels in the presence of 0.1% sodium dodecyl sulfate (Chua 1980). Equal amounts of protein were loaded into each lane, and electrophoresis was carried out at 4 mA constant current for 12 16 h at 4 ~ C. The gels were then either stained, or prepared for electroblotting onto cyanogen bromide paper. For staining the gels were placed in 0.25% Coomassie brilliant blue R, 40% methanol and 7% acetic acid for 1-2 h and then destained in 50% methanol, 7% acetic acid for 2-3 h before being photographed with Polaroid P/N 55 film (Polaroid Corp., Cambridge, MA, USA). Electroblotting. After P A G E the gels were washed twice for ~10 min each in deionized water and twice for 15 rain each in transfer buffer (50 mM Na acetate, 0.01% Na azide). The proteins were then electrophoretically transferred onto cyanogen bromide paper using a Bio-Rad (Richmond, Cal., USA) TransBlot apparatus as described by Nyari et al. (1981). The paper was removed from the Trans-Blot apparatus and placed in a solution of 1% bovine serum albumin (BSA) and 1 M glycine to quench the unused binding sites of the cyanogen bromide paper. The paper was then washed in phosphate-buffered saline, pH7.4, 0.1% BSA, 0.1% non-ionic detergent (NP-40) and 0.02% Na azide. The paper was reacted with 10 lal of rabbit antisera in the same solution at room temperature for 3 h, then washed, and reacted with 140,000 Bq (0.4 laCi) 12sI-Staphylococcus aureus protein-A (Amersham Radiochemicals, Arlington Heights, Ill., USA) for i h at room temperature in the same solution. The paper was washed, dried and exposed to Kodak XAR-5 film (Eastman-Kodak, Rochester, N.Y., USA) with an intensifying screen (Cronex Lighting Plus; Dupont Co., Wilmington, Dd., USA). Antibody production. Female white New Zealand rabbits were twice injected subcutaneously with a 1/1 (v/v) mixture of isolated protein and Freund's adjuvant (complete). The primary injection contained 200-300 lag of purified protein in a concentration of I m g m l - 1 . The second injection contained 100-200 lag of purified protein and was given four weeks after
S.P. Mayfield and W.C. Taylor: Photosynthetic proteins in developing maize leaves
483
the primary injection. Two weeks after the second injection the rabNts were bled and the serum separated from the red blood cells by centrifugation. No further purification of the antisera was necessary. Phosphoenolpyruvate carboxylase, RuBPCase, LHCP, CPI and CF 1 were isolated as described by Harpster et al. 1984). The antibodies to Fe-NADP and plastocyanin were a gift from Richard Malkin (Department of Molecular Plant Biology, University of California, Berkeley). The antibody to PPDK was the gift of Kazuko Aoyagi (Chemistry Department, University of California, Berkeley). All antibodies were prepared in similar fashion. Fe-NADP and plastocyanin were purified from spinach (cv. Resistoflay; Ferry-Morse, Mountain View, Cal., USA). All other proteins were purified from maize_
Antibody titer. Serial dilutions of both membrane- and solubleprotein samples taken from the leaf tip were subjected to PAGE, transferred to cyanogen bromide paper, and probed with the different antisera to determine the lowest level of protein which could be detected with the protein blotting method. In all cases the leaf tip contained the most protein, and this amount was therefore defined as the 100% level. With all proteins except for CPI and LHCP, approx. 1% of the protein content of the tip could be detected. The LHCP antisera detected protein at about 0.2% of the tip content, while the CPI antisera detected only protein that was in concentrations greater then 5% of the tip content.
Results
Profiles of membrane and soluble proteins. Coomassie-blue-stained polyacrylamide gels of membrane proteins extracted from third leaves of maize seedlings show that the major proteins of the basal tissue are not the same as the major proteins in the more mature leaf sections (Fig. 1). Three prominent, low-molecular-weight membrane proteins that appear in the basal tissue at 14.5, 18.2, and 19.5 kdalton are greatly reduced in the leaf-tip tissue, and other proteins not prominent in the basal tissue are prominent in the more mature sections, as are the 27.5 kdalton LHCP polypeptides. The soluble-protein profile follows a similar pattern in that the abundant proteins of the mature tissue are not the same as the abundant proteins of the basal tissue (Fig. 2). The carboxylase enzymes are easily seen in the tip section as prominent bands at 90 kdalton (PEPCase), 59 kdalton (large subunit, LSu, of RuBPCase) and 14 kdalton (small subunit, SSu, of RuBPCase). However, it is very difficult to tell from the stained gel at what point these proteins first appear in the leaf sections. Chlorophyll content of leaf sections. Chlorophylls a and b were found in every leaf section of the young maize leaf, although the amount of chlorophyll increases greatly in the more mature leaf sections. The chlorophyll a/b ratio is 2.5 in the basal tissue and increases gradually to 3.8 in the tip section
Fig. 1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of membrane proteins isolated from: A, base to 1 cm; B, 1-2 cm; C, 2-4 cm; D, 4-6 cm; E, 6 8 cm; F, 8 cm-tip of a 14-cm-long maize leaf. Prominent protein bands that decrease as the tissue matures are found at 19.5 kdalton (I), 18.2 kdalton (II) and 14.5 kdalton (III). LHCP is clearly visible in mature tissue at 27.5 kdalton (IV)
(Table 1). The amount of chlorophyll per g fresh weight and the chlorophyll a/b ratio reported here are in close agreement with other reports (Leech et al. 1973; Antonielli et al. 1981).
The appearance of specific membrane proteins. The appearance of LHCP as visualized by protein blot shows that the protein is present in every leaf section of the developing maize leaf (Fig. 3), increasing in amount from the first (basal) section of the leaf to the tip. The accumulation of LHCP follows that of chlorophyll, but the ratio of LHCP to chlorophyll changes from the base to the tip of the leaf (Table 1). The CPI protein of P S I appears very faintly in the first 2 cm of leaf tissue. We believe that this is a consequence of the reduced sensitivity of detection of this protein on our protein blots and that the protein is present at levels similar to that of LHCP. Several factors contribute to the
484
S.P. Mayfield and W.C. Taylor: Photosynthetic proteins in developing maize leaves
Fig. 3. Autoradlograph of protein blot or SDS-PA(SE ot membrane proteins isolated from: A, base to I cm; B, 1-2 cm; C, 2M cm; D, 4-6 cm; E, 6-8 cm; F, 8 cm-tip of 14-cm maize leaf. The blot was reacted with antisera to CPI, CF 1, and LHCP
Fig. 2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble proteins isolated from: A, base to I cm; B, 1-2 cm; C, 2 4 cm; D, 4-6 cm; E, 6-8 cm; F, 8 cm-tip of 14-cm maize leaf. Carbon fixation enzymes visible in the mature tissue are: PEPCase (I), RUBPCase: LSu (II) and SSu (III). A major protein found in the basal tissue but absent in the leaf tip appears at 80 kdalton (IV) Table 1. Chlorophyll a and b content, chlorophyll a/b ratio, chlorophyll per mg membrane protein, amount of LHCP, and the ratio of LHCP/chlorophyll (total) of maize leaf segments Distance gg from CHt/g base FW (cm)
Chl a/b ratio
0-1 1-2 2-4 4-6 6-8 8-14
2.62 2.78 3.22 3.26 3.60 3.79
29.7 62.3 170.5 377.0 838.7 1416.1
p.g Cht/mg merebrane protein 0.25 1.15 5.02 11.12 22.66 26.02
Relative Amount amount of of LHCP/ LHCP gg Chl
4 6 13 29 83 100
16.00 5.21 2.60 2.60 3.66 3.84
reduced sensitivity of detection of CPI. The CPI migrates as a broad, diffuse band and hence the 125I signal is diffuse. This, coupled with the low titer of the CPI antisera, makes it difficult to visualize the protein in the first two leaf sections. However, upon long exposures we can visualize the CPI
Fig. 4. Autoradiograph of protein blot of SDS-PAGE of soluble proteins isolated from: A, base to I cm; B, 1-2 cm; C, 2~4 cm; D, 4-6 cm; E, 6-8 cm; F, 8 cm-tip of 14-cm maize leaf. The blot was reacted with antisera to PEPCase and RuBPCase. The band below PEPCase reacts with PEPCase antisera in the crude extract, but is not present in purified PEPCase extracts
band in the basal sections. The CPI protein is present in all other leaf segments and appears to increase in concentration in a manner similar to that of the L H C P (Fig. 3). a-Coupling factor 1 (CFj) is also present in every leaf section and increases in concentration from the base to the tip of the leaf (Fig. 3).
The appearance of specific soluble proteins. The chloroplast-encoded large subunit (LSu) of RuBPCase can be found in the distal sections of the maize leaf only (Fig. 4). The two nucleus-encoded enzymes PEPCase and P P D K appear in the same four sections of the leaf (Fig. 5). All three of these enzymes first appear in the third leaf segment,
S.P. Mayfield and W.C. Taylor: Photosynthetic proteins in developingmaize leaves
Fig. 5. Autoradiograph of protein blots of SDS-PAGE of soluble proteins isolated from: A, base to I cm; B, 1 2 cm; C, 2~cm; D, 4-6 cm; E, 6-8 cm; F, 8 cm-tip of 14-cm maize leaf. The blots were reacted with antisera to PPDK, Fe-NADP and plastocyanin (PC) separately. A composite figure is used for ease of presentation
2-4 cm from the base of the leaf. From serial-dilution assays we estimate that all three proteins are present at about 1% of their final concentration in the third leaf section (data not shown) and, as shown in Figs. 4 and 5, they increase in concert with one another as the tissue matures. The electron-transport protein plastocyanin appears in very section of the leaf and increases in concentration as the tissue matures (Fig. 4). Another electron-transport protein, Fe-NADP, can be seen in every sections of the leaf, but increases in concert with the carbon-fixation enzymes from the third section distal (Fig. 5). Discussion
The appearance of proteins involved in light harvesting (LHCP, CPI) and A T P generation (CF1) , along with the presence of chlorophylls a and b in the first cm of the developing leaf, indicates that photophosphorylation may be taking place in this most immature leaf tissue. Baker and Leech (1977) measured both P S I and PS II activities in similar tissue and Miranda et al. (1981) found Oz evolution, although low, also to be present in the basal tissue of maize. If ATP generation through photophosphorylation exists in the basal tissue via P S I and PS II, then electron transport should link the
485
two photosystems at this point as well. Plastocyanin was chosen as a representative of the electron-transport chain between the two photosysterns and, as shown in Fig. 4, plastocyanin is present in every section of the leaf. The light-harvesting and electron-transport proteins seem to increase in concert with one another as the cells mature. Although the maize leaf appears to have the potential for generating ATP from light energy in the basal tissue, the ability to fix carbon in this region is lacking. Neither of the CO 2 fixing enzymes (PEPCase, RuBPCase) are present, nor are there functional stomata and hence little, if any, CO 2 diffusion. Miranda et al. (1981) have shown that Kranz anatomy and functional stomata first appear at the same position in the second leaf of maize, 3-5 cm from the node below the leaf. This is the same position at which we first measure the appearance of the carbon-fixing enzymes and P P D K . That all three enzymes appear only in tissue that has differentiated indicates that cellular differentiation may be a requirement for the accumulation of these enzymes. Dean and Leech (1982) and Viro and Kloppstech (1980) have reported in wheat and barley, respectively, that RuBPCase appears in the basal section of leaves at relatively high levels and increases as the cells mature. This is not what we observe in maize and the difference may be related to the difference in the photosynthetic schemes of the plants. Both barley and wheat are C 3 plants which do not utilize differentiated mesophyll and bundle-sheath cells for photosynthesis. Maize, which possesses differentiated cell types used in the C4 carbon-reduction pathway, might require the differentiation of cell types before the carbon fixing enzymes in this pathway can accumulate (e.g. PEPCase, RuBPCase, PPDK). It has been postulated that prior to the appearance of Kranz anatomy, a C 3 pathway might be used in maize (Waygood et al. 1976). However, we find no evidence for this, but find that both the C 3 enzyme RuBPCase and the C 4 enzyme PEPCase appear in the same leaf sections and increase in concert with one another. Perchorowicz and Gibbs (1980) and Williams and Kennedy (1978) have reported that the base of the maize leaf contains both RuBPCase and PEPCase. This disagreement with our results may be because both Perchorowicz and Gibbs, and Williams and Kennedy used larger sections (2.5 cm and 4 cm, respectively) as compared with ours (1 cm). We first detect the two carboxylases in the third cm of the leaf, and this may be the source of the enzymes measured by the other groups.
486
S.P. Mayfield and W.C. Taylor: Photosynthetic proteins in developing maize leaves
Antonielli et al. (1981) reported that the activity ratio of PEPCase to RuBPCase increases (fourfold) from the sheath to the blade in mature maize plants, while Williams and Kennedy (1978) observed a slightly lower PEPCase/RuBPCase activity ratio in the base (sheath) of a maize seedling as compared with the blade. We directly measured the amount of PEPCase and RuBPCase as protein, not as an enzymatic activity, and find the two enzymes absent in the base, and approximately equal to one another in concentration at all other points in the leaf. Although the presence of an enzyme does not assure an enzymatic activity, the absence of the enzyme does preclude it, and thus it is unlikely there is carbon fixation in the basal sections. The regulation of the appearance of photosynthetic proteins is a complex process which involves protein synthesis in both undifferentiated and differentiated cells. The proteins responsible for carbon fixation appear in an order indicating that cellular differentiation plays a role in their accumulation. This is not the case with the light-harvesting and ATP-generating proteins in maize nor does it seem to be a requirement in grasses that do not use C 4 photosynthesis. We thank Richard Malkin for Fe-NADP and plastocyanin antisera, Kazuko Aoyogi for PPDK antisera, and Pioneer Hi-Bred for B73 seed. We also thank Timothy Nelson for a critical reading of the manuscript. This research was supported by grants from the National Science Foundation (PCM 78-26789) and the Competitive Research Grants Office of the U.S. Department of Agriculture (82-CRCR-I-1083).
References Antonielli, M., Lupattelli, M., Venanzi, G. (1981) Some characteristics of the chlorophyllous parenchyma of maize outside the leaf lamina. Plant Sci. Lett. 21, 107-119 Arnon, D. (1949) Copper enzymes in isolated chloroplast: polyphenol-oxidase in Beta vuIgaris. Plant Physiol. 24, 1-15 Baker, N.R., Leech, R.M. (1977) Development of photosystem I and photosystem II activites in leaves of light grown maize (Zea mays). Plant Physiol. 60, 640-644 Bj6rkman, O., Troughton, J., Nobs, M. (1974) Photosynthesis in relation to leaf structure. In : Basic mechanisms in plant
morphogenesis. Brookhaven Symp. in Biology, vol. XXV, pp. 206-217. U.S. Department of Commerce, Springfield, Va., USA Boffey, S.A., Ellis, R.J., Sellden, G., Leech, R.M. (1979) Chloroplast division and DNA synthesis in light-grown wheat. Plant Physiol. 64, 502-505 Chua, N.-H. (1980) Electrophoretic analysis of chloroplast proteins. Methods Enzymol. 69, 434-446 Dean, C., Leech, R.M. (1982) Genome expression during normal leaf development. Plant Physiol. 69, 904-910 Edwards, G.E., Huber, S.C. (1981) The C 4 pathway. In: The biochemistry of plants, vol. 8, pp. 237-281, Hatch, M.D., Boardman, N.C., eds. Academic Press, New York London Ellis, R.J., Jellings, A.J., Leech, R.M. (1983) Nuclear DNA content and the control of chloroplast replication in wheat leaves. Planta 157, 376-380 Harpster, Mayfield, S.P., Taylor, W.C. (1984) Effects of pigment-deficient mutants on the accumulation of photosynthetic proteins in maize. Plant Molec. Biol. (in press) Leech, R.M., Rumsby, M.G., Thomson, W.W. (1973) Plastid differentiation, acyllipid, and fatty acid changes in developing green maize leaves. Plant Physiol. 52, 240-245 Leese, R.M., Leech, R.M. (1976) Sequential changes in the lipids of developing proplast Idy Pisolated from greening maize leaves. Plant Physiol. 57, 78%794 Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the folin reagent. J. Biol. Chem. 193, 262-275 Miranda, V., Baker, N.R., Long, S.P. (1981) Anatomical variation along the length of the Zea mays leaf in relation to photosynthesis. New Phytot. 88, 595-605 Nyari, L.T., Tan, Y.H., Ehrlich, H.A. (1981) Production and characterization of monoclonal antibodies to human fibroblast (beta) interferon. In: The biology of the interferons, pp. 67-73, DeMaeyer, E., Galasso, G., Schellekenes, H., eds. Elsevier North Holland, New York Perchorowicz, J.T., Gibbs, M. (1980) Carbon dioxide fixation and related properties in sections of the developing green maize leaf. Plant Physiol. 65, 802-809 Sharman, B.C. (1942) Developmental anatomy of the shoot of Zea mays L. Ann. Bot. (London) 6, 245-282 Viro, M., Kloppstech, K. (1980) Differential expression of the genes for ribulose-l,5-bisphosphate carboxylase and lightharvesting chlorophyll a/b protein in the developing barley leaf. Planta 150, 41-45 Waygood, E.R., Law, G. (1976)CO 2 fixation in leaves and chloroplasts of maize. Plant Physiol. 57, S-15 Williams, L.E., Kennedy, R.A. (1978) Photosynthetic carbon metabolism during leaf ontogeny in Zea mays L. : enzyme studies. Planta 142, 269-274 Received I0 September; accepted 4 November 1983
Planta (1984)161:481-486
9 Springer-Verlag 1984
The appearance of photosynthetic proteins in developing maize leaves Stephen P. Mayfield 1 and William C. Taylor 2 , 1 Division of Molecular Plant Biology and 2 Department of Genetics, University of California, Berkeley, CA 94720, USA
Abstract. The appearance of photosynthetic pro-
teins was directly measured in developing maize (Zea mays L.) leaves. Third leaves of 10-14-d-old seedlings were dissected into six successive sections from the basal meristem to the tip of the leaf. The membrane and soluble proteins were separated by polyacrylamide gel electrophoresis and then transferred onto cyanogen bromide paper. After transfer of membrane proteins the paper was reacted with antisera raised against the light-harvesting chlorophyll a/b protein of photosystem II, the chlorophyll a-binding protein of reaction center P-700 of photosystem I and the 0~-subunit of chloroplast-coupling factor 1. The blots of soluble proteins were reacted with antisera raised against the electron-transport proteins plastocyanin and Fe-NADP reductase (EC 1.6.7.1), the carbon-fixing enzymes ribuloseq,5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31), as well as pyruvate orthophosphate dikinase (EC 2.7.9.1). The schedule of appearance of proteins shows that the lightharvesting and ATP-generating proteins are present in the most immature segments at the leaf base and accumulate rapidly as the cells mature. The carbon-reducing enzymes, however, appear only in tissue that has differentiated into mesophyll and bundle-sheath cells. Key words: Chloroplast membrane protein - Leaf
development - Photosynthesis (proteins) - Zea (chloroplast proteins). * To whom correspondence should be addressed Abbreviations: CFl~c~-Subunit of chloroplast coupling factor 1; C h l : c h l o r o p h y l l ; CPI=chlorophyll a-binding protein of reaction center P700 of PSI; LHCP - light-harvesting chlorophyll a/b protein of PSII; LSu=large subunit of RuBPCase; PEPCase = phosphoenolpyruvate carboxylase; P P D K = pyruvate orthophosphate dikinase; PS I, I I - p h o t o s y s t e m I and II, respectively; RuBPCase=ribulose-l,5-bisphosphate carboxylase; SSu = small subunit of RuBPCase
Introduction
Photosynthetic competence requires the light-capturing and energy-transformation mechanism of the chloroplastic photosynthetic membranes (the light reaction), as well as the CO2-fixing ability of the stromal enzymes (dark reaction). To help understand the appearance of these two systems within the developing leaf of maize we monitored the synthesis and assembly of the photosynthetic apparatus in relationship to cell age and stage of differentiation. The leaves of maize develop basipetally with cell division taking place at the basal meristem (Sharman 1942). Cells are diplaced by new divisions, resulting in the youngest cells always being present at the base of the leaf, and successively older cells towards the tip of the leaf. Because of this pattern of development, which results in a gradient of cellular development and differentiation, successive sections of maize leaves provide relatively homogeneous populations of cells (Leese and Leech 1976; Baker and Leech 1977). Plastid differentiation follows a similar pattern, with the base of the immature leaf containing only proplastids while the more mature cells contain fully differentiated chloroplasts (Leech et al. 1973). The base of the immature maize leaf contains both chlorophylls a and b and has photosystem I (PS I) and photosystem II (PS II) activity (Baker and Leech 1977). The basal sections also contain stomata, although they are neither mature nor functional, and CO 2 diffusion may be very limited in this region (Miranda et al. 1981). The ability to evolve 02, implying an active PS II, is present in the basal sections of young maize leaves, although not to the same degree as in the leaf tip (Baker and Leech 1977). Maize leaves, like those of most C 4 plants, have a typical Kranz anatomy in which mesophyll and
482
S.P. Mayfield and W.C. Taylor: Photosynthetic proteins in developing maize leaves
bundle-sheath cells are structurally and functionally differentiated. The initial step of carbon fixation occurs in mesophyll cells where phosphoenolpyruvate carboxylase (PEPCase) fixes CO 2 to form C 4 acids. These are transported to the bundlesheath cells where decarboxylation and refixation of CO 2, via ribulose-l,5-bisphosphate carboxylase (RuBPCase), occurs (for reviews see Bj6rkman et al. 1974; Edwards and Huber 1981). In maize, Kranz anatomy does not appear until after cell expansion and elongation are complete, which in young second leaves occurs 3-5 cm above the basal node (Miranda et al. 1981). Biochemical function within the maize leaf is dependent upon the cell type and thus upon cellular differentiation. To assess the effect of cell maturity and differentiation on the accumulation of photosynthetic proteins, we dissected developing maize leaves into successive sections from the basal node to the tip of the leaf. The appearance of specific proteins was measured immunologically on blots of protein separated by gel electrophoresis. Specific proteins were detected in crude leaf homogenates by their reaction with antisera raised against each protein. The use of crude homogenares allowed for the complete extraction of all protein and avoided the likelihood of differential protein recovery after subcellular fractionation. Proteins representative of both the light and dark reactions of photosynthesis were monitored. For the light reaction we followed the appearance of the light-harvesting chlorophyll a/b protein (LHCP) of PS II, the chlorophyll a-binding protein of reaction center P700 of PSI (CPI), the e-subunit of chloroplast coupling factor 1 (CF 0 and the electron-transport proteins plastocyanin and FeNADP reductase (Fe-NADP). For the dark reaction we followed the carbon-fixation enzymes RuBPCase and PEPCase, and pyruvate orthophosphate dikinase (PPDK).
Material and methods Plant material. Seeds (kernels) of Zea mays L. (inbred B73; Pioneer Hi-bred International, Johnston, IR., USA) were planted onto a 1:1 (v/v) mixture of soil and sand, covered with 1 cm of vermiculite, and placed into a growth chamber under 16 h light (65 W m -z from fluorescent [cool white; Westinghouse, Danvers, Mass., USA] and incandescent lamps) and 8 h dark. Plants were harvested 7-10 d after planting when the third leaves (after the coleopfile) were 12-14 cm in length. The first and second leaves were peeled away and the third and younger leaves were cut into six successive sections from the base of the leaf to the tip. The first and second sections were 1 cm in length while all other sections were 2 cm except for the last section, which was 4-6 cm in length. The tissue
was harvested, sectioned, frozen in liquid nitrogen, and stored at - 7 0 ~ C until needed.
Chlorophyll determination. Leaf sections were ground to a fine powder with dry ice in a mortar and pestle, thawed in 80% acetone, and filtered through Whatman (Clifton, N.J., USA) No. 1 filter paper. Chlorophyll content was determined by the method of Arnon (1949). Protein preparation. The leaf sections were homogenized in 3 : 1 (v/w) homogenization buffer with a Polytron homogenizer (Brinkman Instruments Co., Westbury, N.Y., USA) for 5-10 s. The homogenization buffer contained 0.4 M sucrose, 10 mM MgClz, 100ram 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris), pH 8.0, and 40 mM 2-mercaptoethanol (BME). The homogenate was filtered through two layers of cheesecloth and centrifuged at 10,000 g for 15 rain. The pellet, containing the membrane proteins, was resuspended in a solution containing 2% sodium dodecyl sulfate (SDS), 12% sucrose, 100 mM Tris (pH 8.0) and 40 mM BME, then frozen and stored at - 2 0 ~ C for later use. The supernatant was centrifuged at 15,600 g for 15 rain to remove any remaining membrane proteins and then brought to 2% SDS, 12% sucrose before being frozen and stored at - 20 ~ C. Concentrations of both membrane and soluble proteins were determined by the method of Lowry et at. (1951).
Polyacrylamide gel electrophoresis (PAGE). Protein samples were separated by electrophoresis through 7.5-15% polyacrylamide gels in the presence of 0.1% sodium dodecyl sulfate (Chua 1980). Equal amounts of protein were loaded into each lane, and electrophoresis was carried out at 4 mA constant current for 12 16 h at 4 ~ C. The gels were then either stained, or prepared for electroblotting onto cyanogen bromide paper. For staining the gels were placed in 0.25% Coomassie brilliant blue R, 40% methanol and 7% acetic acid for 1-2 h and then destained in 50% methanol, 7% acetic acid for 2-3 h before being photographed with Polaroid P/N 55 film (Polaroid Corp., Cambridge, MA, USA). Electroblotting. After P A G E the gels were washed twice for ~10 min each in deionized water and twice for 15 rain each in transfer buffer (50 mM Na acetate, 0.01% Na azide). The proteins were then electrophoretically transferred onto cyanogen bromide paper using a Bio-Rad (Richmond, Cal., USA) TransBlot apparatus as described by Nyari et al. (1981). The paper was removed from the Trans-Blot apparatus and placed in a solution of 1% bovine serum albumin (BSA) and 1 M glycine to quench the unused binding sites of the cyanogen bromide paper. The paper was then washed in phosphate-buffered saline, pH7.4, 0.1% BSA, 0.1% non-ionic detergent (NP-40) and 0.02% Na azide. The paper was reacted with 10 lal of rabbit antisera in the same solution at room temperature for 3 h, then washed, and reacted with 140,000 Bq (0.4 laCi) 12sI-Staphylococcus aureus protein-A (Amersham Radiochemicals, Arlington Heights, Ill., USA) for i h at room temperature in the same solution. The paper was washed, dried and exposed to Kodak XAR-5 film (Eastman-Kodak, Rochester, N.Y., USA) with an intensifying screen (Cronex Lighting Plus; Dupont Co., Wilmington, Dd., USA). Antibody production. Female white New Zealand rabbits were twice injected subcutaneously with a 1/1 (v/v) mixture of isolated protein and Freund's adjuvant (complete). The primary injection contained 200-300 lag of purified protein in a concentration of I m g m l - 1 . The second injection contained 100-200 lag of purified protein and was given four weeks after
S.P. Mayfield and W.C. Taylor: Photosynthetic proteins in developing maize leaves
483
the primary injection. Two weeks after the second injection the rabNts were bled and the serum separated from the red blood cells by centrifugation. No further purification of the antisera was necessary. Phosphoenolpyruvate carboxylase, RuBPCase, LHCP, CPI and CF 1 were isolated as described by Harpster et al. 1984). The antibodies to Fe-NADP and plastocyanin were a gift from Richard Malkin (Department of Molecular Plant Biology, University of California, Berkeley). The antibody to PPDK was the gift of Kazuko Aoyagi (Chemistry Department, University of California, Berkeley). All antibodies were prepared in similar fashion. Fe-NADP and plastocyanin were purified from spinach (cv. Resistoflay; Ferry-Morse, Mountain View, Cal., USA). All other proteins were purified from maize_
Antibody titer. Serial dilutions of both membrane- and solubleprotein samples taken from the leaf tip were subjected to PAGE, transferred to cyanogen bromide paper, and probed with the different antisera to determine the lowest level of protein which could be detected with the protein blotting method. In all cases the leaf tip contained the most protein, and this amount was therefore defined as the 100% level. With all proteins except for CPI and LHCP, approx. 1% of the protein content of the tip could be detected. The LHCP antisera detected protein at about 0.2% of the tip content, while the CPI antisera detected only protein that was in concentrations greater then 5% of the tip content.
Results
Profiles of membrane and soluble proteins. Coomassie-blue-stained polyacrylamide gels of membrane proteins extracted from third leaves of maize seedlings show that the major proteins of the basal tissue are not the same as the major proteins in the more mature leaf sections (Fig. 1). Three prominent, low-molecular-weight membrane proteins that appear in the basal tissue at 14.5, 18.2, and 19.5 kdalton are greatly reduced in the leaf-tip tissue, and other proteins not prominent in the basal tissue are prominent in the more mature sections, as are the 27.5 kdalton LHCP polypeptides. The soluble-protein profile follows a similar pattern in that the abundant proteins of the mature tissue are not the same as the abundant proteins of the basal tissue (Fig. 2). The carboxylase enzymes are easily seen in the tip section as prominent bands at 90 kdalton (PEPCase), 59 kdalton (large subunit, LSu, of RuBPCase) and 14 kdalton (small subunit, SSu, of RuBPCase). However, it is very difficult to tell from the stained gel at what point these proteins first appear in the leaf sections. Chlorophyll content of leaf sections. Chlorophylls a and b were found in every leaf section of the young maize leaf, although the amount of chlorophyll increases greatly in the more mature leaf sections. The chlorophyll a/b ratio is 2.5 in the basal tissue and increases gradually to 3.8 in the tip section
Fig. 1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of membrane proteins isolated from: A, base to 1 cm; B, 1-2 cm; C, 2-4 cm; D, 4-6 cm; E, 6 8 cm; F, 8 cm-tip of a 14-cm-long maize leaf. Prominent protein bands that decrease as the tissue matures are found at 19.5 kdalton (I), 18.2 kdalton (II) and 14.5 kdalton (III). LHCP is clearly visible in mature tissue at 27.5 kdalton (IV)
(Table 1). The amount of chlorophyll per g fresh weight and the chlorophyll a/b ratio reported here are in close agreement with other reports (Leech et al. 1973; Antonielli et al. 1981).
The appearance of specific membrane proteins. The appearance of LHCP as visualized by protein blot shows that the protein is present in every leaf section of the developing maize leaf (Fig. 3), increasing in amount from the first (basal) section of the leaf to the tip. The accumulation of LHCP follows that of chlorophyll, but the ratio of LHCP to chlorophyll changes from the base to the tip of the leaf (Table 1). The CPI protein of P S I appears very faintly in the first 2 cm of leaf tissue. We believe that this is a consequence of the reduced sensitivity of detection of this protein on our protein blots and that the protein is present at levels similar to that of LHCP. Several factors contribute to the
484
S.P. Mayfield and W.C. Taylor: Photosynthetic proteins in developing maize leaves
Fig. 3. Autoradlograph of protein blot or SDS-PA(SE ot membrane proteins isolated from: A, base to I cm; B, 1-2 cm; C, 2M cm; D, 4-6 cm; E, 6-8 cm; F, 8 cm-tip of 14-cm maize leaf. The blot was reacted with antisera to CPI, CF 1, and LHCP
Fig. 2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble proteins isolated from: A, base to I cm; B, 1-2 cm; C, 2 4 cm; D, 4-6 cm; E, 6-8 cm; F, 8 cm-tip of 14-cm maize leaf. Carbon fixation enzymes visible in the mature tissue are: PEPCase (I), RUBPCase: LSu (II) and SSu (III). A major protein found in the basal tissue but absent in the leaf tip appears at 80 kdalton (IV) Table 1. Chlorophyll a and b content, chlorophyll a/b ratio, chlorophyll per mg membrane protein, amount of LHCP, and the ratio of LHCP/chlorophyll (total) of maize leaf segments Distance gg from CHt/g base FW (cm)
Chl a/b ratio
0-1 1-2 2-4 4-6 6-8 8-14
2.62 2.78 3.22 3.26 3.60 3.79
29.7 62.3 170.5 377.0 838.7 1416.1
p.g Cht/mg merebrane protein 0.25 1.15 5.02 11.12 22.66 26.02
Relative Amount amount of of LHCP/ LHCP gg Chl
4 6 13 29 83 100
16.00 5.21 2.60 2.60 3.66 3.84
reduced sensitivity of detection of CPI. The CPI migrates as a broad, diffuse band and hence the 125I signal is diffuse. This, coupled with the low titer of the CPI antisera, makes it difficult to visualize the protein in the first two leaf sections. However, upon long exposures we can visualize the CPI
Fig. 4. Autoradiograph of protein blot of SDS-PAGE of soluble proteins isolated from: A, base to I cm; B, 1-2 cm; C, 2~4 cm; D, 4-6 cm; E, 6-8 cm; F, 8 cm-tip of 14-cm maize leaf. The blot was reacted with antisera to PEPCase and RuBPCase. The band below PEPCase reacts with PEPCase antisera in the crude extract, but is not present in purified PEPCase extracts
band in the basal sections. The CPI protein is present in all other leaf segments and appears to increase in concentration in a manner similar to that of the L H C P (Fig. 3). a-Coupling factor 1 (CFj) is also present in every leaf section and increases in concentration from the base to the tip of the leaf (Fig. 3).
The appearance of specific soluble proteins. The chloroplast-encoded large subunit (LSu) of RuBPCase can be found in the distal sections of the maize leaf only (Fig. 4). The two nucleus-encoded enzymes PEPCase and P P D K appear in the same four sections of the leaf (Fig. 5). All three of these enzymes first appear in the third leaf segment,
S.P. Mayfield and W.C. Taylor: Photosynthetic proteins in developingmaize leaves
Fig. 5. Autoradiograph of protein blots of SDS-PAGE of soluble proteins isolated from: A, base to I cm; B, 1 2 cm; C, 2~cm; D, 4-6 cm; E, 6-8 cm; F, 8 cm-tip of 14-cm maize leaf. The blots were reacted with antisera to PPDK, Fe-NADP and plastocyanin (PC) separately. A composite figure is used for ease of presentation
2-4 cm from the base of the leaf. From serial-dilution assays we estimate that all three proteins are present at about 1% of their final concentration in the third leaf section (data not shown) and, as shown in Figs. 4 and 5, they increase in concert with one another as the tissue matures. The electron-transport protein plastocyanin appears in very section of the leaf and increases in concentration as the tissue matures (Fig. 4). Another electron-transport protein, Fe-NADP, can be seen in every sections of the leaf, but increases in concert with the carbon-fixation enzymes from the third section distal (Fig. 5). Discussion
The appearance of proteins involved in light harvesting (LHCP, CPI) and A T P generation (CF1) , along with the presence of chlorophylls a and b in the first cm of the developing leaf, indicates that photophosphorylation may be taking place in this most immature leaf tissue. Baker and Leech (1977) measured both P S I and PS II activities in similar tissue and Miranda et al. (1981) found Oz evolution, although low, also to be present in the basal tissue of maize. If ATP generation through photophosphorylation exists in the basal tissue via P S I and PS II, then electron transport should link the
485
two photosystems at this point as well. Plastocyanin was chosen as a representative of the electron-transport chain between the two photosysterns and, as shown in Fig. 4, plastocyanin is present in every section of the leaf. The light-harvesting and electron-transport proteins seem to increase in concert with one another as the cells mature. Although the maize leaf appears to have the potential for generating ATP from light energy in the basal tissue, the ability to fix carbon in this region is lacking. Neither of the CO 2 fixing enzymes (PEPCase, RuBPCase) are present, nor are there functional stomata and hence little, if any, CO 2 diffusion. Miranda et al. (1981) have shown that Kranz anatomy and functional stomata first appear at the same position in the second leaf of maize, 3-5 cm from the node below the leaf. This is the same position at which we first measure the appearance of the carbon-fixing enzymes and P P D K . That all three enzymes appear only in tissue that has differentiated indicates that cellular differentiation may be a requirement for the accumulation of these enzymes. Dean and Leech (1982) and Viro and Kloppstech (1980) have reported in wheat and barley, respectively, that RuBPCase appears in the basal section of leaves at relatively high levels and increases as the cells mature. This is not what we observe in maize and the difference may be related to the difference in the photosynthetic schemes of the plants. Both barley and wheat are C 3 plants which do not utilize differentiated mesophyll and bundle-sheath cells for photosynthesis. Maize, which possesses differentiated cell types used in the C4 carbon-reduction pathway, might require the differentiation of cell types before the carbon fixing enzymes in this pathway can accumulate (e.g. PEPCase, RuBPCase, PPDK). It has been postulated that prior to the appearance of Kranz anatomy, a C 3 pathway might be used in maize (Waygood et al. 1976). However, we find no evidence for this, but find that both the C 3 enzyme RuBPCase and the C 4 enzyme PEPCase appear in the same leaf sections and increase in concert with one another. Perchorowicz and Gibbs (1980) and Williams and Kennedy (1978) have reported that the base of the maize leaf contains both RuBPCase and PEPCase. This disagreement with our results may be because both Perchorowicz and Gibbs, and Williams and Kennedy used larger sections (2.5 cm and 4 cm, respectively) as compared with ours (1 cm). We first detect the two carboxylases in the third cm of the leaf, and this may be the source of the enzymes measured by the other groups.
486
S.P. Mayfield and W.C. Taylor: Photosynthetic proteins in developing maize leaves
Antonielli et al. (1981) reported that the activity ratio of PEPCase to RuBPCase increases (fourfold) from the sheath to the blade in mature maize plants, while Williams and Kennedy (1978) observed a slightly lower PEPCase/RuBPCase activity ratio in the base (sheath) of a maize seedling as compared with the blade. We directly measured the amount of PEPCase and RuBPCase as protein, not as an enzymatic activity, and find the two enzymes absent in the base, and approximately equal to one another in concentration at all other points in the leaf. Although the presence of an enzyme does not assure an enzymatic activity, the absence of the enzyme does preclude it, and thus it is unlikely there is carbon fixation in the basal sections. The regulation of the appearance of photosynthetic proteins is a complex process which involves protein synthesis in both undifferentiated and differentiated cells. The proteins responsible for carbon fixation appear in an order indicating that cellular differentiation plays a role in their accumulation. This is not the case with the light-harvesting and ATP-generating proteins in maize nor does it seem to be a requirement in grasses that do not use C 4 photosynthesis. We thank Richard Malkin for Fe-NADP and plastocyanin antisera, Kazuko Aoyogi for PPDK antisera, and Pioneer Hi-Bred for B73 seed. We also thank Timothy Nelson for a critical reading of the manuscript. This research was supported by grants from the National Science Foundation (PCM 78-26789) and the Competitive Research Grants Office of the U.S. Department of Agriculture (82-CRCR-I-1083).
References Antonielli, M., Lupattelli, M., Venanzi, G. (1981) Some characteristics of the chlorophyllous parenchyma of maize outside the leaf lamina. Plant Sci. Lett. 21, 107-119 Arnon, D. (1949) Copper enzymes in isolated chloroplast: polyphenol-oxidase in Beta vuIgaris. Plant Physiol. 24, 1-15 Baker, N.R., Leech, R.M. (1977) Development of photosystem I and photosystem II activites in leaves of light grown maize (Zea mays). Plant Physiol. 60, 640-644 Bj6rkman, O., Troughton, J., Nobs, M. (1974) Photosynthesis in relation to leaf structure. In : Basic mechanisms in plant
morphogenesis. Brookhaven Symp. in Biology, vol. XXV, pp. 206-217. U.S. Department of Commerce, Springfield, Va., USA Boffey, S.A., Ellis, R.J., Sellden, G., Leech, R.M. (1979) Chloroplast division and DNA synthesis in light-grown wheat. Plant Physiol. 64, 502-505 Chua, N.-H. (1980) Electrophoretic analysis of chloroplast proteins. Methods Enzymol. 69, 434-446 Dean, C., Leech, R.M. (1982) Genome expression during normal leaf development. Plant Physiol. 69, 904-910 Edwards, G.E., Huber, S.C. (1981) The C 4 pathway. In: The biochemistry of plants, vol. 8, pp. 237-281, Hatch, M.D., Boardman, N.C., eds. Academic Press, New York London Ellis, R.J., Jellings, A.J., Leech, R.M. (1983) Nuclear DNA content and the control of chloroplast replication in wheat leaves. Planta 157, 376-380 Harpster, Mayfield, S.P., Taylor, W.C. (1984) Effects of pigment-deficient mutants on the accumulation of photosynthetic proteins in maize. Plant Molec. Biol. (in press) Leech, R.M., Rumsby, M.G., Thomson, W.W. (1973) Plastid differentiation, acyllipid, and fatty acid changes in developing green maize leaves. Plant Physiol. 52, 240-245 Leese, R.M., Leech, R.M. (1976) Sequential changes in the lipids of developing proplast Idy Pisolated from greening maize leaves. Plant Physiol. 57, 78%794 Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the folin reagent. J. Biol. Chem. 193, 262-275 Miranda, V., Baker, N.R., Long, S.P. (1981) Anatomical variation along the length of the Zea mays leaf in relation to photosynthesis. New Phytot. 88, 595-605 Nyari, L.T., Tan, Y.H., Ehrlich, H.A. (1981) Production and characterization of monoclonal antibodies to human fibroblast (beta) interferon. In: The biology of the interferons, pp. 67-73, DeMaeyer, E., Galasso, G., Schellekenes, H., eds. Elsevier North Holland, New York Perchorowicz, J.T., Gibbs, M. (1980) Carbon dioxide fixation and related properties in sections of the developing green maize leaf. Plant Physiol. 65, 802-809 Sharman, B.C. (1942) Developmental anatomy of the shoot of Zea mays L. Ann. Bot. (London) 6, 245-282 Viro, M., Kloppstech, K. (1980) Differential expression of the genes for ribulose-l,5-bisphosphate carboxylase and lightharvesting chlorophyll a/b protein in the developing barley leaf. Planta 150, 41-45 Waygood, E.R., Law, G. (1976)CO 2 fixation in leaves and chloroplasts of maize. Plant Physiol. 57, S-15 Williams, L.E., Kennedy, R.A. (1978) Photosynthetic carbon metabolism during leaf ontogeny in Zea mays L. : enzyme studies. Planta 142, 269-274 Received I0 September; accepted 4 November 1983
