David Ross grant to C. Y. T. Journal Paper No. 6375 of the Purdue. University Agricultural Experiment Station. 2 Present address: Department of Vegetable ...
Plant Physiol. (1977) 59, 733-737
Storage Protein Synthesis in Maize III. DEVELOPMENTAL CHANGES IN MEMBRANE-BOUND POLYRIBOSOME COMPOSITION AND IN VITRO PROTEIN SYNTHESIS OF NORMAL AND OPAQUE-2 MAIZE' Received for publication August 5, 1976 and in revised form November 16, 1976
RICHARD A. JONES,2 BRIAN A. LARKINS, AND C. Y. TSAI Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907 ABSTRACT Zein synthesis accompanied an increase in large poyribosomes of maize (Zea mays) endosperm cells. The two classes of polyribosomes (free and membrane-bound) had dissimilar size class distributions. Membrane-bound polyribosomes were predominantly rge size classes, which were not found in free polyribosomes. The ratio of lare membrane-bound polysomes to total membrane-bound polysomes was highest when zein was being synthesized. Appearance of the large polysomes correlated with the onset of zein accumulation in vivo. These large size casses were nearly absent in the opaque-2 mutant at all stages of endosperm development. Similaly, rRNA content was reduced in the mutant from that in normal endosperm development. These differences
were associated with reduced in vitro synthesis and in vivo accumulation of zein.
Developing maize endosperm is highly specialized for the synthesis of storage proteins, principally zein. Unlike storage proteins of legumes which are found in extracts of all organs of the seed (11), the two major zein proteins are found only in endosperm tissue primarily below the subaluerone layers (7). Furthermore, zein is localized mainly in cytoplasmic organelles termed protein bodies (14). Zein accumulation commences about 16 days after pollination and is approximately 50% complete by 28 days (13). This protein is characterized by its solubility in 70% ethanol and its distinct amino acid composition. Genetic probes with specific effects on the accumulation of zein are also available (9). Reduction in zein content in these mutants has been implicated as their primary role in improving the nutritional quality of maize seed (12). While a dramatic reduction in protein body size has been noted in the opaque-2 mutant (14), the mechanism regulating zein biosynthesis has not been characterized. In this communication, we report the relationship between polysome composition and ability to synthesize zein in vitro during seed maturation of normal and opaque-2 maize.
stages of kernel development and frozen in liquid N2 immediately after harvesting in the field. Polyribosome Isolation. Methods for preparing maize polyribosomes have been described (8). Polysome pellets were suspended in buffer B and analyzed by sucrose density gradient centrifugation (8). Gradients were scanned at 254 nm with an ISCO model UA-5 absorbance monitor. Areas under replicate polysome profiles were determined from the average of three measurements of two different curves with a planimeter. In Vitro Protein Synthesis. Protein synthesis was conducted by incubating maize polysomes in an in vitro system derived from wheat germ (General Mills). The S-1003 supernatant fraction was prepared according to the procedure of Marcus (10). Transfer RNA was isolated from maize kernels 22 days after pollination by the procedure of Brunngraber (1). The incubation mixture contained in a final volume of 0.28 ml: 35 mm tris-acetate (pH 8); 1 mm ATP; 35 AM GTP; 11 mm creatine phosphate; 16 mg creatine phosphate kinase; 4 mm Mg-acetate; 48 mm KCI; 3 mm dithiolthreitol; 45 JM each of 19 amino acids; 20 Ag tRNA; 0.125 AuCi of '4C-leucine (364 mCi/,umol, Schwartz Bioresearch Inc.); 0.12 ml wheat germ S-100; and 1 to 2 A260 units of polysomes. Reaction mixtures were incubated at 30 C for 15 min. Hot 5% trichloroacetic acid-insoluble protein was collected on Whatman GF1A filters, washed with 20 nl cold 5% trichloroacetic acid, dried under a heat lamp (10), and counted in Omnifluor-toluene at 80% efficiency with a Beckman LS-100 scintillation counter. Ethanol-soluble protein was extracted (2) from the reaction mixtures after addition of absolute ethanol to a final volume of 70% and heating the stoppered tubes for 45 min at 60 C. The ethanolic extract was deposited on filter paper discs (Whatman 3MM) and sequentially immersed for 10 min in 200 volumes of 5 % NaCl, distilled H2O with two changes, and finally washed with anhydrous ethyl ether. The air-dried discs were placed in scintillation vials for counting as described above. The values for incorporation were the averages of three experiments. Determination of rRNA Levels. Methods for preparing maize rRNA have been described (4). Solubilized RNA was analyzed by sucrose density gradient centrifugation. Duplicate gradients were scanned at 254 nm and the amount of ribosomal RNA was
determined by planimetry. MATERIALS AND METHODS Plant Material. Ears from a maize (Zea mays) inbred line (W22) and its isogenic opaque-2 version (W2202) were obtained from self-pollinated plants. Ears were taken at appropriate
RESULTS Zein Accumulation during Endosperm Development. Zein was present in detectable amounts approximately 16 days after pollination (Fig. 1). In normal maize, zein was rapidly accumulated in the endosperm shortly after this period. By 28 days after pollination, zein had reached approximately 50% of the level achieved at maturity. Zein content increases up to 40 days after
I This work was supported by a grant from the Lilly Endowment and a David Ross grant to C. Y. T. Journal Paper No. 6375 of the Purdue University Agricultural Experiment Station. 2 Present address: Department of Vegetable Crops, University of California, Davis, Calif. 95616. 3Abbreviation: S-100: post 100,000g supernatant. 733
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Physiol. Vol. 59, 1977
principal size class in normal maize, were absent during all stages of mutant endosperm development (Fig. 4). Analysis of the profiles (Figs. 4 and 5) indicated that large membrane-bound polysomes (i.e. polysomes bearing five or more ribosomes) represented 93% of the total membrane-bound polysomes in normal (Table I), but only 84% of the total polysomes in the mutant. Little change was observed in either of these proportions during development. The proportion of large unique size classes (i.e. containing greater than 10 ribosomes) in
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pollination in the normal line. In contrast, the opaque-2 mutant accumulated little zein 20 days after pollination and had essentially completed zein accumulation by 30 days. The dramatic reduction in total zein by the mutant is therefore due both to temporal and kinetic differences from the normal counterpart. Developmental Changes in rRNA Content. Ribosomal RNA levels of normal maize endosperm increased steadily during development (Fig. 2), and plateaued around 25 days after pollination. This preceded maximum protein content (Fig. 1). Comparable levels of rRNA were initially present in both the normal and mutant on a kernel basis. However, rRNA levels increased more slowly and plateaued several days earlier in the mutant. When ribosomal RNA content was expressed on a gram fresh weight basis, the developmental patterns of normal and mutant were similar (Fig. 2B). Highest concentrations occurred at 12 days after pollination, leveled off at 16 days, and then declined over the next 2 weeks. Ribosomal concentration in the mutant at day 16 was reduced 30% from that of the normal. Isolation of Polysomes from Dissected Endosperms. In previous studies, whole kernels were utilized for polysome isolation (4, 8). Since zein is synthesized only in the endosperm, it was of interest to determine what contribution the embryo made to the polysomes of whole kernels. The embryo contributed minimal amounts of polysomal material (Fig. 3). However, removal of the embryo resulted in the loss of the large size classes from the membrane-bound fraction of the endosperm (cf. Fig. 4). Since the embryo is essentially devoid of polysomes, and its removal leads to the loss of the large polysome size classes in the endosperm, we used intact kernels for the subsequent isolation and analysis of polysome changes during development. Developmental Changes in Membrane-bound Polysomes. Representative profiles of the membrane-bound polysomes at different stages of normal and mutant endosperm development were obtained (Fig. 4). The periods examined represented specific stages of zein accumulation, i.e. A: prior to detectable zein accumulation (12 days after pollination); B: onset of zein accumulation (16 days); C: active zein accumulation (22 days); and D: period of declining zein accumulation (28 days). At 12 days, the large size classes were not as distinct as at later stages of endosperm development in normal maize (Fig. 4). The appearance of the large size classes correlated with the pattern of zein accumulation. These large unique polysomes, which were the
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FIG. 2. Ribosomal RNA content of normal and mutant maize during endosperm development. rRNA extractions were made by homogenizing kernels in GPS buffer and centrifuging the extract for 10 min at 37,000g. The supernatant was layered on linear sucrose gradients (75300 mg/ml) and centrifuged for 4 hr at 45,000 rpm in an SW 50.1 rotor. The area of absorbance monitored at 254 nm is expressed in planimeter units. Total rRNA in normal (A) and opaque-2 (a) expressed on a kernel basis (A) or gram fresh weight basis (B). A
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Physiol. Vol. 59, 1977
ZEIN SYNTHESIS IN MAIZE
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the membrane-bound fraction from normal increased during development (60% at 12 days to 71% at 28 days). These large size classes actively synthesize zein (6). Although the proportion of these large size classes was higher in the mutant at 12 days (64% large polysomes), it decreased during seed development, becoming only 45% at 28 days. Less total A260 absorbing material was recovered in the mutant membrane-bound polysome fraction than in normal. The profiles reflect this decrease. Comparison of in Vitro Protein Synthesis during Development. At comparable stages of kernel development, the membrane-bound polysomes incorporated more '4C-leucine than the free polysomes on an equal A260 unit basis (Table II). "4CLeucine incorporation by the free polysomes was greatest at the early developmental stages, and then declined. At 35 days after pollination, 40% less label was incorporated than at 12 days (9,842 cpm versus 5,848 cpm) on an equal A260 unit basis. Ten per cent of the acid-insoluble protein was ethanol-soluble at any stage of development. The membrane-bound polysomes supported greater '4C-leucine incorporation at 22 days after pollination and then declined (Table II). A major portion of the protein synthesized was similar to native zein (4, 8). The proportion of ethanol-soluble protein to acid-insoluble protein synthesized in vitro increased during kernel development. Zein represented 32% of the acidinsoluble protein at 12 days and increased to 49% by 35 days. The free polysome fraction from opaque-2 also incorporated less "4C-leucine than the membrane-bound fraction (Table III), although a higher proportion of ethanol-soluble (zein) protein was synthesized by free polysomes of opaque-2 than normal. However, the membrane-bound polysomes from opaque-2 synthesized less zein than normal at all stages of development (cf. Tables II and III). In opaque-2, the proportion of zein synthesized in vitro increased during the initial 22 days after pollination and then declined. In normal, this proportion increased steadily during development. At 35 days after pollination, the membrane-bound polysomes from opaque-2 incorporated 989 cpm into zein, while those of normal incorporated 3,627 cpm. The
BOTTOM ) and opaque-2 (- -) maize. Develop-
reduced synthesis of zein was a reflection of the absence of the large size-classes of membrane-bound polysomes in the mutant (Fig. 4). Changes in in Vitro Protein Synthesis during Kernel Development. The pattern of in vitro protein synthesis by free and membrane-bound polysomes was similar during normal and mutant kernel development (Fig. 5). Protein synthesis was high at early stages of development and began to decline by 22 days after pollination. However, in opaque-2, a 40% reduction in protein synthetic activity occurred during early developmental stages in comparison with normal. The low levels of zein synthesized by free polysomes of normal or mutant kernels fluctuated little during seed development. Mutation at the opaque-2 locus markedly reduced the capacity of membrane-bound polysomes to incorporate 14C-leucine into acid-insoluble protein when expressed on a kernel basis (Fig. SB). This was 50% of the incorporation in normal (4,042 cpm versus 8,120 cpm) at 22 days after pollination. Zein synthesis in vitro increased rapidly from 12 days to 22 days after pollination. This early increase in zein synthesis preceded the period of most rapid accumulation of zein in vivo (Fig. 1). SDS-polyacrylamide gel analysis of the zein synthesized in vitro at 12 days indicated that little of the two major zein proteins was synthesized (data not shown). Membrane-bound polysomes from the mutant supported less in vitro zein synthesis than those from normal at early stages of development. Zein synthesis increased in normal kernels from 12 to 22 days after pollination, but in opaque-2 it increased only after 16 days. The reduced synthesis of zein in vitro at early stages of development of opaque-2, when compared to normal, correlated with the reduced and delayed accumulation of this protein in vivo (Fig. 1). DISCUSSION A prerequisite for recovery of undegraded polysomes from stored kernels was immediate freezing of ears in liquid N2 after harvesting (8). Removal of the endosperm, the tissue in which
736
JONES, LARKINS, AND TSAI
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FIG. 5. In vitro protein synthesis by free (A) and membrane-bournd (B) polysomes during development of normal (U and A) and opaque (@ and x) kernels. Protein synthesized was fractionated into aci idinsoluble (U and 0) and ethanol-soluble (A and x). Incorporation is expressed on a kernel basis. Ratio of Large to Total Polysomes of the Membrane-Bound Fraction from Developing Kernels of Normal and Opaque-2 Maize. Developmental l1ormal Opaque-2 Stage Polysomes > S Polysomes > 10 Polysomes > 5 Polysomes > 10 DAY Total P Total P Total P Tbtal P Table I.
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zein is synthesized, resulted in loss of large size classes in normal maize. Polysome profiles from isolated normal endosperms were similar to those obtained from whole kernels of opaque-2 (cf. Figs. 2 and 4C). The lability of the large size classes may be due to increased RNase action caused by tissue dehydration (3), or temperature elevation during dissection, but not during polysome isolation, as the buffer effectively inhibited RNase activity (4, 8). Stored kernels with ruptured seed coats invariably contained degraded polyribosomes, suggesting that dehydration may decrease polyribosome stability during storage. During cereal seed development, rapid synthesis of a limited number of proteins occurs. This facilitates study of storage
Plant Physiol. Vol. 59, 1977
protein synthesis in a cell-free system. During kernel development, zein is synthesized primarily by membrane-bound polysomes. The ability of free polysomes to synthesize small amounts of zein may represent steady-state levels of transported zein mRNA or an artifact of polysome isolation procedure. The developmental pattern of zein synthesis in vitro by normal membrane-bound polysomes correlated with the pattern of zein accumulation in vivo (cf. Figs. 1 and 5). The major discrepancy in the two patterns occurred at 12 days after pollination when more zein was synthesized in vitro than could be detected in vivo. This may simply reflect the difference in sensitivity of the two methods for determining zein. Synthesis of zein at 12 days supports the observation of Khoo and Wolf (7), who detected putative protein bodies at 12 days. It appears that protein body formation is initiated between 12 and 16 days after pollination. Zein synthesis with opaque-2 membrane-bound polysomes was reduced considerably from that in normal (cf. Tables II and III). The reduction was 75% at 16 days and 65% at 22 days after pollination. The developmental pattern of zein synthesized in vitro correlated with zein accumulated in vivo. It is apparent that the mutant not only reduced and delayed the synthesis of this protein, but also ceased zein synthesis much earlier than in normal maize. This decreased "availability" of zein mRNA in the mutant may reflect reduced levels of the message or its altered translation. To determine whether transcriptional or translational controls are affected by the mutant, it is necessary to isolate and purify the mRNA quantitatively and study its properties in vitro. Analysis of the polysome profiles from normal maize indicated that the proportion of large size classes in the membrane-bound fraction increased during kernel development (Fig. 4 and Table I). These size classes synthesize zein (6), and do not appear to be caused by aggregation (8). Large polysomes are associated with the endoplasmic reticulum which are forming protein bodies (5). Presence of the large unique size classes during development led to an increased zein synthetic activity. It is estimated from the depth in the gradient that the large polysome size class bears 22 ribosomes. A messenger RNA of this length would be sufficient to code for a protein of 180,000 daltons. Considering the small mol wt (about 20,000 daltons) of the zein proteins, and the polysome size, the mRNA may be redundant or code for both proteins. This could account for the rapid synthesis of zein (approximately 375 ,ug/day) over a short period. Incorporation of C-Leucine by Free and Membrane-Bound Polysoms from Developing Kernels of Normal Maize. (Values are the means of four values obtained in three experiments.) EthanolTotal AcidEthanol-Soluble Polysome Developmental Insoluble Acid-Insoluble Stage Soluble Type Ratio Days _m/A260/hr cem/260/h!r 12 Free 9,842 736 0.07 16 823 9,412 0.08 22 808 0.09 8,766 28 818 6,888 0.12 35 434 5,848 0.07 12 Membrane14,586 4,656 0.32 Bound 16 15,338 5,239 0.34 22 15,952 6,894 0.43 28 13,730 6,047 0.44 35 0.49 7,441 3,627 Table II.
Incorporation of 14C-Leucine by Free and Membrane-Bound Polysomes from Developing Kernels of 0paqu-2 Maize. Values are the means of four values obtained in three experiments. Total AcidEthanolPolysome Developmental Ethanol-Soluble Insoluble Soluble Acid-Insoluble Type Stage Ratio Days c_sVA260/hr cpm/A260/hr 12 Free 5,639 668 0.11 16 705 5,086 0.14 22 5,216 1,029 0.20 28 744 5,207 0.14 35 312 4,802 0.06 Membrane12 Bound 14,246 2,570 0.18 16 8,463 2,208 0.26 22 11,922 4,101 0.34 28 12,900 2,898 0.22 35 989 9,460 0.10 Table III.
Plant Physiol. Vol. 59, 1977
ZEIN SYNTHESIS IN MAIZE
The near absence of the large membrane-bound polysome size classes during development in the mutant correlated with reduced synthesis of zein in vitro (Fig. 4 and Table II). The absence of these size classes is not due to RNase action during polysome isolation (4, 6). Furthermore, the large size classes are absent in normal when zein is not being synthesized, i.e. in embryos and in endosperms during germination (unpublished observation). The rRNA content of maize endosperm was also affected by the opaque-2 mutation and was reflected in a reduced rate of zein accumulation. In early stages of normal and mutant endosperm development, the rRNA increase paralleled that of zein. At 22 days in the mutant, rRNA levels were reduced 30% from that present in normal and began to decline several days prior to the decline in rRNA levels in normal maize. Therefore, part of the reduced synthetic capacity in the opaque-2 mutant is reflected in a reduced level of ribosomal material. LITERATURE CITED 1. BIUNNGRABEER EF 1962 A simplified procedure for the preparation of soluble RNA from rat liver. Biochem Biophys Res Commun 8: 1-3 2. DALBY A 1974 Rapid method for determination of zein content of whole maize seed or
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isolated endosperms. Cereal Chem 51: 586-592 3. Hsiso TC 1970 Rapid changes in levels of polyribosomes in Zea mays in response to water stress. Plant Physiol 46: 281-285 4. JONES, RA, BA Lurzs, CY TsAM 1976 Reduced synthesis of zein in vitro by a high lysine mutant of maize. Biochem Biophys Res Commun 69: 404-410 5. JONES RA, BA LAmiNs, CY TSAI 1976 Regulation of zein synthesis in maize endosperm. Plant Physiol 57: 5-36 6. JONES RA, BA LIUNS, CY TsAi 1977 Storage protein synthesis in maize. II. Reduced synthesis of a major zein component by the opaque-2 mutant of maize. Plant Physiol 59: 525-529 7. KHoo V, MJ WOLF 1970 Origin and development of protein granules in maize endosperm. Am J Bot 57: 1042-1050 8. LARK,s BA, CE BRAcKErt, CY Tsm 1976 Storage protein synthesis in maize. Isolation of zein-synthesizing polysomes. Plant Physiol 57: 740-745 9. LEE KH, RA JONES, A DALBY, CY TsM 1976 Genetic regulation of storage protein content in maize endosperm. Biochem Genet 14: 641-650 10. MAucus A 1974 The wheat embryo cell-free system. Methods Enzymol 12: 749-761 11. MILLERD A 1975 Biochemistry of legume seed proteins. In Annual Reviews of Plant Physiol Vol 26. Annual Reviews, Palo Alto pp 53-72 12. NELSON OE 1969 Genetic modification of protein quality in plants. In Advances in Agronomy Vol 21. Academic Press, New York p 171 13. TsAi CY, A DALBY 1974 Comparison of the effect of shrunken-4, opaque-2, opaque-7, and floury-2 genes on the zein content of maize during endosperm development. Cereal Chem 51: 825-829 14. WOLF MJ, V KHOO, HL SECKnGER 1967 Sub-cellular structure of endosperm proteins in high-lysine and normal corn. Science 157: 556-557
