Glutamine Metabolism in Corn Kernels Cultured In Vitro - NCBI

Plant Physiol. (1985) 77, 520-523 0032-0889/85/77/0520/04/$0 1.00/0

Glutamine Metabolism in Corn Kernels Cultured In Vitro' Received for publication July 11, 1984 and in revised form November 30, 1984

SANTOSH MISRA2 AND ANN OAKS* Biology Department, McMaster University, Hamilton, Ontario, Canada L8S 4K1 ABSTRACI The fate of glutamine, the major source of nitrogen supplied to the developing maize endosperm, has been examined in endosperm tissues of corn caryopsis grown under sterile conditions. In the culture system, [U4"Cglutamine was included in the medium or was injected directly into the endosperm. Samples were harvested at intervals up to 168 hours. Protein and starch fractions were then separated and analyzed for their '4C content. At 168 hours, 31% of the total label incorporated in the endosperm was in zein, 15% in glutelin, and 24% in starch. When individual amino acids and sugars in the endosperm powders were analyzed, the 14C still remaining in the glutamine accounted for only 12 to 14% of the total radioactivity.

Developing cereal kernels depend upon vegetative tissues for carbohydrate, nitrogen, and other nutrients (8). In corn, glutamine is a major form of reduced nitrogen either transported to the developing seed (1) or recovered from endosperm tissue (1 1, 12). It is also the major amino acid in the storage protein zein (17). The concentration of free glutamine in the endosperm decreases at a time when zein levels are increasing (12). The major fate of imported glutamine could, therefore, be its direct incorporation as glutamine into endosperm proteins. Because callus cultures derived from maize endosperm tissue will grow with glutamine, asparagine, or NH4' as the only nitrogen source (4, 15, 20), it is clear that this tissue has the capacity to both catabolize and synthesize glutamine. Also, the levels of glutamate synthase, an enzyme involved in the degradation of glutamine, reach relatively high levels in maize endosperm tissue at a time when total endosperm nitrogen is increasing (14, 18). In this paper, we have examined the fate of ['4C]glutamine in the developing endosperm of corn. A caryopses culture initially developed by Gengenbach (5) and later modified by Shimamoto and Nelson (16) was employed. [14C]Glutamine was included either in the media or was injected directly into the endosperm of developing caryopses. Our results show that glutamine is extensively metabolized in the endosperm tissue. The metabolic products of glutamine contribute both to the synthesis of amino acids in storage proteins and to the synthesis of endosperm carbohydrates. MATERIALS AND METHODS Samples of Zea mays (hybrid var W64A x W182E) were purchased from the Wisconsin Seed Foundation, Madison, WI. Corn was grown in field plots in the Royal Botanical Gardens in ' Supported by research grants from National Sciences and Engineering Research Council-Canada (A28 18). 2 Present address: Biology Department, University of Calgary, Calgary, Alberta T2N lN4 Canada.

Hamilton, and was hand pollinated when the silks first appeared. It was also grown under growth chamber conditions as described previously (12). Caryopses Culture. Ears of maize were harvested 5 to 7 d after pollination and were cut into blocks containing 10 caryopses per block as described by Gengenbach (5) and Shimamoto and Nelson (16). The blocks were surface sterilized with 1% NaOCI for 1 min and then rinsed thoroughly with sterile distilled H20. Five of these blocks were transferred to a medium containing [U- 4C]glutamine (5 uCi/20 ml; 40 mCi/mmol). Alternatively, [U-14C]glutamine was injected directly into the endosperm (1 Al or 0.05 uCi/endosperm) 10 d after the initiation of the culture. The caryopses were incubated for specific intervals up to 168 h at 28C. At the end of the experiment, the blocks were harvested. Endosperm, embryo, and cob tissues were separated, frozen in liquid nitrogen, and stored at -20C until further analysis. These samples were then lyophilized for 2 d, and the dry weight was recorded. The tissue was then ground to a fine powder in a Prolabo (Dangomau) ball mill. The total radioactivity in each sample was assayed according to the method of Shimamoto and Nelson (16). The agar medium was eluted overnight with 50 ml of water at 4°C. This extract was used for estimating the radioactivity left in the medium at the end of the experiment. Fractionation of Endosperm Reserves. A modified method of Sodek and Wilson (19) was employed to progressively extract the various endosperm fractions. Two replicate samples of 50 to 100 mg each were extracted in the following manner: extraction in 0.1 M NaCl for 1 h at 4°C was followed by centrifugation for 10 min at top speed (2575g) in a clinical centrifuge. The pellet was washed three times with water and the washings were combined with the original supernatant solution. This constituted the combined water-soluble and salt-soluble fraction, i.e. albumins, globulins, free amino acids, organic acids, and sugars. This combined fraction was lyophilized and then resuspended in 70% ethanol (v/v), which dissolved the amino acids, sugars, and organic acids. The remaining albumin and globulin proteins were rinsed 3 times in 70% ethanol, and separated by centrifugation. The residue remaining after the extraction with 0.1 M NaCl was treated with 0.1 N HCI for 1 h at 100°C in order to hydrolyze the starch. After centrifugation, the pellet was washed three times with water and the washings were combined with the supernatant, and was used to assay starch. The residue was then shaken with 70% ethanol containing 1 mm 2-mercaptoethanol for 1 h at 60°C and was then centrifuged at 12,000g for 5 min to recover the supernatant. The pellet was washed twice with ethanol and the washings were combined with the ethanol supernatant solution (the zein fraction). Finally, the glutelins were extracted by incubating the pellet with 0.1 N NaOH for 30 min at 40°C. The supernatant was recovered by centrifugation. The pellet remaining after extraction of the major protein fractions was digested in 6 N HCI at 1 15C and 15 lbs pressure for 12 h. To determine radioactivity in each fraction, 50-Ad aliquots were mixed with 10 ml of scintillation cocktail. Hydrolysis of Endosperm Proteins. To recover glutamine in the protein digests, a modified method of Winkler and Schon

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GLUTAMINE METABOLISM IN CORN KERNELS

(22) was used for the enzymic hydrolysis of proteins. Fifty to one hundred mg of endosperm powder was pretreated for 1 h at 40°C with 0.1 N NaOH (1.8 ml) containing 2% (w/v) SDS. The pH of the pre-mix was adjusted to 8.3 using 2 N HCI and 2.0 ml of sodium-borate (0.1 M) buffer (pH 8.3) was added. To this buffered pre-mix, 20 mg of dialyzed pronase and 2.5 mg of carboxypeptidase A were added. Ethanol at a final concentration of 10%, required to dissolve the zein efficiently, and 20 Al of chloroform were also added. The mixture was then incubated at 4O°C for 48 h in a shaking water bath. The samples were then spun for 10 min at 2575g. The supernatant and the residue were stored separately at -20°C. Amino Acid Analysis. The soluble fraction containing amino acids, sugars, and organic acids was first passed over a Dowex50 x 8 (200-400 mesh) H+ resin (1 x 5 cm). After washing the column with 50 ml of water to remove sugars, the amino acids were eluted with 50 ml of 2 N NH40H. Each fraction was taken to dryness under vacuum at 40°C and then made up to a known volume. Orthophthaldialdehyde derivatives of glutamine, glutamate, asparagine, and aspartate were separated by reversed phase chromatography in an HPLC system (Beckman, model 334) as described by Winspear and Oaks (23). Other amino acids were collected in bulk. Each of these fractions was taken to dryness in scintillation vials and after addition of 10 ml of scintillation fluid (toluene-Triton X- 114 [1:1, v/v] containing 0.2% Omniflor), was counted in a liquid scintillation counter (Beckman, model LS250).

RESULTS Increase in Dry Weight of Tissue with Time of Incubation. Table I shows the increase in dry weight of endosperm, embryo, and cob tissue during the 7-d incubation period. The average weight of endosperm increased from 26 ± 2.4 mg at day 1 to 45 ± 3.6 mg at day 7. The average weight of embryo also increased from 0.80 ± 0.13 mg to 2.8 ± 0.25 mg, whereas the dry weight of cob tissue remained fairly constant (175 ± 23 to 187 ± 15 mg). The dry weight increase observed in endosperm tissue was comparable to that observed in kernels developing on the cob under growth chamber conditions. Cully et al. have also found an almost normal growth response in caryopsis cultures (3). Total Uptake of lU-'4CiGlutamine. The total uptake of [U'4C]glutamine increased for 96 h and then remained constant (Fig. 1). There was a parallel decrease in the "C content in the medium. Radioactivity in endosperm and embryo showed a continuous increase over a period of 168 h, whereas it remained fairly constant in the cob tissue after the initial 2 d (Fig. 2). Most of the radioactivity was in the water-soluble fraction of the endosperm at 15 h. With time, relatively moye of the endosperm radioactivity appeared in the zein fraction (Fig. 3). Incorporation into this fraction was almost linear from 24 to 168 h. At 168 h, about 37% of the total label was in the zein fraction, 15% in

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FIG. 1. Samples were harvested 5 to 7 d after pollination. Incubation on nutrient media containing [U-'4C]glutamine (0.25 uCi/ml; 20 ml) was performed as described in "Materials and Methods." Seven caryopses were harvested at each sampling time and the mean values of five replicates are shown at each point. The cpm were recorded for the powder prepared from seven caryopsis (- *) or for the total media (O-O). For the media, the scale was reduced by one-half.

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FIG. 2. Kinetics of uptake of [U-'4C]glutamine by cob, endosperm, and embryo tissue. Sample treatment is as described in Figure 1. (@-.4), Cob tissue; (0-O), endosperm; (O-O), embryo.

glutelin, 24% in starch, and 19% remained in the free amino acid fraction (Fig. 3). About 30% of the total radioactivity in zein was in the acidic amino acids and their amides and, of this, 10% was in glutamine (Table III). In the total endosperm powder, glutamine also accounted for about 1 1% of the total radioactivity, whereas in the cob tissue it accounted for 35% of the total Table I. Changes in Dry Weight ofEndosperm, Embryo, and Cob radioactivity. Tissue during a 7-Day Growth Period Analysis of Cob Tissue. Since all the label entering the kernel Endosperm, embryo, and cob tissue were separated, frozen in liquid must pass through the cob, this tissue was also examined for and as described in "Materials and Methods."

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24 26±2.4 0.80±0.13 175±23 27 ± 2.7 147 ± 33 48 0.90 ± 0.07 96 33 ± 3.0 1.90 ± 0.78 197 ± 34 45 ± 3.6 2.80 ± 0.25 168 187 ± 15 were 'Seven endosperms used to calculate the dry weights and three to five replicate samples were assayed at each point.

incorporation of 14C. Results in Table II show that glutamine was converted to sugars and organic acids in cob tissue. These two fractions accounted for 39% of the total radioactivity recovered. Glutamine was the major amino acid in the amino acid fraction (Table III). Metabolic products of glutamine produced in the cob tissue could be translocated to the endosperm and hence could confound interpretations ofendosperm metabolism. To rule out this possibility, glutamine was injected directly into the endosperm. Fate of '4C Injected into the Endosperm. An analysis of the various endosperm fractions obtained 168 h after injection of

MISRA AND OAKS

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FIG. 3. Kinetics of incorporation of [U-'4C]glutamine into endo), Amino acids; (x-x), zein; (O-O), sperm components. ( starch; (E-A), glutelin; (-.), albumins and globulins; ( -), organic acids and sugars.

Table II. Distribution of[U-'4CJGlutamine in Tissue Extracts from Cultured Caryopses Fifty mg of freeze dried powder was used for extraction of starch, proteins, and water-soluble components. Extraction of various components is described in "Materials and Methods." Radioactivity is expressed in cpm/50 mg endosperm or cob tissue powder. The experiment was terminated after 168 h with [U-_4C]glutamine. Cob Fraction Endosperm cpm x 10-3 % cpm x 10-3 % 39.6 62.2 20.0 35.0 Starch 37.0 33.0 Zein 8.5 7.5 Glutelin 8.8 7.8 1.1 0.3 Albumin + globulin 10.5 33.2 11.8 103.2 Amino acids 4.9 4.4 38.7 120.2 Organic acids + sugars 2.2 2.0 Residue 112.8 310.2 Total recovered 87.0 94.0 % Recovery

glutamine into the endosperm confirmed that glutamine was not only used for the synthesis of amino acids in the endosperm but also that a large fraction was converted into carbohydrates (Table IV). The distribution of radioactivity in the various fractions after 168 h of incubation was 21% in starch, 38% in zein, 8% in glutelin, and 13% in free amino acid fraction. In zein hydrolysates, glutamine accounted for 14% of the total label (data not shown). This distribution pattern is similar to the pattern obtained when ['4C]glutamine was supplied through the nutrient media. In both experiments, label in glutamine in the endosperm tissue accounted for only about 12 to 14% of the total label incorporated into this tissue. DISCUSSION When ['4C]glutamine was supplied to wheat stems, it contributed significantly to the synthesis of starch (48% of total radioactivity in meal) and gluten (34%), the major storage protein (7, 9). In gluten, 50% of the 14C was recovered in glutamine and glutamate. However, radioactivity was also found in proline, arginine, aspartic acid, threonine, glycine, serine, and alanine.

Plant Physiol. Vol. 77, 1985

Table III. Distribution of the '4C in the Amino Acids of the Free Amino Acid and Zein Hydrolysate Fractions Caryopsis were grown for 168 h after the addition of [U-'4C]glutamine to the medium. Amino acids were eluted from the Dowex-50 (HI) column with 2 N NH40H, and were redissolved in 30% methanol. The samples were then applied to an HPLC reverse phase column as described in "Materials and Methods." Values in parentheses represent values relative to the total recovered radioactivity. Zein was hydrolyzed using commercial pronase and carboxypeptidase A preparation, and the resultant free amino acids were treated with 30% methanol as described in "Materials and Methods." Zein Free Amino Acids Hydrolysate Amino Acids Endo(EndoCob sperm) sperm cpm x 10-2 388.8 (35) Glutamine 13.0 (11) 27.8 (10) 132.0 17.6 17.8 Glutamate 147.2 13.9 19.6 Aspartate 163.2 22.7 7.8 Asparagine Neutral and basic 57.2 amino acids 200.3 180.0 268.0 110.9 Total recovered 1,031.5 94 92 93 Recovery (%) Table IV. Distribution ofRadioactivity in Endosperm of Cultured Maize Caryopsis Label was supplied in the media as [U-'4C]glutamine (0.25 'UCi/ml media; 20 mls) or by injection of [U-"C]glutamine (0.05 MCi) into the endosperm. In each case, the kernels were analyzed 168 h after the ['4C] glutamine was supplied. '4C Injected '4C Supplied to MeFraction dium into Endosperm cpm x 10-3/ % of cpm x 10-3/ % of 100 mg total 100 mg total 74.2 24.7 20.0 Starch 21.3 57.8 Amino acids 19.3 12.7 13.0 Sugars + or24.6 8.0 6.6 7.1 ganic acids Albumin + 17.9 5.9 7.6 8.1 globulin 91.7 30.7 Zein 37.6 40.2 45.0 Glutelin 15.0 7.3 8.0 1.0 0.3 0.6 Residue 0.6 93 Total 300

Harvey also found a considerable conversion of acetate or proline carbon into both starch and storage proteins in developing maize kernels (6). The distribution of "'C in protein amino acids was the same with either precursor. In wheat also ["'C]proline fed to developing spikes was metabolized to glutamate and contributed significantly to the synthesis of both starch and gluten (9). Thus, it appears that glutamine and proline could support a de novo synthesis of amino acids in cereals and could also contribute carbon to the synthesis of starch. However, since the protein precursors used in these studies were supplied via the stem and since incubations were carried out over several weeks nothing can be said concerning either the rates of conversion or the sites of conversion. Sodek injected "1C precursors (aspartic acid, alanine, acetate) into developing endosperms of maize (17). In each case, all the protein amino acids became labeled. Label was also found in sugars and organic acids. Thus, he showed that endosperm tissue had the capacity to synthesize a broad spectrum of

amino acids. The relative efficiency of movement of amino acids from the

GLUTAMINE METABOLISM IN CORN KERNELS maternal tissue to the endosperm is much lower for amino acids than for sugars (16). Studies done with proline, leucine, and phenylalanine showed that each amino acid was metabolized in the cob tissue. When [ 4C]proline was fed to caryopses, only 18% of the "'C was recovered as proline from the cob tissue, while sugars and various other amino acids accounted for 60% of total label. In endosperm extracts, 30% of the total label was recovered in proline, suggesting a selective transport of proline to the endosperm tissue. Not all amino acids are metabolized since Cully et al. (3) found no evidence of metabolism of [35S]methionine in their caryopsis cultures. In the present study, we found that ["'C]glutamine supplied in the medium was extensively metabolized in the cob tissue. Sugars, organic acids, and amino acids accounted for 71% of the incorporated radioactivity. When ["'C]glutamine was injected directly into the immature endosperm, 21% of the "1C was found in starch, 38% in zein, 8% in glutelin, and 13% in the free amino acid fraction. In zein hydrolysates, glutamine accounted for 10% of the total radioactivity. The distribution was essentially similar with both types of "'C administration. Thus, glutamine is extensively metabolized in the endosperm tissue. Glutamine, whether supplied by the transport system or synthesized in the endosperm tissue, could be converted to glutamate via glutamate synthase (14, 18) and finally to a-ketoglutarate by the action of glutamate dehydrogenase or a transaminase. As with a few other specialized tissues, for example, castor bean endosperm (2) or maize scutellum (13), the cereal endosperm is able -to convert organic acids to sugars. This conversion can account for up to 30% of the glutamine carbon supplied (Table II). A similar conversion is seen when proline or acetate are the "4C precursors (6, 9, 17). Our results show that glutamine carbon is almost equally partitioned to zein and carbohydrates (Table II). A similar partitioning is observed under field conditions. For example, when zein synthesis is limited either genetically or by low nitrogen nutrition, the synthesis of starch is also inhibited (10, 21). Thus, caryopsis culture permits normal endosperm development, normal synthesis of storage proteins, and normal partitioning of carbon into starch and protein in the kernels (21). Since the composition of the media can be altered with ease, caryopsis cultures are of potential use in studying the effect of various nutrients or hormones on the growth and metabolism of the developing kernel.

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LITERATURE ClTE;D 1. ARRUDA P, WT DASILVA 1979 Amino acid composition of vascular sap of maize ear peduncle. Phytochemistry 18: 409-410 2. BEEVERS H 1981 Role of glyoxylate cycle. In Abraham Marcus, ed, The Biochemistry of Plants, A Comprehensive Treatise. Academic Press, New York, pp 117-129 3. CULLY DE, BG GENGENBACH, TA SMITH, I RUBENSTEIN, TA CONNELLY, WD PARK 1984 Endosperm protein synthesis and L-[35S]methionine incorporation in maize kernels cultured in vitro. Plant Physiol 74: 389-394 4. FARRAR KR, LS GANUGAPATI 1970 Growth of maize endosperm tissue in vitro. J Sci Food Agric 21: 329-333 5. GENGENBACH BG 1977 Development of maize caryopses resulting from in vitro pollination. Planta 134: 91-93 6. HARVEY BMR 1973 Hydrolysis of endosperm proteins in germinating maize. PhD thesis. McMaster University, Hamilton, Ontario, pp 1-200 7. KOLDERUP F 1979 Interconversion of amino acids in maturing wheat grain. In FAO/IAEA Symposium on Seed Protein Improvement in Cereals and Legumes, Vol I. International Atomic Energy Agency, Vienna, pp 187-202 8. LESAR EL, DM PETERSON 1981 Growth and composition of kernels developing on excised oat panicles in liquid culture. Crop Sci 21: 742-747 9. MCCONNELL WB 1969 Studies of wheat plants using '4C labelled compounds. XXII. Incorporation into wheat proteins. Can J Biochem 47: 19-23 10. MERTZ ET 1976 Case histories of existing models. In Genetic Improvement of Seed Proteins. National Academy of Science, Washington, DC, pp 57-70 1 1. MISRA S 1983 Glutamine and asparagine metabolism in developing endosperm of corn. PhD thesis. McMaster University, Hamilton, Ontario, pp 1-216 12. MISRA S, A OAKS 1981 Enzymes of nitrogen assimilation during seed development in normal and high lysine mutants in maize Zea mays (W64A). Can J Bot 59: 2725-2743 13. OAKs A, H BEEVERS 1964 The glyoxylate cycle in maize scutellum. Plant

Physiol 39: 431-434 14. OAKs A, K JONES, S MISRA 1979 A comparison of glutamate synthase obtained from maize endosperm and roots. Plant Physiol 63: 793-795 15. SHANNON JC, JW LIN 1977 A simplified medium for the growth of maize (Zea mays L) endosperm tissue in suspension culture. Physiol Plant 40: 285291 16. SHIMAMOTo KO, OE NELSON 1981 Movement of '4C-compounds from maternal tissue into maize seeds grown in vitro. Plant Physiol 67: 429-432 17. SODEK L 1976 Biosynthesis of lysine and other amino acids in the developing maize endosperm. Phytochemistry 15: 1903-1905 18. SODEK L, WV DASILVA 1977 Glutamate synthase: A possible role in nitrogen metabolism of the developing endosperm. Plant Physiol 60: 602-605 19. SODEK L, CM WILSON 1971 Amino acid composition ofproteins isolated from normal, opaque-2 and floury-2 corn endosperm by a modified Osborne procedure. J Agric Food Chem 19: 1144-1150 20. STRAUS J 1960 Maize endosperm tissue culture in vitro. III. Development of a synthetic medium. Am J Bot 47: 641-647 21. TSAI CY, DM HUBER, HL WARREN 1980 A proposed role ofzein and glutelin as nitrogen sinks in maize. Plant Physiol 66: 330-333 22. WINKLER U, WJ SCHON 1979 Enzymatic hydrolysis of seed proteins: A procedure for evaluating the nutritive value. In Seed Improvement in Cereals and Grain Legumes, Vol I. International Atomic Energy Agency, Vienna, pp 343-351 23. WINSPEAR MJ, A OAKS 1983 Automated pre-column amino acid analysis by reverse phase HPLC. J Chromatogr 270: 378-382