Jul 30, 1984 - asparaginase (EC 3.5.11), ammonia-assimilating enzymes and aspartate and alanine aminotransferases (EC 2.61.1 and EC 2.6.1.1.2). Aspara ...
Plant Physiol. (1985) 77, 382-388 0032-0889/85/77/0000/00/$0 1.00/0
Nitrogen Nutrition and Metabolic Interconversions of Nitrogenous Solutes in Developing Cowpea Fruits' Received for publication July 30, 1984
MARK B. PEOPLES, CRAIG A. ATKINS, JOHN S. PATE*, AND DAVID R. MURRAY2 Department of Botany, University of Western Australia, Nedlands, WA 6009, Australia ABSTRACT Budgets for import and utilization of ureide, amides, and a range of amino acids were constructed for the developing first-formed fruit of symbiotically dependent cowpea (Vigna unguiculata [L.] Walp. cv Vita 3). Data on fruit total N economy, and analyses of the xylem and phloem streams serving the fruit, were used to predict the input of various solutes while the compositions of the soluble and protein pools of pod, seed coat, and embryo were used to estimate the net consumption of compounds. Ureides and amides provided virtually all of the fruit's N requirements for net synthesis of amino compounds supplied inadequately from the parent plant. Xylem was the principal source of ureide to the pod, while phloem was the major source of amides to pod and seed. All fruit parts showed in vitro activity of urease (EC 3.5.1.5), allantoinase (EC 3.5.2.5), asparaginase (EC 3.5.11), ammonia-assimilating enzymes and aspartate and alanine aminotransferases (EC 2.61.1 and EC 2.6.1.1.2). Asparagine:pyruvate aminotransferase (EC 2.6.1.14) was recovered only from the pod. The pod was initially the major site for processing and incorporating N; later seed coats and finally embryos became predominant. Ureides were broken down mainly in the pod and seed coat. Amide metabolism occurred in all fruit organs, but principally in the embryo during much of seed growth. Seed coats released N to embryos mainly as histidine, arginine, glutamine, and asparagine, hardly at all as ureide. Amino compounds delivered in noticea'bly deficient amounts to the fruit were arginine, histidine, glycine, glutamate, and aspartate, while seeds received insufficient arginine, histidine, seine, glycine, and alanine. Quantitatively based schemes are proposed depicting the principal metabolic transformation accompanying N-flow between seed compartments during development.
synthesis, and consumption are related to changes in in vitro activities of enzymes of N metabolism in developing tissues of the fruit, emphasizing particularly the fruit's use of amides and ureides (1, 3, 6, 14, 15) as a general source of amino groupings for its N nutrition. A recently described cryopuncturing technique (9) is used to sample phloem sap entering fruits, and an in vivo seed cup leakage technique (14, 16) and assays of embryo sac fluid are employed to identify the solutes traversing the apoplastic junction between seed coat and embryo. A picture is thus established of the successive net transfers of nitrogenous solutes between different parts of the fruit and seed and the principal net transformations of amino compounds preceding their incorporation into storage pools and protein. MATERIALS AND METHODS Plant Material and Harvest. The nodulated plants of cowpea
(Vigna unguiculata [L.] Walp. cv Vita 3, inoculated with Rhizobium CB756) used in this investigation were taken from the same population as used in a previous study on the C, N, and H20 economy of the first-formed fruit of the species (12). Samples of 15 fruits were removed at intervals after anthesis, chilled on ice, and immediately separated into pod, seed coat, and embryo. Fresh weights were recorded, and samples of material subjected to enzyme assay or solute analysis. Collection and Analysis of Peduncle Tracheal Sap and Phloem Exudate. At intervals during fruit development, tracheal (xylem) sap was extracted by mild vacuum from peduncles, and phloem exudate collected by cryopuncturing the fruit stalk with a needle cooled in liquid N2 (12). Samples of sap were assayed for ureide and amino acids as previously described (9). Extraction and Analysis of the Soluble and Insoluble N Components of Fruit Tissues. Pod, embryos, and seed coats were homogenized with 80% (v/v) ethanol and their soluble and insoluble components separated by centrifugation. The supernatants were evaporated to dryness, partitioned between ether and water to remove pigments and lipids, and the resulting aqueous extracts assayed for ureides and amino acids (9). Total N of the ethanol-insoluble pellet (see above) was determined using a standard Kjeldahl procedure, and its total protein N and protein amino acid composition assessed as the total and individual weights of amino acid residues released following acid digestion in 6 N HCI ( 10WC, 48 h), with or without prior treatment with BTI3. The BTI treatment converted Asn and Gln to the corresponding diamines, which coeluted with Lys during amino acid analysis. The difference between Asp and Glu levels
A recent study (12) of the fruit of cowpea ( Vigna unguiculata [L.] Walp.) has modeled quantitatively its economy of carbon, nitrogen, and water and defined the respective roles of xylem and phloem in supplying the fruit with these materials from the parent plant. This paper extends information on the fruit's nitrogen nutrition by constructing balance sheets for net import and metabolism of ureide and a range of amino compounds by pod and seed parts during fruit development. The information utilized includes data from the above mentioned model of fruit total N economy, and assays for individual nitrogenous compounds in the xylem and phloem streams supplying the fruit and in the soluble and insoluble pools of N in the pod, seed coat, embryo sac fluid and embryo. These budgets for solute intake, in hydrolysates formed with and without BTI treatment was used as a measure of Asn and Gln residues in the protein samples (see ' Supported by the Australian Research Grants Scheme, and the Wheat 2). Measurement of Apoplastic Leakage from Seed Coats. The Industry Research Council of Australia. 2 Biology Department, University of Wollongong, P.O. Box 1144, Wollongong NSW 2500, Australia.
3 Abbreviation: BTI, bis( 1, l-trifluoracetoxy)iodobenzene. 382
383
NITROGEN NUTRITION OF DEVELOPING COWPEA FRUITS PEDUNCLE TRACHEAL XYLEM SAP
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FIG. 1. Changes with age in ethanol-insoluble nitrogen (A, B, and C) and protein amino acid composition (D, E, and F) of pod, seed coats and embryos of the first-formed fruits of effectively nodulated plants of cowpea ( Vigna unguiculata [L.] Walp. cv Vita 3). The amino components marked 'others' included Pro, Val, Cys, and Met. All data plotted cumulatively.
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FIG. 3. Changes during fruit development of cowpea in the levels of ureides and amino acids (A, B, and C) and in amino acid composition (D, E, and F) of peduncle tracheal (xylem) sap, cryopuncture phloem exudate, and seed embryo sac fluid of first-formed fruits of effectively nodulated plants of cowpea (V. unguiculata [L.] Walp. cv Vita 3). The xylem and phloem data represent mean composition of sap collected at a range of times day and night at 3-d intervals from 2 d after anthesis until fruit maturity. Data plotted cumulatively. Amino components marked 'others' in D and E include Pro, Gly, Ala, Cys, Met, Tyr, Phe, Lys, and NH3: in F include Thr, Ser, Gly, Cys, Iso, Leu, Tyr, Phe, Abu,
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nitrogenous solutes passing from seed coat to developing seeds assayed using the recently described seed cup leakage techZ E 0.2 nique (14, 16). A rectangular incision was made in the ventral mid section of an attached fruit and the resulting flap of pod wall removed to expose a seed. The distal half of the seed was 4 16 20 4 8 12 16 20 then cut away, and the embryo of the attached remaining half SEED COATS carefully removed. The resulting attached empty half seed coat ~~~~~~~~~~~E 60 -H cup was rinsed with buffer (5 mm Mes-Tris [pH 6.0], 0.5 mm ~0.3 ~~~~~~~~~6 CaCl2, 100 mm sucrose [111]), and after discarding this wash and refilling the seed cup with buffer, the cut away part of the pod ASP E wall was wrapped with parafilm to maintain a humid atmosphere 0.1 in the fruit. After 2 h the buffer solution from the seed coat cup was collected and assayed for solutes (9). 4 8 12 16 20 4 8 12 16 20 Extraction of Enzymes of N Metabolism. Samples of pods and EMBRYOS embryos were homogenized in a chilled mortar and pestle with acid-washed sand and four volumes of ice-cold 30 mm TricineKOH buffer (pH 8.0), containing 20 mm MgCl2, 2 mm DTT, E 0 2< we ~~~~~~~~~6 ° +~~~~8 ACIDSI 2% (w/v) insoluble PVP. Homogenates were centrifuged and FIG. 2. Changes wth age in ttal ureide ad aminoaci40 con t LYS E50.2(25,000g for 10 min at 4C) and the supernatants treated with PEG 6000 to extract asparagine:pyruvate aminotransferase (2), with (NH4)2SO4 to isolate asparaginase activity (2), or desalted 1L6 20' 4on columns of Sephadex G-25, equilibrated in 15 mm TricineKOH buffer (pH 8.0) containing 20 mM MgCl2 and 2 mM DTT prior to assay for other enzyme activities. FIG. 2. Changes with age in total ureide and amino acid (A, Because of their high phenolic content, seed coats were exB, and C) and amino acid composition (D, E, and F) of the ethanolsoluble fractions of pod seed coats and embryos of the first-formed fruits tracted in buffer containing 30% (w/w) PEG 4000. After centrifof effectively nodulated plants of cowpea (V. unguiculata [L.] Walp. cv ugation at 4°C, the pellets of cellular debris and precipitated Vita 3). Amino acids marked 'others' include Pro, Gly, Val, Cys, Met, protein were retained, resuspended in desalting buffer, and reIle, Leu, Tyr, Phe, and amino butyric acid (Abu). All data plotted centrifuged. The resulting supernatant was used for assay. Treatment of pod or embryo tissue in this way did not enhance the cumulatively. ASP ~~~~~~~~i 60
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contents
Plant Physiol. Vol. 77, 1985
PEOPLES ET AL.
384
Table I. Nitrogenous Solutes Leaked into Attached Half-Seed Coat Cup ofAttached Fruits of Cowpea (V. unguiculata [L.] Walp cv Vita 3) during Mid Pod Fill (14 to 16 Days after Anthesis) Leakage Ratea Solute Exp. 2C Exp. 1b /lg N halfseed coat cup-' h-' 1.23 1.37 Gin 0.85 1.09 Asn 0.56 0.52 His 0.46 0.52 Arg 0.39 0.40 Ser 0.30 0.32 Ala 0.2 1 0.24 Lys 0.20 0.21 Thr 0.19 0.20 Leu 0.17 0.19 Pro 0.16 0.18 Phe 0.15 0.17 Ile 0.13 0.16 Val 0.11 0.16 Glu 0.09 0.11 Tyr 0.08 0.10 Cys + Met 0.07 0.09 Gly 0.12 0.06 Ureides 0.05 0.06 NH3 0.03 0.05 Asp 0.01 0.02 y-Abu
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FIG. 5. Changes with fruit age in activities of enzymes of ammonia assimilation (A, B, and C) and transamination (D and E) in tissue extracts of pod, seed coats, and embryos of first-formed fruits of symbiotically dependent cowpea (V. unguiculata [L.] Walp. cv Vita 3).
described elsewhere (15). Total allantoinase activity was the sum of the soluble and insoluble components. Urease activity (EC 3.5.1.5) was measured by the release of '4CO2 from ['4C]urea (3). Activity in extracts of all fruit tissues was completely inhibited 5.56 6.22 Total by the specific urease inhibitor acetohydroxamate (Ref. 5; aSee text and References 14 and 16 for details of technique for Sigma). collecting leakage products. NAD:glutamate oxidoreductase (EC 1.4.1.2) (2), aspartate b Pooled leakage products collected for 2 h at noon from a single seed aminotransferase (EC 2.6.1.1) (8), and alanine aminotransferase from each of nine separate fruits. (EC 2.6.1.2) (8) activities were determined as extract-dependent c As footnote b, but with seven fruits. linear rates of decrease in optical density at 340 nm. Glutamate synthase was measured with either methyl viologen as an electron donor (EC 1.4.7.1) (2), or as glutamine-dependent NADH oxidation (EC 1.4.1.14) (8). Glutamine synthetase (EC 6.3.1.2) was assayed in a biosynthetic reaction based on glutamyl hydroxamate synthesis (2), E E while asparagine:pyruvate aminotransferase (EC 2.6.1.14) activ.E ity was determined by production of 2-ketosuccinamic acid (2), E and asparaginase (EC 3.5.1.1) by the production of ['4C]Asp from ['4C]Asn (2). All assays were conducted at 30°C. .-
RESULTS Changes in Nitrogenous Constituents of Component Parts of Developing Fruits (Figs. 1 and 2). The total insoluble N and protein N of pod and seed coats increased rapidly during the first .IE half of fruit development and then declined by more than 50% E up to fruit maturity (Fig. 1, A and B). Embryos increased in total E and protein N throughout their development (Fig. IC). Protein accounted for 83% to 86% of total ethanol-insoluble N fractions of pod, seed coats or embryos, except during the last 3 d of the 8 12 16 20 20 fruit's life when the respective values all exceeded 90% (Fig. 1). DAYS AFTEP ANTHESIS The amino acid composition of protein of fruit tissues showed FIG. 4. Changes with age in activities of enzymes of ureide metabo- little change during development (Fig. 1), although embryo prolism (A and B) and of asparagine utilization (C and D) in tissue extracts tein was especially rich in the basic amino acids, Arg and His. of pod, seed coats, and embryos of first-formed fruits of symbiotically Free amino acids and ureides together constituted only very small fractions (less than 10%) of the total N of pod, seed coats, dependent cowpea (V. unguiculata [L.] Walp. cv Vita 3). or embryos. The ratio of amino acid N to ureide N was high in all parts of the fruit throughout most of development. The pod levels of enzyme activity recovered in extracts. Enzyme Assays. Allantoinase (EC 3.5.2.5) was assayed by showed a rise and then a fall in soluble N paralleling its build up measuring the glyoxylate freed from enzymically derived allan- and subsequent mobilization of solute reserves. Seed coats lost toic acid (6) with soluble and insoluble activity determined as amino acid N, but not ureide N, in later seed development. E
4
NITROGEN NUTRITION OF DEVELOPING COWPEA FRUITS
385
Table II. Balance Sheets for Net Intake and Utilization of Ureide and Amino Compounds in Developing Fruits of Cowpea (V. unguiculata [L.I Walp cv Vita 3) over the Period 0 to 11 Days after Anthesis Input Input Gain in Gain as Net Balance % Met by Compound by Soluble Poola Proteina in Fruitb Synthesis by Phloema Xylem ain Fruitc 0.17 2.48 0 +12.00 9.69 Ureided 0 4.58 0.79 0.18 1.11 + 4.08 Glnd 0 Asnd 3.59 2.00 0.24 1.91 + 3.44 0 0.11 0.02 0 + 0.35 0.26 0 Abude 0.01 0.51 + 0.34 0.78 0.08 0 prod + 0.24 Vald 0.65 0.02 0.92 0.53 0 0.42 NDf ND 0.35 + 0.07 Cys + Metd 0 - 0.02 0.03 0.01 0.39 Tyr 0.35 5 3.21 - 0.90 1.13 27 1.24 0.06 Arg - 0.44 0.43 0.02 1.41 Ile 0.56 31 0.02 1.74 - 0.76 Thr 0.35 0.65 43 - 1.47 0.21 0.07 2.89 His 1.28 50 ND - 0.74 0.03 1.43 0.72 51 Lys 0.28 0.02 0.74 2.30 - 1.30 Leu 56 ND 0.01 1.19 - 0.69 Phe 0.51 58 0.47 0.20 0.03 2.00 - 1.36 67 Ala - 2.22 0.23 0.03 3.12 Ser 0.70 70 0.18 0.07 2.23 - 1.64 71 0.48 Glu - 1.72 0.42 0.02 2.32 0.20 73 Asp 0.03 0.01 2.24 - 2.17 0.05 96 Gly a Data of Figures 1 to 3, and assuming ratio of phloem:xylem import is 11:9 (see 12). b Derived as input in xylem and phloem minus incorporation into soluble pool and protein. Positive values signify delivery in excess, negative values suggest part of requirement met by synthesis within fruit. c Derived as (net balance of fruit . [net gains in soluble pool + protein]) x 100. Compounds arranged in increasing extent of their requirement being met by synthesis in fruit as opposed to vascular intake. d Delivered in excess in xylem and phloem. 'Abu, -y-aminobutyric acid. f ND, not detected.
Embryos increased steadily in amino acid and ureide content until seed maturity, when over 80% of the soluble N of the embryo was as amino acids as opposed to ureides (Fig. 2). The composition of the pools of free amino acids varied markedly between fruit tissues (Fig. 2). Amides (Asn and Gln) were dominant constituents of the pod, accounting for approximately 60% of total soluble N until day 16 after which Asn declined, while the proportions as ammonia increased. Amides accounted for less than 40% of the soluble amino acid N of seed coats, with the remaining N distributed over a wide range of compounds. His accounted for more than 20% of the soluble amio acid N in early embryo development, with a further 20 to 40% as amides. The His and Gln content declined after day 8, while Arg increased from less than 10% to almost 40% of the embryo's soluble amino acid N by maturity. Changes in Nitrogenous Solutes of Peduncle Tracheal Xylem Sap, Cryopuncture Phloem Exudate, and Embryo Sac Fluid during Fruit Development (Fig. 3). The overall concentration of nitrogenous solutes in phloem exudate (Fig. 3B) was from 2 to 10 times higher in phloem exudate than in tracheal xylem sap (Fig. 3A), yet ureides represented only 9 to 12% of phloem-borne N versus 56 to 63% of tracheal sap N. Asn, Gln, and Arg were the prominent amino compounds of tracheal sap (Fig. 3D). The amides also predominated in phloem sap, contributing about 40% of its total amino acid N (Fig. 3E). Phloem contained a wide range of other amino acids whose relative composition remained fairly constant, but unlike tracheal xylem sap, phloem contained only relatively small proportions of N as Arg. The liquid accumulating in embryo sacs of developing seeds was collected over the period from 5 to 8 d after anthesis. Amounts obtained ranged from 0.06 to 0.46 ul/seed, depending on seed age. The fluid was rich in amino acids, but contained
only a small proportion (3-6%) of its total N as ureide (Fig. 3C). His comprised 30 to 55%, and the amides around 20% of the total N present as amino compounds in the fluid (Fig. 3F). Significant levels of Ala and Pro were also present. Free ammonia was detected in only trace amounts. Analysis of Solutes Released from Attached Seed Coat Cups (Table I). A wide range of amino acids was recovered from half seed cups (Table 1). Rates of leakage of Gln, Asn, His, Arg, Ser, and Ala were highest, followed by Lys, Thr, Leu, Pro, Phe, Ile, Val, and Glu, and a number of other amino compounds at even lower rates. Ureides accounted for only 1 to 2% of the total N released. Changes in Enzyme Activities Associated with N Metabolism in Developing Fruits (Figs. 4 and 5). Enzymes of ureide breakdown (allantoinase, urease) showed increasing activities in the pod early in fruit development and then declined progressively (Fig. 4, A and B). Both enzymes were present in seeds. Seed coats at first contained the bulk of the recovered activities, but later the embryos showed by far the greater activities. Enzymes of Asn utilization (asparaginase and asparagine aminotransferase) (Fig. 4, C and D) were detected in pod extracts, with maximum levels being recorded before the main phase of seed filling. Only asparaginase was present in seed tissues and remained high in the embryo throughout the period ofrapid protein accumulation. Enzymes of ammonia assimilation were recovered from all parts of the developing fruit (Fig. 5, A-C). Early in development, the pod showed high levels of glutamine synthetase, methyl viologen- and NADH-dependent glutamate synthase, and glutamate oxidoreductase activity. These enzymes all declined in the pod in later fruit growth, but increased progressively in seed coats, and then especially in embryos. During the final 4 to 5 d of seed development, the embryo's activities of glutamine syn-
386
PEOPLES ET AL.
Table III. Balance Sheetsfor Net Intake and Utilization ofUreide and Amino Compounds in Maturing Fruits of Cowpea (V. unguiculata [L.] Walp cv Vita 3) over the Period 12 to 22 Days after Anthesis Compound
Input Input Gain (+) or Gain Net % Met by in as Balance Synthesis by by Loss Phloem' Xylem' Pool' Protein' in Fruitb in Fruitc
Ureided Asnd Glnd Lys"
7.24 10.88 +0.08 0 +18.04 0 13.87 2.12 -0.14 3.56 +12.57 0 7.86 + 9.49 16.47 0.73 -0.15 0 NDf 0.97 + 1.85 0 2.83 +0.01 0.52 -0.01 0 + 0.66 Abudee 0.13 0 ND 1.02 + 0.18 0 Cys + Metd 1.20 ND +0.01 3.71 - 0.98 2.74 ND 26 Pro -0.02 4.40 - 1.54 Ser 2.55 0.29 35 4.67 - 2.12 -0.01 45 Leu 2.28 0.26 2.18 - 1.04 0.02 +0.01 47 Tyr 1.13 47 1.67 -0.01 3.63 - 1.70 Ile 0.25 Val 2.12 0.29 -0.01 4.63 - 2.21 48 Thr 1.92 0.13 -0.01 4.21 - 2.15 51 +0.01 3.19 - 1.67 52 Phe 1.47 0.06 Ala 1.77 0.12 4.62 - 2.71 -0.02 59 His 2.32 ND -0.04 7.79 - 5.43 70 71 3.11 1.26 +0.12 14.80 -10.55 Arg 6.32 - 4.58 0.41 Glu 1.31 -0.02 73 84 0.72 0.35 -0.01 6.60 - 5.52 Asp 4.57 - 4.42 96 0.10 0.06 +0.01 Gly a Data of Figures 1 to 3, and assuming ratio of phloem:xylem import is 4:1 (see 12). b Derived as input in xylem and phloem minus incorporation into soluble pool and protein. Positive values signify delivery in excess, negative values suggest part of requirement met by synthesis within fruit. 'Derived as (net balance of fruit . [net gains in soluble pool + protein]) x 100. Compounds arranged in increasing extent of their requirement being met by synthesis in fruit as opposed to vascular intake. d Delivered in excess in xylem and phloem. I Abu, y-aminobutyric acid. f ND, not detected.
thetase and glutamate synthase decreased sharply, but high levels of glutamate oxidoreductase were retained until embryo maturity. Methyl viologen-linked glutamate synthase activity was restricted to the pod. Aspartate and alanine aminotransferase activities of the fruit (Fig. 5, D and E) closely reflected tissue total N levels, with maximum activities being attained between days 9 and 13 in the pod, at day 13 in the seed coat, and not until fruit maturity in the embryo. Balance Sheets for Import, Synthesis, and Consumption of Ureide and Specific Amino Compounds during Fruit Development (Tables II and III). The approach used was to assume that xylem and phloem supplied the fruit with all of its recorded increment of N, that proportional intake through xylem and phloem was as indicated in an earlier study (12), and that these transport channels delivered ureides and amino acids to the fruit in the relative amounts suggested from analyses of tracheal (xylem) sap and cryopuncture (phloem) sap (Fig. 3). Two stages offruit development were considered, namely pod and early seed growth (0-11 d after anthesis) and seed filling (12-22 d). The ratios for phloem to xylem import of N for these stages were 1 1:9 and 4:1, respectively (12). Using data on changes in amounts of specific nitrogenous compounds in the soluble and insoluble pools of N in the fruit (Fig. 2), and the fruit's net increment in total N (Figs. I and 2), estimates were made of the fruit's net requirement for each compound. These estimates were then compared with the amounts of the same compounds predicted
Plant Physiol. Vol. 77, 1985
to have been imported by xylem and phloem, thus making it possible to assess whether the fruit was in net positive or negative
balance in respect of each compound. Tables II and III summarize the data for the two phases of fruit development. During both stages, ureides, Asn, and Gln arrived in considerable excess of the amounts required for direct incorporation of the same compounds into soluble pools or protein, and since the surpluses involved were quite large relative to the net N increment of the fruit, catabolism of these compounds was suggested to constitute the primary source of N for net synthesis of other amino compounds supplied inadequately through xylem and phloem. Other minor compounds supplied to the fruit in excess were Cys, Met, and y-aminobutyrate (Abu) (0-1 1 and 12-22 d), Pro and Val (0-1 1 d), and Lys (12-22 d), but the small surpluses involved had little impact on the fruit's total N budget. All other compounds were in negative net balance, indicating partial synthesis within the fruit. In certain cases (e.g. Arg, His, Glu, Asp, and Gly [12-22 d]), 70% or more of the net requirement for the compound in question was estimated to have been met by synthesis within the fruit as opposed to import from the parent plant. In other cases (Ile, Tyr, Arg [0-1 1 d]; Ser, Pro [12-22 d]) proportional amounts synthesized by the fruit were much less (
