Urate-mediated regulation of urate oxidase in - Springer Link

Protoplasma(1998) 202:17-22

PROTOPLASMA 9 Springer-Verlag 1998 Printed in Austria

Urate-mediated regulation of urate oxidase in Chlamydomonas reinhardtii J. M. Alamillo, A. R. Franco, J. Cfirdenas, and M. Pineda* Departamento de Bioqufmicay BiologfaMolecular, Facultad de Ciencias, Universidad de C6rdoba, C6rdoba Received November 19, 1997 Accepted January 20, 1998

Summary. Expression of uricase (urate oxidase) from Chlamydomonas reinhardtii has been investigated by using specific polyclonal antibodies. By Western blot analyses performed under nondenaturing conditions, a 124 kDa protein band corresponding to active uricase was detected in protein extracts from cells cultured with urate or nitrogen-starved cells. This protein band was absent in cells cultured with ammonium.Besides the 124 kDa band, the antibodies also reacted with a massive protein band, with an apparent molecular mass of 500 kDa, that was detected in all nutritional conditions assayed. In vitro, inactive uricase from cells grown with ammonium was activated by incubation in presence of urate. The appearanceof uricase activity in vitro coincided with a decrease of the 500 kDa protein and an increase of the 124 kDa band corresponding to the active enzyme. We suggest that a posttranslational regulation of uricase synthesis takes place in C. reinhardtii, and that urate may be responsible for the assembly or maturation of inactive precursors to form the active uricase.

Keywords: Chlamydomonas; Posttranslational regulation; Uricase. Introduction Regulation of urate oxidase (uricase; urate: oxygen oxidoreductase, EC 1.7.3.3), the enzyme that mediates the conversion of uric acid into allantoin, has been studied in several microorganisms and in root nodules of leguminous plants. In Neurospora crassa, synthesis of uricase seems to be regulated at the transcriptional level, requires uric acid, and is repressed by a m m o n i u m (Wang and Marzluf 1979). In Bacillus fastidiosus, the expression of uricase also requires urate, is maximal under limited oxygen concentrations, and is repressed by ammonium, allantoin, and *Correspondence and reprints: Departamento de Bioqufmica y Biologfa Molecular, Facultad de Ciencias, Universidad de C6rdoba, Avda. San Alberto Magno s/n, E-14071 C6rdoba, Spain.

allantoate even in the presence of urate (Bongaerts et al. 1978). In root nodules of leguminous plants, uricase represents the second most abundant cytoplasmic protein (nodulin-35) playing an important role in symbiotic nitrogen fixation (Bergmann et al. 1983) and in both nodules and peroxisomes development (Lee et al. 1993). Immunological studies have recently shown that soybean nodule uricase is also expressed during germination and early seedlings growth (Tajima et al. 1991, 1993; Damsz et al. 1994) and that uricase accumulated during the pod-filling stage (Takane et al. 1997). Uricase has been purified from adult leaves of chickpea, broad bean, and wheat. The three plant uricases were found to be tetramers of similar molecular mass (120-130 kDa) and to exhibit similar catalytic and kinetic properties, and were expressed at very low levels in leaves (Montalbini et al. 1997). Nothing is known about expression regulation of these uricases. Synthesis of nodulin-35 takes place during the nodule ontogeny in uninfected cells of Glycine max and Phaseolus vulgaris (Bergmann et al. 1983, Nguyen et al. 1985, Sfinchez et al. 1987, Papadopoulou et al. 1995), and the enzyme synthesis is regulated by oxygen at the m R N A level in Glycine max callus tissue (Larsen and Jochimsen 1986, Xue et al. 1991). However, little is known about the regulation of uricase synthesis in the green alga Chlamydomonas reinhardtii. This enzyme has been purified and characterized as a copper protein of 124 kDa consisting of four identical subunits (Alamillo et al. 1991), whose activity is regulated by its substrates, urate and oxygen (Alamillo et al. 1992), and whose synthesis seems to be repressed by a m m o n i u m (Pine-

18

da et al. 1984, 1987), but it is still unknown whether this regulation takes place at the transcriptional or translational level. In this paper we present data suggesting the constitutive synthesis of inactive uricase precursors in C. reinhardtii, and we show that uric acid plays a crucial role in the posttranslational assembling or maturation of these precursors to form the active, 124 kDa uricase tetramer. Material and methods Cell culture and preparation of extracts Cells of Chlamydomonas reinhardtii 6145c (from the collection of Dr. R. Sager, Sidney Farber Cancer Center, New York) were grown in minimal liquid medium (Sueoka 1960), under saturating light conditions (15-20 W/mZ), with 4 mM ammonium chloride as a nitrogen source as previously described (Pineda et al. 1984). Cells at A660= 1.5 were harvested by centrifugation at 4,000 g for 10 min, the pellet was washed with distilled water and resuspended cells were transferred to a minimal medium with different nitrogen sources where they were allowed to grow until mid-logarithmic phase of growth, unless otherwise stated. Cells were then harvested and washed as above, and centrifuged at 20,000 g for 20 rain. The cell pellet was broken by freezing at -40 ~ and thawing with gentle stirring in 0.1 M Tris-glycine buffer, pH 8.5, (2 ml/g fresh weight) and centrifuged at 27,000 g for 10 min. The resulting supernatant was used as crude extract.

Antisera preparation Uricase from C. reinhardtii (120-130 ~tg) purified to homogeneity (Alamillo et al. 1991) was emulsified in one volume of complete Freund's adjuvant and injected into New Zealand white rabbits. Two weeks later, the animals received a second injection and after two more weeks they were injected with a booster containing 80-100 ~g of protein emulsified in incomplete Freund's adjuvant. Blood (20-30 ml) was collected ten days after the last injection. New boosting injections were given at 1-month intervals, and the rabbits were bled ten days after each injection. Immunoglobulins were purified by adding solid ammonium sulfate up to 40% saturation and, after 30 min at 4 ~ centrifugation of the suspension at 12,000 g for 10 min. The supernatant was discarded and the resulting precipitate washed at 4 ~ with 1.75 M ammonium sulfate until a white pellet, containing immunoglobulins, was formed. This white pellet was dissolved in 1/3 of the initial volume with 17.5 mM phosphate buffer, pH 7, and dialyzed against the same buffer. Precipitated lipoproteins were removed by centrifugation as above. Antibodies were further purified in a DEAE-Sephacel chromatography column (9 X 1.5 cm) previously equilibrated with 17.5 mM phosphate buffer, pH 7. Antibodies were not retained by the column and were recovered in the first protein-containing fractions. The antibody solution was concentrated by ultrafiltration under vacuum and kept at -20 ~

Gel immunodiffusion and immunoelectrophoresis Double immunodiffusion experiments were performed according to Ouchterlony and Nilson (1973). Rocket immunoelectrophoresis was carried out according to Weeke (1973) in glass plates (94 • 84 mm)

J.M. Alamillo et al.: Regulation Of uricase in Chlamydomonas covered with 1% agarose in 0.1 M Tris-glycine buffer, pH 8.6. Agarose was melted at 90 ~ and allowed to cool down to 56 ~ Then, antibodies (200-250 ~tg) were added and the emulsion spread onto the glass plates. Antigens were placed in wells on the edge of the plates and electrophoresis was performed in a cooling chamber for 3-4 h at 220 V. Gels were washed for 48 h with saline phosphate buffer and stained for protein or uricase activity (Pineda et al. 1984).

Molecular mass determination Apparent molecular mass of uricase and its precursors was determined by electrophoresis on polyacrylamide gels under nondenaturing (7% in acrylamide) and denaturing (10% in acrylamide) conditions according to Laemmli (1970). The following standards were used as markers (in kDa): bovine thyroglobulin (669); horse spleen apoferritin (443); sweet-potato ~-amylase (229); muscle rabbit myosin (205); bovine serum albumin (132 and 66, dimer and monomer, respectively); E. coli [3-galactosidase (116); rabbit muscle phosphorylase b (97.4); hen ovalbumin (45); rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (36); bovine erythrocytes carbonic anhydrase (29); bovine pancreas trypsinogen (24); soybean trypsin inhibitor (20.1); and bovine milk c~-lactalbumin (14.2).

Western blotting and uricase immunodetection After electrophoresis, proteins were transferred to nitrocellulose membranes according to Towbin etal. (1979). Membranes were washed thoroughly, incubated at 4 ~ overnight with a 1 : 125 solution of antiuricase antibodies and, after several washes, incubated with rabbit antiimmunoglobulins attached to either alkaline phosphatase or horseradish peroxidase. Immunodetection with phosphatase or peroxidase was performed following the protocols from Bio-Rad (Richmond, CA, U.S.A.) or Amersham (Little Chalfont, U.K.), respectively.

Urate determination Intracellutar urate was determined either enzymatically by the colorimetric assay of Fossati et al. (1980) or by HPLC using an analytical polypore H column (220 • 4.6 mm) with a precolumn containing the same stationary phase. As mobile phase, H2804 0.014 N was used. Chromatography was carried out at a flow-rate of 0.5 ml/min and chromatograms were recorded at 292 nm.

Results and discussion Specificity

of

antibodies

against

uricase

from

Chlamydomonas reinhardtii was tested by the Ouchterlony double diffusion analysis (Fig. 1 A). The precipitin lines exhibited uricase activity (results not shown), which indicates that immunoprecipitates were formed by the specific reaction of antibodies with uricase. Uricase activity decreased in crude extracts by precipitation when increasing volumes of antiserum were incubated with a fixed amount of enzyme extract (Fig. 1 B). Complete inactivation was only achieved when identical volumes of both enzyme extract and antiserum were mixed, which indicates that antiserum is of low titer.

19

J. M. Alamillo et al.: Regulation of uricase in Chlamydomonas

Table 1. Uricase protein and activity in Chlamydomonas cells cultured under different nitrogen conditions

Culture conditions

1 mM urate + 4 mM ammonium + 7 ~tM cycloheximide + 10 mM chloral hydrate 4 mM ammonium No nitrogen source

E c

Uricase rocket height

enzyme activity

(cm)

(pkat/mg)

6.3 5.4 5.4 5.6 5.3 5.8

142.0 8.4 0.0 5.0 8.4 76.8

Cells grown in media with ammonium were washed and transferred to the indicated culture conditions. After 11 h, uricase protein and activity were determined in crude extracts as described in Material and methods

0.8

I,I

cO < cb 0

0"-'--0

F.D

,

0.0

0

B

I

i

I

30

60

90

ANTIBODIES

(/~I)

Fig. 1. A Ouchterlony's double immunodiffusion of uricase from C: reinhardtii. The center well contained 12 ~xl (38 ~tg) of crude extract from cells grown with urate as sole nitrogen source. The outer wells contained 12 ~xl of the indicated dilutions of antiserum or nonimmune serum (N/). Gel was stained for protein with Coomassie brilliant blue. B Immunoprecipitafion of uricase from C. reinhardtii. Identical aliquots of crude extracts (100 p~g protein) containing uricase activity were incubated at 4 °C for 24 h with the indicated volumes of antiserum in a final volume of 0.2 ml. Uficase activity was measured in the incubation mixture (O), in the supernatant of samples centrifuged at 10000 g for 10 min (O), and in controls with nonimmune serum (zS)

Uricase activity in Chlamydomonas cells grown in media containing ammonia or protein synthesis inhibitors (cycloheximide or chloral hydrate) was low or negligible when compared with that of cells grown with urate or subjected to nitrogen starvation (Table 1). However, in all these cases, except for urate, similar amounts of uricase protein were present as determined by quantitative rocket immunoelectrophoresis (Table 1). This suggested that Chlamydomonas uricase was also synthesized in the presence of ammonia, although in an inactive form, which coincides with our previous findings of negligible levels of uricase activity in cells grown with ammonium (Pineda et al. 1984, 1987). In cells cultured with urate, levels

of uricase activity showed a high increase (18-fold) with respect to ammonium-cultured cells. However, only a small increase in uricase protein levels was found (Table 1). This strong effect on enzyme activity, whereas only slightly inducing the amount of uricase protein, suggested the constitutive synthesis of inactive uricase forms or precursors. When analyzed by denaturing Western blot, a protein band at 32 kDa, which corresponds to the size of active uricase subunits (Alamillo et al. 1991), was found in extracts obtained from cells cultured with urate or in the absence of nitrogen source. This protein was not detected in extracts from ammoniumgrown cells (Fig. 2). Since cells cultured with ammonium have almost negligible uricase activity, this result is consistent with repression of uricase synthe-

Fig. 2. Immunological detection of uricase proteins of crude extracts from C. reinhardtii cells after denaturing electrophoresis in the presence of SDS. Extracts from cells cultured with ammonium (1), urate (2), or subjected to nitrogen starvation (3) were used

20

Fig. 3. Detectionof uricase protein (A) and uricase activity (B) after nondenaturing electrophoresisof crude extracts from C. reinhardtii. A Electrophoresiswas performedwith extracts (10 ~xgprotein) from cells culturedfor 9 h with ammonium (1), urate (2), urate plus chloral hydrate (3), urate plus ammonium (4) or subjected to nitrogen starvation (5). After transfer to nitrocellulosefilters, uricase protein was immunodetectedas describedin Material and methods. B Uricase activityafter electrophoresisof extractsfrom urate-growncells

sis in the presence of ammonium (Pineda et al. 1984, 1987). However, a second protein band at about 60 kDa reacted with the antibodies in all three culture conditions (Fig. 2). Therefore, it is possible that uricase-related proteins could be also present in ammonium-grown cells. It is unclear whether this second band of about twice the actual size of uricase monomers could represent a precursor form that needs to be processed or it could be formed by the covalent dimerization of the subunits. The presence of possible uricase precursors was tested by Western blot analysis under nondenaturing conditions (Fig. 3 A). Under these conditions, several proteins were recognized by antiuricase antibodies in extracts from cells grown in media containing ammonium, urate, urate with chloral hydrate, urate with ammonium, or subjected to nitrogen starvation (Fig. 3 A). The smallest protein band (124 kDa) was more intense in cells grown in media with urate or lacking a nitrogen source than in ceils cultured with urate and either chloral hydrate or ammonium, and in cells grown only with ammonium. The largest protein band (ca. 500 kDa) was always present, and other, less intense bands of intermediate size were detectable mainly in cells cultured with urate or subjected to nitrogen starvation. When extracts from urate-grown cells were subjected to electrophoresis under nondenaturing conditions and gels were stained for uricase activity, both the smallest protein band and those of

J. M, Alamilloet al.: Regulationof uricase in Chlamydomonas intermediate size exhibited enzyme activity (Fig. 3 B). However, activity was rapidly detectable in the smallest protein band and only after a long incubation period in those of intermediate size. This is consistent with the largest protein band being formed by aggregation of either inactive uricase precursors or inactive uricase subunits. The presence of intermediate bands could be explained as intermediary forms of the enzyme that are generated during an activation process and, in consequence, they are not completely active, or as smaller aggregation forms able to retain partial activity. This is supported by the fact that a long incubation period was required to detect enzymatic activity in those intermediary forms. Pineda et al. (1984) have demonstrated the existence of uricase activity in cells of C. reinhardtii grown with adenine, guanine, hypoxanthine, xanthine, urate, allantoin, and allantoate, whereas with ammonium or nitrate uricase activity was negligible. Extracts from cells grown with adenine, guanine, hypoxanthine, xanthine, allantoin, and allantoate contained proteins of 500 and 124 kDa which reacted with uricase antisera, whereas those grown with nitrate or ammonia had mainly the 500 kDa inactive protein (data not shown). This corroborates that the largest protein is formed under all conditions studied. These results agree with those previously reported (Pineda et al. 1984) on the regulation of uricase in Chlamydomonas by ammonia with the proviso that regulation seems to take place not only at the level of enzyme synthesis but also posttranslationally by activation of a preformed enzyme. Nodulin-35 (uricase II) from soybean root nodules is synthesized very early in infection and it does not become enzymatically active until approximately 14 days after infection (Bergmann et al. 1983). However, this enzyme has been expressed and assembled into a functional tetrameric holoenzyme in E. coli and no posttranslational modifications seem to be required for activity (Suzuki and Verma 1991). Unlike C. reinhardtii, Bacillus fastidiosus uricase was repressed by allantoin or allantoate, even in the presence of uric acid, which induced the synthesis of the enzyme (Bongaerts et al. 1978). In Chlamydomonas, analyses of uricase mRNA are required to assess whether uricase expression is also regulated at a transcriptional level. Urate played a key role in the conversion of the inactive precursor of uricase into its enzymatically active form. Addition of urate to crude extracts from cells lacking uricase activity led to a recovery of the activity (Fig. 4) in parallel with a decrease in the largest

J. M. Alamillo et al.: Regulation of uricase in Chlamydomonas

A m

Table 2. Intracellular urate concentration in C. reinhardtii cells grown with a m m o n i u m and subjected to nitrogen starvation

B

60 )ira I-

r~ la.i

.r r

/

~mO

9176

30

w

0

I

I

2

3

v

1

Starvation time

Urate concentration (nmol/mg protein)

(b)

enzymatic method

HPLC

0 1 2 4

0.8 8.7 2.9 2.6

1.2 7.3 2.8 2.5

Cells grown with a m m o n i u m were washed and transferred to minim u m medium with no nitrogen source. At the indicated times, cells were harvested, washed with distilled water and broken by freezing and thawing. After centrifuging, urate was determined in supernatants as described in Material and methods

B

0

21

TIME (h) Fig. 4. In vitro activation of uricase by urate. Urate was added to extracts from cells grown with 4 m M a m m o n i u m at a final concentration of 0.2 raM, the mixture was incubated at 25 ~ and uricase activity was determined spectrophotometrically at the indicated times. 100% activity corresponded to the uricase activity of extracts from cells grown With urate (134 pkat/mg). Inset Detection of uricase-related proteins in Western blots with 25 ~tg of protein taken at the beginning of the experiment (A) and after 2 h incubation (B)

protein band and an increase of the smaller band detected by the antiserum (Fig. 4). Data of Table 1 and the observed lack of uricase activity in media containing urate plus ammonium can be explained by the inhibition of urate transport into the cells when ammonia is present in the medium at concentrations above 0.1 mM (Pineda and Cfirdenas 1985, Pineda et al. 1987). This can account for the lack of activation under these conditions and for the appearance of activity when external ammonia is exhausted and thus urate can enter the cells. The effect of nitrogen starvation on uricase activity can be explained by the results shown in Table 2. Significant amounts of urate were found when the intracellular concentration of urate was measured in nitrogen-starved cells, which can account for the uricase activity observed in Chlamydomonas cells under these conditions. Thus, in absence of a nitrogen source urate is formed, presumably by the degradation of nucleic acids, reaching concentrations high enough to activate the uricase. Urate can be the metabolite responsible for the maturation or activation of uricase in starved cells, and in cells grown with adenine, guanine, hypoxanthine, and

xanthine. It can be also responsible for the activity found in cells grown with allantoin and allantoate, since enough time (20-24 h) has to elapse before the cells start using these compounds as nitrogen sources (Pineda et al. 1984, Piedras 1995), which forced the cells to remain under nitrogen starvation conditions. From these results, the role of ammonium on uricase regulation needs to be reassessed. The limiting factor in the regulation of Chlamydomonas uricase activity is not the presence of ammonium but the absence of urate. Uricase could be constitutively formed as an inactive precursor that needs urate to become active. This also explains the low basal levels of uricase detected in cells grown with ammonium or nitrate, since under these conditions nucleotides exchange is taking place and there is a very low intracellular concentration of urate. The results presented here indicate that Chlamydomonas uricase is posttranslationally regulated, although other levels of regulation cannot be ruled out, and that uric acid plays a major role in this regulation by activating inactive precursors of this enzyme.

Acknowledgements This work was supported by grants from DGES (PB96-0504-CO202) and Junta de Andalucla (CVI-115), Spain, and from HCM-networks, EC(ERB40SPL921476). The secretarial assistance of C. Santos and I. Molina is acknowledged.

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22 Bergmann H, Preddie E, Verma DPS (1983) Nodulin-35: a subunit of specific uricase (uricase II) induced and localized in the uninfected cells of soybean nodules. EMBO J 2:2333-2339 Bongaerts GPA, Uitzetter J, Brouns R, Vogels GD (1978) Uricase of Bacillus fastidiosus: properties and regulation of synthesis. Biochim Biophys Acta 527:348-358 Damsz B, Dannenhoffer JM, Bell JA, Webb MA (1994) Immunocytochemical localization of uricase in peroxisomes of soybean cotyledons. Plant Cell Physiol 35:979-982 Fossati P, Prencipe L, Berti G (1980) Use of 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone chromogenic system in direct enzymic assay of uric acid in serum and urine. Clin Chem 26:227-231 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Larsen K, Jochimsen BU (1986) Expression of nodule-specific uricase in soybean callus tissue is regulated by oxygen. EMBO J 5: 15-19 Lee N-G, Stein B, Suzuki H, Verma DPS (1993) Expression of antisense nodulin-35 RNA in Vigna aconitifolia transgenic root nodules retards peroxisome development and affects nitrogen availability to the plant. Plant J 3:599-606 Montalbini P, Redondo J, Caballero JL, Cfirdenas J, Pineda M (1997) Uricase from leaves: its purification and characterization from three different higher plants. Planta 202:277-283 Nguyen T, Zelechowska M, Foster V, Bergmann H, Verma DPS (1985) Primary structure of the soybean nodulin-35 gene encoding uricase II localized in the peroxisomes of uninfected cells of nodules. Proc Natl Acad Sci USA 82:5040-5044 Ouchterlony O, Nilson L-A (1973) Immunodiffusion and immunoelectrophoresis. In: Weir DM (ed) Handbook of experimental immunology. Blackwell, Oxford, pp 19.1-19.34 Papadopoulou K, Roussis A, Kuin H, Katinakis P (1995) Expression pattern of uricase II gene during root nodule development in Phaseolus vulgaris. Experientia 51: 90-94 Piedras P (1995) Metabolismo de los ureidos alantofna y alantoato en Chlamydomonas reinhardtii. PhD thesis, Department of Biochemistry and Molecular Biology, University of C6rdoba, C6rdoba, Spain

J. M. Alamillo et al.: Regulation of uricase in Chlamydomonas Pineda M, Cfirdenas J (1985) The urate uptake system in Chlamydomonas reinhardtii. Biochim Biophys Acta 820:95-99 - Fernandez E, Cfirdenas J (1984) Urate oxidase of Chlamydomonas reinhardtii. Physiol Plant 62:453-457 Cabello P, Cfirdenas J (1987) Ammonium regulation of urate uptake in Chlamydomonas reinhardtii. Planta 171:496-500 Sfinchez F, Campos F, Padilla J, Bonneville J-M, Enr/quez C, Caput D (1987) Purification, cDNA cloning, and developmental expression of the nodule-specific uricase from Phaseolus vulgaris L. Plant Physiol 84:1143-1147 Sueoka N (1960) Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 46:83-91 Suzuki H, Verma DPS (1991) Soybean nodule-specific uricase (nodulin-35) is expressed and assembled into a functional tetrameric holoenzyme in Escherichia coli. Plant Physiol 95: 384-389 Tajima S, Ito H, Tanaka K, Nanakado T, Sugimoto A, Kouchi H, Okazaki K (1991) Soybean cotyledons contain a uricase that cross-reacts with antibodies raised against the nodule uricase (Nod 35). Plant Cell Physiol 32:1307-1311 - Tanaka K, Takane K, Sugimoto A, Okazaki K, Kouchi H (1993) Soybean nodule uricase gene (nodulin 35) is expressed in cotyledons during seed development and early germination. In: Palacios R, Mora J, Newton WE (eds) New horizons in nitrogen fixation. Kluwer, Dordrecht, p 373 Takane K, Tanaka K, Tajima S, Okazaki K, Koouchi H (1997) Expression of a gene for uricase II (Nodulin-35) in cotyledons of soybean plants. Plant Cell Physiol 38:149-154 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350-4354 Wang LC, Marzluf GA (1979) Nitrogen regulation of uricase synthesis in Neurospora crassa. Mol Gen Genet 176:385-392 Weeke B (1973) Rocket immunoelectrophoresis. Scand J Immunol 2 Suppl 1:37-46 Xue Z, Larsen K, Jochimsen BU (1991) Oxygen regulation of uricase and sucrose synthase in soybean callus tissue is exerted at the mRNA level. Plant Mol Biol 16:899-906 -