Báo cáo khoa học: Regulation of maize lysine metabolism and endosperm protein synthesis by opaque and floury mutations potx

Eur J Biochem 270, 4898–4908 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03890.x Regulation of maize lysine metabolism and endosperm protein synthesis by opaque and floury mutations ´ Ricardo A Azevedo1, Catherine Damerval2, Jacques Landry3, Peter J Lea4, Claudia M Bellato5, Lyndel W Meinhardt6, Martine Le Guilloux2, Sonia Delhaye3, Alejandro A Toro1, Salete A Gaziola1 and Bertha D A Berdejo1 Departamento de Gene´tica, Escola Superior de Agricultura Luiz de Queiroz, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil; Station de Ge´ne´tique Ve´ge´tale INRA/UPS/INA-PG/CNRS UMR 8120, La Ferme du Moulon, Gif-sur-Yvette; 3INRA, Laboratoire de Chimie Biologique, INA-PG, F78850 Thiverval-Grignon, France; 4Department of Biological Sciences, University of Lancaster, Lancaster, UK; 5Centro de Energia Nuclear na Agricultura, Universidade de Sa˜o Paulo, Sa Paulo, Brazil; and 6Departamento de ˜o Gene´tica e Evoluca˜o, Universidade Estadual de Campinas, Campinas, Brazil ¸ The capacity of two maize opaque endosperm mutants (o1 and o2) and two floury (fl1 and fl2) to accumulate lysine in the seed in relation to their wild type counterparts Oh43+ was examined The highest total lysine content was 3.78% in the o2 mutant and the lowest 1.87% in fl1, as compared with the wild type (1.49%) For soluble lysine, o2 exhibited over a 700% increase, whilst for fl3 a 28% decrease was encountered, as compared with the wild type In order to understand the mechanisms causing these large variations in both total and soluble lysine content, a quantitative and qualitative study of the N constituents of the endosperm has been carried out and data obtained for the total protein, nonprotein N, soluble amino acids, albumins/globulins, zeins and glutelins present in the seed of the mutants Following two-dimensional PAGE separation, a total of 35 different forms of zein polypeptides were detected and considerable differences were noted between the five different lines In addition, two enzymes of the aspartate biosynthetic pathway, aspartate kinase and homoserine dehydrogenase were analyzed with respect to feedback inhibition by lysine and threonine The activities of the enzymes lysine 2-oxoglutate reductase and saccharopine dehydrogenase, both involved in lysine degradation in the maize endosperm were also determined and shown to be reduced several fold with the introduction of the o2, fl1 and fl2 mutations in the Oh43+ inbred line, whereas wild-type activity levels were verified in the Oh43o1 mutant Maize production is the highest of all crop plants and serves as an important source of dietary protein for human and livestock consumption However, the nutritional quality is not adequate, due to the lack of the essential amino acids lysine and tryptophan in the seed proteins [1] Zeins, which account for 50–70% of the endosperm proteins in maize seeds, have a characteristic amino acid composition, being rich in glutamine and hydrophobic amino acids, whilst being very poor in lysine and tryptophan [2] Based on their solubility, genetic properties, and the apparent molecular masses, zeins have been classified into a- (22 and 19 kDa), the most abundant, b- (14 kDa), c- (27 and 16 kDa) and d-zein (10 kDa) [3] Four main strategies have been attempted in order to obtain plants with a high lysine seed content: plant breeding, characterization of naturally occurring mutants, induction of biochemical mutants and the production of transgenic plants [4,5] Perhaps the most exciting result obtained during this research was the identification of the high-lysine opaque (o2) maize mutant [6] Unfortunately, the highlysine trait was negatively correlated with other agronomic characteristics, such as resistance to plant pathogens and yield [1] More recently, quality protein maize (QPM) varieties have been produced which maintain the high-lysine and high-tryptophan characteristics conditioned by the o2 mutation in a modified-vitreous endosperm, with favorable agronomic characteristics [7–9] The amino acid lysine is derived from aspartate and the biosynthetic pathway involves the action of several strongly regulated enzymes [10] The enzyme aspartate kinase (AK; EC 2.7.2.4), which converts aspartic acid into b-aspartyl phosphate, can exist in at least two distinct isoforms, one (or two) sensitive to lysine feedback inhibition and the other sensitive to threonine feedback inhibition, the latter being a bifunctional polypeptide with the threonine-sensitive homoserine dehydrogenase isoenzyme (HSDH; EC 1.1.1.3) [11] ´ Correspondence to R A Azevedo, Departamento de Genetica, Escola Superior de Agricultura Luiz de Queiroz, Universidade de Sao Paulo, ˜ Piracicaba CEP 13418–900, SP, Brazil Fax: + 55 19 3433 6706, Tel.: + 55 19 3429 4475, E-mail: raazeved@esalq.usp.br Abbreviations: AK, aspartate kinase; DHDPS, dihydrodipicolinate synthase; HSDH, homoserine dehydrogenase; LOR, lysine 2-oxoglutarate reductase; N, nitrogen; NPN, nonprotein nitrogen; PVPP, insoluble polyvynylpyrrolidone; SDH, saccharopine dehydrogenase; SAA, soluble amino acids Enzymes: AK (EC 2.7.2.4); HSDH (EC 1.1.1.3); LOR (EC 1.5.1.8); SDH (EC 1.5.1.9); DHDPS (EC 4.2.1.52) (Received September 2003, accepted 22 October 2003) Keywords: lysine metabolism; maize; storage proteins Ó FEBS 2003 Lysine metabolism in maize mutants (Eur J Biochem 270) 4899 The AK isoenzymes have been characterized at both the biochemical and molecular level in several plant species [4,5,10,12], and shown to be a major factor in the regulation of the carbon flux through the aspartate pathway [4,10] HSDH catalyses the conversion of aspartate semialdehyde to homoserine in the presence of the coenzymes NADH or NADPH and is present in plant species in two isoforms, resistant and sensitive to threonine inhibition [10] The first enzyme unique to lysine synthesis, dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) has also been extensively studied and characterized in plants catalyzing the condensation of pyruvate and aspartate semialdehyde into dihydrodipicolinic acid [4] DHDPS is also subject to feedback inhibition by micromolar concentrations of lysine [4] Several mutants that overproduce and accumulate threonine have been obtained by selection on media containing amino acids or their analogues and this phenomenon has been shown to be due to alteration in the feedback pattern of the lysine-sensitive AK isoenzyme [4] However, in the case of cereal seeds, the mutants failed to accumulate lysine in higher concentration [10,13,14] The development of plant transformation techniques has allowed the production of transgenic plants expressing the enzymes of lysine biosynthesis that are insensitive to feedback regulation analogous to the biochemical mutants Again, most of the plants did not exhibit significant accumulation of lysine in the seed [4,12] Positive results were however, obtained with barley, canola and soybean transgenic seeds in which dramatic increases in the lysine content were observed [5,15] Very little was known about lysine catabolism in plant until recently [5,12,16] The first two enzymatic steps are catalyzed by the bifunctional protein lysine 2-oxoglutarate reductase–saccharopine dehydrogenase (LOR–SDH; EC 1.5.1.8 and EC 1.5.1.9, respectively) LOR–SDH protein has been studied in some plant species [17–21] where the activity was particularly high in the endosperm tissue in cereal crops [17,18] The regulation of the LOR activity has been shown to be complex, involving several distinct mechanisms [5,12,16] Recent studies have confirmed that in order to obtain lysine overproduction in cereal seeds, manipulation of lysine degradation is needed [5,12,16] This suggestion is supported by five main points [5]: (a) The cereal mutants or transgenic plants not exhibit significant accumulation of lysine in the seeds; (b) LOR–SDH activities are endosperm specific in cereal crops only; (c) LOR–SDH activities are drastically reduced in the high-lysine o2 maize mutant as compared with the wild-type; (d) lysine catabolism intermediates accumulate in the seeds of lysine overproducing plants of soybean and canola, indicating reduced LOR–SDH activities; and (e) LOR–SDH activities are lower in legume plants and rice, which is the cereal crop with the highest concentration of lysine in the seed The product of the o2 gene is specifically expressed in the endosperm and the protein was shown to activate the transcription of the 22 kDa a-zein [22] and 14 kDa b-zein genes [23], together with the b-32 [24] and cyPPDK1 (one of two cytosolic isoforms of pyruvate orthophosphate dikinase) genes [25] Other possible direct or indirect target genes of the o2 factor have been shown to belong to various metabolic pathways [26–28] In the o2 mutant, LOR–SDH mRNA and protein quantities were severely reduced (about 90%), and the expression pattern during grain development was markedly modified [29] The genomic sequence of the gene and its 5¢ regulatory regions revealed the presence of o2 boxes in the upstream promoter, confirming the hypothesis of a transcriptional control of the Lor/Sdh gene by the o2 protein [16] These large effects suggest that o2 protein may play an important role in the developing grain, as a coordinator of the expression of storage protein, and nitrogen and carbon metabolism genes [30] Although there is now plenty of information available about o2, information related to lysine metabolism for several other similar mutants that have been classified as high-lysine and exhibit the opaque phenotype are very scarce A comprehensive investigation into these mutants was initiated, with the aim of obtaining new insights into the regulation of lysine metabolism in maize During the course of this work Hunter et al [31] published an analysis of some of these mutants Our work now extends the studies of Hunter et al [31] and provides further insights into the complex but critical regulation of lysine accumulation within the maize seed, reporting for the first time the biochemical characterization of these mutants using proteomic and enzymological approaches Experimental procedures Maize mutants Seeds of the mutant genotypes opaque (o1 and o2) and floury (fl1 and fl2) and the respective wild type, Oh43 +, were kindly provided by the Maize Genetics Cooperation Seed Stock Center (Urbana, IL, USA) Plants of all genotypes were grown in the glasshouse at ESALQ-USP, Brazil and self-pollinated Maize ears were harvested 20 days after pollination (DAP) directly into liquid nitrogen and stored at ) 80 °C until used for enzyme extraction The experiments were repeated over three summer seasons (1999–2000, 2000–01 and 2001–02) Preparation of endosperm samples Endosperms were isolated from mature grains previously soaked in water for 30 by peeling off the outer tegument and excising the germ After freeze drying, the endosperms were ground to a powder using a ball mill Fractionation of nitrogen (N) constituents The isolation of endosperm N constituents was undertaken in duplicate as previously described by Landry et al [32] Quantitation of N constituents For an accurate quantitation, the nonprotein nitrogen (NPN) and protein content were determined by the ninhydrin assay of a-amino N released after alkaline digestion (3 M NaOH, 130 °C, 45 min) for the TCA, E1,2, E4 extracts [32] or acid digestion (6 M HCl, 110 °C, 18 h) for the E3 extract and residues, according to Landry et al [33] Soluble amino acids (SAA) were quantitated by ninhydrin without previous digestion of the TCA extracts [32] 4900 R A Azevedo et al (Eur J Biochem 270) Amino acids analysis Soluble amino acids from mature seeds were extracted and analyzed exactly as described by Gaziola et al [9] As the OPA-lysine derivative is rapidly degraded, a second analysis was performed using a 15-min elution time Four replicates were analyzed Protein extraction and two-dimensional polyacrylamide gel electrophoresis of zein polypeptides The procedure for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis of zein isoforms followed that published previously by Consoli and Damerval [34] Briefly, three sets of three mature kernels were combined for each genotype and individually analyzed, generating three independent replicates Embryos and pericarp were manually excised, and the endosperms were crushed in liquid nitrogen for each genotype The proteins were resuspended in a urea-Triton X-100–2 ME buffer Isoelectric focusing was performed in 10-cm long rod gels in a pH gradient ranging from 5.5 to 8.5 Approximately 40 mg of total proteins were loaded on each gel The SDS dimension was separated using a 14% acrylamide slab gel, and staining was adapted from the colloidal Coomassie blue method of Neuhoff et al [35] Images of the 2D patterns were recorded and image analysis and spot detection were carried out as described by Consoli and Damerval [34] Specific zein protein extraction was previously used to confirm the zein identity of the polypeptide spots visualized in 2D gels [34] Statistical analyses of zein isoform amounts A previous analysis of colloidal Coomassie blue staining intensity as a function of protein loading was carried out for zein spots and it was demonstrated that for 86% of the isoforms, a linear relationship was obtained [34] Differences in total zein amounts loaded onto the gels were compensated by scaling the raw integrated optical density of every spot i in each gel j according to Consoli and Damerval [34] One-way analysis of variance with the genotype as the factor, were then performed for each spot on their scaled integrated optical density, and a significant effect was retained at P < 0.05 Enzymes partial purification and assays All procedures were carried out at °C unless stated otherwise Four replicates each composed of five selected maize ears, which were harvested (20 DAP), combined, and mixed, were used for enzyme analysis For the extraction of AK, frozen seeds were extracted in five volumes of buffer A [50 mM Tris/HCl, 200 mM KCl, 0.1 mM phenylmethanesulfonyl fluoride, 0.1 mM EDTA, mM dithiothreitol, mM L-lysine, mM L-threonine, 10% (v/v) glycerol and 5% (w/v) insoluble polyvynylpyrrolidone (PVPP), pH 7.4] The extract was filtered through three layers of miracloth, and centrifuged at 16 000 g for 30 to remove the cellular debris Solid ammonium sulfate was added slowly to 30% saturation with gently stirring for at least 30 The suspension was centrifuged at 16 000 g for Ó FEBS 2003 30 and the supernatant subjected to a second ammonium sulfate precipitation at 60% saturation for 30 with continuous stirring Precipitated protein was recovered by centrifugation at 16 000 g for 30 and the protein pellets were dissolved in a small volume of buffer B [25 mM Tris/HCl, mM dithiothreitol, 0.1 mM L-lysine, 0.1 mM L-threonine and 10% (v/v) glycerol, pH 7.4] The sample was loaded onto a Sephadex G25 column (2.5 · 20 cm) equilibrated with five column volumes of buffer B and run under gravity The desalted sample was collected and assayed for AK activity AK activity was assayed routinely in a final volume of 500 mL as described by Brennecke et al [28] Controls containing lysine and threonine were included to identify the isoenzymes sensitive to lysine and threonine Activity was expressed as nmolỈmin)1Ỉmg)1 protein Four replications were carried out for each assay For the extraction of HSDH, frozen seeds were extracted in five volumes of buffer C [50 mM Tris/HCl, 200 mM KCl, 0.1 mM phenylmethanesulfonyl fluoride, mM EDTA, mM dithiothreitol, mM L-threonine, 10% (v/v) glycerol and 5% (w/v) PVPP, pH 7.5] The extract was filtered through three layers of miracloth, and centrifuged at 16 000 g for 30 to remove completely cellular debris from the extract Solid ammonium sulfate was added slowly to 30% saturation with gently stirring for at least 30 The suspension was centrifuged at 16 000 g for 30 and the supernatant subjected to a second ammonium sulfate precipitation at 60% saturation for 30 with continuous stirring Precipitated protein was recovered by centrifugation at 16 000 g for 30 and the protein pellets were dissolved in a small volume of buffer D [25 mM Tris/HCl, mM EDTA, mM dithiothreitol, 0.1 mM L-threonine and 10% (v/v) glycerol, pH 7.5] The sample was loaded onto a Sephadex G25 column (2.5 · 20 cm) equilibrated with five column volumes of buffer D and run under gravity The desalted sample was collected and assayed for HSDH activity HSDH activity was assayed routinely spectrophotometrically at 340 nm in a final volume of 1.1 mL at 30 °C as described by Azevedo et al [11] The effect of threonine on the HSDH activity was determined by the addition (10 mL of a M solution) of the amino acid to the assay mixture Activity was expressed as nmolỈmin)1Ỉmg)1 protein Four replications were carried out for each assay For the extraction of LOR–SDH, frozen seeds were extracted in five volumes of buffer E [100 mM potassium phosphate, 50 mM KCl, mM EDTA, mM dithiothreitol, 0.1 mM phenylmethanesulfonyl fluoride, 10% (w/v) glycerol and 5% (w/v) PVPP, pH 7.0] The homogenate was first filtered through three layers of miracloth and then centrifuged at 15 000 g for 30 to remove cellular debris The supernatant was adjusted to 30% ammonium sulfate saturation by gently stirring for at least 30 The suspension was centrifuged at 15 000 g for 30 and the supernatant subjected to a second ammonium sulfate precipitation at 55% saturation for 30 with continuous stirring After centrifugation at 15 000 g for 30 min, the sedimented proteins were dissolved in 10 mL of buffer E (minus phenylmethanesulfonyl fluoride and PVPP) The sample was then loaded onto a Sephadex G50 column (2.6 · 20 cm) previously equilibrated with buffer F Ó FEBS 2003 Lysine metabolism in maize mutants (Eur J Biochem 270) 4901 [100 mM Tris/HCl, mM dithiothreitol, mM EDTA and 10% (v/v) glycerol, pH 7.4] and run under gravity The desalted sample was collected and assayed for LOR and SDH activities LOR activity was routinely assayed spectrophotometrically in a mL cuvette at 30 °C by following the change in absorbance at 340 nm over a 15-min period, with appropriate adjustments for a lysine-free blank as described by Gaziola et al [18] Activity was expressed as nmol NADPH oxidizedỈmin)1Ỉmg)1 protein Four replications were carried out for each assay SDH activity was measured spectrophometrically in a mL cuvette by following the rate of substrate-dependent reduction of NAD+ to NADH monitored at 340 nm at 30 °C over a 15-min period, with appropriate adjustments for a saccharopine-free blank as described by Gaziola et al [18] Activity was expressed as nmol NAD+ reducedỈ min)1Ỉmg)1 protein Four replications were carried out for each assay Protein determination Protein concentrations of the samples were determined as described by Bradford [36] using bovine serum albumin as a standard Results Distribution patterns of N constituents Table provides data concerning the percentage contribution of the main N constituents present in opaque (o), floury (fl) and wild-type (+) endosperms The amounts of SAA and NPN were of the same magnitude for the wildtype inbred line and all mutants The albumins and globulins of the mutants exhibited variable amounts, ranging from a value similar to that of the wild-type inbred line (Oh43fl1), to a value 3.5-fold higher (Oh43o2) The same mutant genotypes also marked the boundaries of variation of zein for the mutants, with the Oh43o2 endosperm being the poorest in zeins, whereas Oh43fl1 exhibited the highest amounts of zeins, but still lower than that of the wild-type Oh43+ In general, the mutants had protein distribution patterns varying between that of Oh43fl1, similar to that of the wild type, and that of Oh43o2 From these results it was possible to assess the importance of lysine-rich nonzeins with accuracy, because of the quantitation of nonprotein N and the exhaustive extraction of zeins Thus, the ratio of the nonzein content of the mutants compared with that of Oh43+ varied from 1.2 to 1.5 for most mutants, whereas for Oh43o2, a ratio of 2.6 was calculated Soluble lysine concentration The Oh43o2 mutant exhibited the highest relative concentration of soluble lysine followed by the Oh43fl2 mutant, whereas the Oh43o1 mutant exhibited the lowest relative concentration of soluble lysine, but still higher than that of the wild-type counterpart (Table 1) The Oh43o2 mutant also exhibited the highest absolute concentration of soluble lysine (7.35 nmolỈmg)1 dry weight) followed by the Oh43fl2 mutant (5.29 nmolỈmg)1 dry weight), whereas the Oh43o1 mutant exhibited the lowest absolute concentration of soluble lysine (1.65 nmolỈmg)1 dry weight), but still higher than that of the wild-type counterpart (0.82 nmolỈmg)1 dry weight) However, the total SAA pool also varied among the genotypes, indicating clear differences between the mutations The total SAA pool was increased slightly following the introduction of the mutations o2, fl1 and fl2, but was reduced by 20% by the o1 mutation (Table 1), however, the relative soluble lysine concentrations were increased considerably following the introduction of each mutation (Table 1) 2D-page Thirty-five zein polypeptides were detected in wild type Oh43+ and Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2 mutants Four polypeptides were identified as c27 kDa zeins, 10 as a22 kDa zeins, 15 as a19 kDa zeins, two as b14 kDa zeins, two as c16 kDa zeins and two as d10 kDa zeins, according to their apparent molecular masses in the SDS dimension (Fig 1) Between 20 and 31 zein isoforms were detected according to the genotype (Table 2) The mutations decreased the number of zein isoforms detected on the 2D gels as compared with the wild-type, indicating a decrease in zein amount and diversity (Fig 2) The effect of each mutation on the amount of every isoform was tested using analyses of Table Quantitation of N constituents Percentage contribution of N constituents present in opaque (o), floury (fl) and wild-type (Oh43+) endosperms Data expressed as percentage (± standard deviation) of endosperm total N N constituents: SAA, soluble amino acids; NPN, nonprotein N; A + G, albumins + globulins corresponding to E1,2 – NPN; non- zeins corresponding to glutelins (Glu) + albumins + globulins; P, endosperm total proteins expressed as percentage of dry matter Soluble lysine is expressed as percentage of total soluble amino acids pool (± standard deviation) followed by the percentage increase in soluble lysine in relation to the wild type N constituents Genotypes SAA Oh43+ Oh43o1 Oh43o2 Oh43fl1 Oh43fl2 0.75 0.60 0.95 0.82 0.94 NPN (0.07) (0.07) (0.00) (0.07) (0.14) A+G Zeins 1.6 2.4 2.9 1.9 2.7 3.2 4.0 11.1 3.7 6.7 77.4 68.6 41.5 71.9 64.8 17.8 25.0 44.5 22.5 25.8 Non-zeins Glu (0.28) (0.20) (0.35) (0.28) (0.35) (1.8) (0.4) (1.2) (0.8) (0.2) (1.0) (0.7) (1.6) (1.1) (0.6) P% DM Lysine % increase 21.0 29.0 55.6 26.2 32.5 10.8 11.6 8.7 12.8 11.2 0.33 0.53 2.70 0.83 1.46 – 61 718 151 342 (0.6) (0.5) (1.2) (0.8) (0.2) (0.02) (0.02) (0.07) (0.02) (0.10) 4902 R A Azevedo et al (Eur J Biochem 270) Ó FEBS 2003 Fig Two-dimensional separation of zein isoforms isolated from the endosperms of maize seeds in the Oh43 background, using isoelectric focusing and SDS/PAGE Wild-type Oh43+ key isoforms are indicated with black arrows The white arrow points to an isoform appearing specifically in the fl2 mutant Molecular masses and pH range are indicated along the gel variance on standardized spot volumes (see Methods) The o2 and fl2 mutations had large effects, as about 60% of the isoforms differed in amount as compared with the wild type Conversely, Oh43o1 and Oh43fl1 mutants exhibited zein patterns and contents similar to those of their wild-type counterpart, in agreement with the data of Table The pattern of c27 kDa isoforms was the most strongly affected by the fl2 mutation, as all the polypeptides disappeared, in contrast, fl1 had little effect Among the 10 a22 kDa zein class isoforms, only one appeared in a similar amount in all of the mutants and wild type (a22z2, Fig 1, 2) The mutations generally decreased the amount of the isoforms as compared with the wild type Among the 15 a19 kDa zein class isoforms, five were unaffected in the mutants The Oh43fl2 mutant exhibited the strongest effect on this zein class, altering the amount of nine isoforms, amongst which, two occurred specifically in this mutant (e.g a19z114, Fig 1, 2) The mutations fl2 and o2 had a parallel effect on b14 kDa and d10 kDa zein isoforms, but the effect of fl2 was less pronounced than that of o2 In all, the o2 mutation markedly altered the pattern of low molecular mass zeins, as compared with the wild type Lysine metabolism In this study, the enzymes AK, HSDH, LOR and SDH were extracted initially from the developing seeds (16, 20 and 24 DAP) of the wild type, which indicated that the main peak of activity of AK (4.32, 7.91 and 3.10 nmolỈmin)1Ỉmg)1 protein at 16, 20 and 24 DAP, respectively), HSDH (5.24, 16.31 and 6.16 nmolỈmin)1Ỉmg)1 protein at 16, 20 and 24 DAP, respectively), LOR (2.05, 3.53 and 2.18 nmolỈmin)1Ỉmg)1 protein at 16, 20 and 24 DAP, respectively) and SDH (2.37, 3.51 and 2.08 nmolỈ min)1Ỉmg)1 protein at 16, 20 and 24 DAP, respectively) was at 20 DAP The activities of the enzymes involved in lysine metabolism have been studied in maize endosperm, exhibiting a peak of activity between 16 and 24 DAP depending on the enzyme [9,28] In this study, the activities of the enzymes AK, HSDH, LOR and SDH were determined in extracts isolated from the wild-type 16, 20 and 24 DAP and the maximum activity for all enzymes was confirmed as 20 DAP Based on this peak of enzyme activity, all genotypes were subsequently analyzed at 20 DAP The activity of AK varied considerably among all genotypes, ranging from 2.78 nmolỈmin)1Ỉmg)1 protein in Oh43fl1)15.29 nmolỈmin)1Ỉmg)1 protein in the Oh43fl2 (Table 3) The Oh43o1 and Oh43fl2 mutants exhibited higher activities (12 and 85%, respectively) when compared with the wild type, whereas the mutants Oh43o2 and Oh43fl1 exhibited lower activities (40 and 66%, respectively) The inhibition by lysine was shown to be reduced in all four mutants when compared with the wild type (60.8% inhibition), ranging from 28.9% in the Oh43fl1 mutant to 55.6% in the Oh43o1 mutant The inhibitory effect of threonine on AK activity was much lower when compared with the effect of lysine, resulting in 11.1% inhibition of AK activity in the wild type, 15.6% in the Oh43fl2 mutant and 3.34% in the Oh43fl1 mutant, whereas a slight stimulation of AK activity was induced by threonine in the Oh43o2 mutant When both amino acids were added together, a more intense inhibitory effect was observed, with the wildtype Oh43+ exhibiting the highest inhibitory effect (92.5%) and the Oh43fl1 the lowest (41.8%) (Table 3) Ó FEBS 2003 Lysine metabolism in maize mutants (Eur J Biochem 270) 4903 Table Two dimensional separation of zeins isolated from maize endosperms Thirty-five zein isoforms were revealed Mean values of spot volumes are indicated Each isoform number is prefixed by the name of the zein class Statistical analyses were performed to test for significant differences in isoform amounts, and genotypes sharing a same letter did not differ significantly (a indicates an amount significantly greater than b) Spot name WT o1 o2 c27z45 c27z38 c27z28 c27z21 a22z4 a22z37 a22z31 a22z3 a22z2 a22z18 a22z12 a22z115 a22z11 a22z1 a19z93 a19z9 a19z8 a19z7 a19z6 a19z5 a19z43 a19z30 a19z23 a19z20 a19z19 a19z17 a19z15 a19z114 a19z10 c16z44 c16z13 b14z33 b14z14 d10z16 d10z109 2262.89(a) 8423.21(a) 7009.92(a) 29520.46(a) 51353.85(a) 4126.22(a) 7140.23(a) 26824.27(a) 26413.60(a) 9363.84(a) 18884.57(a) 23365.92(a) 81282.15(a) 51483.41(a) 25934.51(c) 65109.57(a) 65917.49(a) 92886.09(a) 2339.87(a) 7646.58(b) 10831.41(b) 16394.55(a) 11402.26(b) 19406.44(a) 12065.24(a) 45064.00(a) 4072.74(a) 31426.21(b) 7480.22(a) 71467.31(ab) 43782.08(bc) 688.72(a) 8712.80(a) 10278.86(b) 50168.37(a) 4266.46(a) 24194.50(a) 32646.71(a) 6607.25(a) 19780.78(a) 18853.00(a) 73752.64(ab) 43735.68(a) 18027.17(c) 50561.34(a) 78276.12(a) 99287.19(a) 0 22279.91(a) 19023.76(a) 20261.95(a) 4832.44(a) 43462.53(a) 2180.72(a) 37841.49(b) 7535.85(a) 83184.64(a) 52390.91(bc) 3303.82(b) 0 8342.94(a) 41558.61(a) 0 4797.66(b) 26387.42(a) 13033.15(a) 0 44042.71(b) 50534.32(a) 72930.41(a) 70261.18(a) 79297.66(a) 79080.27(a) 17683.49(a) 8024.70(b) 2706.90(b) 10550.99(b) 0 51245.73(a) 118849.72(a) 39052.79(c) 109105.04(a) 20394.88(a) The activity of HSDH varied considerably among all genotypes, ranging from 17.4 nmolỈmin)1Ỉmg)1 protein in Oh43+ to 38.4 nmolỈmin)1Ỉmg)1 protein in Oh43o2 (Table 3) All mutants exhibited higher activities when compared with the wild-type, with the Oh43o2 mutant exhibiting a 2.2-fold higher HSDH activity The effect of threonine was tested on HSDH activity, exhibiting an inhibitory effect in the wild-type and Oh43o2, Oh43fl1 and Oh43fl2 mutants, but stimulating HSDH activity in the Oh43o1 mutant (Table 3) Table shows the activities of the enzymes LOR and SDH, both involved in lysine degradation, which were also measured for all genotypes Large variations were observed for LOR activity, varying from 0.49 nmol NADPH oxidized min)1Ỉmg)1 protein in Oh43fl2)3.83 nmol NADPH oxidized min)1Ỉmg)1 protein in Oh43o1, which was even higher than the activity in the wild type (3.50 nmol NADPH oxidized min)1Ỉmg)1 protein) (Table 3) The Oh43o2 mutant exhibited a sixfold reduction of LOR activity, a reduction that was even higher (7.1-fold) in the Oh43fl2 fl1 25013.17(a) 8406.33(a) 16517.54(ab) 60342.63(a) 5461.84(a) 24681.31(a) 32919.48(a) 7301.23(a) 25436.17(a) 23972.68(a) 101923.68(a) 42954.38(a) 33846.97(bc) 50230.13(a) 75690.34(a) 63968.14(a) 6122.26(b) 8822.52(b) 12926.97(a) 19055.38(ab) 0 51198.85(a) 44790.63(b) 6828.42(a) 73327.86(ab) 27284.15(c) fl2 0 0 0 25758.31(a) 28709.67(a) 0 31431.73(a) 19571.06(a) 88989.89(a) 47289.65(a) 52618.48(a) 47679.73(b) 46370.74(a) 62619.74(a) 61067.52(a) 0 27136.28(a) 15690.45(a) 54852.13(a) 18178.69(ab) 22304.39(a) 46990.69(a) 23034.16(b) 51025.04(bc) 75793.39(ab) mutant Reduction of LOR activity was also observed in the Oh43fl1 mutant (40% lower) when compared with the wild type, whereas in the Oh43o1 mutant the activity was slightly higher than the wild type Similar reductions in SDH activity along with LOR, were also induced by the o2, fl1 and fl2 mutations and thus the LOR/SDH ratio did not exhibit major variations Discussion The opaque and floury mutations and their respective wildtype (Oh43+) were obtained from the Maize Genetics Cooperation Seed Stock Center (USA) and cultivated in Brazil for three successive summer seasons Very little variation among the genotypes was observed for time of flowering indicating a similar developmental behavior, which would be expected as all mutants are in the same genetic background This also allowed the self-pollination and production of seeds for all genotypes The content of the various N constituents in the endosperm is dependent on Ó FEBS 2003 4904 R A Azevedo et al (Eur J Biochem 270) Fig Mutants Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2 The arrows point to the isoforms indicated on the wild-type gel (Fig 1) Table Determination of activity of enzymes involved in lysine metabolism AK specific activity (nmolỈmin)1Ỉmg protein)1), HSDH specific activity (nmolỈmin)1Ỉmg protein)1), LOR specific activity (nmol NADPH oxidizedỈmin)1Ỉmg protein)1) and SDH specific activity (nmol NAD + reducedỈmin)1Ỉmg protein)1) were determined in extracts of 20 DAP maize endosperms and following the addition of lysine (L) and/or threonine (T) Standard deviation (SD) values were all below 5% for the L, T and LT treatments Genotypes Oh43 + Enzyme AK Control (SD) % inhibition by + % inhibition by + % inhibition by + HSDH Control (SD) % inhibition by + LOR (SD) SDH (SD) LOR/SDH ratio a mM L mM T mM LT mM T Oh43o1 Oh43o2 Oh43fl1 Oh43fl2 8.282 (0.314) 60.8 11.1 92.5 9.240 (0.371) 55.6 13.5 78.3 4.956 (0.121) 42.2 + 4.73a 83.2 2.783 (0.120) 28.9 3.34 41.8 15.290 (0.414) 37.5 15.6 49.5 17.4 (0.71) 31.0 3.505 (0.121) 3.490 (0124) 1.00 27.6 (0.88) + 26.1 3.830 (0.133) 3.050 (0.090) 1.25 38.4 (1.43) 25.0 0.590 (0.016) 0.705 (0.071) 0.83 19.8 (0.57) 6.1 2.115 (0.030) 1.870 (0.097) 1.13 21.0 (0.57) 17.1 0.490 (0.013) 0.890 (0.033) 0.55 Indicates activation of enzyme activity genetic and environmental factors With the view of dissociating these two factors, the present results were compared with data taken from the literature and concerning the same genotypes, but cultivated at diverse locations: Bergamo, Italy [37]; Orsay, France [32]; LaFayette, USA [38]; and Tucson, USA [31] Furthermore, for a better comparison, the genotypes were ranked according to an increasing content of zeins (Table 4): (a) Zein percentages Ó FEBS 2003 Lysine metabolism in maize mutants (Eur J Biochem 270) 4905 Table Zein and lysine determinations in distinct studies Percentage of zein and protein lysine in maize seeds References: PS: present study; Misra et al [38]; Di Fonzo et al [37]; Landry et al [32]; Hunter et al [31] Lysine percentage true proteins (estimated), values in parentheses correspond to lysine percentage crude proteins (assayed) Genotype Zeins Lysine percentage [Reference] W64Ao2 Oh43o2 Oh43o2 Oh43o2 W64Afl2 Oh43fl2 W64Ao1 W64+ W22+ Oh43fl2 Oh43o1 W22+ Oh43fl1 W22+ Oh43+ Oh43+ 28.7 41.5 47.1 49.3 50.0 64.8 66.4 67.8 65.5 65.7 68.6 68.7 71.9 74.3 77.4 77.5 3.8 3.78 3.14 (3.5) 2.8 2.34 1.7 1.5 – (2.3) 2.08 (2.3) 1.87 1.68 1.49 (1.6) 31 PS 32 38 31 PS 31 31 37 38 PS 38 PS 32 32 38 ranged from 28.7% (W64Ao2) to 77.5% (Oh43+) The difference between the minimum and maximum percentages was almost the same as that found by Balconi et al [39] between Illinois low protein (40%) and Illinois high protein (74.5%) genotypes, taking into account that these values are slightly (5%) underestimated as E4 proteins were excluded from zeins by the authors (b) The effect of environmental conditions upon the content of zeins for a given genotype can result in a discrepancy of 8–9% in the case of Oh43o2, W22o2 and W22 + or be negligible in the case of Oh43fl2 or Oh43+ (c) For a given mutant gene the genetic background can have a considerable impact upon the zein content, however, this is not always the case as can be seen with W64Ao1 and Oh43o1 (d) More generally, the gradual increase in zein content would indicate a progressive change in the relative proportions of soft and hard endosperms, respectively, poor and rich in zeins Therefore, the effect of one gene upon the distribution pattern of protein fractions cannot be generalized from that found for only one genetic background The opaque and floury mutants used in this study have been classified as high-lysine endosperm mutants, however, such higher concentrations of lysine can be due to alterations in the storage protein fractions and/or in the concentration of soluble lysine in the endosperm In previous studies, the soluble lysine concentration has been shown to be increased in the o2 maize mutant when compared with the wild-type maize [9,30,31] Estimating the percentage of lysine in true proteins by assuming the lysine content of nonzeins is independent of genotype and equal to g per 100 g of proteins, and based on the distribution of the endosperm proteins, the mutants exhibited higher concentrations of total lysine when compared with their wild-type counterpart We have also observed a significant variability in the absolute and relative soluble lysine concentrations among the mutants analyzed The o2 mutation led to an increase in the total SAA pool and in the soluble lysine concentration in the endosperm, confirming the previous reports for this mutant [2,13,30,31], although such increases may vary depending on the genetic background to which the gene is introduced [30,31] In the other mutants, distinct responses were observed in relation to lysine concentration, showing that the mutants Oh43fl1 and Oh43fl2, exhibited increases in total SAA and soluble lysine concentration, in a similar way to the Oh43o2 mutant, leading to higher lysine concentrations in the endosperm, but not to the same extent However the Oh43o1 mutant, exhibited a lower concentration of total SAA, but an increased concentration of relative and absolute contents of soluble lysine, which on balance indicates that the Oh43o1 mutant has a small significant increase (101%) in soluble lysine The results observed for the o1 mutation are similar to that reported by Hunter et al [31], who observed an amino acid composition similar to the wild-type counterpart On the other hand, Balconi et al [39] reported an increased concentration of total lysine in the o1 mutant to the same extent as that for the o2 mutant In general, all mutants can be classified as high-lysine mutants, but the increases in lysine observed were not as great as that observed for the o2 mutation Hunter et al [31] used one dimensional SDS/PAGE to compare qualitative and quantitative differences in zein patterns among a range of opaque mutants Except for o2, little effect of the mutations was observed The analysis was refined by immunoblotting with specific antisera, which demonstrated that in o2 there was a decreased amount a22 kDa, b14 kDa and d10 kDa isoforms, whereas in fl2 the a22 kDa zeins were reduced Using 2D electrophoresis, we were able to observe complex patterns of alterations in the mutants as compared with the wild type The various isoforms detected are not due to artifacts during protein isolation and/or fractionation, but to genetic differences in charge and amino acid content [40] A given mutation can increase or decrease the relative amount of different isoforms belonging to the same class of zein, indicating very specific effects In the Oh43 background fl1 and o1 mutations had very little effect A similar low effect was also observed for o1 in the W64A background [31] The mutations o2 and fl2 had their largest effects on the a22 kDa and c27 kDa zeins, mostly decreasing the amount of the isoforms present The effect of o2 on b14 kDa isoforms was consistent with a regulatory role of this transcriptional activator on these zein genes [23] In contrast to Hunter et al [31], we found that o2 increased rather than decreased the relative amount of the d10 kDa isoforms This may be due to a specific effect of the background, as we used Oh43 while Hunter et al [31] used W64A A large background effect on the range of o2 effects had already been observed (e.g [34]) The enzymes of lysine metabolism have been studied and characterized in several plant species [10] As wild-type maize and the o2 mutant were the only sources of information in the literature as far as lysine metabolism is concerned, we have used them as controls for our analysis of the other mutants The data in Table provide evidence that there is a wide variation in terms of AK activity, with several-fold variation in AK activity among the genotypes studied, which, in the case of the low rates in the Oh43o2 mutant, agreed with previous results published by other 4906 R A Azevedo et al (Eur J Biochem 270) authors [9,28] Two mutants, Oh43o1 and Oh43fl2 exhibited increases in AK activity, whereas the mutants Oh43o2 and Oh43fl1 exhibited a reduction in AK activity when compared with their wild type, Oh43+ AK activity has been shown to be determined by the action of at least two separate isoenzymes, one that is sensitive to lysine inhibition and the other sensitive to threonine inhibition [10] Furthermore, in higher plants, the lysine-sensitive isoenzyme normally accounts for 50–80% of the total AK activity, with the exception of AK activity in coix endosperm, in which the threonine-sensitive isoenzyme predominates [41] Independent of the mutation, in the Oh43 genetic background, lysine produced the stronger inhibition of AK activity, suggesting that the lysine-sensitive isoenzyme is predominant in this genetic background and that such distribution of isoenzymes activities is not affected by any of the introduced mutations Threonine inhibition of AK was low in all the lines, but there was evidence of a further reduction, caused by the o2 and fl1 mutations However, lysine inhibition was reduced in all the mutants when compared with the wild-type, particularly in fl1 and fl2 The presence of AK activity more insensitive to lysine and threonine inhibition was confirmed when both amino acids were tested together, resulting in less than 50% inhibition of the total AK activity in fl1 and fl2, with lesser reductions being detected in the opaque mutants Stimulation of HSDH activity by threonine was observed for the Oh43o1 mutant, however, no major effects on AK activity were observed in this mutant, which might indicate a specific effect of the o1 gene on the HSDH domain of the bifunctional polypeptide Apart from these results, HSDH activity does not appear to be under any particular influence from the mutations analyzed All the genotypes tested exhibited variations for threonine inhibition, suggesting the presence of both HSDH isoenzymes It has been suggested that HSDH does not have a regulatory role in the biosynthesis of lysine, although this enzyme shares the same substrate (aspartate semialdehyde) with DHDPS, which could eventually be a key point in determining the flux of carbon through the pathway, leading to threonine or lysine biosynthesis [10] Although a recent study using transgenic Arabidopsis thaliana expressing bacterial DHDPS and having knockout mutation for lysine catabolism produced high increases in soluble lysine and methionine [42], no evidence of an increase in soluble methionine was detected in the opaque and floury mutants analyzed in this work (data not shown) Evidence has been obtained from biochemical and molecular analyses that AK activity is possibly regulated by the o2 gene [13], intensifying its effect on the total pool of SAA and free threonine accumulation in maize endosperm [13] Moreover, one of the genes encoding a lysine-sensitive AK isoenzyme was linked to the o2 gene in chromosome [13] Wang et al [43] also observed that AK activity varied in its sensitivity to lysine inhibition, even between distinct lines in which the o2 was introduced Furthermore, several quantitative trait loci for SAA content have been identified, one of them linked to another AK-HSDH encoding gene [43] The analysis of o2 mutants has indicated that the lysine-sensitive AK isoenzyme, but not the bifunctional threonine-sensitive AK-HSDH isoenzyme, is affected by the mutation [43] Ó FEBS 2003 The enzymes of lysine catabolism, LOR and SDH, were also analyzed in all genotypes and exhibited significant alteration in activity depending on the mutant LOR and SDH were initially identified as one bifunctional enzyme containing both enzyme domains [12,17,18], whilst later monofunctional LOR and SDH enzymes were identified [12] The results reported in the literature generally indicated that SDH activity is more stable than LOR activity [5] The dramatic sixfold reduction of LOR activity in the mutant Oh43o2 also confirmed the results observed for the effect of the o2 gene on LOR activity [9], which is due to a decreased mRNA and enzyme protein synthesis [29] SDH activity was also influenced by the o2 gene, exhibiting a 4.9-fold decrease in enzyme activity, which is a greater reduction when compared with previous work with this mutant [9] The reduction in LOR and SDH activities observed for the Oh43o2 mutant was also observed in Oh43fl1 and Oh43fl2 mutants, with the latter exhibiting a LOR activity even lower that of the Oh43o2 mutant (7.1-fold) Our results suggest that the catabolism of lysine catalyzed by the enzyme LOR, may be under the regulation of the opaque and floury mutations This is in addition to the biosynthetic enzymes AK and to a lesser extent HSDH discussed previously The way this pleiotropic regulation can take place may be different according to the mutation It has been shown in previous studies in which the LOR and SDH enzymes were isolated and characterized, that the LOR has an essential role in the regulation of lysine catabolism, as this enzyme is modulated by Ca2+, ionic strength and protein phosphorylation/dephosphorylation in several plant species [19,29,44,45], however, such modulation effects not appear to influence SDH activity Pleiotropic regulation is also supported by the effect of the mutations on the storage proteins analyzed by 2D-PAGE In parallel with their considerable effect on LOR and SDH activity, both o2 and fl2 induced large alterations in the synthesis pattern of a22 kDa and c27 kDa zeins Furthermore, the Oh43o2 and Oh43fl2 mutants also exhibited higher concentrations of soluble lysine in the endosperm, not only based on its concentration, but accompanying the effect of each mutation on the concentration of the total pool of SAA Curiously, the Oh43o1 mutant, which has been classified as high-lysine, did not exhibit major effects on the catabolism of lysine in the endosperm, which suggests that the high lysine concentration cannot be explained by an altered lysine catabolism in this mutant Although there was a slight increase in the concentration of soluble lysine in the Oh43o1 mutant when compared with the other mutants, the lysine degradation enzyme pattern as well as the AK activity, was shown to be at the same level of the wild-type counterpart Furthermore, Hunter et al [31] who also analyzed this mutant, but in a different genetic background, could not find any important effect of this mutation The mutants Oh43o1 and Oh43fl1 exhibited little effect either on the zein polypeptides or on LOR and SDH activities The analysis of other mutations in the same phenotypic class as the o2 gene indicates that the mutations may strongly influence lysine metabolism and storage protein synthesis and accumulation in maize Many of the zein polypeptides have been shown to vary in these mutants and Ó FEBS 2003 Lysine metabolism in maize mutants (Eur J Biochem 270) 4907 a new range of studies must be carried out to determine the precise molecular regulation of the synthesis of these polypeptides by such mutations It is also clear that future studies on the effect of these mutations should also be carried out on the activity of the DHDPS enzyme, which has been shown to be a key regulatory step in lysine biosynthesis [5,10], but has only been tested in the o2 mutant so far [43] Acknowledgements This work was financed by grants to RAA from Fundacao de Amparo ¸ ˜ ` a Pesquisa Estado de Sao Paulo, Brazil (FAPESP 98/12461–0 and ˜ 01/13904–8) and the British Council (RAA and PJL) The authors also wish to thank the Conselho Nacional de Desenvolvimento Cientı´ fico e ´ Tecnologico (CNPq, Brazil) and FAPESP for 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Experimental procedures Maize mutants Seeds of the mutant genotypes opaque (o1 and o2) and floury (fl1 and fl2) and the respective wild type, Oh43 +, were kindly provided by the Maize Genetics Cooperation... present in opaque (o), floury (fl) and wild-type (+) endosperms The amounts of SAA and NPN were of the same magnitude for the wildtype inbred line and all mutants The albumins and globulins of the... true proteins by assuming the lysine content of nonzeins is independent of genotype and equal to g per 100 g of proteins, and based on the distribution of the endosperm proteins, the mutants exhibited