Lysine biosynthesis and nitrogen metabolism in quinoa (Chenopodium quinoa): Study of enzymes and nitrogen-containing compounds

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Plant Physiology and Biochemistry 46 (2008) 11e18 www.elsevier.com/locate/plaphy

Research article

Lysine biosynthesis and nitrogen metabolism in quinoa (Chenopodium quinoa): Study of enzymes and nitrogen-containing compounds Vanderlei A. Varisi a, Liliane S. Camargos b, Leandro F. Aguiar b, Renata M. Christofoleti a, Leonardo O. Medici c, Ricardo A. Azevedo a,* a

Departamento de Gene´tica, Escola Superior de Agricultura Luiz de Queiroz, Universidade de S~ao Paulo, Piracicaba, CEP 13418-900, SP, Brazil b Departamento de Cieˆncias Naturais, Universidade Federal do Mato Grosso do Sul, MS, Brazil c Departamento de Cieˆncias Fisiolo´gicas, Universidade Federal Rural do Rio de Janeiro, Serope´dica, RJ, Brazil Received 2 March 2007 Available online 6 October 2007

Abstract Aspartate kinase (AK, EC 2.7.2.4), homoserine dehydrogenase (HSDH, EC 1.1.1.3) and dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) were isolated and partially purified from immature Chenopodium quinoa Willd seeds. Enzyme activities were studied in the presence of the aspartate-derived amino acids lysine, threonine and methionine and also the lysine analogue S-2-aminoethyl-L-cysteine (AEC), at 1 mM and 5 mM. The results confirmed the existence of, at least, two AK isoenzymes, one inhibited by lysine and the other inhibited by threonine, the latter being predominant in quinoa seeds. HSDH activity was also shown to be partially inhibited by threonine, whereas some of the activity was resistant to the inhibitory effect, indicating the presence of two isoenzymes, one resistant and another sensitive to threonine inhibition. Only one DHDPS isoenzyme highly sensitive to lysine inhibition was detected. The results suggest that the high concentration of lysine observed in quinoa seeds is possibly due to a combined effect of increased lysine synthesis and accumulation in the soluble form and/or as protein lysine. Nitrogen assimilation was also investigated and based on nitrate content, nitrate reductase activity, amino acid distribution and ureide content, the leaves were identified as the predominant site of nitrate reduction in this plant species. The amino acid profile analysis in leaves and roots also indicated an important role of soluble glutamine as a nitrogen transporting compound. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Aspartate kinase; Dihydrodipicolinate synthase; Homoserine dehydrogenase; Lysine; Nitrate reductase

1. Introduction Chenopodium quinoa, commonly known as quinoa, belongs to the plant group known as pseudocereal, genus Chenopodium, family Chenopodiaceae [10] mainly grown and cultivated in the Andean region of South America. Quinoa has a high worldwide potential as a crop and has been described as an important source of protein for humans due to the digestibility and balanced essential amino acid composition [42].

* Corresponding author. Fax: þ55 19 3433 6706. E-mail address: [email protected] (R.A. Azevedo). 0981-9428/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2007.10.001

Nutritional composition analysis of quinoa seeds has revealed protein concentrations ranging from 7.47% to 22.08% with an average of 13.81% [9]. The balanced composition of quinoa proteins has also been described as similar to the casein protein [41]. Furthermore, there are significant quantities of oil, vitamins, minerals such as calcium and magnesium, and starch (60e70%) in the seeds [41]. On the other hand, the presence of saponins has been described as a negative factor affecting the consumption of quinoa due to the bitter taste produced [46]. Nitrate is normally the main source of nitrogen (N) available to higher plants [29] and a deficiency in N is the major limiting factor of productivity in many crops in world

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V.A. Varisi et al. / Plant Physiology and Biochemistry 46 (2008) 11e18

agriculture using low inputs [37]. N is an important component of amino acids, nucleic acids, coenzymes and other plant metabolites [1]. Amino acids are the main constituent of proteins and the major N-containing compounds in plants providing substrates for a wide range of cellular metabolism such as energy generation and cell wall synthesis, which are important in the growth and developmental processes of plants [7,19]. Nitrate taken up by plants is initially reduced to nitrite in a reaction catalyzed by nitrate reductase (NR, EC 1.6.6.1). Nitrite is then reduced to ammonium by nitrite reductase (NiR, EC 1.7.7.1), which is assimilated into glutamine (Gln) and glutamate (Glu) by the enzymes glutamine synthetase and glutamate synthase (GSeGOGAT; EC 6.3.1.2 and EC 1.4.7.1, respectively) [31]. NR activity is considered a limiting factor for nitrate assimilation in plants and distinct environmental conditions such as light and some metabolites such as sugars and amino acids, may affect the enzyme activity at both the transcriptional and posttranslational levels [30]. Amino acids are synthesized in a complex network controlled by enzymes, intermediate metabolites and final end products. Among the amino acids termed essential, lysine (Lys), threonine (Thr), methionine (Met) and isoleucine (Ile) are synthesized by the aspartate metabolic pathway in three distinct branches [4]. Aspartate kinase (AK), homoserine dehydrogenase (HSDH) and dihydrodipicolinate synthase (DHDPS) are considered to be key enzymes in Lys biosynthesis [5] whereas lysine ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) are important in controlling Lys catabolism [6,8,22,26,39,47]. The first enzymatic reaction of the aspartate pathway is catalyzed by AK, which has been isolated, purified and characterized at both biochemical and molecular levels from several plant species [19,34]. At least two isoenzymes of AK are normally present in plants, one as a monofunctional polypeptide sensitive to Lys inhibition and another as a bifunctional polypeptide containing both AK and HSDH domains, sensitive to Thr [4]. A very recent report has revealed that maize AK may also be involved in the control of germination efficiency [2]. HSDH is the first enzyme in the branch leading to Thr and Met synthesis, sharing with DHDPS the same substrate, baspartyl semialdehyde (ASA). Two isoenzymes, one sensitive and another resistant to Thr feedback inhibition, have been described in several plant species [19,34]. In the Lys branch of the pathway, DHDPS catalyses the condensation of pyruvate and ASA to produce 4-hydroxy-2,3,4,5-tetrahydrodipicolinate. Although quinoa has received a great deal of attention particularly in recent years and considering the fact that the seeds are a highly nutritious food product with a higher tryptophan (Trp) and Lys concentration [17], the regulation of Lys and nitrogen metabolism is totally unknown. In this study, we report the isolation and activity patterns of enzymes involved in Lys biosynthesis in developing quinoa seeds. Other aspects related to N assimilation and amino acid content have also been investigated. Such information will serve for future studies concerning seed nutritional quality and genetic manipulation for increased amino acid synthesis and accumulation.

2. Results 2.1. AK, HSDH and DHDPS activities during the development of quinoa seeds AK, HSDH and DHDPS enzymes exhibited very similar activity trends in the three developmental stages analyzed (Table 1). AK activity from 20 DAA immature seeds was further characterized in relation to the aspartate-derived amino acids and AEC. Lys at 1 mM and 5 mM inhibited AK activity by 19.5% and 23.8%, respectively (Fig. 1). Thr was also shown to inhibit AK activity at 1 mM (45.2%) and 5 mM (49.7%), whereas the combination of both amino acids added to the assay mixture at 1 mM and 5 mM, caused an inhibition of 37.6% and 47.6%, respectively (Fig. 1). Met and AEC did not produce any significant changes in AK activity at all concentrations tested (Fig. 1). The effect of these compounds on HSDH activity from 20 DAA immature seeds was also tested (Fig. 2). Lys, Met and AEC did not affect HSDH activity, whereas Thr was effective in inhibiting HSDH activity at 1 mM (46.9%) and 5 mM (63.9%) (Fig. 2), which was not significantly altered when added in combination with Lys. The addition of Lys to the assay mixture induced a strong inhibition on DHDPS activity from 20 DAA immature seeds at 1 mM (65.3%) and 5 mM (69.9%), whilst AEC inhibited DHDPS activity by 28.7% and 60.1% at 1 mM and 5 mM, respectively (Fig. 3). The addition of Thr and Met to the DHDPS assay did not alter DHDPS activity (Fig. 3). 2.2. NR activity, nitrate concentration and ureides’ compounds NR activity was higher in leaves than in roots in all stages analyzed (Fig. 4A), but in both tissues NR activity decreased during development (Fig. 4A). The nitrate concentration increased during development and significant differences between leaves and roots were observed only at stage 2 (Fig. 4B). Ureides were detected in leaves and roots of quinoa in the three stages studied (Fig. 5). The highest concentration of allantoate was observed at stage 3 in both tissues (Fig. 5A), whereas the allantoin concentration remained constant during vegetative development (Fig. 5B). 2.3. Amino acid composition The concentration of total soluble amino acids remained unaltered during development with leaves exhibiting a higher Table 1 AK, HSDH (nmol min1 mg1 protein) and DHDPS (units mg1 protein) (SD) specific activities from C. quinoa seeds at three stages of development Enzymes

AK HSDH DHDPS

Seed development stages 16 DAA

20 DAA

24 DAA

9.57 (0.53) a 4.63 (0.09) a 6.80 (0.36) b

9.51 (0.15) a 4.44 (0.15) a 7.72 (0.20) a

9.66 (0.41) a 4.55 (0.26) a 7.28 (0.20) ab

Values for the same enzyme sharing the same letters did not differ significantly at 0.05 (Tukey test).

10

*

8

* *

6

*

*

*

4 2 0

13

9

12

DHDPS activity (units.mg-1 protein)

AK activity (nmol.min-1.mg-1 protein)

V.A. Varisi et al. / Plant Physiology and Biochemistry 46 (2008) 11e18

C

K1

K5

T1

T5

KT1 KT5

M1

8 7 6

*

5 4 3

*

K1

K5

2

*

*

*

1 0

M5 AEC1 AEC5

*

C

T1

T5

KT1

KT5

M1

M5 AEC1 AEC5

Fig. 1. Effect of the addition of amino acids (K, lysine; T, threonine and M, methionine) and S-2-aminoethyl L-cysteine (AEC) at 1 mM and 5 mM concentrations on AK activity from 20 DAA immature seeds. Asterisk (*) indicates significant difference when compared to the control at 0.05 (Dunnet test).

Fig. 3. Effect of the addition of amino acids (K, lysine; T, threonine and M, methionine) and S-2-aminoethyl L-cysteine (AEC) at 1 mM and 5 mM concentrations on DHDPS activity from 20 DAA immature seeds. Asterisk (*) indicates significant difference when compared to the control at 0.05 (Dunnet test).

total concentration than roots (Fig. 6). In leaves, Glu was the main amino acid accounting for 35.66% of the total soluble amino acids pool, while in roots Gln was predominant accounting for 28.13% of the total soluble amino acids pool (Table 2). In seeds, Glu was also the main soluble amino acid corresponding to 21.46%, whereas the aspartate-derived amino acids Lys, Thr and Met accounted for 1.72%, 5.09% and 4.89%, respectively (Table 2). However, when the amino acids incorporated into protein were analyzed, glutamate and glutamine (Glx) were predominant corresponding to 20.86% of the total protein amino acids, while Lys accounted for 4.26% (Table 3).

Lys and Thr synthesis. These investigations are essential if the synthesis and accumulation of amino acids and storage proteins are to be understood and they should eventually lead to genetic manipulation of quinoa and other plant species. The activities of AK, HSDH and DHDPS were detected in the three seed developmental stages analyzed, exhibiting very

Leaves Roots

Aa

Ab 120

80 Ba 40

Bb Bc Stage 1

Stage 2

Stage 3

120

6

B 100

5 4 3

*

* *

2

*

Bb

80 Ab 60

Aa

Aa

Leaves Roots Aa

Ac

40 20

1 0

Aa

160

0

Nitrate (µmol.g-1· FW)

HSDH activity (nmol.min-1.mg-1 protein)

The key enzymes controlling the aspartate metabolic pathway in plants have been isolated, purified and some regulatory properties characterized [4,7,48]. However, in quinoa, the pathway has never been investigated as far as we are aware, thus this report is likely to be the first one to have isolated and analyzed the activity of the three enzymes involved in

A NR (µmol. NO2-. g-1 FW. h-1)

3. Discussion

200

C

K1

K5

T1

T5

KT1

KT5

M1

M5 AEC1 AEC5

Fig. 2. Effect of the addition of amino acids (K, lysine; T, threonine and M, methionine) and S-2-aminoethyl L-cysteine (AEC) at 1 mM and 5 mM concentrations on HSDH activity from 20 DAA immature seeds. Asterisk (*) indicates significant difference when compared to the control at 0.05 (Dunnet test).

0

Stage 1

Stage 2

Stage 3

Fig. 4. Variation in the level of NR activity (A) and nitrate content (B) in root and leaf of Chenopodium quinoa during three vegetative stages. Tissues with the same capital letters did not differ at 0.05 (Tukey test). Stages with the same lowercase letters did not differ at 0.05 (Tukey test).

V.A. Varisi et al. / Plant Physiology and Biochemistry 46 (2008) 11e18

14 1800

Allantoate (nmol.g-1 FW)

1600

A

Leaves Roots

1400 Aa 1200 1000 Ab

800

Ab

Ba

Bb 600

Bc

400 200 0

Stage 1

Stage 2

Stage 3

1800

Allantoin (nmol.g-1 FW)

1600

B

Leaves Roots Aa

1400 Aa

1200

Aa

Aa

Table 2 Soluble amino acids (%) (SD) in three different tissues of C. quinoa Amino acids

Leaf

Root

Seed

Aspartate 17.61 (1.89) b 9.69 (0.43) c 6.71 (0.85) d Glutamate 35.66 (2.09) a 18.81 (0.36) b 21.46 (1.80) a Asparagine 1.20 (0.21) f 4.09 (0.62) ef 2.36 (0.32) hij Serine 7.70 (0.69) dc 5.02 (0.58) e 3.21 (0.48) fghij Glutamine 9.39 (0.01) c 28.13 (0.67) a 4.30 (0.34) defgh Histidine N.D. N.D. N.D. Glycine 3.81 (0.04) ef 5.06 (0.53) e 6.28 (1.17) de Threonine 5.78 (1.69) de 5.21 (0.33) e 5.09 (0.97) def Arginine 3.92 (0.20) ef 0.78 (0.06) jk 9.92 (0.26) c Alanine 6.90 (0.21) dce 4.54 (0.11) e 17.54 (0.71) b Tyrosine 1.85 (0.72) f 7.65 (0.22) d 4.15 (1.58) efghi Methionine 1.47 (0.26) f 2.03 (0.16) ghi 4.89 (0.39) defg Valine 1.06 (0.06) f 3.10 (0.26) fg 5.31 (0.58) def Phenylalanine 0.67 (0.25) f 0.33 (0.11) k 1.37 (0.23) j Isoleucine 1.01 (0.03) f 2.62 (0.32) gh 3.14 (0.28) fghij Leucine 0.79 (0.07) f 1.78 (0.26) hij 2.54 (0.28) ghij Lysine 1.19 (0.06) f 1.16 (0.08) ijk 1.72 (0.16) ij Values for the same column sharing the same letter did not differ significantly at 0.05 (Tukey test). N.D., not detected.

Aa

1000 Aa 800 600 400 200 0

Stage 1

Stage 2

Stage 3

Fig. 5. Variation in the level of allantoate (A) and allantoin (B) in Chenopodium quinoa in three vegetative stages. Tissues with the same capital letters did not differ at 0.05 (Tukey test). Stages with the same lowercase letters did not differ at 0.05 (Tukey test).

similar levels, indicating that any of the three studied stages could be used as source of activity to study the regulatory properties of the enzymes. A similar result has recently been reported for AK and HSDH from sorghum seeds [19], while

in coix the highest activities for all enzymes of the aspartate pathway were observed at the corresponding developmental stage 2 [34]. In maize, the highest level of AK and HSDH activities was observed at 16 DAP, decreasing to nearly zero at 28 DAP [25], which followed the pattern of storage protein synthesis and accumulation. Based on this result, 20 DAA immature seeds were used on the following analyses of AK, HSDH and DHDPS. Since the addition of both Lys and Thr was able to partially inhibit AK activity, it is most likely that at least the two well described isoenzymes of AK, the Lys-sensitive and the Thrsensitive forms, are also present in quinoa seeds as have been observed in other plant species [7]. It is also clear that Thr was a more effective inhibitor of AK activity than Lys, indicating that the Thr-sensitive AK is predominant in quinoa seeds. In fact, this result was first clearly reported in coix immature seeds [34] and later in sorghum seeds [19] and it is

3.0 Aa

Amino acids (µmol.g-1 FW)

Aa 2.5

Aa

Leaves Roots

2.0

1.5

Ba Ba

1.0

Ba

0.5

0.0

Stage 1

Stage 2

Stage 3

Fig. 6. Variation in the level of total soluble amino acids of Chenopodium quinoa in three vegetative stages. Tissues with the same capital letters did not differ at 0.05 (Tukey test). Stages with the same lowercase letters did not differ at 0.05 (Tukey test).

Table 3 Amino acids incorporated into protein (%) (SD) in C. quinoa mature seeds Amino acids

Seed

Aspartate þ asparagine Glutamate þ glutamine Serine Histidine Threonine þ glycine Arginine Alanine Tyrosine Methionine Valine Phenylalanine Isoleucine Leucine Lysine

10.04 20.86 7.90 8.06 14.99 8.66 7.59 2.33 0.39 3.51 2.69 3.62 5.09 4.26

(0.36) (0.86) (0.09) (1.58) (0.42) (0.22) (0.23) (0.15) (0.04) (0.33) (0.23) (0.36) (0.22) (0.87)

c a d d b dc d g h efg fg efg e ef

Values sharing the same letter did not differ significantly at 0.05 (Tukey test).

V.A. Varisi et al. / Plant Physiology and Biochemistry 46 (2008) 11e18

likely to be correlated with the flux of carbon and nitrogen through the aspartate pathway leading mainly to the synthesis of Lys. For HSDH, the only clear significant effect was the inhibition induced by Thr, whereas all other compounds did not affect the enzyme activity. Although Thr was effective in causing HSDH inhibition at 1 mM, HSDH activity could not be inhibited completely by this amino acid even at 5 mM. This result suggests that two isoenzymes of HSDH are also present in quinoa, one sensitive and another resistant to Thr inhibition [7]. Such a result was further confirmed by the addition of Thr and Lys together to the assay mixture, which did not alter the inhibition observed by Thr. Interestingly, the 46% and 63% inhibition observed at 1 mM and 5 mM Thr, respectively, indicates that the Thr-sensitive isoenzymes predominate in quinoa seeds, which correlates with the high Thr-sensitive AK activity already discussed, suggesting that the bifunctional Thr-sensitive AKeHSDH polypeptide identified in other plant species may also exist and predominate in quinoa seeds. Such an increasing inhibitory effect at the higher Thr concentration used is also particularly important since HSDH and DHDPS share the same substrate, ASA. Thus, with any cell Thr accumulation may change the flux through the pathway by a stronger inhibition of HSDH activity, possibly diverting C and N to the Lys biosynthetic branch of the pathway, which is mainly regulated by DHDPS. As observed in other plant species studied [7], DHDPS was inhibited by Lys suggesting the existence of at least one DHDPS isoenzyme, which is strongly inhibited by Lys. This result is further supported by the inhibition also observed when AEC was tested, confirming that Lys exerts a feedback inhibition on DHDPS activity controlling its own synthesis as described previously in different plant species [7]. AEC has been shown to be able to inhibit both AK [19] and DHDPS [24] activities and has also been shown to be able to substitute for Lys in proteins [4]. The addition of AEC to the assay did not affect significantly AK or HSDH activities, but was able to inhibit DHDPS activity, although not in the same extent as that obtained with Lys particularly at 1 mM. Such a result deserves further investigation, since AEC in some plants did not produce any effects as an inhibitor and was unable to substitute for Lys as a substrate for LOR [13]. The effect of Met, another aspartate-derived amino acid, on AK and HSDH activities has been described previously in other plant species [19,35] and although in sorghum seeds Met was able to inhibit AK activity [19], in quinoa seeds no effect was observed on any of the enzymes tested. Although some amino acids have been described to exert an important role in the regulation of the aspartate pathway by the feedback inhibition of AK, HSDH and DHDPS, this mechanism is not well understood in plants [23]. The sensitivity of these enzymes to inhibition by Lys or Thr is determined by domains that apparently bind these amino acids [23]. The site of nitrate reduction has been described to be shoots [14,36,43] or roots [38], depending on the plant species and exogenous nitrate availability [18]. Based on NR activity, quinoa performed a great proportion of nitrate reduction in the leaves, which may be advantageous for this species in using directly the product of photosynthesis. Although, NR is

15

regulated by a variety of factors, nitrate has been described as a primary signal for NR control [16]. An increase in nitrate concentration did not cause an induction of NR activity in quinoa, it is therefore possible that only certain levels of nitrate may be responsible for the induction of NR effect. Chen et al. [16] reported that high levels of nitrate cause an induction of NR activity in the leaf blades of rape, cabbage and spinach, however, such NR activity increases were not significant among the highest nitrate concentrations tested. It has been described that the activity of NR may be affected by an increase in amino acids such as Gln and Asn, or that these amino acids may affect the uptake of nitrate causing an indirect effect on NR activity [3,44,45]. As discussed before, in quinoa an increase in the nitrate concentration leads to a reduction in NR activity. Since it has been reported that Gln at approximately physiological levels can inhibit NR activity in maize roots directly rather than by altering nitrate uptake [45], it is possible to suggest that such an amino acid may be a candidate factor responsible for the reduction in NR activity observed in leaves and roots of quinoa. The effect of amino acids was also suggested in Canavalia ensiformis, in which the predominant site of nitrate reduction was shown to change during development. Nitrate reduction occurred in the leaves during the vegetative growth period, but in the roots during flowering, and such a change appeared to be correlated with the Gln content [14]. The presence of important levels of allantoate and allantoin in the roots and leaves of C. quinoa has also been detected. In plants, the ureides allantoin and allantoate are formed during purine metabolism and in some legumes both compounds play an important role as nitrogen transport compounds [20]. Ureides are probably present in all plants since they are intermediates in the catabolism of purines, however, they may not necessarily accumulate to concentrations above the level of detection. Quinoa appears to be a non-legume plant species, which accumulate ureides to significant concentrations, exhibiting a tremendous potential to be used in studies involving such compounds in non-legume plant species. The amino acids’ profile observed in the leaves and roots suggests an important function of Gln as a N transport compound in quinoa as has been described for other species [31]. Interestingly, Asn another key transport amino acid [32] was present at much lower concentrations. Glu was the predominant soluble amino acid in all tissues, confirming the important homeostatic role of the amino acid in plants [21]. In the seeds, important amounts of the aspartate-derived amino acids Thr, Met, Ile and Lys were observed in both the soluble and protein forms. In the case of Lys, the results may indicate a reduced activity of the enzymes LKR and SDH causing a reduction in Lys catabolism as observed in other plant species [5,25,33]. In addition, Glu and Gln were the predominant amino acids incorporated into proteins. Quinoa also exhibited high Lys and Thr concentrations in proteins, confirming the previous report by Ruales and Nair [42], who observed high concentrations of aspartic acid (predominant), Lys and Thr in quinoa seeds after acid hydrolysis. Furthermore, the storage protein fractions albumins and globulins are also predominant

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V.A. Varisi et al. / Plant Physiology and Biochemistry 46 (2008) 11e18

in quinoa, whereas the deficient Lys storage protein fraction, prolamin, accounts for insignificant amounts of the total protein pool [9]. Such a result does not follow the normal distribution of storage proteins of cereal crops such as maize, in which prolamins account for 55e70% of the total storage proteins of the seed [27,28]. Quinoa is a species that is gaining importance as a source of protein for humans due to the digestibility and the balanced essential amino acid composition. Particularly, the predominance of the Thr-sensitive AK isoenzyme and the strong inhibition of the HSDH enzyme by Thr may be an important characteristic in C. quinoa, since Thr accumulation may change the C flux through the pathway by an inhibition of HSDH activity leading to the Lys biosynthesis and consequently, higher availability of Lys to be incorporated into proteins, particularly albumins and globulins, which may explain the important amount of Lys found in this species. However, further investigations involving the characterization of LKR and SDH, which are involved in Lys catabolism in quinoa seeds may help to elucidate the regulatory aspects of Lys catabolism in this plant species. 4. Methods 4.1. Material C. quinoa BRS Piabiru seeds were provided by Dr. Carlos Roberto Spehar, Centro de Pesquisa Agropecua´ria dos Cerrados, EMBRAPA e Empresa Brasileira de Pesquisa Agropecua´ria, Brazil. Seeds were grown in a glasshouse at ESALQeUSP, Brazil, under natural light conditions, with temperature ranging from 25 to 35  C, in 1.2 l pots with two plants per pot containing sand. A volume of 0.5 l of Hoagland’s solution containing 15 mM nitrate was reapplied every 2 days. In the first experiment, to analyze nitrogen assimilation, plants were harvested during the vegetative development at 20, 28 and 36 days after germination (stages 1, 2 and 3). In a second experiment, for evaluation of the aspartate metabolic pathway enzymes, seeds were harvested during development at stages 16, 20 and 24 days after anthesis (DAA), placed in liquid nitrogen and stored at 80  C. All experiments were carried out using three repetitions from independent biological harvests. 4.2. Extraction and partial purification of AK, HSDH and DHDPS For extraction of AK and HSDH enzymes all procedures were carried out at 4  C. Immature seeds harvested at 16, 20 and 24 DAA were extracted in 5 volumes of 50 mM TriseHCl buffer (pH 7.4) containing 200 mM KCl, 0.1 mM phenylmethanesulphonyl fluoride, 0.1 mM EDTA, 1 mM dithiothreitol, 2 mM L-Lys, 2 mM L-Thr, 10% (v/v) glycerol and 5% (w/v) insoluble polyvinylpyrrolidone. The extract was filtered and centrifuged at 12,000 g for 30 min to remove the cells’ debris from the extract. Solid ammonium sulphate was added to 30% of saturation by stirring for 30 min. The sample was centrifuged at 12,000 g for 30 min and the supernatant was subjected to

a second ammonium sulphate saturation at 60%. The sample was precipitated by centrifugation at 12,000 g for 30 min and the pellet was dissolved with a small volume of 25 mM Trise HCl buffer (pH 7.4) containing 1 mM dithiothreitol, 0.1 mM L-Lys, 0.1 mM L-Thr and 10% (v/v) glycerol. The sample was loaded on a Sephadex G-25 column equilibrated with the same buffer and the desalted sample was collected and used to measure AK and HSDH activities. For extraction of DHDPS enzyme all procedures were carried out at 4  C. Immature seeds harvested at 16, 20 and 24 DAA were extracted in 5 volumes of 100 mM TriseHCl buffer (pH 7.5) containing 1 mM phenylmethanesulphonyl fluoride, 2 mM EDTA, 1.4% (w/v) ascorbic acid and 5% (w/v) insoluble polyvinylpyrrolidone. The extract was filtered and centrifuged at 12,000 g for 30 min to remove the cells’ debris from the extract. Solid ammonium sulphate was added to 30% of saturation by stirring for 30 min. The sample was centrifuged at 12,000 g for 30 min and the supernatant was subjected to a second ammonium sulphate saturation at 60%. The sample was precipitated by centrifugation at 12,000 g for 30 min and the pellet was dissolved with a small volume of 100 mM TriseHCl buffer (pH 7.5) containing 2 mM EDTA and 1.4% (w/v) ascorbic acid. The sample was loaded on a Sephadex G-25 column equilibrated with the same buffer and the desalted sample was collected and used to measure DHDPS activity.

4.3. AK, HSDH and DHDPS assays AK activity was assayed in a final volume of 500 ml as described by Brennecke et al. [12]. Controls containing 1 mM and 5 mM L-Lys, L-Thr, L-Lys plus L-Thr were include to ensure that the activity measured was due to AK and to identify the isoenzymes sensitive to Lys and Thr. The effect of 1 mM and 5 mM L-Met and AEC on AK activity was also investigated. Activity was expressed as nmol min1 mg1 protein. HSDH activity was assayed spectrophotometrically at 340 nm in a final volume of 1.0 ml as described by Azevedo et al. [5]. HSDH activity was determined in the presence of 1 mM and 5 mM L-Lys, L-Thr, L-Lys plus L-Thr, L-Met and AEC. Activity was expressed as nmol min1 mg1 protein. DHDPS activity was assayed in a final volume of 250 ml as described by Wallsgrove and Mazelis [50] with modifications according to the results obtained in this investigation. The assay mixture comprised 25 ml of 100 mM TriseHCl buffer (pH 8.0), 25 ml of 100 mM pyruvate, 25 ml of 10 mM ASA (neutralized just before the use with NaOH), 125 ml enzyme extract and water. The assay was incubated at 35  C for 1 h and stopped by the addition of 1 ml of stop buffer (0.22 M citric acid, 0.55 M sodium phosphate and 0.25 mg mL1 of o-aminobenzaldehyde). The mixture was incubated at 35  C for 1 h to allow color formation. After centrifugation at 12,000 g for 10 min, the absorbance was determined at 520 nm. DHDPS activity was determined in the presence of 1 mM and 5 mM L-Lys, L-Thr, L-Lys plus L-Thr, L-Met and AEC. One unit of enzyme activity is arbitrarily defined as the amount of enzyme that produces a change in A520 of 0.001 min1.

V.A. Varisi et al. / Plant Physiology and Biochemistry 46 (2008) 11e18

17

4.4. NR activity determination

References

NR activity was determined as previously described by Radin [40]. Discs of fresh leaves and roots were previously vacuum infiltrated and incubated for 1 h in the dark at 30  C in a solution containing nitrate. The absorbance was determined at 540 nm.

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4.5. Extraction of N-containing compound and amino acids incorporated into protein The non-protein nitrogen (including soluble amino acids) from fresh leaves and roots and mature seeds were extracted in 10 volumes of a solution containing methanol/chloroform/ water (6/2.5/1.5, v/v/v) as described by Azevedo et al. [5]. For the extraction of amino acids incorporated into proteins in seeds, 10 volumes of 0.1 M NaOH was added to the pellet obtained in the first step and the mixture was homogenized. After centrifugation at 4000 g for 40 min, the supernatant was transferred to a new tube. HCl (4 mL, 6 N) was added to a solution containing 10 mg of protein. The solution was incubated at 105  C for 22 h. 4.6. Nitrate, total amino acid, allantoate and allantoin determinations Nitrate concentration was determined by the method of Cataldo et al. [15]. The assay mixture comprised 100 ml of sample and 400 ml of 5% salicylic acid. The assay was incubated for 20 min at room temperature and 9.5 ml of 2 N NaOH were added to the mixture. The absorbance was determined at 410 nm. Soluble amino acid and protein amino acid contents were determined by the ninhydrin assay as described by Azevedo et al. [5] and allantoate and allantoin were determined by the method of Vogels and Vanderdr [49]. 4.7. Protein determination The protein concentration was determined by the method of Bradford [11] using bovine serum albumin as a standard. 4.8. Amino acid analysis For the quantification of amino acids, the samples were analyzed as o-phthaldialdehyde (OPA) derivatives by analytical high-performance liquid chromatography (HPLC) as previously described by Azevedo et al. [5]. Acknowledgements This work was funded by the Fundac¸~ao de Amparo a` Pesquisa do Estado de S~ao Paulo (FAPESP, Grant no. 04/ 16039-4). The authors also thank the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico for the fellowship and scholarship, and the Coordenac¸~ao de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES, Brazil) for scholarship, and Dr. Carlos Roberto Spehar for providing quinoa seeds.

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