Genotypic variability of nitrogen metabolism enzymes in nodulated roots of Vicia faba

0038-0717(93)EOOlO-J

GENOTYPIC ENZYMES

Soil Bid. Bioehem. Vol. 26, No. 6, pp. 785-789, 1994 Coovrieht CT, 1994 ElsevierScienceLtd

Printed’& drea;Britain. All rights reserved 0038-0717/94$7.00+ 0.00

VARIABILITY OF NITROGEN METABOLISM IN NODULATED ROOTS OF ~1CW FABA

JUAN M. CABA, CARMEN LLUCH and FRANCISCOLIGERO* Grupo de Investigation

Fijacion de Nitrogeno, Departamento de Biologia Vegetal, Universidad de Granada, Granada 18071, Spain (Accepted 9 October 1993)

Summary-Twenty-nine genotypes of Viciafaba were grown in the presence or absence of NO; to study variability in root and nodule nitrate reductase (NR, EC 1.6.6.1) and nodule cytosolic glutamine synthetase (GS, EC 6.3.1.2) activities. These V. fuba lines apparently lack constitutive root NR activity (NRA), whereas inducible activity was detected in all of them except line VF17. Although a marked genotypic variability was found for this activity (F-value = 400) the major factor affecting its expression was nitrate (95% of the total effect observed). Most of the lines, however, showed appreciable rates of constitutive NRA in their nodules, that further declined with 4 mM NO,. High genotypic variability was also found for the level of GS activity from the plant fraction of V. faba nodules. This activity, little affected by 2 mM NO,, was generally declined by 4 mM for a week at the end of the experiment. No correlation was found between the activities measured here and measurements of symbiotic performance. Nodule NR, however, correlated inversely with both GS and nodule protein content.

INTRODUCTION In legumes, nitrogen can be incorporated by the assimilation of soil nitrogen, mostly in the form of nitrate, and by atmospheric N, fixation when the plant is in symbiosis .with bacteria of the genera Rhizobium or Bradyrhizobium. A full understanding of the relationship between NO; reduction and N, fixation is needed to help maximize the use of both N sources by nodulated legumes. Nitrate reductase (NR) and nitrogenase co-exist in legume root nodules, and it has been suggested that nodule nitrate reduction contributes to N economy in the plant (Randall et al., 1978; Vance and Heichel, 1981). On the other hand, nodule nitrate reduction has also been claimed to be a major factor in the inhibitory effect of NO; on N2 fixation (Heckmann et al., 1989; Kanayama et al., 1990). Temperate legumes reduce a large proportion of the NO; in the roots, a fact that may lead to higher amounts of photosynthate being translocated toward the root system (Wallace, 1986; Chalifour and Nelson, 1988a). This in turn may improve the supply of carbohydrates to the nodules, to fuel both N, fixation and NO; reduction. The ammonium thus produced is assimilated into glutamate through the cytosolic glutamate synthase cycle (Robertson and Farnden, 1980). Marked variability for the activities of this pathway was observed in alfalfa (Groat et al., 1984; Egli et al., 1989) clover (Cresswell et al., 1992) and pea (Rosendahl et al., 1989) germplasms, where they also *Author for correspondence.

correlated positively with N, fixation. Moreover, in bean, GS has been suggested to be a rate limiting step (Hungria et al., 1991; Pacovsky and Fuller, 1991). In previous studies with six inbred lines of V. faba, we found high genotypic variability for NO; reduction (Caba et al., 1990a) and ammonium assimilation (Caba et al., 1993) activities. In the present work we studied root and nodule NR and cytosolic glutamate synthetase (GS) activities, and their relationships with symbiotic performance, in a larger number of lines.

MATERIALS AND METHODS Plant material and growth conditions Of the 29 genotypes of V. faba used for this study, 28 inbred lines were kindly provided by Dr A. Martin (CSIC, Cordoba, Spain). The remaining genotype was the commercial cultivar Alborea (Semillas Pacific0 S.A., Sevilla, Spain). The procedure for seed germination, inoculation and plant growth was as described by Caba et al. (1990a). Plants grew with nutrient solution (Rigaud and Puppo, 1975) containing 2 mM KNO,. One week before flowering, nitrate concentration was raised to 4 mM in half of the plants. As controls, one set of plants from each genotype was grown continuously on solution without NO,. All genotypes were harvested at the beginning of flowering (on different days, but at the same physiological stage) (Caba et al., 1990a). Root systems were rinsed with tap and distilled water and nodule and root samples were kept on ice until 785

JUAN M. CABA et

786

al.

enzyme extraction. Plant dry weight was recorded after drying for 24 h at 70°C.

Table 2. Nitrate reductase activity bmol NO; (g fr. wt))’ h- ‘1 in nodules of different lines of V. fiba inoculated with R. legumi~wrwn GRLl9 and treated with 0. 2 or 4 mst KNO,

Enzyme extraction and assays

Line

Extraction and in vitro assays for root and nodule NR (EC 1.6.6.1) were described by Caba et al. (1990a). Nodule GS (EC 6.3.1.2) was determined by the y-glutamyl hydroxamate (y GH) semibiosynthetic assay, according to Caba ef al. (1993). The reaction was linear for at least 30min, and a total time of 15 min was used in all assays. Two blanks, minus L-glutamate and minus enzyme, and a zero-time control were included. Soluble protein in nodule extracts was determined by the Bradford method (1976), using bovine serum albumin as the standard (lo-501.18 protein).

VF4 VF16 VF17 VF21 VF22 VF31 VF38 VF40 VF44 VF46 VF49 VF51 VF59 VF60 VF72 LSD (0.05)

INO, 1OW

INO;

0

2

4

0.33

0.28

0.08

0.71 0.03 0.89 0.45 0.49 0.65 0.25

0.90 0.03 0.68 0.61 0.47 0.52 0.13

0.44 0.02 0.52 0.62 0.35 0.42

0 0.35 0.29 1.07 1.82 1.68 0.25

0.04 0.08 0.30 1.58 1.40 2.22 0.26

0.11 0 0.13 0.35 0.94 0.98 1.40 0.14

Line VF81 VFW9 VF125 VF127 VF132 VFl47 VF166 VF168 VF172 VF175 VFI88 VF242 VF247 Alborea

1(mM)

0

2

4

0.02 0 0.11 0.37 I .28 0.02 0.17 0.05 0.79 0.05 0.42 0.24 0.14 0.79

0.02 0.05 0.08 0.57 I .46 0.04 0.20 0.20 0.69 0.06 0.50 0.22 0.13 0.67

0.05 0.07 0.05 0.30 0.95 0.09 0.16 0.12 0.49 0.04 0.29 0.15 0.12 0.5 I

0.19

Statistical design and analysis

The experiment had a randomized block design, and was repeated at least once. Data, expressed as the means of four replicates, are representative of two experiments. The results were subjected to a 2-way analysis of variance (ANOVA) and the least significant difference (LSD) test between means.

RESULTS

AND

DISCUSSION

When plants grew on solution without NO;, only 10 lines showed in vitro NR activity (NRA) in the roots (Table l), which was assumed to represent the constitutive NR. This finding should be viewed with caution, however, as activity approached the limit of detection except in VF72, confirming the results of Caba et al. (1990a). Constutitive NR activity has been described in leaves of soybean (Streit et al., 198?), field bean (Chalifour and Nelson, 1988a; J. M. Caba and F. Ligero, unpubl.) and alfalfa (Deroche and Babalar, 1987). In the last study the authors also reported constitutive activity in roots, although Arrese-Igor et al. (1991) detected activity only in Table 1.Nitrate reductase activity bmol NO; (g fr. wt))’ h -‘I in roots of different lines of V. faba inoculated with R. leguminosarum GRL19 and treated with 0, 2 or 4 mM KNO, [NO; 1 (mW Line VF4 VFl6 VF17 VF21 VF22 VF3l VF38 VF40 VF44 VF46 VF49 VFSI VF59 VF60 VF72 LSD (0.05)

ING;

0

2

4

0 0 0.07 0 0 0 0.07 0.07 0 0 0 0 0 0 0.11

1.06 0.91 0.07 2.21 0.67 0.54 0.75 0.19 0.58 0.46 0.23 1.16 0.20 0.40 0.77

1.41 0.78 0.08 2.10 1.21 0.94 1.33 0.46 1.30 1.20 0.97 2.79 0.32 0.65 2.42

Line VF81 VF109 VFIZS VF127 WI32 VF147 VFl66 VF168 WI72 VFl7.5 VFISS VF242 VF241 Alborea 0.1 I

1(m@

0

2

4-

0.04 0 0 0.03 0 0 0.06 0 0 0 0.05 0.03 0.06 0

1.23 0.60 1.30 1.31 0.61 0.46 0.58 0.38 1.25 0.16 1.04 0.61 0.13 0.30

2.71 2.15 2.07 3.07 1.70 3.54 0.99 0.97 2.25 0.48 3.76 I.66 0.61 0.67

nodules. Sawhney et al. (1991) also found constitutive activity in pigeon pea throughout the plant. In plants grown on 2 mM NO; solution, NRA was detected in the roots of all lines, although it varied widely from Iess than 0.1 (VF17) to 2.27 (VF2 I) p mol NO; (g FW)-’ h-’ (P < 0.05, Table 1). This mainly inducible activity was not related to constitutive NRA. Further stimulation of activity, ranging from 38 to 600% for VF4 and 147 respectively, was achieved by increasing the NO; supply to 4 mM during the last week of culture. This response was not observed, however, in lines VFI 6, 17, and 2 1. Nitrate treatment was the factor that most strongly affected NRA, accounting for >95% of the total effect observed (based on analysis of variance). However, the influence of plant genotype on NRA expression in roots of V.&&z was highly signi~cant (P < 0.001). An additional finding that should be highlighted is the apparent lack of inducible NRA in roots of line VF17; even higher concentrations of NO; (data not shown) did not induce NR. The pattern of NRA expression in nodules of the same lines differed markedly in both response to NO; and dependence on piant genotype (Table 2). Except for VFI09 and 44, all lines expressed NRA in the plant fraction of their nodules in the absence of NO; (termed constitutive activity), which varied from GO.05 (VF17, 81, 147, 168, and 175) to > 1.6 (VF59 and VF60) I.tmol NO; (g fr. wt)-’ h-‘. From the resutts in Table 2 we inferred that in most inbred lines and in cv. Alborea, nodules express only constitutive NR, as no significant stimulation was obtained after they were fed with 2 mM NO; from sowing, at least under our experimental conditions. Moreover, this activity was reduced by NO; after both long-term 2 mM treatment (VF21, VF46, and VF59) and shortterm 4 rnM treatment (VF59, 132, 172, and 158 and Alborea). These results confirm previous observations in fewer genotypes (Caba et al., 1990a), in which 8 mM NO; caused a dramatic decline (55%) of nodule activity.

Variability of nitrogen metabolism in Vi& fubiu When plants grew on 2 tTIM NO; solution, nodule NRA was significantly stimulated only in lines VFI 6, 51, 60, and 127, but this effect was inverted by 4 mM NO< applied at the end of the experiment (Table 2). Constitutive and inducible nodule NRA have been described in a number of legumes (Streeter, 1985; Deroche and Babalar, 1987; Ligero et al., 1987; Chalifour and Nelson, 1988b; Arrese-Igor et al., 1989; Becana et al., 1989; Kanayama et al., 1990; Sawhney et al., 1991), although contradictory data have been reported for some of the species. The diversity of experimental conditions as well as host genotype-bacterial strain combinations used is a plausible explanation for this situation. Moreover, the physiological significance of the nodule’s ability to reduce nitrate has been debated for years (Vance and Heichel, 1981; Arrese-Igor et al., 1991). Regardless of this controversy, on a whole plant basis the nodule contributes appreciably to the root system’s potential to reduce nitrate in V. f&z, a species with root-based nitrate reduction (Sutherland et al., 1984; Wallace, 1986). In the presence of 2 mM NO;, total nodule activity represented over 10% of root activity in 10 lines, reaching up to 60% in VF59 and 60, which belong to medium- and high-symbiotic performance groups respectively (Caba et al., 1994). On the other hand, lines unable to reduce NO; in nodules, according to the criteria of Caba et al. (1990a), also differed in N, fixation or NO; utilization efficiencies, as judged by their responses to NO; supply (Caba et al., 1993). In the plant fraction of nodules, ammonium, a by-product of nitrogenase and of nitrate reduction, is assimilated into glutamine by cytosolic GS, a key reaction in the overall symbiotic process. This process has been reported to be a limiting factor in Nz fixation in bean (Hungria et al., 1991; Pacovsky and Fuller, 1991). Preliminary results suggested that GS might be associated with symbiotic efficiency in V. fuba genotypes (Caba et al., 1993). All genotypes used in this study expressed nodule GS activity, at rates between 101 (VF22) and 327 (VF109) pmol yGH (g fr. wt))’ h-’ (Table 3) when grown under fully symbiotic conditions (solution without NO;). This activity was little affected in either direction when plants were supplied with 2 mM NO; from the time of sowing, although in many cases the differences were statistically significant (P < 0.05). Line VF147 was the most markedly affected, showing 60% stimulation. In contrast, negative effects were never >20%. increasing NO; from 2 to 4 mM for a week caused a significant, generally greater decline in activity in 17 lines (Table 3), VF22 being inhibited by up to 66% as reported by Caba et af. (1993). Inhibition of nodule GS by NO; (Groat and Vance, 1981; VCzina et al., 1988), stimulation (Sawhney et al., 1985) or no apparent effect (Schuller et al., 1986) have been reported in other legumes. A more interesting result may be the high genotypic variability observed in nodule GS activity

181

Table 3. Glutamine synthetase activity bmol ?GH (g fr. wt)-’ b ‘1 in nodules of different lines of V. .fahainoculated with R. kgumin~~arum GRLl9 and treated with 0, 2 or 4rn~ KNO,

[NO; I bW

[NO; l(mM) Line VF4 VF16 VF17 VF2l VF22 VF3l VF38 VF40 VF44 VF46 VF49 VF5l VF59 VF60 VF72 LSD (0.05)

0

2

4

I45 173 197 177 101 204 210 I55 174 234 291 205 140 196 296

I50 140 179 157 III 196 190 127 21 I 211 283 161 II0 198 319

153 II8 184 161 60 179 139 I35 194 195 213 174 81 214 315

Line VF8l VF109 VP125 VFl27 VF132 VF147 VF166 VF168 VF172 VFl75 VF188 VF242 VF247 Alborea

0

2

4

287 327 223 207 152 I41 235 236 I91 167 198 215 164 I36

284 317 186 205 139 225 I91 199 170 136 210 218 217 139

286 317 170 I85 136 I95 197 210 146 142 186 198 178 II9

II

expression in V. faba, which represents over 65% of the total effect (ANOVA). There are few physiological and biochemical studies of nodule activities in large numbers of genotypes. To our knowledge, similar studies have been done only in alfalfa (Groat et al., 1984), a species in which lower amounts of variability were found. V. faba nodules have normal GSnl and GSn2 (Caba et al., 1990b); however, the specific polypeptide composition in a particular association, and in different germplasm sources, as well as the effect of NO; on isoenzyme composition and activity, are not yet known. When activity was expressed on a unit-noduleprotein basis (deduced from Tables 3 and 4, data not shown), specific activity paralleled activity gg’ fresh weight in most lines, so that genotypic variability and responses to NO; remained similar, although the rank order of activity may have changed. However, valid comparisons of different germplasm sources to elucidate the physiological significance of specific activity requires knowledge of the enzyme : non-enzyme protein ratios. Table 4. Soluble protein content [mg (g fr. wt)-‘1 in nodules of different lines of V. faba inoculated with R. k-guminosarum GRL19 and treated with 0, 2 or 4rn~ KNO,

[NO, I(r@

[NOTI Line VF4 VF16 VF17 VF2l VF22 VF3l VF38 VF40 VF44 VF46 VF49 VF5l VF59 VF60 VF72 LSD (0.05)

0

2

4

13.4 IO.1 II.5 10.5 5.3 9.6 II.5 II.0 9.4 12.7 13.5 IO.1 9.7 9.6 10.9

13.0 9.7 IO.5 9.2 6.1 9.7 9.2 II.1 II.0 8.4 12.0 9.2 9.5 9.7 12.0

10.9 8.6 10.6 8.1 5.6 8.5 9.2 9.7 12.1 8.7 II.4 8.7 9.5 II.0 10.6

Line VF8l VF109 VFl25 VF127 VF132 VF147 VF166 VF168 VF172 VFl75 VF188 VF242 VF247 Alborea 0.6

0

2

4

13.4 12.7 14.8 13.2 9.2 8.0 10.4 15.2 9.4 9. I 9.1 12.1 5.8 9.3

13.1 II.1 10.5 II.5 9.3 II.9 8.3 12.0 8.4 8.3 II.9 12.7 6.0 7.8

II.9 II.9 11.3 9.7 8.6 7.7 9.8 13.7 7.0 8.6 9.0 13.4 4.6 7.8

JUAN M. CABA et al.

788 Table 5. Simple correlation R. leguminosorunr GRLl9

mg N per plant Days of culture Root NR Nodule

NR

Nodule

GS

Protein content

matrix among parameters determined in different lines of V. faba inoculated with with 0. 2 or 4 nw KNO,. Values in italics correspond to plants grown without nitrate. *I’ s 0.05: +*P Q 0.01: ***P 4 0.001

and treated

Plant dry weight

mg N per plant

Days of culture

Root NR

Nodule NR

Nodule GS

0.909*** 0.922**’ 0.686*** 0.638’** -0.108 0.009 - 0.023 - 0.024 0.048 0.119 0.122 0.128

0.544*** 0.500*** 0.004 0.032 -0.144 0.234 0.147 0.199 0.158 0.200

-0.234’ 0.017 0.018 0.066 -0.160 - 0.144 -0.036 - 0.151

-0.094 - 0.295 0.008 0.254 -0.169 - 0.028

-0.333** - 0.345 -0.210* - 0.248

0.579*** 0.612*”

The inbred lines of V.fuba we investigated showed very high genotypic

variability

for root and nodule

NR as well as for nodule cytosolic GS activities. Moreover, these lines also varied markedly in symbiotic efficiency (Caba et al., 1994). As seen in Table 5, however, no correlation was observed between these nitrogen metabolism activities of nodulated roots and measurements of symbiotic performance. On the other hand, nodule NRA correlated inversely with nodule GS and soluble protein content, while the two latter were directly and significantly correlated. These findings minimize the significance of these metabolic variables as potential traits of interest in breeding programs for this crop. However, the possibility remains that different experimental approaches, i.e. host genotype-bacterial strain combinations, genotypes of different growth habit, and time-course studies of activities, may reveal different relationships. Acknowledgements-We thank Dr A. Martin for providing Vicia faba pure lines, and MS Karen Shashok for revising the English style. Financial support was obtained from the Plan Andaluz de Investigacibn, CICYT, and INIA.

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