The effect of nitrogen source on GS activities and polypeptides

in 5 mM NO3 (Figure 5.4B and D). The total GS activities in each tissue were

also measured (Figure 5.5).

The most significant effect of the N source on GS was that GS2 was present in roots of plants grown on NO3 but not detectable in NH^^-grown roots. Both

GS2 and GS l polypeptides were present in the stems and leaves of L. japonicus under all three N-regimes, and the N source had little effect on the relative proportions of the two proteins in these tissues.

In L. japonicus, the absence of GS2 protein in the roots of plants grown on 5 mM N H / was reflected in the total GS activity, which was significantly less (at the 95% confidence level) than those grown on 5 mM NO3 {ca 50%; Figure

5.5A). The GS activity in roots grown on 0.2 mM NO3 was also significantly

lower than those on 5 mM NO3 , and surprisingly, so too was the activity in roots

grown on 20 mM NO3 . However there was no significant effect of the N source on leaf or stem GS activity. There was little or no change in GS activity in roots, stems or leaves of L. corniculatus grown under the three N conditions (Figure 5.5B).

When separated by 2-D electrophoresis (Figure 5.6), at least two of the charge variants that were detectable in roots of L. japonicus grown on 5 mM NO3*

were also identifiable in roots grown on 0.2 mM NO3 , and in the radicle

germinated on sterile distilled water. However most of the GS2 charge variants were absent in roots grown on NH^^, with the exception of the charge variant

closest to the more basic end of the lEF gradient, which was present in both L. japonicus and L. corniculatus. Therefore, either the GS2 polypeptide is present

constitutively at low levels in the roots of plants grown on N H /, or NO; was not entirely excluded from the treatment.

5 .4 .4 The presence o f a putative vegetative storage protein in the leaves o f L. japonicus.

The total proteins in the leaf tissue of L. japonicus plants grown on either 20 mM NO; or 0.2 mM NO; were separated by 2-D electrophoresis, and detected by silver staining (Figure 5.7). It was apparent that one protein, although present at low levels in leaves grown under the low NO; regime, was expressed

abundantly in the leaves grown on high N O ;. This protein was estimated to be approximately 22 kD in size, and may be a vegetative storage protein as described by Staswick (1990).

5.4.5 The nitrate and amino acid composition o f both species grown on different nitrogen sources

The differences in enzyme activities between the different N treatments and between the two species, prompted further investigation of their respective N metabolism. High performance liquid chromatography was used to determine the amino acid composition of the leaf, stem and root of plants grown on either 2 0

mM N O ;, 5 mM NO; or 5 mM NH^^. These were tabulated along with the concentrations of NO; found in each tissue, and, in L. japonicus, the NH*^ levels (Table 5.2 and 5.3).

root asparagine levels were (as a proportion of the total amino N) 63%, 54% and 85 % respectively, and although the actual concentration of asparagine was much higher in tissues of L. japonicus grown on than on NOg" (e.g. ten-fold higher in the roots of NH^^-grown plants), the percentage of total amino N represented by asparagine in plants grown on 5 mM NOg was very similar to that in those grown on 5 mM (e.g. 59%, 57% and 69% in leaf stem and root respectively). This implies that asparagine is the major N transport compound in L. japonicus

regardless of N source.

The other major amino acids in the leaves and stems under all the N regimes were glutamate, aspartate, serine, GABA and alanine. The relative proportions of these amino acids (as a percentage of total amino N) were very similar in plants grown under each N regime. Glutamate levels were particularly high, comprising 13-24% of the total reduced N in the leaves and ca 10% in the stem in all three treatments, and the concentration of GABA was at its highest in the stem, comprising 4-8% of the total amino N.

The total concentration of amino N in the roots was 10-fold greater in the NH4+ treatment than under the two NO, treatments, primarily due to the extremely

high level of asparagine (85% of the total). The roots grown on also had very high levels of glutamine (ca 8 % of the total N), an amino acid not detected in

roots of plants grown on NO3 , and present at levels six- to nine-fold higher than in

NH/-grown leaf and stem tissue. The other major amino acids in the roots were

glutamate, aspartate and serine, although they were present at lower levels than in the other tissues.

B) The major N compounds found in L. corniculatus.

As was the case in L. japonicus, asparagine was the predominant amino acid in L. corniculatus tissues (Table 5.3). However the proportion of amino N contributed by asparagine was actually higher in the leaf and stem of plants grown on 5 mM NO3 (40% and 64% respectively) than in those grown on 5 mM NH^+

(15% and 38%) and this was reflected in the concentrations of asparagine in these two tissues, i.e. greater than 3 ^mol.g'fwt in NO;-grown plants and approximately

1 ^mol.g ’fwt in NH/-grown plants. The asparagine content in the roots of all three treatments was very similar (75-83% of total amino N). The asparagine concentrations in the roots of the NO,-grown L. corniculatus plants were two- to four-fold higher than those in L. japonicus, but the concentration in NH^^-grown roots represented less than a third of what was found in roots of NH^^-grown L. japonicus.

The other major amino acids in the leaves and stems were, as in L.

japonicus, glutamate, aspartate, serine, alanine and GABA. Alanine comprised up to 16% of the amino N in the leaves of L. corniculatus, in contrast to only 4-8% in leaves and stems of L. japonicus. Unlike in L. japonicus, where the

concentration of GABA was highest in the stem (up to 8 %), GABA was more

Glutamine levels in roots of L. corniculatus grown on (13% of total N) were higher than was found in L. Japonicus (8 % of total N), and glutamine was again

virtually undetectable in the NO,-grown plants.

C) The concentration o f NOj in both species

As might be expected, the concentration of NO; in both species when grown on 20 mM and 5 mM NO; was veiy high (see Table 5.2 and 5.3), comprising 90-96% of the total N (soluble in methanol) in L. japonicus and 83- 96% in L. corniculatus. The NO; concentrations in the leaf, stem and root of L. corniculatus grown on 5 mM NO; were only 35-50% that found in L. japonicus. In plants grown on 20 mM NO; the concentration of NO, in the leaf of L.

corniculatus was also 50% lower than that found in L. japonicus, but the stem and root NO; concentrations in the two species were very similar. This suggests that in the 5 mM NO; treatment the NO; was either not being transported as efficiently to L. corniculatus shoots, or was being diluted by growth (L. corniculatus being much larger than L. jcponicus), or was being assimilated rather than stored. The higher shoot NR activity in L. corniculatus (see Chapter 5.3.1) suggested that assimilation was the reason. In the NH^^ treatment, low levels of NO; were found in tissues of both species, although these were just at the limits of detection.

5.4.6 Differences between the two species in protein concentration, fresh