(Received for publication, January 11, 1996)
From the
The primary metabolic fate of phenylalanine, following its
deamination in plants, is conscription of its carbon skeleton for lignin,
suberin, flavonoid, and related metabolite formation. Since this accounts
for 30-40% of all organic carbon, an effective means of
recycling the liberated ammonium ion must be operative. In order to
establish how this occurs, the uptake and metabolism of various
N-labeled precursors (
N-Phe,
NH
Cl,
N-Gln, and
N-Glu)
in lignifying Pinus taeda cell cultures was investigated, using a
combination of high performance liquid chromatography,
N NMR,
and gas chromatography-mass spectrometry analyses. It was found that the
ammonium ion released during active phenylpropanoid metabolism was not
made available for general amino acid/protein synthesis. Rather it was
rapidly recycled back to regenerate phenylalanine, thereby providing an
effective means of maintaining active phenylpropanoid metabolism with no
additional nitrogen requirement. These results strongly suggest that, in
lignifying cells, ammonium ion reassimilation is tightly
compartmentalized.
The successful colonization of
land by vascular plants, from their aquatic forerunners, was in large
measure due to elaboration of the phenylpropanoid/phenylpropanoid-acetate
pathways. At this critical juncture in evolution, phenylalanine (tyrosine)
became the portal entry of phenols into lignins, lignans, flavonoids,
suberins, and proanthocyanidins. Vascular plants thus have a very high
Phe/Tyr turnover, since 30-40% of all assimilated carbon in
photosynthesis is of phenylpropanoid/phenylpropanoid-acetate origin(1, 2, 3, 4, 5) .
Phenylpropanoid metabolism is not only a feature of normal developm ent, but can also be induced. For example, when loblolly pine (Pinus taeda) cell suspension cultures are exposed to high levels of sucrose(6) , there is an induction of lignin synthesis. Curiously, little attention has been paid to the issue of the relationship between phenylpropanoid and nitrogen metabolism(7) . This is surprising since there are many indications from physiological studies of significant metabolic relations between nitrogen depletion and the build-up of aromatic compounds, e.g. Lotus pendunculatus produces flavolans under nitrogen-limiting conditions, but not when nitrogen is provided(8) .
Scrutiny of the prearomatic pathway, leading to Phe/Tyr, and subsequent phenylpropanoid/phenylpropanoid-acetate metabolism reveals some noteworthy features. First, prephenate accepts an amino group from glutamate via transamination, constituting the point whereby nitrogen is introduced(9, 10, 11, 12, 13, 14, 15) . Second, when Phe/Tyr are committed to phenylpropanoid metabolism, rather than to protein or alkaloid synthesis, nitrogen (as the ammonium ion) is immediately removed via the appropriate lyase reaction (16, 17, 18) (Fig. S1). Third, for every mole of cinnamate (p-hydroxycinnamate) formed, an equimolar amount of ammonium ion is generated. Consequently, an efficient means of nitrogen recycling must exist within cells undergoing active phenylpropanoid metabolism, otherwise severe nitrogen deficiency would result. A possible mechanism for recycling is shown in Fig. S2, where the ammonium ion released during lysis is metabolized via glutamine synthetase/glutamate synthase to generate glutamate(19, 20, 21, 22) , thereby permitting arogenate synthesis, and hence completion of the cycle.
Scheme 1: Biosynthetic routes leading to the formation of the aromatic amino acids, tyrosine (Tyr) and phenylalanine (Phe), from prephenate.
Scheme 2: Proposed scheme for nitrogen recycling in P. taeda during active phenylpropanoid metabolism.
The operation of
such a phenylpropanoid-nitrogen cycle during lignification was established
using actively lignifying P. taeda cell cultures. This was
carried out by examining the uptake, metabolism, and product formation of
several N-labeled precursors, in the presence and absence of
specific enzyme inhibitors, as described below.
HPLC was performed on a Waters model 600E
system controller, fitted with a Waters Ultra Wisp model 717 sample
processor, and a photodiode array detector (Waters model 996) equipped
with a NEC Power Mate 2 personal computer. HPLC separations were carried
out using a Pico-Tag column (3.9 300 mm), with detection at 254
nm, using the procedure of Hagen et al.(33) . The
eluent system consisted of two solvent systems: eluent 1, 975 ml of 0.07
N sodium acetate, titrated to pH 6.5 with glacial acetic acid,
and mixed with 25 ml of acetonitrile; and eluent 2, 450 ml of
acetonitrile, 150 ml of methanol, and 400 ml of Milli-Q water measured
separately and then mixed. Elution was carried out in several steps at a
flow rate of 1 ml min
as follows: 100% eluent 1 to 13.5
min; 13.5 min, step to eluents 1:2 (97:3); 13.5-24 min, concave
curve (Waters no. 8) to eluents 1:2 (94:6); 24-30 min, convex curve
(Waters no. 5) to eluents 1:2 (91:9); 30-50 min, linear change to
eluents 1:2 (66:34); 50-62 min, and held at this condition;
62-62.5 min, linear change to 100% eluent 2; 62.5-66.5 min,
held at 100% eluent 2; 66.5-67 min, linear change to 100% eluent 1;
67-87 min, held at 100% eluent 1. The total run time between
injections including column reequilibration was 87 min. Quantitation of
the Phe, Gln, and Glu pools in the different amino acid samples was made
from calibration curves obtained from derivatizing and analyzing known
amounts of each amino acid.
GC-MS was performed on a Hewlett
Packard 5989A GC-MS system operating in the EI-mode. All separations were
performed on a 15 m 0.25-mm (internal diameter) DB-5MS column
(0.25-µm filter), with helium as a carrier gas at 5 p.s.i. The source
temperature was 250 °C, and the injector and interface temperatures
were at 275 °C, with an electron multiplier voltage of 2200 V for all
applications. For analysis of the N-DMTBS amino acid samples, the oven
temperature program was raised from 175 °C (5 min) to 275 °C at 10
°C/min and then held for 12.5 min. The mass range scanned was
50-650 atomic mass units for electron ionization studies. The
specific isotopic abundance of Phe, Gln, and Glu was determined by
plotting extracted ion current profiles and calculating ratios from the
following ion clusters: Phe (
N:
N, 336:337); Gln
(
N
N:
N
N:
N
N, 431:432:433);
and Glu (
N:
N, 432:433). The following equations
for specific isotopic calculations (isotope = I) were used to
substract the contribution of natural abundance
N,
C,
H,
O,
O,
Si, and
Si as follows (34) .
Phe-2TBDMS: (M
, m/z 393);
[M
- C
H
(m/z
57)] =
C
H
NO
Si
(m/z
336)/C
H
NO
Si
(m/z 337);
N = I
;
N = I
-{17
0.011
(
C) + 30
0.00015 (
H) + 0.0037
(
N) + 2
0.0004 (
O) + 2
0.051 (
S)} I
= I
- 0.298 I
. Gln-3TBDMS: (M
,
m/z 488); [M
-
C
H
(m/z 57)] =
C
H
N
O
Si
(m/z
431)/C
H
N
NO
Si
(m/z
432)/C
H
N
O
Si
(m/z 433;
N
= I
;
N
N = I
- {19
0.011 (
C) + 43
0.00015 (
H)
+ 2
0.0037 (
N) + 3
0.0004
(
O) + 3
0.051 (
Si)}
I
= I
- 0.377 I
;
N
= I
- 0.373
N
N -
{0.021(
C
) + 3
0.002
(
O) + 3
0.034 (
Si) + 0.0078
(
Si
)} I
=
I
- 0.373
N
N - 0.137
I
. Glu-3TBDMS: (M
m/z 489;
[M
- C
H
(m/z
57)] =
C
H
NO
Si
(m/z
432)/C
H
NO
Si
(m/z 433;
N = I
;
N
= I
- {19
0.011 (
C)
+ 42
0.00015 (
H) + 0.0037 (
N)
+ 4
0.0004 (
O) + 3
0.051
(
Si)} I
= I
-
0.374 I
.
The apparent metabolic fate of the ammonium ion released during
metabolism of N-Phe was examined, under conditions where
cells were undergoing active lignin synthesis. Thus, 10 mM
N-Phe (99.9 atom %
N) was administered to
lignifying P. taeda cell cultures, these then being allowed to
metabolize over a 4-day period, with cells removed at 24-h intervals for
N NMR spectroscopic analyses. At t = 24 h,
three clearly resolved signals (data not shown) were observed at
91.1, 18.3, and 16.7 ppm. These resonances were assigned to the
-amide nitrogen of Gln,
-nitrogen atoms of Gln and Glu, and the
amino group of Phe, respectively, based on chemical shifts of authentic
standards and previously published data(24, 25, 26, 27, 28, 29, 30, 31, 32) . By 96 h, additional resonances at
19.9
and 13.2 ppm were also evident (Fig. 1A); these were
attributed to alanine and serine as previously assigned in nitrogen
metabolism studies with white spruce (Picea glauca) buds(24) . Importantly, no resonances at any stage corresponding to
NH
were observed, in accordance
with earlier observations, such as with potato (Solanum
tuberosum) slices, where it did not reach detectable levels (7) during active phenylpropanoid-glutamine synthetase/glutamate
synthase metabolism.
Figure 1:
Representative N-NMR spectra of P.
taeda cellular amino acid extracts obtained after administration of
A, 10 mM
N-Phe (96 h); B, 10
mM
N-Phe and 0.1 mML-AOPP (96 h);
C, 10 mM
N-Phe and 5 mM MSO (72
h); and D, 10 mM
N-Phe and 5 mM
AZA (72 h).
Subsequent GC-MS and HPLC analyses of the
extracts confirmed and extended these observations (see Table 1).
Thus, both isotopic enrichment and total amounts (micrograms/g fresh
weight) of each principal metabolite (Gln and Glu) were determined,
following incubation of N-Phe (99.9 atom %, 10 mM)
with P. taeda cell cultures, for periods up to 96 h. As can be
seen, the phenylalanine present in the soluble pool in the cells was
90 atom %
N-enriched at all intervals sampled (24, 48,
72, and 96 h). But its amount decreased from
677 µg/gfw
(t = 24 h) to
25 µg/gfw (t = 96
h) as a result of the utilization of its carbon skeleton for
phenylpropanoid metabolism. However, the Gln/Glu pools were enriched by
only
43-53 atom %
N, with both nitrogens of Gln
labeled in relatively equal amount. In contrast to that of Phe pool sizes,
the relative amounts (micrograms/gfw) of Gln/Glu dropped by only about 50%
over the duration of the 96-h experiment.
To prove unambiguously
that the glutamine synthetase/glutamate synthase pathway was assimilating
the ammonium ion released during lignification, incubations of 10
mMN-Phe (99.9 atom %
N) were repeated,
but now in the presence of specific inhibitors of phenylalanine ammonia
lyase, glutamine synthetase, and glutamate synthase, respectively. Thus,
when incubations were conducted with lignifying P. taeda cell
cultures in the presence of 0.1 mML-AOPP(35) , a known PAL inhibitor, the major resonance now observed was
that of unmetabolized
N-Phe (Fig. 1B).
Small resonances were also noted at
18.3 ppm, suggesting that
phenylpropanoid metabolism was not completely inhibited by
L-AOPP, in accordance with previous observations(35, 40) . This was confirmed by quantitative measurements
which revealed that the amounts of Phe (
90%
N enriched)
remained essentially constant (2926-2105 µg/gfw) throughout the
96-h duration of the experiment. Indeed, the employment of L-AOPP
resulted in considerable PAL inhibition as evidenced by the 4- to 100-fold
increase in Phe levels over the 24-96-h time frame examined
(cf.Table 1, Phe with and without L-AOPP). But
PAL was not inhibited fully, as revealed by the small isotopic enhancement
of both Gln (19-20%) and Glu (22-25%), respectively (Table 1). Nevertheless, these results clearly showed that overall PAL
inhibition by L-AOPP adversely affected metabolic flux into
Gln/Glu. Interestingly, it had little effect upon the pool sizes of each
amino acid relative to that observed previously during active
lignification.
The effects of treating lignifying P. taeda
cell cultures with 5 mM MSO(36) , a glutamine synthase
inhibitor, was investigated. The results in Fig. 1C show
only resonances corresponding to N-Phe and
NH
at
16.7 and 0.0 ppm;
signals due to either
N-Gln,
N-Glu,
N-Ala, and
N-Ser were absent, indicating that
their metabolism from
N-Phe was now inhibited. Quantification
of both isotopic enrichment and total amounts (micrograms/g fresh weight)
supported this conclusion (Table 1). The levels of
N-Phe (
90 atom %
N) rapidly decreased from
2492 µg/gfw (at 24 h) down to
34 µg/gfw within 96 h, due
to the action of PAL. However, as suggested by the
NH
resonance at 0.0 ppm, no significant
incorporation into
N-Gln occurred, as confirmed from its low
isotopic enrichment (2-3%). Glutamine synthase inhibition had no
apparent effect on the relative pool sizes of Glu which again remained
comparable to those previously noted, although, by contrast, the Gln
levels dropped to being near undetectable. Interestingly, the Glu isolated
was
5-10% enriched, perhaps suggesting that a small amount of
Glu formation might occur via transamination of Phe, as suggested
earlier(41) . In summary, this experiment revealed that
inhibition of glutamine synthase prevented an effective assimilation of
NH
, released during Phe
deamination, into either Gln, Glu, or any other amino acid.
The
effects of treating lignifying P. taeda cells with
N-Phe, in the presence of AZA(37) , an inhibitor of
glutamate synthase, was also examined. The results, illustrated in Fig. 1D, show that the predominant resonances were due to
N-Phe and
N-Gln (
-amide), with only a very
small signal at
18.3 ppm, due to either
H
N-
N-Gln, or
N-Glu if incomplete
inhibition occurred. This interpretation was confirmed by quantification,
as shown in Table 1; azaserine treatment had little effect upon
carbon metabolism into cinnamate, as evidenced by rapid depletion of Phe
from
1162 to 70 µg/g fresh weight over the 96-h duration.
Glutamine levels were now higher (315-120 µg/gfw) than
previously noted, due to glutamate synthase inhibition, with the
N-Gln essentially being only singly labeled (at the
-amide-nitrogen); this established that further cycling of the
nitrogen through glutamate synthase to ultimately afford the corresponding
(
N,
N) double-labeled species was greatly
reduced. Significantly, the effect of AZA on Glu levels resulted in a
4-9-fold depletion from previous levels.
Taken together,
these results indicated that the primary metabolic fate of the nitrogen of
Phe, following its release as ammonium ion during active phenylpropanoid
metabolism, was sequential assimilation into glutamine and then glutamate.
There was no evidence for ammonium ion assimilation by glutamine
dehydrogenase, since in the presence of either MSO or AZA, neither of
which inhibits glutamine dehydrogenase, essentially no detectable
incorporation of NH
into Glu
occurred.
While the above experiments provided convincing evidence
for the metabolic sequence N-Phe
NH
N(
-amide)-Gln
N
-Glu, they did not
establish that the ammonium ion, liberated during deamination, was
recycled back to Phe during active phenylpropanoid metabolism.
Consequently, the fate of exogenously provided
NH
Cl,
N(
-amide)-Gln, and
N-Glu to lignifying P. taeda cell cultures was next
investigated, to ascertain whether Phe would accumulate in a
N-enriched form. Experiments were carried out in the presence
and absence of the PAL inhibitor, L-AOPP, as before.
P. taeda cell cultures were first administered 10 mMNH
Cl for 96 h, with samples removed at 24-h
intervals and analyzed. As can be seen in Fig. 2A,
N NMR spectroscopic analyses of the extracts were devoid of
any signal corresponding to
N-Phe. Instead, a range of
resonances attributed to
N-Gln,
N-Glu,
NH
Cl, the
and
,
` nitrogens of
arginine (
66.8 and 53.4 ppm),
N-Ser, the
-nitrogen
of proline (
32.4 ppm), and the side chain amino groups of Lys
(
), Orn (
), and
-aminobutyric acid at
11.7 ppm were
evident. Thus, when an ammonium source, such as NH
Cl, was
administered to lignifying P. taeda cell cultures, it was made
available to general pools for amino acid/protein synthesis, i.e.
its fate differed from that of
N-Phe-derived ammonium ion
which resulted in the enhancement of
N signals only in Glu,
Gln, Ala, and Ser. This strongly suggests that the ammonium ion generated
during lignin synthesis is tightly compartmentalized, and not made
available for general amino acid metabolism/protein synthesis.
Figure 2:
Representative N-NMR spectra of P.
taeda cellular amino acid extracts obtained after administration of
A, 10 mM
NH
Cl (72 h); and
B, 10 mM
NH
Cl in the presence
of 0.1 mML-AOPP (96 h).
Interestingly, as can be seen from Table 2, administration of
NH
Cl (10 mM) to the lignifying P.
taeda cell cultures resulted in a rapid 6-fold increase in Gln levels
(495 µg/gfw) followed by its gradual decline to
98 µg/gfw
over 96 h, with both nitrogens being enriched (79-52%). This was
expected since an increased availability of
NH
stimulates glutamine synthetase, with a
concomitant increase in Gln accumulation(42, 43) . By
contrast, both the amount and isotopic enrichment of Glu were only
slightly elevated over previous levels (see Table 1). As before,
the
N-Phe pool size was very small, although significantly it
was now partially enriched (19-37%
N), thus providing
the first hint of evidence in support of the proposed
phenylpropanoid-nitrogen cycle.
Incubation of the lignifying P.
taeda cell cultures with NH
Cl (10
mM), in the presence of L-AOPP (0.1 mM), was
next carried out. As can be seen in Fig. 2B and Table 2, the most notable feature was the steady growth of
N-Phe (201-1301 µg/gfw), this ultimately resulting
in the dominant signal (at
16.7 ppm) in the NMR spectrum.
Additionally, all three metabolites, Glu (48-70%
N),
Phe (51-64%
N), and Gln (68-89% total
N) were isotopically enriched. Thus, proof that
NH
Cl was normally destined for the phenylpropanoid pathway was
in hand.
Existence of this phenylpropanoid-nitrogen cycle was
proven further by incubating the lignifying cells with
N(
)-Gln (10 mM) and
N-Glu (10
mM), respectively, in the presence and absence of L-AOPP
(0.1 mM). As noted previously for
NH
Cl
metabolism, administration of
N(
)-Gln (10 mM)
did not result in any resonance corresponding to
N-Phe at any
time points examined. Only signals due to
N-Gln and
N-Glu were evident by 24 h (data not shown), with prolonged
incubation (72-96 h) resulting in additional resonances for
N-Ala,
N-Ser, and
N-Asn (Fig. 3A), respectively.
(The
N-Asn resonance was
not unexpected, since the Gln pool can serve as amino donor to aspartate,
which in turn undergoes transamination to give Asn-amino N; hence it is
also assumed that the resonance at
18.3 ppm contains a minor
contribution due to Asp(
) and Asn(
) (43) .)
Figure 3:
Representative
N-NMR spectra of P. taeda cellular amino acid
extracts obtained after administration of A, 10 mM
N-Gln (96 h); and B, 10 mM
N-Gln in the presence of 0.1 mML-AOPP (96
h).
Determination of pool sizes and isotopic enrichment as before verified
that Phe concentrations were low (7-13 µg/gfw) but enriched
(23-28% N), whereas
N-Gln and
N-Glu levels remained fairly constant (cf.
NH
Cl
metabolism); note also that the isotopic
enrichments of both were lower than before, with the
N-Gln
being predominantly singly labeled.
In a somewhat analogous manner,
administration of N-Glu (10 mM) to P. taeda
cell cultures, gave amino acid extracts devoid of
N-Phe
resonances at any of the sampling points, whereas signals due to
N-Glu,
N-Ala,
N-Ser, and
N-Pro were readily detected (Fig. 4A). This
was further verified by quantification which again revealed a small Phe
pool size (6-10 µg/gfw of 42-47%
N
enrichment) (Table 2).
Figure 4:
Representative N-NMR spectra of P.
taeda cellular amino acid extracts obtained after administration of
A, 10 mM
N-Glu (72 h); and B, 10
mM
N-Glu in the presence of 0.1 mML-AOPP (96 h).
On the other hand, when P.
taeda cells were incubated with N(
)-Gln and
N-Glu in the presence of 0.1 mML-AOPP,
resonances due to L-Phe (
16.7 ppm) were now readily evident
(Fig. 3B and 4B). This was further proven by
quantification which revealed a
100-fold increase in its amount
(
1012-1074 µg/gfw from 72-96-h duration) with an
isotopic enrichment of 29-75%
N, i.e. again
confirming the proposed phenylpropanoid-nitrogen cycle.
Resonances
due to N-Gln,
N-Glu,
N-Ala, and
N-Ser were also observed, as well as minor signals due to
N Pro, Lys, Orn, and
-aminobutyric acid. Moreover,
although Gln pool sizes were close to those previously noted (Table 2), there was a significant increase in the extent of formation of
double-labeled
N-Gln. This is presumed to result via
limited catabolism of Glu to ammonium ion and 2-oxoglutarate, with
subsequent reassimilation by glutamine synthetase to yield
N(
),
N(
)-Gln.
With the combined use of HPLC,
N NMR, and GC-MS, the mechanism for recycling of ammonia
during phenylpropanoid metabolism in lignifying P. taeda
(loblolly pine) cell cultures was unambiguously established. The use of
enzyme inhibitors was necessary to elucidate this mechanism, with
metabolite accumulations being consistent with the known mode of action of
L-AOPP, MSO, and AZA; namely inhibition of PAL, glutamine
synthetase, and glutamate synthase, respectively. The ammonium ion
generated during active phenylpropanoid biosynthesis is first incorporated
into the
-amide of glutamine, followed in turn by the
-amino
position of glutamate, which then acts as an amino donor for a range of
transamination products, including the aromatic amino acids, arogenate,
and phenylalanine. This nitrogen cycle explains how optimum use is made of
the plant's available nitrogen, so that active phenylpropanoid
metabolism and lignification can continue, even at low nitrogen
levels.