J. Plant Physiol. 158.

593 598 (2001)

Urban & Fischer Verlag
http://www.urbanfischer.de/journals/jpp

Proline metabolism in response to highest nitrogen dosages in green

bean plants (Phaseolus vulgaris L. cv. Strike)
Esteban Snchez*, Luis Ramn Lpez-Lefebre, Pablo Carlos Garca, Rosa Mara Rivero, Juan Manuel Ruiz, Luis Romero
Department of Plant Biology, Faculty of Sciences, University of Granada, 18071-Granada, Spain

Received October 9, 2000 Accepted January 2, 2001

Summary
The objective of the present work was to determine what impact extremely high nitrogen dosages
would have on proline metabolism in order to use this amino acid as a bioindicator of N status of
green bean plants (Phaseolus vulgaris L. cv. Strike). In this effort, we identified the most favourable
pathway of proline synthesis under our experimental conditions. The N was applied to the nutrient
solution in the form of NH4NO3 at 5.4 mmol/L (N1, optimal level), 11.6 mmol/L (N2), 17.4 mmol/L (N3),
and 23.2 mmol/L (N4). Our results indicate that the application of high N dosages in Phaseolus is
characterized by the accumulation of NO3 , NH4 + and proline in root and foliar organs. However,
although the enzymes in charge of proline biosynthesis, ornithine--aminotransferase (OAT, EC
2.6.1.13) and 1-pyrroline-5-carboxylate synthetase (P5CS, EC 2.7.2.11/1.2.2.41) vary in behaviour
depending on the N status, in our experiment, this amino acid appears to be synthesized mainly by
the enzyme ornithine--aminotransferase. This suggests predominance of the ornithine pathway over
the glutamine pathway. Finally, under our experimental conditions, proline can be defined as a good
indicator of N excess of green bean plants.
Key words: Phaseolus vulgaris green bean nitrogen toxicity proline metabolism
Abbreviations: BSA bovine serum albumin. EDDHA Ethylenediamine-di(o-hydroxyphenylacetic
acid. EDTA ethylenediaminetetraacetic acid. OAT ornithine--aminotransferase. P5CS 1-pyrroline-5-carboxylate synthetase. PDH Proline dehydrogenase. PVPP insoluble polyvinylpyrrolidone

Introduction
It is well known that proline accumulates in plants during
adaptation to various types of environmental stresses such
as drought, salinity, high and low temperatures, nutrient deficiency, exposure to heavy metals and high acidity, and that
these types of stress limit plant distribution and productivity
* E-mail corresponding author: lromero@ugr.es

(Shevyakova 1984, Lalk and Drffling 1985, Paquin 1985,

Machackova et al. 1989, Delauney and Verma 1993, Zaifnejad et al. 1997, Hare et al. 1999).
Different roles have been proposed for proline accumulation as an adaptive response; it has been suggested that
proline may function as an osmoticum (Wyn Jones et al.
1977), a sink of energy and reducing power (Blum and Ebercon 1976), a nitrogen-storage compound (Ahmad and Hellebust 1988), a hydroxy-radical scavenger (Smirnoff and Cum0176-1617/01/158/05-593 $ 15.00/0

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Esteban Snchez et al.

Figure 1. The proline biosynthetic and degradation pathway. Enzymes: (1) ornithine--aminotransferase, (2) pyrroline-5-carboxylate synthetase, (3) pyrroline-5-carboxylate reductase,
(4) proline dehydrogenase, and (5) pyrroline
5-carboxylate dehydrogenase. GSA: glutamic-semialdehyde and P5C: pyrroline-5-carboxylate.

bes 1989), and a compatible solute that protects enzymes

(Schobert and Tschesche 1978, Paleg et al. 1981, 1984, Charest and Phan 1990). It may also play a role in the regulation
of cellular redox potentials (Saradhi and Saradhi 1991).
In plants, proline is synthesized from either glutamate or
ornithine (Fig. 1) (Delauney et al. 1993). The first two steps of
the proline biosynthesis from glutamate are catalysed by a
single bifunctional enzyme, 1-pyrroline-5-carboxylate synthetase (P5CS, EC 2.7.2.11/1.2.1.41) which produces glutamic-semialdehyde (GSA). This GSA is spontaneously converted
into pyrroline-5-carboxylate (P5C), which is then reduced by
P5C reductase (P5CR, EC 1.5.1.2) to proline (Fig. 1.) (see
Zhang et al. 1995).
Plants also synthesize proline from ornithine, by ornithine-aminotransferase (Fig. 1) (OAT, EC 2.6.1.13). If the -amino
group of ornithine is transaminated, the product would be
-keto--amino-valerate, which cyclizes to 1-pyrroline-2carboxylate (P2C) and is then reduced to proline (Fig. 1). Alternatively, trans-amination of the -amino group yields GSA,
which is converted to proline via P5C (see Delauney and
Verma 1993).
On the other hand, the metabolism and accumulation of
proline also depends on its degradation, which is catalysed
by the mitochondrial enzyme proline dehydrogenase (Fig. 1)
(PDH, EC 1.5.99.8; see Hare et al. 1999).
In present-day agriculture, the main types of stress commonly resulting from the heavy use of inorganic fertilizers are
related to the nutritional status of certain nutrients, primarily

nitrogen (N), given its extensive use (Ruiz and Romero 1998,
1999). Rabe (1990) reviewed the influence of numerous kinds
of abiotic and biotic stress on the composition of N-containing compounds in plants. The amino compounds most often
accumulated in foliar tissue as a function of stress include
glutamine, asparagine, arginine, citruline, ornithine and principally proline. In general, although only scant literature is
available on the subject, it appears that the correlation between the availability of N and the accumulation of proline is
usually positive (Lovatt 1990, Rabe 1990, Andersen et al.
1995).
The objective of the present work was to determine the effect of abiotic stress, such as extremely high nitrogen dosages, on proline metabolism in order to use this amino acid
as a bioindicator of the N status of Grench Bean plants. In
addition, we have identified the most favourable pathway of
proline synthesis under our experimental conditions.

Materials and Methods

Crop design
Seeds of Phaseolus vulgaris cv. Strike were sown and grown in a
chamber under controlled environmental conditions, with relative humidity of 60 80 %, temperature 30/20 C (day/night), and a photoperiod of 16/8 h in a photosynthetic photon flux density of 350 mol m 2 s 1
(measured at the top of the plants with a 190 SB quantum sensor, LICOR Inc., Lincoln, NE). Four plants were grown in 8-L pots (25 cm

Proline metabolism and extreme nitrogen dosages

upper diameter, 17cm lower diameter, 25 cm in height), filled with vermiculite. For 30 days before the experimental treatments, the plants
received a nutrient solution of 5.4 mmol/L NH4NO3, 1.6 mmol/L K2HPO4,
0.3 mmol/L K2SO4, 4 mmol/L CaCl2 2H2O, 1.4 mmol/L MgSO4 H2O,
5 mol/L Fe-EDDHA, 2 mol MnSO4 H2O, 1 mol/L ZnSO4 7H2O,
0.25 mol/L CuSO4 5H2O, 0.3 mol/L Na2MoO4 2H2O, and 5 mol/L
H3BO3. The nutrient solution (pH 6.0 6.1) was renewed every 3 days.
At 30 days after sowing, the different N treatments in the form of
NH4NO3 were applied for 30 days (until harvest), 5.8 mmol/L (N1, the
optimum level according to Carbonell-Barrachina et al. (1997),
11.6 mmol/L (N2), 17.4 mmol/L (N3), and 23.2 mmol/L (N4). This experimental design was a complete randomized block with 6 replicates (individual pots) with 24 plants per treatment.

Plant sampling
The plants were sampled at 60 days after sowing, at full pod development. All the root and leaf samples were taken in the mature state.
The material was rinsed three times in distilled water after disinfecting
with non-ionic detergent at 1 % (Wolf 1982) and then blotted on filter
paper. A subsample of fresh roots and leaves were used for the analysis of P5CS, OAT, PDH, NO3 , NH4 + , and proline, with triplicate assays for each extraction.

Plant analysis
Frozen tissues (three samples of approximately 5 g fresh weight per
treatment) were then ground to a fine powder in a chilled mortar and
pestle, in the presence of washed quartz sand (0.3 g [g fresh weight] 1,
with liquid nitrogen, and then homogenized with an ultraturax (CATX620) in the appropriate extraction buffer. Ratios for buffer volume: g
fresh weight were 2 : 1. All operations were carried out at 4 C (Lutts et
al. 1999).
Extraction procedure used for 1-pyrroline-5-carboxylate synthetase (P5CS, EC 2.7.2.11/1.2.2.41) and proline dehydrogenase (PDH,
EC 1.5.99.8) in 50 mmol/L Tris-HCl buffer (pH 7.4) containing 7mmol/L
MgCl2, 0.6 mol/L KCl, 3 mmol/L EDTA, 1 mmol/L dithiothreitol, and 5 %
(w/v) insoluble polyvinylpyrrolidone. Homogenates were filtered
through two layers of Miracloth (Calbiochem) and the filtrate was centrifuged at 39,000 gn for 20 min. The supernatants were desalted on a
sephadex G-25 column (Pharmacia Biochem PD-10) and eluted with
50 mmol/L Tris-HCl (pH 7.4) containing 10 % glycerol. The solution
used for extraction of ornithine--aminotransferase (OAT, EC 2.6.1.13)
consisted of 100 mmol/L K-Pi buffer pH 7.9 with 1 mmol/L EDTA, 15 %
glycerol, and 10 mmol/L 2-mercaptoethanol. The extract was centrifuged at 15,000 gn for 15 min and the supernatant was treated with
60 % (NH4)2SO4 for 45 min (Lutts et al. 1999).
The P5CS activity was assayed following Zhang et al. (1995). The
reaction mixture for P5CS contained the following in final volume of
0.5 mL (pH 7.0): 50 mmol/L L-glutamate, 20 mmol/L MgCl2, 10 mmol/L
ATP, 100 mmol/L hydroxamate-HCl, 50 mmol/L Tris. The reaction was
started by the addition of 0.5 mL of enzymatic extracts. After 5 min at
37 C the reaction was stopped by the addition of 0.5 mL of the stop
buffer (2.5 % of FeCl3 plus 6 % of trichloracetic acid, dissolved in
100 mL de HCl 2.5 N [p/p/v]). The Pi concentrations were determined
using a sensitive malachite-green assay (Geladopoulus et al. 1991).
The PDH was assayed by following the NAD + (or NADP + ) reduction
at 340 nm in a 0.15 mol/L Na2CO3-HCl buffer (pH 10.3) containing
15 mmol/L L-proline and 1.5 mmol/L NAD + or NADP + . OAT was

595

assayed according to Charest and Phan (1990) in 0.2 mol/L Tris-KOH

buffer (pH 8.0) containing 5 mmol/L ornithine, 10 mmol/L -ketoglutarate, and 0.25 mmol/L NADH in a volume of 2 mL; the decrease in
absorbance of NADH was monitored at 340 nm after initiating the
reaction with the addition of 0.2 mL of the enzyme extract. Protein content was measured following the method of Bradford (1976), with BSA
as a standard.
Free proline was quantified in 95 % ethanol extracts from roots and
leaves. A freshly harvested sample of 0.5 g from both types of tissues
was crushed in 5 mL 95 % (v/v) ethanol. The insoluble fraction of the
extract was washed twice with 5 mL of 70 % ethanol. All soluble fractions were centrifuged at 3,500 gn for 10 min. The supernatants were
collected and stored at 4 C for proline determination (Irigoyen et al.
1992). The free-proline content was measured according to the
method described by Paquin and Lechasseur (1979).
For the quantification of NO3 and NH4 + , at each sampling, portions were ground, with a ratio of 1 : 5 (w/v), in a mortar at 0 C in
50 mmol/L KH2PO4 buffer (pH 7.5), containing 2 mmol/L DTT and 1 %
(w/v) insoluble PVPP. The homogenate was filtered and centrifuged at
3,000 gn for 5 min, after which the supernatant was centrifuged at
30,000 gn for 20 min. The resulting extract was used to measure the
NO3 and NH4 + concentrations (Farnden and Roberston 1980, Groat
and Vance 1981, Lillo 1984, Kaiser and Lewis 1984, Singh and Srivastava 1986). The determination of the NO3 in the enzyme extract was
measured by spectrophotometry as described by Cataldo et al.
(1975). The NH4 + in the resulting extract was determined by spectrophotometry as described by Krom (1980).

Statistical analysis
Standard analysis of variance techniques were used to assess the significance of treatment means. The data shown are mean values SE.
Differences between treatment means were compared using the LSD
at the 0.05 probability level. A correlation analysis was also made between the difference variables. Levels of significance are represented
by * at P < 0.05, ** at P < 0.01, *** at P < 0.001, and ns: not significant.

Results and Discussion

An adequate N supplement is a key factor for growth and
productivity in most crops. Generally, plant growth slows under a N supply exceeding 10 mmol/L, a value considered on
the thereshold of toxicity for some species (Ulrich and Fong
1973, Benton Jones 1997, Cao and Tibbets 1998). Therefore,
in our experiment, all treatments except for N1 were considered a priori to be toxic, as the N concentration applied varied from 11.6 mmol/L (N2) to 23.4 mmol/L (N4). The effectiveness of the N treatments in our experiment is reflected in the
production of root (P < 0.001) and foliar biomass (P < 0.001),
which diminished progressively as the N dosage increased
(Fig. 2). That is, N4 presented the markedly less root and foliar biomass of 18 % and 62 %, respectively, than N1, indicating that the N4 treatment was super-optimum or toxic, whereas
N1 stimulated of growth.
Two metabolic pathways, glutamate and ornithine metabolism, are very important in proline formation (Fig. 1) (Kavi Kish-

596

Esteban Snchez et al.

Figure2. Root and shoot biomass in green bean

plants in response to the N application (N1:
5.8 mmol/L, N2: 11.6 mmol/L, N3: 17.4 mmol/L,
and N4: 23.2 mmol/L). Data are means s.e.
(n = 6).

Table 1. Response of proline metabolism and the accumulation of NO3 and NH4 + in the roots and leaves of green bean plants subjected to different N treatments (N1: 5.8 mmol/L, N2: 11.6 mmol/L, N3: 17.4 mmol/L, and N4: 23.2 mmol/L).
Treatments

P5CS

OAT

Proline

N1
N2
N3
N4
LSD at 0.05
Significance

0.83 0.04
0.77 0.05
0.66 0.01
0.65 0.07
0.03
**

1.95 0.01
2.21 0.02
2.55 0.02
4.83 0.03
0.94
*

53.3 9.6
63.2 6.5
71.2 5.4
84.4 9.8
10.7
**

N1
N2
N3
N4
LSD at 0.05
Significance

0.20 0.01
0.19 0.01
0.17 0.01
0.16 0.01
0.01
**

1.03 0.01
1.43 0.01
1.63 0.02
1.91 0.02
0.78
*

246.7 15.5
360.2 16.5
365.5 22.5
588.0 15.5
37.4
**

PDH

NO3

NH4 +

Roots
0.083 0.006
0.066 0.005
0.059 0.003
0.058 0.004
0.009
*

4.67 0.33
4.90 0.31
5.16 0.22
3.64 0.27
0.15
**

1.41 0.08
1.48 0.07
1.49 0.07
1.61 0.07
0.05
**

0.028 0.001
0.027 0.001
0.024 0.002
0.021 0.002
0.001
**

7.50 0.67
7.96 0.90
10.67 0.67
11.04 0.59
0.33
**

1.52 0.05
1.79 0.05
2.05 0.08
2.26 0.05
0.05
**

Leaves

Data are means s.e. (n=6). The least significant difference (LSD) is given vor each treatment. Levels of significance were represented by * at
P < 0.05, ** at P < 0.01, *** at P < 0.001, and ns: not significant by ANOVA at the 0.05 probability level. 1-pyrroline-5-carboxylate synthetase
(P5CS) expressed as mol NAD(P)H oxidized (mg 1 proteins min 1); Ornitine--aminotransferase (OAT) expressed as mol NADH oxidized
(mg 1 proteins min 1); Proline dehydrogenase (PDH) expressed as mol NAD(P) + reduced (mg 1 proteins min 1); Proline expressed as g g 1
fresh weight; nitrates (NO3 ) and ammonium (NH4 + ) expressed in mol g 1 fresh weight.

or et al. 1995). With respect to the metabolic pathway of glutamate for the biosynthesis of proline, in our experiment, the N
dosage significantly affected the root and foliar activity of
P5CS (P < 0.01; Table 1), with the lowest activities evident in
the N4 treatment, registering 22 % and 17 % lower, respectively, than the highest activity at N1.

With regard to the metabolic pathway of ornithine, the N treatments also significantly influenced the activity of OAT. In our experiment, the application of the highest N dosages boosted the
activities of root and foliar OAT (P< 0.05; Table 1), presenting the
highest activities at N4. These were, respectively, 59 % and
46 % higher than for the lowest activities, found at N1.

Proline metabolism and extreme nitrogen dosages

The levels of root and foliar proline (P < 0.01; Table 1) presented their highest concentrations in both organs appeared
under treatment N4. Our results reveal an inverse correlation
between the enzymatic activity of P5CS and the proline concentration, both in the roots and the leaves (P5CS-proline, r =
0.85**), while both organs showed a correlation which was
directly correlated with the OAT activity and the proline concentration (roots, r = 0.80***, leaves, r = 0.75**).
In short, we suggest that the biosynthesis and accumulation of root and leaf proline in our experiment, due perhaps
to the high N dosages, is mediated principally by the ornithine pathway. Our results agree with those of Rhodes et al.
(1986) and Delauney and Verma (1993), who mentioned that
the glutamate pathway for proline biosynthesis is predominant under stress conditions such as high salinity and N
limitation. On the contrary, in plants administered excess N
in our experiment, the P5CS root and foliar activities
dropped (Table 1), while the OAT root and foliar activities
(Table 1) significantly rose, consistent with the interpretation
that the ornithine pathway is enhanced under these conditions.
The other important factor that controls proline levels in
plants is degradation. That is, L-proline is oxidized to P5C in
plant mitochondria by PDH (Fig. 1) (Rayapati and Stewart
1991). In our experiment, the PDH root (P < 0.05) and foliar activities (P < 0.01; Table 1) diminished as the N dosage was
augmented, presenting minimum activities at N4, with a drop
of 31% and 24 % with respect to the highest activity at N1. As
indicated above, the concentrations of root and foliar proline
(Table 1) registered their highest concentrations at N4. This
result can probably be explained by the inverse correlation
between proline and PDH activity, both in the roots and in the
leaves (r = 0.75* and 0.88***, respectively).
One of the factors which can be a determinant in the regulation processes of proline synthesis is availability and concentration of inorganic nitrogenous forms (NO3 and NH4 + ) in
plants (Delauney et al. 1993). In higher plants, the physiological significance of proline accumulation has yet to be completely determined. Apart from acting as an osmolite, proline
accumulation has other potentially important cell functions.
Proline may act as a N source in the cell under stress conditions, where the accumulation of this nitrogenous compound
could be utilized as a form of stored N (Dandekar and Uratsu
1988). In our experiment, the application of high dosages of N
augmented the NO3 concentrations in the roots and leaves
(P < 0.001; Table 1), the highest appearing in the N3 and N4
treatments, respectively. With respect to the NH4 + values, in
our experiment, the highest NH4 + concentrations in both organs were found in the N4 treatment (P < 0.01; Table 1) and
the lowest in the N1. Thus, our results indicate a positive and
significant correlation between the NO3 and NH4 + and proline contents (root: NH4 + -proline, r = 0.73**; leaf: NO3 -proline, r = 0.74**; NH4 + -proline, r = 0.85***), and a negative and
significant correlation between NO3 contents and root-proline accumulation (NO3 -proline, r = 0.50*).

597

In short, the application of high N dosages in Phaseolus is

characterized by the accumulation of NO3 , NH4 + and proline
in root and foliar organs. However, although the enzymes in
charge of proline biosynthesis, ornithine--aminotransferase
and 1-pyrroline-5-carboxylate synthetase, vary in behaviour
depending on the N status, in our experiment, this amino acid
appears to be synthesized mainly by the enzyme ornithine-aminotransferase, suggesting predominance of the ornithine
pathway over the glutamine pathway. Finally, under our experimental conditions, proline can be defined as a good indicator of N excess of green bean plants.

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