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New Phytol. (1999), 142, 451–462

Nitrogen reserve status affects the

dynamics of nitrogen remobilization and

mineral nitrogen uptake during recovery of

contrasting cultivars of Lolium perenne from

defoliation

S. LOUAHLIA " , J . H. MACDUFF # , A. OURRY "*, M. HUMPHREYS #

J. BOUCAUD"

"UA INRA 950 Physiologie et Biochimie VeU geU tales, Institut de Recherche en Biologie

AppliqueU e, UniversiteU , 14032 Caen Cedex, France

# Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth,

Ceredigion, Dyfed SY23 3EB, UK

Received 26 October 1998; accepted 15 March 1999

Nitrogen (N) allocated to leaf growth in forage grasses and legumes following severe defoliation is predominately

mobilized from the remaining root and leaf sheath tissues, since both N uptake from the soil and N#fixation are

severely down-regulated for several days. The hypothesis that a low N reserve status at the time of defoliation

limits N remobilization and leaf regrowth was tested with contrasting cultivars of Lolium perenne (cvs Aberelan

and Cariad) in flowing solution culture. Plants were grown under ‘high’ or ‘ low’ (uptake of N decreased by 50%)

regimes of N supply for 10 d before a single severe defoliation. Labelling with "&N was used to assess the

importance of N reserves, including putative vegetative storage proteins, relative to N translocated from

concurrent uptake, as a source of leaf N during regrowth. Leaf regrowth, N uptake and N mobilization were all

affected by previous N supply. Low plant N status at the time of defoliation increased regrowth dry weight of

‘Aberelan’ by 10% and translocation of N absorbed from the medium by 23%, while mobilization of N reserves

was decreased by 56%. On the contrary, regrowth dry weight of ‘Cariad’ was decreased by 23%, and

translocation of N absorbed by 21% in low plant N status, compared with high plant N status. Concentrations

of soluble protein in roots and remaining leaf sheaths decreased after defoliation in plants only under optimal N

supply. Analysis of soluble proteins in sheath material by SDS–PAGE suggested that three polypeptides (55, 36.6

and 24 kDa) might function as vegetative storage proteins, although they were of low abundance in plants,

subjected to monthly harvests, grown in controlled conditions and in the field. The apparent antagonism between

uptake of NH%

+or NO$

− by roots and mobilization of N reserves is discussed together with evidence for functional

vegetative storage proteins in L. perenne.

Key words: ammonium, defoliation, Poaceae, Lolium perenne, nitrate, vegetative storage proteins, fructans.

Mechanical defoliation and grazing induce major

modifications in the carbon (C) and N metabolism of

grasses and forage legumes during their subsequent

recovery (Richards, 1993; Volenec et al., 1996). For

example, uptake of mineral N decreases rapidly and

Abbreviations: DP, degree of polymerization; FSC, flowing

solution culture; VSP, vegetative storage protein; WSC, water

soluble carbohydrate.

*Author for correspondence (fax 00 33 2 56 53 60; e-mail

ourry!ibba.unicaen.fr).

acutely (Macduff & Jackson, 1992), influx of nitrate

and ammonium decreases while their efflux from the

roots to the external medium appears to increase. It

is not understood how this is regulated and exactly

which signals and transduction pathways are

involved (Macduff & Jackson, 1992).

It is generally accepted (Richards, 1993; Volenec

et al., 1996) that adaptation to defoliation in many

species involves a capacity for mobilization of N

compounds stored in perennial tissue such as roots,

stubble or crown, enabling N to be supplied to

growing leaves despite the down-regulation of N

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452 S. Louahlia et al.

uptake by the roots. Several studies (Phillips et al.,

1983; Thornton et al., 1993, 1994; Thornton &

Millard, 1996) have shown that different grass

species, including Lolium perenne, Festuca rubra,

Agrostis castellana, Poa trivialis, Bromus mollis are

able to remobilize internal N to sustain early

regrowth. In certain species such as Medicago sativa

(Ourry et al., 1994) or the annual grass Lolium

temulentum (Ourry et al., 1996) it has been argued

that the status of N reserves at the time of defoliation

may be a crucial determinant of regrowth rate. This

is based on a view that growth is severely decreased

by a shortage of organic N, delaying refoliation and

recovery of N#

fixation and}or N uptake. It follows

that large differences in forage regrowth yields might

result from relatively moderate initial differences in

availability of N reserves. However, this has not

been demonstrated convincingly for an agro-

nomically important and perennial species of grass

such as L. perenne. Thornton et al. (1993, 1994) and

Thornton & Millard (1996) have argued that reliance

on mobilized N during recovery from defoliation

tends to be higher in those grass species that are

abundant in low input, ‘extensive’ grassland farming

systems. As a consequence they suggested that a high

propensity for N storage in grasses is an adaptation

to environments poor in N.

Given that the free nitrate pool is only modest in

most plants, and functions as a short-lived storage

compartment (!24 h), it is organic forms of N such

as amino acids and proteins that constitute the main

N storage components within the plant (e.g. Millard,

1988). This is supported by the decline in soluble

protein concentrations observed during the first few

days following shoot removal in M. sativa

(Hendershot & Volenec, 1993), Trifolium repens

(Corre et al., 1996) and L. perenne (Ourry et al.,

1989). Staswick (1989, 1990, 1994) has proposed

several criteria by which the involvement of specific

proteins in N storage may be judged. Accordingly,

vegetative storage proteins (VSP, see Abbreviations

section) are supposed to be prominent (accounting

for at least 5% of total soluble proteins), readily

hydrolysed (since their degradation products are

used by growth of tissues other than those containing

VSP) and devoid of any catalytic activity.

Several polypeptides fulfilling these criteria have

been identified in T. repens and M. sativa, and have

been designated as functional VSPs. These are

relatively abundant proteins (Hendershot & Volenec,

1993; Corre et al., 1996) that are hydrolysed to a

greater extent than are the majority of soluble

proteins, and then accumulate during the later stages

of refoliation. As with many of the VSPs identified in

woody species (Ste!pien et al., 1994), it is notable that

these VSPs in forage legumes accumulate during

autumn and winter, and are then almost entirely

hydrolysed when growth resumes in spring

(Bouchard et al., 1998). Rather surprisingly, no

polypeptides conforming to the criteria for VSPs

have been identified in other forage legumes such

as Trifolium pratense or Melilotus officinalis

(Cunningham & Volenec, 1996; Li et al., 1996).

Furthermore, to our knowledge, there have been no

attempts to assess the presence or absence of VSPs in

grasses.

The aim of the present experimental work with

L. perenne was to resolve these uncertainties. The

specific objectives were, first, to determine whether

the N status of plants at the time of defoliation

determines regrowth rate of the shoot and the rate of

recovery in the uptake of mineral N supplied as

NO$

− plus NH%

+. As a corollary, the extent to which

rates of remobilization and uptake of these two forms

of N are inversely related during recovery from

defoliation should be forthcoming. To provide a

measure of intra-specific variation, two contrasting

varieties of perennial ryegrass were compared:

‘Aberelan’, known to have a high nitrogen use

efficiency (NUE) (Wilkins et al., 1997), and ‘Cariad’,

known to have a tendency to accumulate higher than

average concentrations of water soluble carbo-

hydrates (WSC) in its leaves and sheaths (Radojevic

et al., 1994). The second objective was to determine

whether specific polypeptides in L. perenne conform

to the criteria for VSPs, with N mobilization

triggered by defoliation or the onset of spring

growth.

A steady-state "&N-labelling technique in flowing

solution culture (FSC), which provided precisely

defined high and low regimes of N supply before

defoliation, was used to allow detailed resolution of

the dynamics of N mobilization and mineral N

uptake during a 21 d recovery period. A comp-

lementary plot experiment assessed the seasonal

variation in putative VSPs of L. perenne under field

conditions.

Plant culture

For the experiment in flowing solution culture

(FSC), seeds of L. perenne L., cvs Cariad and

Aberelan were imbibed for 48 h on tissue paper

saturated with 10 mol m−$ CaSO%. Each cultivar was

then sown separately into two culture units of an

FSC system, incorporating automatic control of

concentrations of NO$

−, NH%

+, K+ and H+ in

solution (Clement et al., 1974; Hatch et al., 1986).

Each culture unit contained 200 dm$ of recirculating

nutrient solution and 24 culture vessels, each with 10

plants. The FSC system was located in a glasshouse,

solution temperature was maintained at 20³0.5°Cand air temperature at 20³2 : 15³1°C day : night

(16.00–06.00 hours) throughout the experiment. The

plants were established under natural illumination

until day 14 after sowing. Supplementary light

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N reserves and regrowth 453

(08.00–16.00 hours) of 200 µmol m−# s−" PAR was

provided between days 14 and 25 by a single 400 W

SON-T lamp (Philips) suspended 1.5 m above the

surface of each culture unit. On day 25 after sowing

all natural light was excluded and illumination was

then provided (including during the treatment

period) by paired 400 W SON-T and HPI}T lamps

(Philips Lighting Ltd, Croydon, Surrey, UK) giving

500 µmol m−# s−" PAR at canopy height.

Until day 38 after sowing, the concentrations of

nutrients in solutions were maintained by regular

manual additions at (mmol m−$) : NO$

−, 250; NH%

+,

250; K+, 250; Mg#+, 100; Ca#+, 424; HPO%

−, 50;

SO%

#−, 550; Fe#+, 5.4; with micronutrients as given

by Clement et al. (1978). Thereafter, the automatic

nutrient control system measured the concentrations

of K+, NO$

− and NH%

+ in each culture unit every 27

min and maintained the treatment ‘set-point ’

concentrations of these ions (to be described later),

by automatic resupply of K#SO

%, (NH

%)#SO

%or

Ca(NO$) at rates equal to their depletion. Net uptake

of these ions was calculated on an hourly basis from

the amounts required to maintain the set-point

concentrations in the flowing solutions. All other

nutrients were resupplied automatically in fixed

ratios to the net uptake of NO$

− and NH%

+ (for one

mol of N, 0.645, 0.057, 0.045, 0.00075 and 0.522 mol

of, respectively, S, Mg, P, Fe and Ca were auto-

matically resupplied). Solution pH was automati-

cally maintained at 6.0³0.1 by delivery of H#SO

%or

Ca(OH)#.

Experimental treatments

In order to condition the N status of the plants at the

time of defoliation, treatments consisting of two

different rates of N supply (‘high N’ and ‘low N‘)

were imposed on day 38 after sowing and continued

for 10 d. The supply of N was defined in terms of

constant concentrations in the flowing nutrient

solutions. The aim was that the ‘ low N’ plants

should absorb approx. 50% of the total N absorbed

by ‘high N’ plants during this period. Based on

previous experience (Wild et al., 1987), the N supply

to ‘high N’ plants (one culture unit of each cultivar)

was maintained at 20 mmol m−$ NH%NO

$, whereas

the ‘ low N’ plants (one culture unit per cultivar)

received 5 mmol m−$ NH%NO

$. In reality the net

uptake of N over 10 d by ‘low N’ ‘Cariad’ was 58%

of the amount absorbed by ‘high N’ plants, whereas

the corresponding figure for ‘ low N’ plants of

‘Aberelan’ was 44%.

All plants were defoliated on day 48 after sowing,

referred to hereafter as day 0. At 1 h before

defoliation (17.00 hours) the automatic resupply of

N was terminated and the solution concentrations

allowed to deplete to !1 mmol m−$ NH%NO

$. Plants

were defoliated by cutting the shoot 4 cm above the

shoot-root junction. Automatic N supply to all four

culture units was immediately resumed at 20 mmol

m−$ "&NH%

"&NO$(5.00 atom % "&N), irrespective of

previous N supply, and continued over 14 d of

regrowth.

Plants were harvested at intervals during the

regrowth period: four culture vessels from each unit

on days 0 (6 h before defoliation), 2 and 4; then three

vessels on days 7, 11 and 14. On each occasion the

plants within a specific culture vessel were bulked

before dividing into roots, sheath, laminae inside the

sheath beneath the defoliation height (4 cm), and

regrowing leaves. Fresh weights were recorded,

after which fractions were freeze-dried, reweighed,

ground to a fine powder, and kept at ®80°C until

analysis.

Field experiment

Seasonal changes in VSP content of L. perenne were

measured in a plot experiment located at the INRA

experimental station in NW France. Three plots (8

m#) of L. perenne cv. Magella were established and

fertilized with 6 g m−# (as NH%NO

$) during March

1997. Following the resumption of growth in the

spring the plots were defoliated on three occasions

(22 Apr., 6 June, 10 July 1997). Whole plants were

harvested at monthly intervals between November

1996-July 1997. The leaf sheath fraction was freeze-

dried, ground to a fine powder and stored at ®80°Cuntil analysis.

Chemical analysis and calculation of N

remobilization

The determination of total N and "&N in plant

samples from the FSC experiment was performed by

a continuous flow isotope mass spectrometer (20–20,

Europa Scientific Ltd, Crewe, UK) linked to a C}N

analyser (Roboprep CN, Europa Scientific Ltd). All

mineral N absorbed from the flowing solutions

during regrowth was "&N labelled. Consequently,

patterns of net translocation of unlabelled N ("%N

absorbed before defoliation) between plant parts

were used to calculate N remobilization to regrowing

tissues. The N in regrowing leaves derived from the

mobilization of N reserves from roots and sheaths

was calculated by subtracting from total N

("%N­"&N), first, the "&N content derived from

uptake of "&NH%

+ or "&NO$

− and, second, the initial

"%N content of laminae remaining after defoliation

(i.e. leaf tissue remaining inside the sheath between

the shoot-root junction and the point of defoliation).

Electrophoretic analysis of soluble proteins

In both experiments, the soluble proteins were

extracted from 100 mg of freeze-dried roots or

sheath at 4°C with 5 ml of 50 mM Tris buffer (pH

7.5) containing 2 mM phenylmethylsulphonyl

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454 S. Louahlia et al.

fluoride, 10 µM leupeptin, 1 mM EDTA and

0.1% (v}v) 2-mercaptoethanol. After centrifugation

(12 000 g, 10 min), using an aliquot of the supernatant

the soluble proteins were precipitated with 7.2%

(w}v) trichloroacetic acid and their content was

determined by the method of Lowry et al. (1951).

Bovine serum albumin (BSA) was used as the protein

standard. Nucleic acids in the remaining supernatant

were precipitated using protamine sulphate (1 mg

ml−") for 15 min. The nucleic acid pellet was

discarded after centrifugation (28000 g, 10 min).

Soluble proteins were separated into five sub-

samples, each receiving 0.015% (w}v) sodium

deoxycholate. After 10 min, the soluble proteins

were precipitated at 4°C with 7.2% (w}v) trichloro-

acetic acid. The protein pellet was rinsed with

acetone. For SDS–PAGE analysis, one pellet of

soluble protein was resuspended in 100 µl of the

buffer described by Leammli (1970), denatured for 5

min at 100°C, and centrifuged (12000 g, 3 min).

SDS-electrophoresis used a 15% duracryl running

gel with a stacking gel containing 5.5% acrylamide.

Two wells were used for loading proteins with

known molecular masses. The gels were run for 4 h

at a constant 500 V, and silver stained as described

by Lopez et al. (1991), then scanned and quan-

titatively analysed using a computerized image

analysis system (Millipore Bioimage).

For two-dimension gel electrophoresis, one pellet

of soluble protein was resuspended in the buffer

described by O’Farrell (1975) and used to run 2-D

SDS–PAGE gels, loaded with 124 µg of protein,

according to a modified procedure from O’Farrell

(1975) on an Investigator System (Millipore Corp.).

First-dimension isoelectric focusing was in a 4.1%

acrylamide tube gel containing 9.5 M urea, 2% (v}v)

Triton X100, 5 mM (3-((3-cholamidopropyl)-

dimethylammonio)-1-propanesulphonate), and 2%

(v}v) Millipore 2-D optimized carrier ampholytes

(pH 3–10). After 2 h of prefocusing to 1500 V with

current limited to 110 µA per tube, protein samples

(20 µl) were loaded on the basic end of the tube gel

and focused with 200 V per tube for 17.5 h. The gels

were removed and pre-equilibrated for 2 min in a

375 mM Tris-HCl buffer (pH 8.6), containing 3%

(w}v) sodium dodecyl sulphate and 50 mM dithio-

threitol. Separation of the isoelectrofocused proteins

in the second dimension was as for the one-

dimensional gels, using a 15% duracryl running gel,

run for 5 h at 500 V. Gels were subsequently silver

stained as described by Lopez et al. (1991).

The two-dimensional gels were analysed by

computerized image analysis (Millipore Bio Image

Millipore Corp., Ann Arbor, MI, USA). Gels were

scanned, and the individual staining intensities of

each polypeptide was then expressed as a percentage

of the total staining intensity of the gel. For each gel,

silver staining intensities of unknown proteins were

estimated using silver staining intensities of proteins

of known molecular weight and isoelectric point,

which were added to each gel in constant amounts of

proteins.

Analysis of soluble sugar

Soluble carbohydrates from freeze-dried samples

were extracted with 80% (v}v) ethanol (30 ml g−" d.

wt) for 1 h at 80°C. After centrifugation (12000 g, 10

min) the supernatant contained fructose, glucose,

sucrose and fructans of 4–7 degree of polymerization

(DP). Fructans of DP "7 were extracted by

resuspending the initial insoluble residue in water

for 1 h at 60°C. After centrifugation (12000 g, 10

min) the supernatants were pooled and evaporated to

dryness under vacuum. The dried extracts were

redissolved in water and filtered through a 0.45 µm

membrane. Aliquots of carbohydrate extracts were

passed through a column containing cation exchange

resin (DOWEX Hydrogen form, mesh 200–400,

Sigma Corp., St Louis, MO, USA) and anion

exchange resin (Amberlite IRA 416, formate form,

Serva, Germany) to remove charged compounds

(Smouter & Simpson, 1991). The columns were

eluted with water.

Samples were concentrated under vacuum, dis-

solved in water and aliquots were taken for analysis

by HPLC. Samples (20 µl) were first injected

through a cation exchange column (Sugar-PAK,

300¬6.5 mm, Millipore Waters, Milford, MA,

USA). The sugars were eluted at 85°C in water at a

flow rate of 0.5 ml min−" and detected with a

differential refractometer (156 Index Detector,

Beckman Instruments, San Ramon, CA, USA).

After separation, the sugars were quantified using

mannitol as an internal standard.

Regrowth, N uptake and N mobilization

There were marked differences between the two

cultivars in several aspects of their response to the

N-treatments. Dry weight of plants, tiller numbers,

and tissue concentrations of N and C immediately

after defoliation (day 0) are given in Table 1. ‘Low

N’ supply during the 10 d before defoliation did not

affect sheath or root dry weight significantly in cv.

Cariad, although the root dry weight of cv. Aberelan

was increased by 25%. The number of tillers per

plant was also unaffected by the regime of N

pretreatment, but was higher for ‘Cariad’ than

‘Aberelan’, accounting for the cultivar differences in

root and sheath dry weight.

As anticipated, ‘ low N’ pretreatment decreased

the total N and soluble protein concentrations in the

plants by the time of defoliation, except for soluble

protein concentrations in the roots of cv. Aberelan.

By contrast, the total concentration of WSC was

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N reserves and regrowth 455

Table 1. Dry weight, tiller number and concentration of nitrogen, soluble proteins, glucose, fructose, sucrose,

fructans (low and high DP) and total water soluble carbohydrates (WSC) in roots and leaf sheath of Lolium

perenne cv. Aberelan and cv. Cariad immediately after defoliation (day 0)

‘Aberelan’ ‘Cariad’

High N Low N High N Low N

Roots Sheath Roots Sheath Roots Sheath Roots Sheath

Dry weight

(mg per plant) 47.8 (4.1) 34.9 (2.2) 60.0 (5.4) 34.7 (2.9) 71.8 (7.6) 49.3 (6.9) 77.8 (12.3) 45.1 (6.7)

Tillers (per plant) 2.8 (0.1) 2.5 (0.2) 3.5 (0.3) 3.1 (0.4)

concentration

(mg g−" d. wt)

N 41.9 (0.6) 45.6 (0.6) 28.3 (0.3) 32.5 (1.4) 41.6 (0.4) 44.1 (0.3) 24.4 (0.4) 29.7 (0.9)

Soluble proteins 15.5 (0.8) 19.3 (0.5) 15.4 (1.1) 11.7 (0.4) 16.8 (0.1) 13.7 (1.7) 18.6 (0.2) 10.3 (0.4)

Glucose 10.5 (0.9) 10.8 (0.5) 8.2 (0.4) 16.4 (4.1) 10.2 (0.4) 18.4 (0.3) 7.2 (0.3) 18.4 (2.2)

Fructose 19.1 (0.8) 9.4 (0.4) 19.6 (1.0) 9.8 (0.9) 15.6 (0.5) 13.0 (0.5) 16.8 (0.2) 13.2 (2.5)

Sucrose 17.6 (0.3) 38.3 (0.4) 20.4 (0.1) 33.68 (1.0) 15.5 (1.1) 30.7 (1.9) 16.0 (0.8) 28.9 (1.3)

Fructans 13.4 (2.0) 31.5 (4.6) 19.0 (3.5) 98.3 (10.9) 8.0 (0.7) 21.5 (2.4) 9.1 (0.7) 47.0 (3.9)

Total WSC 60.7 (1.7) 90.0 (4.2) 67.2 (4.7) 158.2 (24.9) 49.3 (2.5) 83.6 (4.7) 49.1 (1.4) 107.5 (8.5)

During the 10 d before defoliation ‘high N’ plants received an optimal N supply and ‘ low N’ plants were supplied at

a rate resulting approx. 50% of the total N uptake achieved by ‘high N’ plants. Values in parentheses are SE of the mean

(n¯4).

higher in ‘ low N’ plants, particularly for fructans in

the leaf sheaths, which were "200% and 100%

higher in ‘Aberelan’ and ‘Cariad’, respectively.

Interestingly, the concentrations of the other com-

ponents of the WSC pool were largely unaffected by

N supply, except for an increase of 50% in glucose

concentrations in ‘ low N’ cv. Aberelan. Growth

before defoliation was lower for ‘Aberelan’ than

‘Cariad’ and the higher concentrations of WSC at

defoliation in ‘Aberelan’ may also reflect this.

Previous work has shown, that when growth is

similar, ‘Cariad’ generally has a higher WSC content

than ‘Aberelan’ (e.g. Evans et al., 1996).

Leaf regrowth following defoliation occurred at a

lower rate in ‘Aberelan’ (Fig. 1a) than ‘Cariad’ (Fig.

1c), irrespective of prior N nutrition, the effect being

related to differences in the number of tillers per

plant (Table 1). The cultivars also showed

differences in the effect of prior N supply on the

amount of N in regrowing leaves that was derived

from mineral N uptake following defoliation. For

‘Aberelan’ this was higher in ‘ low N’ plants than in

‘high N’ plants at a given regrowth dry weight (Fig.

1a), whereas there was no such effect in ‘Cariad’

(Fig.1c).

For both cultivars, and irrespective of N pre-

treatments, remobilized N was the predominant

source of N allocated to leaves during early regrowth,

accounting for "80% and 60% in ‘high N’ plants,

and 70% and 50% in ‘low N’ plants on day 2 and

day 4, respectively.

Low N pretreatment decreased the total N content

at the time of defoliation (Table 1), and therefore that

potentially available for remobilization; this decrease

was substantial in ‘Aberelan’ (Fig. 1b) but marginal

in ‘Cariad’ (Fig. 1d). This is illustrated by the data

after 14 d of regrowth. For ‘Cariad’ the dry weight

of leaves, leaf N derived from uptake and leaf N

derived from remobilization in ‘ low N’ plants were,

respectively, 23%, 21% and 27% lower than the

corresponding values recorded for ‘high N’ plants.

In ‘Cariad’ remobilization of N from reserves

accounted for 18% and 17% of total leaf N, in ‘ low

N’ and ‘high N’, respectively. By contrast, ‘ low N’

plants of ‘Aberelan’ (Fig. 1a,b), showed a 10%

(­23 mg d. wt per plant) increase in leaf dry weight

and a 23% (­2.9 mg N per plant) increase in the

amount of leaf N derived from uptake compared

with the corresponding ‘high N’ plants. The ‘low

N’ pretreatment decreased remobilization of N by

56% (®1.4 mg N per plant) in ‘Aberelan’ ; N

reserves only contributed 6.8% of leaf N in ‘low N’

plants compared with 17% in ‘high N’ plants.

These results suggest that ‘Aberelan’ compensated

for the lower concentration of N reserve by in-

creasing the uptake and translocation of N more

rapidly following defoliation than ‘Cariad’, with the

added benefit of increasing the rate of leaf regrowth

over that achieved by the ‘high N’ plants. This

stimulatory effect of N limitation before defoliation,

on subsequent early growth rates, relative to those in

N-sufficient plants, was confirmed in a separate

experiment with ‘Aberelan’ (data not presented).

The time-course for total mineral N uptake following

defoliation, calculated from "&N labelling data (Table

2), revealed marked cultivar differences in the

dynamics of the ‘ low N’ effect. ‘Low N’ plants of

both cultivars had proportionately similar increases

in uptake relative to ‘high N’ plants during the first

2 d and it was only subsequently that they diverged

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456 S. Louahlia et al.

20

15

10

5

00 100 200 300 400

Regrowth dry weight (mg per plant)

Lab

elle

d N

fro

m u

pta

ke (

mg

per

pla

nt)

0 100 200 300 400

20

15

10

5

0

0500

1

2

3

4

5

0

1

2

3

Un

lab

elle

d N

fro

m m

ob

iliza

tio

n (

mg

per

pla

nt)

0 10050 150 200 250 0 10050 150 200 250

(a) (b)

(c) (d)

Fig. 1. Effect of N supply before defoliation of Lolium perenne ‘Aberelan’ (a,b) and ‘Cariad’ (c,d) on the source

of N allocated to regrowing leaves plotted as a function of leaf dry weight. Successive points refer to days 0,

2, 4, 7, 11 and 14 after defoliation. N from uptake of NH%NO

$after defoliation (a,c) was labelled with "&N; N

remobilized from roots and sheath tissue (b,d) was unlabelled. During the 10 d before defoliation ‘high N’

plants (filled squares) received an optimal N supply whereas ‘ low N’ plants (open squares) were supplied at

a rate resulting in approx. 50% of the total N uptake achieved by ‘high N’ plants. Vertical and horizontal bars

indicate³SE of the mean when larger than the symbol, n¯4 for the first three harvests and n¯3 for the

remaining harvests. Note the different scales on the horizontal axis for a,b and for c,d.

200

150

100

50

00 2 4 6 8

Days of regrowth

Cu

mu

late

d µ

mo

l nit

rate

or

amm

on

ium

per

pla

nt

10 12

200

150

100

50

0

500

750

1000

1250

250

1000

1250

1500

(a) (b)

(c) (d)

14 0 2 4 6 8 10 12 14

250

250

300

750

500

0

200

400

600

0

200

400

600

800

Fig. 2. Effect of previous N supply on cumulative net uptake of NO$

− (a,c) and NH%

+ (b,d) from 20 mmol m−$

NH%NO

$by Lolium perenne ‘Aberelan’ (a,b) and ‘Cariad’ (c,d) during 14 d of regrowth following defoliation.

During the 10 d before defoliation ‘high N’ plants (filled squares) received an optimal N supply whereas ‘ low

N’ plants (open squares) were supplied at a rate resulting in approx. 50% of the total N uptake achieved by

‘high N’ plants. Uptake was calculated from the amount of each N form delivered to flowing nutrient solutions

in order to maintain constant concentrations, and was highly correlated (r !0.99) with values obtained by "&N

labelling of the solutions. Note that the numbers in the centre apply both to the right hand axis of a and c as

well as to the left hand axis of b and d.

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N reserves and regrowth 457

Table 2. Total uptake of N from 20 mmol m−$ NH%NO

$by Lolium perenne cv. Aberelan and cv. Cariad during

14 d of regrowth after defoliation, calculated from "&N labelling data

Cumulative N uptake (mg N g−" root d. wt)

‘Aberelan’ ‘Cariad’

Days after

defoliation High N Low N % high N High N Low N % high N

2 15.6 (0.6) 26.3 (1.2) ­69 12.6 (0.4) 21.26 (0.9) ­69

4 36.4 (3.7) 46.6 (1.8) ­28 36.9 (5.4) 38.0 (1.4) 0

7 96.0 (1.6) 97.6 (3.2) 0 100.2 (6.7) 86.9 (1.4) ®13

11 180.8 (5.8) 187.5 (3.5) 0 198.5 (5.0) 169.6 (5.9) ®14

14 212.2 (3.9) 212.4 (5.7) 0 225.0 (4.9) 200.2 (8.3) ®11

During the 10 d before defoliation,‘high N’ plants received an optimal N supply and ‘ low N’ plants were supplied at

a rate resulting in approx. 50% of the total N uptake achieved by ‘high N’ plants. Values in parentheses are SE of the

mean (n¯4 for the first three harvests and n¯3 for the remaining harvests). Uptake by ‘ low N’ plants is also expressed

as a proportion of uptake by ‘high N’ plants.

20

10

50 3 6 9

Days of regrowth

So

lub

le p

rote

ins

(mg

g–1

d. w

t)

12

20

15

(a) (b)

(c) (d)

30

25

10

0 3 6 9 12

15

25

Fig. 3. Changes in concentrations of soluble protein in sheath (a,c) and roots (b,d) of Lolium perenne ‘Aberelan’

(a,b) and ‘Cariad’ (c,d) during 14 d of regrowth after defoliation. During the 10 d before defoliation ‘high N’

plants (filled squares) received an optimal N supply whereas ‘ low N’ plants (open squares) were supplied at

a rate resulting in approx. 50% of the total N uptake achieved by ‘high N’ plants. Vertical and horizontal bars

indicate³SE of the mean when larger than the symbol, n¯4 for the first three harvests and n¯3 for the

remaining harvests.

in response. In terms of the net effect over 14 d, ‘ low

N’ pretreatment decreased total N uptake by 11% in

‘Cariad’ compared with ‘high N’ plants, but had no

effect on ‘Aberelan’.

The extent to which ‘low N’ treatment before

defoliation affected the subsequent uptake of NH%

+

and NO$

−, from the 20 mmol m−$ NH%NO

$supply,

varied with cultivar and time (Fig. 2). For example,

during the early phase of regrowth (0–4 d) of

‘Aberelan’, ‘ low N’ showed increased uptake of

both NH%

+ and NO$

− relative to ‘high N’ plants

(Fig. 2a,b), whereas ‘ low N’ ‘Cariad’ increased only

NO$

− uptake (Fig. 2c). Further, the relative en-

hancement in NO$

− uptake by ‘low N’ compared

with ‘high N’ plants persisted over 14 d in ‘Cariad’,

but was reversed after day 4 in ‘Aberelan’, although

the uptake of NH%

+ remained higher by ‘low N’

plants of both cultivars. The total uptake of NH%

+

over the experimental period was approx. twofold

higher than that of NO$

−, irrespective of cultivar and

N treatment before defoliation.

Soluble protein and profiles after SDS–PAGE

In both cultivars, ‘ low N’ pretreatment decreased

the concentrations of soluble proteins present at the

time of defoliation in sheath tissues (Fig. 3a,c) but

had hardly any effect in roots (Fig. 3b,d). The

Printed from the CJO service for personal use only by...

458 S. Louahlia et al.

45

kDa

31

21.5

14.5

D0 D2 D4 D7 D11 D14

(a)

55 kDa

36.6 kDa

24 kDa

45

kDa

31

21.5

14.5

D0 D2 D4 D7 D11 D14

(b)

Fig. 4. The effect of ‘high N’ (a) or ‘ low N’ (b) pretreatment before defoliation of Lolium perenne ‘Aberelan’

on subsequent changes in SDS–PAGE profiles of soluble proteins extracted from sheath tissue on days 0, 2,

4, 7, 11 and 14. Each well was loaded with soluble proteins extracted from 1.8 mg tissue d. wt, and the position

of molecular weight markers is indicated on each side. Arrows indicate polypeptides of 55, 36.6 and 24 kDa

affected by defoliation.

43

kDa

30

20.1

14.1

(a)

43

kDa

30

20.1

14.1

(b)

4.8 6.7IEF

MW4.8 6.7

IEF

MW

Fig. 5. Two-dimensional gel electrophoresis of soluble proteins extracted from ‘high N’ sheath tissue of Loliumperenne ‘Aberelan’, on day 0 immediately after defoliation (a) and 4 d later (b). Each gel was loaded with 124

µg of soluble protein and proteins of 55, 36.6 and 24 kDa affected by defoliation are indicated by arrows. The

position of isolectric points and molecular weight markers are indicated on the side of each gel.

relative insensitivity of roots to ‘ low N’ pretreatment

in this respect extended to trends in soluble protein

concentrations over the subsequent 14 d. These were

of broadly similar direction and magnitude in both

‘low N’ and ‘high N’ plants (Fig. 3). By contrast,

the concentrations of soluble protein in sheaths of

‘ low N’ ‘Aberelan’ and ‘Cariad’ increased pro-

gressively over the 14 d recovery period, whereas in

Printed from the CJO service for personal use only by...

N reserves and regrowth 459

25

So

lub

le p

rote

ins

(mg

g–1

d. w

t)

10

5

0

15

20

08 N

ov.

06 D

ec.

13 J

an.

20 F

eb.

20 M

ar.

22 A

pr.

22 M

ay

06 J

un

e

10 J

uly

1996–97

Mo

nth

ly m

ean

air

tem

per

atu

re (

°C)

Fig. 6. Seasonal changes in the concentration of total

soluble proteins (open squares) extracted from leaf sheaths

of Lolium perenne ‘Magella ’ grown under field conditions

and the monthly mean air temperature (filled circles).

Arrows indicate harvests. Vertical bars indicate³SE of the

mean for n¯3, when larger than the symbol.

‘high N’ plants they decreased until day 4, indicating

net hydrolysis, and then increased, the overall trend

being similar to for ‘Aberelan’ and ‘Cariad’.

The net hydrolysis of soluble protein in the

‘reserve’ tissues of ‘high N’ plants over the first 4 d

following defoliation was substantial. Concentra-

tions decreased by 32% in leaf sheath (Fig. 3a) and

by 15% in root tissue (Fig. 3b) of ‘Aberelan’ and by

47% in the sheath (Fig. 3c) and 25% in roots (Fig.

3d) of ‘Cariad’. The SDS–PAGE profiles of soluble

protein extracts of leaf sheath tissue from ‘Aberelan’

at different times after defoliation (Fig. 4) showed

that defoliation induced specific hydrolysis of several

polypeptides. In ‘high N’ plants (Fig. 4a) poly-

peptides with molecular weights of 55, 36.6 and 24

kDa, were evident at the time of defoliation and then

faded from the SDS–PAGE profile over the next 2–4

d, before reappearing and accumulating after day 7.

The same three polypeptides were present in lower

abundance in the tissues of ‘ low N’ ‘Aberelan’ on

the day of defoliation (Fig. 4b) and accumulated

between days 7 and 11 to concentrations above those

recorded on day 0.

Whilst the pattern of net hydrolysis followed by a

net accumulation of these particular polypeptides in

‘high N’ plants, together with their relative initial

scarcity in ‘ low N’ plants, suggested that they were

candidate VSPs, their silver staining intensities in

SDS–PAGE were low. Analysis of two-dimensional

gel profiles of soluble proteins extracted from sheath

tissue on day 0 (Fig. 5a) and day 4 after defoliation

(Fig. 5b) confirmed that these polypeptides were not

prominent in plants grown in FSC.

Under field conditions the concentrations of

soluble protein in the leaf sheath fraction (Fig. 6) in-

creased during autumn and winter, reaching a maxi-

mum during February and thereafter decreasing

31

14.5

21.5

08 N

ov.

06 D

ec.

13 J

an.

20 F

eb.

20 M

ar.

22 A

pr.

22 M

ay

10 J

uly

1996–97

45

kDa

55 kDa

36.6 kDa

24 kDa

Fig. 7. Seasonal changes in SDS–PAGE profile of soluble

proteins extracted from leaf sheaths of Lolium perenne‘Magella ’, grown under field conditions. Each well was

loaded with 10.23 µg of soluble protein, and the position of

molecular weight markers is indicated on each side. Arrows

indicate polypeptides of 55, 36.6, 24 kDa affected by

defoliation.

during Spring as growth resumed. The trend in the

concentrations of soluble protein during the year was

inversely related to the trend in monthly mean air

temperatures (Fig. 6), with a linear correlation

coefficient of determination of 0.88.

The 82% decrease in protein content of sheath

tissue between February and July, suggested a high

rate of protein hydrolysis. However, as the sheath

fraction was a composite of tissues with very different

ages, it is probable that much of this decline in

protein content was associated with senescence. The

presence of the same 55, 36.6 and 24 kDa poly-

peptides in plants grown in FSC was confirmed by

SDS–PAGE of proteins extracted from the field-

grown material (Fig. 7), but the profiles did not

provide convincing evidence of any seasonal pattern

in abundance.

Plant N status and the source of N for leaf growth

after defoliation

For numerous forage species (Ourry et al., 1989;

Richards, 1993; Thornton et al., 1993, 1994;

Thornton & Millard, 1996; Volenec et al., 1996),

mobilization of N compounds from tissues remain-

ing after severe defoliation plays an important role in

providing N for subsequent leaf growth. Whilst the

Printed from the CJO service for personal use only by...

460 S. Louahlia et al.

present results confirm this to be true for L. perenne,

more significantly they demonstrate for this species

that plant N status at the time of defoliation, which

is indicative of the potential availability of N reserves

for mobilization, is an important determinant of the

subsequent magnitude and dynamics of N

mobilization into the regrowing leaves. Further, they

point to the existence of substantial intra-specific

genotypic variation in the interactions between N

status, N mobilization and the rate of leaf growth

following defoliation. Decreased remobilization of N

in ‘low N’ plants was more acute in ‘Aberelan’ than

in ‘Cariad’ (Fig. 1). It is unlikely that this particular

difference was associated with variation in the

concentrations of tissue soluble carbohydrate be-

tween the two cultivars. With the notable exception

of fructans, there were only modest variations under

the present experimental conditions.

Low N status at the time of defoliation lessened the

extent to which mineral N uptake was subsequently

down-regulated (Fig. 2). This is consistent with

previous observations for L. perenne supplied solely

with NO$

− (Macduff et al., 1989). However, the

present results suggest that the extent of this N

status-related attenuation differs for the NH%

+ and

NO$

− uptake systems, and may also vary between

cultivars. Relative to the ‘high N’ plants, ‘ low N’

‘Aberelan’ exhibited significant NH%

+ and NO$

−

uptake during the first 4 d of recovery, whereas only

NO$

− uptake continued in ‘ low N’ ‘Cariad’ (Fig. 2).

The basis for these differences is unclear, but it is

tempting to regard the ability of ‘ low N’ ‘Aberelan’

to sustain NH%

+ uptake as having a direct bearing on

the higher dry weight of leaves of ‘ low N’ compared

with ‘high N’ plants of this cultivar (Fig. 1). The

capability to sustain uptake of NH%

+ following

defoliation presumably compensated the decrease in

N reserves resulting from ‘low N’ pretreatment

(Table 1).

The ability of L. perenne ‘Aberelan’, to com-

pensate for decreased availability of remobilizable N

by enhancing the rate of recovery in mineral N

uptake (Table 2), contrasts with that reported for L.

temulentum (Ourry et al., 1996) and M. sativa (Ourry

et al., 1994; Avice et al., 1997). In the latter species,

no such compensation was evident and the greater

delay in recovery of N uptake and N#

fixation may

account for their relatively slower refoliation, and

lower dry weight yields compared with L. perenne.

In this context it may be significant that L.

temulentum is an annual species, of little agronomic

significance and regarded as adapted to conditions of

relatively low N availability. On the other hand,

cultivars of L. perenne, such as those used in the

present study are usually the result of performance

selection under a relatively high plane of N nutrition.

These inter-specific differences in the ability to use

alternative sources of N following defoliation, would

appear to be consistent with the view that N storage

by grasses is a primary adaptation to environmental

conditions of low or fluctuating availability of N

(Thornton et al., 1993, 1994; Thornton & Millard,

1996); with N reserves constituting a buffer system

against periods of prolonged N starvation.

Are mobilization of N reserves and uptake of mineral

N coregulated following defoliation?

The generally inverse relationship between rates of

remobilization of stored N and uptake of mineral N

following defoliation stress, illustrated by the present

data, prompts several questions concerning how

these changes in internal N fluxes might be regu-

lated. Of particular interest is whether or not N

mobilization and N uptake are subject to a common

set of regulatory signals and, if so, how these up-

regulate mobilization almost coincidentally with the

down-regulation of N uptake following defoliation.

A definitive answer to these questions requires far

more detailed study of N dynamics during the first

24 h after defoliation than is reported here. However,

there is sufficient relevant information to suggest the

following crude ‘supply and demand’-based model

may apply at the whole plant level. Severe defoliation

has the immediate effect of removing a large

proportion of the plant’s ‘sink capacity’ for N

assimilates. A sudden perturbation in ‘demand’

would be expected to cause a rapid increase in

concentrations of amino acids in the root system,

and}or in the flux of amino acids cycling through the

remaining vascular system. Given that NH%

+ and

NO$

−uptake are considered to be subject to various

forms of negative feedback regulation, by amino

acids amongst other effectors (Ourry et al., 1997),

this would explain the very rapid down-regulation of

N uptake following defoliation (Macduff & Jackson,

1992). The down-regulation of uptake may then be

reinforced and sustained by the amino acid flux from

hydrolysis of reserve proteins in the roots and sheath

tissue, until such time as the net consumption rate of

amino acids for protein synthesis in new leaf tissue

exceeds their mobilization-related production in the

source tissues.

According to this scheme, the attenuation, or

absence, of down-regulation of N uptake in ‘ low N’

status plants would occur initially because there is a

lower (or zero) increase in amino acid concentrations

following removal of ‘sink’ tissues. This would be a

consequence of the prevailing shortfall in the uptake

of N relative to assimilatory N demand for tissue

synthesis. Subsequently, as a consequence of the

smaller N reserve pool in ‘ low N’ plants, there

would be a lower rate of amino acid production from

protein hydrolysis and other N mobilization pro-

cesses and, more speculatively, a much shorter

residence time for these amino acids at sites of

regulatory action on the N uptake systems. This

model could also explain the apparent over-com-

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N reserves and regrowth 461

pensation observed in ‘ low N’ ‘Aberelan’ (Fig. 1).

However, it does not account for the dramatic up-

regulation of N mobilization from source tissues

following defoliation, the exact timing of which in

relation to the initiation of the decline in mineral N

uptake remains uncertain. Intuitively it is unlikely

that N remobilization is stimulated directly by an

‘amino acid surge’ in the vascular system of the

plant following defoliation, as anticipated in the

model outlined: other signalling systems uncon-

nected with plant N status are more probable in our

opinion (e.g. aspects of soluble carbohydrate status).

Moreover, there are grounds for expecting the

opposite effect. For example, a temporary increase

in plant N status, denoted by increased vascular

amino acid concentrations, might down-regulate N

mobilization if there is no immediate requirement

for the resulting amino acids associated with tissue

synthesis. It is significant that apparent ‘antagonism’

between N uptake and mobilization of N stores is a

widespread phenomenon in the plant kingdom

(Imsande & Touraine, 1994), and is of particular

significance at certain ontogenetic stages, such as

seed setting.

There are clearly various other hypothetical

explanations for aspects of the N dynamics observed

following defoliation of grasses. The down-regu-

lation of N uptake per se might trigger the

mobilization of N from reserves: the plant in effect

mistaking the ensuing post-defoliation stress for a

shortfall in external N supply that requires tem-

porary buffering from N reserves. Another possible

explanation for the continuation of mineral N uptake

by ‘low N’ plants, at least in the short-term following

defoliation, invokes the ‘N demand’ constituted by

replenishment of the vacuolar nitrate pool, pre-

viously depleted during ‘low N’ pretreatment

(Macduff et al., 1989). However, it is less straight-

forward to explain continued uptake of NH%

+ by this

mechanism.

Are VSPs involved in the storage and remobilization

of N in perennial ryegrass?

VSPs have been characterized in two species of

forage legumes: M. sativa (Hendershot & Volenec,

1993) and T. repens (Corre et al., 1996). In both

species they account for at least 20% of total soluble

protein, in taproots and stolons. They have at least

two functions, which are usually separated in time:

the provision of reduced N for early regrowth

following defoliation, and also for spring growth. As

far as we are aware, functional VSPs have not been

reported in grasses such as L. perenne. The three

polypeptides (of 55, 36.6 and 24 kDa) identified in

the present study (Figs. 4, 5) satisfied some of the

criteria for classification as VSPs. They were nearly

fully hydrolysed during early regrowth and accumu-

lated in tissue during autumn and winter. Moreover,

under conditions of ‘ low N’ supply (Fig. 4b) they

did not accumulate to the same extent as in ‘high N’

plants (Fig. 4a), suggesting that their synthetic rate is

somehow coupled with plant N status. Taken

together, these characteristics might suggest that

they have a functional role as VSPs. However, they

were not found to be prominent polypeptides

(accounting for !8% of total silver staining in-

tensity), and accordingly do not satisfy all of the

criteria for VSP status as prescribed by Staswick

(1989, 1990, 1994). It is possible, but unlikely, that

these polypeptides belong to the same hetero-

polymer, in which case their VSP status would be

less doubtful. Similar conclusions were reached by

other workers searching for VSPs in M. officinalis

and T. pratense (Cunningham & Volenec, 1996; Li et

al., 1996).

This work was partly funded through an INRA}BBSRC

grant to A. O. The authors thank Mrs Micheline Meyer

(ATOS, University of Caen, France) for her invaluable

help with electrophoresis, Dr Terence N. Ledger for

valuable discussions and improvement of the manuscript

and Mr D. Perrin for his efficient collaboration for field

work.

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