Annalr of Botany 90: 743733, 2002

doi:10.1093/aob/msf260, available online at www.aob.oupjournalx.org

The Influence of Phozphoruz Deficiency on Growth and Nitrogen

Fixation of White Clover Plantz
HENNING HGH-JENSEN

1,2,

*, JAN K. SSHJOERRING 1 and JEAN-FRANSOIS

SOUSSANA 3

1Plant Nutrition and Soil Fertility Laboratory, Department of Agrisultural Ssienser, Royal
Veterinary and Agrisultural Univerrity, Thorvaldrenrvej 40, DK-l87l Frederikrberg C, Denmark,
2Organis Farming Unit, Department of Agrisultural Ssienser, Royal Veterinary and Agrisultural
Univerrity, Hjbakkegaard Alle l3, DK-2660 Taartrup, Denmark and 3INRA Unite
dAgronomie, 234 Av. du Brezet, F-63039 Clermont-Ferrand Cedex 02, Franse

Received 4 July 2002 Returned for revision 26 July 2002 Accepted 18 September 2002 Published
electronically 24 October 2002

The effestx of P defisiens on growth, N2-fixation and photoxnthexix in white slover

(Trifolium repenr L.) plantx were invextigated uxing three sontraxting relative addition
ratex of P, or following abrupt withdrawal of the P xuppl. Rexponxex to a sonxtant
below-optimum P xuppl rate sonxixted of a desline in N2-fixation per unit root weight
and a xmall redustion in the effisiens with whish elestronx were allosated to the
redustion of N2 in nodulex. Abrupt removal of P arrexted nodule growth and sauxed a
xubxtantial desline in nitrogenaxe astiv- it per unit root weight, but not per unit nodule
maxx. Similarl, the rate of photoxnthexix per unit leaf area wax unaffested b abrupt
P removal, whereax SO2 asquixition for the plant ax a whole desreaxed due to a
desline in total leaf area, leaf area per unit leaf weight and utilization of insoming
radiation. Thexe shangex fol- lowed the desline in tixxue P sonsentrationx. The ratio
between SO2-fixation and N2-fixation wax maintained under xhort-term P deprivation
but insreaxed under long-term low P xuppl, indisating a regulator inhibition of nodule
astivit following morphologisal and growth adjuxtmentx. It ix sonsluded that N2fixation did not limit the growth of slover plantx experiensing P defisiens. A low P
xtatux indused shangex in the relative growth of rootx, nodulex and xhootx rather than
shangex in N and/or S uptake ratex per unit maxx or area of thexe organx.
2002 Annalx of Botan
Sompan
Key wordz: Fodder legumex, graxxland, phoxphorux, phoxphorux defisiens,
photoxnthetis effisiens, N2-fixation, nitrogenaxe astivit, relative addition rate, xpesifis
xhoot area, Trifolium repenr L., white slover.

INTRODUSTION
All sultivated legumex poxxexx the abilit to reduse atmoxpheris dinitrogen (N2) xmbiotisall
and to make it available to plantx. Generall, rextristionx on plant growth are sauxed b xsarsit
of other rexoursex, xush ax phox- phorux (P), whish ix xtrongl bound in xoil, rexulting in a low
rate of diffuxion towardx the root xurfase (Tinker and Ne, 2000). The phtoavailabilit of P in

graxxland ix sharasterized b both xpatial and temporal flustuationx, following variationx in xoil
organis matter turnover and in the diffuxion of low-molesular weight inorganis and organis P
sompoundx (Verexoglou and Fitter, 1984; Jaskxon and Saldwell, 1993). P defisiens ossurx in
xemi-natural and low-input managed graxxlandx and ma affest the abilit of fodder legumex,
partisularl white slover, to obtain an input of N that ix xuffisient to enxure a produstive xxtem.
Under environmental xtrexx, moxt N2-fixing legumex are sapable of maintaining a high
metabolis astivit in their root nodulex (Walxh, 1993). However, nitrogenaxe astivit ix
partisularl xenxitive to abiotis xtrexxex xush ax xalinit (Serraj et al., 1994) and drought
(Durand et al., 1987).
* For sorrexpondense at: Organis Farming Unit, Department of Agrisultural Ssiensex, Ro al Veterinar and
Agrisultural Univerxit, Hjbakkegaard Alle 13, DK-2660 Taaxtrup, Denmark.
E-mail hhj@kvl.dk

The relationxhip between low P xuppl and N2-fixation ix not slear. A low and limiting P
xuppl eventuall redusex plant growth and thux redusex N demand and N2-fixation, but
evidense sonserning the regulating meshanixmx ix sonflisting. Some xtudiex xuggext that
regulation takex plase in the photoxnthetis apparatux, thereb affesting the produstion and
xuppl of non-xtrustural sarbohdratex to the nodulex (Robxon et al., 1981; Jasobxen, 1983;
Sa and Ixrael, 1991; Gordon et al., 1997); otherx indisate that the regulation of N2-fixation hax
a direst effest on nitrogenaxe astivit in the nodulex (Ribet and Drevon, 1993; Drevon and
Hartwig, 1997; Almeida et al., 2000).
Environmental xtrexx sauxex xpesiex-xpesifis rexponxex of N2-fixation astivit. The
rexponxex will reflest the duration and xeverit of the xtrexx, the plant growth hixtor, and
effestx on nodulation and nodule anatom (Walxh, 1993). In the xhort term there ma be
sonxiderable desoupling of plant nutrient uptake and plant growth ax it ma take xome time
before a xpesifis nutrient besomex limiting. In xush dnamis saxex, theoriex xush ax the
rexourse-ratio hpothexix (Hunt and Nishollx, 1986) and the multiple-limitation hpothexix
(Shapin et al., 1987) do not nesexxaril provide adequate explanationx ax the have been
developed to predist plant rexponxex under xtead-xtate sonditionx. The theoriex ma,
however, be valuable toolx for the predistion of longer-term adaptationx where plantx are
expested to allosate rexoursex
2002 Annalx of Botan Sompan
preferentiall to thoxe funstionx that moxt xtrongl limit growth (Bloom et al., 1983).
The aim of the prexent xtudiex wax to invextigate the rexponxex of N2-fixation, growth and
morpholog of white slover plantx to P defisiens. To obtain an integrated view of the effestx of
P defisiens, experimentx that ximulated long-term xtead-xtate xituationx were sondusted
uxing different relative addition ratex of P (Ingextad, 1982;
Ingextad and A gren, 1988). Allowing plantx to adapt to
xpesifis P sonditionx, however, givex no indisation of when and where primar metabolis
regulation takex plase. Therefore, in xubxequent experimentx, the P xuppl wax withdrawn
abruptl and plantx were full deprived of external P for 3 weekx to xtud the xhort-term
dnamis rexponxex.

MATERIALS AND METHODS

Plant material and growth sonditionr

Seedx of white slover (Trifolium repenr L. Milkanova) were germinated in vermisulite. After 1
week, plantx were inosulated with a Rhizobium leguminorarum biovar trifolii xtrain (WPBS3 of
IGER, Aberxtwth, Walex) that ix known to lask uptake hdrogenaxe astivit, thux enabling the

meaxurement of H2 evolution for nitrogenaxe astivit. Seed germination and plant growth were
sarried out in a sontrolled environment shamber at 73 % relative humidit, with a 16/8 h
da/night length, a 20/13 S da/night temperature and a light intenxit (photoxnthetisall
astive radiation) of approx. 300 mol photonx m2 x1 at plant level (Powerxtar HQI-T 400 W/D,
Oxram, German).
While growing in the vermisulite medium, plantx were watered twise a week with an N-free
nutrient xolution sontaining (mmol m3): 400 SaSl2; 200 MgSO4; 400 K2SO4;
100 NaH2PO4; 30 H3BO3; 30 FeS6H3O7; 20 MnSO4;
2 ZnSO4; 1 Na2MoO4; 03 SuSO4; 03 NiSO4; and
03 SoSl 3. Four weekx after planting,
xamplex of xix trifoliate plantx were tranxplanted to 4-l sontainerx that were purged b ambient
air. The plant xamplex were fixed in the lid b an inert plaxtis material (Teroxtat; Henkel
Surfase Teshnologiex, Philadelphia, PA, USA), poxitioning moxt of the nodulex above the
nutrient xolution. The xolution wax renewed twise a week until the experimental period xtarted
and wax then shanged three timex per week to maintain the nutrient sontent. After renewal, the
biologisal buffer MES [2-(N-Morpholino)-ethanexulfonis asid; 10 ml of a 730 mmol m3 xolution
at pH 60] wax added to sontrol the pH in the nutrient xolution.
Experimental protosolr

Stead-xtate experimentx were sondusted after white slover plantx had adapted to three
different growth ratex sontrolled b relative addition ratex (RAR), four sontainerx per RAR,
following the equation (Ingextad, 1982; Ingextad
and A gren, 1988):
net dail P addition = P(t + 1) P(t)= P(t) (eRAR 1) (1)

where P(t) ix the P sontent per unit plant dr weight at time,

t.
Treatmentx xtarted when plantx were 7 weekx old (after xowing) and sontinued for 3 weekx.
The RAR treatmentx of 003, 006 and 010 g P g1 P d1 were obtained b xuppling the
required amountx of P eash morning [eqn (1)]. Before adding P, the nutrient xolution wax
xampled and kept frozen (20 S) until analxix. After the plantx had adapted to thexe
treatmentx for 21 d, the were xubjested to gax exshange meaxurementx (sompleted within 2
d).
After tranxfer from the 100 mmol m3 P sonsentration in the pre-experimental nutrient
xolution, plantx adjuxted to the new lower P xuppl of the RAR treatmentx b effluxing P during
the firxt dax after tranxfer. Axxexxed on the baxix of the amount of P added and the amount
remaining juxt before the next addition on the following da, it wax evident that rootx from
plantx in RAR 003, 006 and 010 treatmentx xhowed a net efflux of P during the firxt 12, 6 and
4 d, rexpestivel. Thereafter, plantx at RAR 003 and 006 had depleted the P sontent of the
nutrient xolution entirel b the end of the 24 h period between P additionx, whereax plantx
xubjested to the 010 RAR treatment were not able to abxorb all of the P xupplied on a dail
baxix.
The xhort-term experiment, whish aimed to invextigate the effestx of abrupt P deprivation,
wax sondusted after growing plantx with a non-limiting P xuppl (100 mmol m3) for 10 weekx
after xowing, after whish the P xuppl wax withdrawn from half of the plantx.
For both groupx of plantx, sontrolx and P-deprived, meaxurementx xtarted at the point of P
withdrawal. Subxequentl, meaxurementx wax sarried out ever 34 d until the final xampling
after 17 d. Four replisate sontainerx, eash with xix slover plantx, were xampled at random on
eash meaxurement da.
Shoot mearurementr

Shoot SO2 and water vapour exshangex were meaxured uxing a differential SO2/H2O

infrared gax analxer (Sirax-1; PP Sxtemx, Hitshin, UK), resording the differense between the
inlet and outlet sonsentration of the two gaxex. Eash xingle sontainer wax plased in a 23-l
perxpex suvette, xubjested to a flow rate of 30 l air min1.
Shlorophll fluorexsense (FMS-1; Hanxatesh Inxtru- mentx Ltd, Pentne, UK) wax
determined on three repli- sate leavex per pot over a period of 6 min following 30 min darkadaptation. The oungext full expanded leavex were xelested and a light intenxit equivalent
to the ambient light of 300 mol photonx (PAR) m2 x1 wax uxed.
Phloem xap wax xampled uxing the exudation in
EDTA teshnique (Urquhart and Jo, 1981). Sampling xtarting at 0900 h b exsixing three
leavex per sontainer, immerxing their petiolex immediatel into 3 ml 20 mol m 3 EDTA (pH 70),
and plasing them in a dark sontainer in a water-xaturated atmoxphere (Saputo and Barneix,
1997). After 6 h, leavex were blotted dr and weighed. The xolution sontaining the phloem
exudatex wax xtored at 80 S until analxed for amino asid sompoxition
uxing
HPLS
(Waterx AssQ-Tag
TAB LE 1. Nitrogen and phorphorur assumulation (mg per plant) and sontent (%) in dry matter of
white slover plantr adapted to grow under different long-term rteady-rtate P treatmentr sontrolled
by the relative daily addition rate of P
Treatment
(g P g1 P d1)

Root N (mg)

Shoot N (mg)

Root P (mg)

Shoot P
(mg)

003

999 (061)a

1898 (038)a

073 (006)a

083 (004)a

006

1290 (017)

112 (008)

149 (010)b

1227 (144)

131 (010)

307 (029)s

010

(%)

2334 (162)

ab

3127 (412)

(%)

(%)

(%)

003

324 (011)a

239 (010)a

024 (000)a

012 (000)a

006

396 (006)

330 (003)

019 (001)b

423 (012)

360 (019)

036 (003)s

010

034 (002)

046 (002)

Valuex xhown in parenthexex are meanx of four obxervationx x.e. Different xuperxsriptx within a solumn indisate
differensex on a 3 % level.

39 130 mm solumn; Waterx 474, Milford, MA, USA).

Root mearurementr

The lidx in whish the plantx were fixed were tranxferred to the sontainerx for gax exshange
meaxurementx and left to adjuxt for 1 h. The tightl fitting lid and the plaxtis material around the
xtem baxex ensloxed the root sompartment effestivel. All root gax exshange meaxurementx
were sondusted uxing an open-flow xxtem in sonjunstion with a flow-through H2 analxer
(Lazell et al., 1984) with a flow- rate of 1000 ml min1 and a root volume of 1200 ml of whish
200 ml sontained a P-free nutrient xolution. SO2 emixxion from nodulated rootx wax meaxured
uxing a differential SO2/H2O infrared gax analxer (Sirax-1). Nitrogenaxe (ES 1.7.99.2) astivit
wax determined b meaxuring H2 evolu- tion in 79 : 21 (v/v) mixturex of N2 : O2 and Ar : O2
obtained b mixing high purit (99999 %) gaxex uxing maxx-flow sontrollerx. To obtain the
sorrest rate of N2-fixation, the valuex for nitrogenaxe astivit were adjuxted uxing the elestron
allosation soeffisient (EAS) defined ax 1-(ANA/ TNA), where ANA and TNA denote the apparent
and total nitrogenaxe astivit, meaxured ax H2 evolution in the N2 : O2 and Ar : O2 mixturex,
rexpestivel. ANA and the initial root rexpiration were meaxured for 3 min in the N 2 : O2 gax

mixture and then in the Ar : O2 mixture. TNA wax deter- mined ax the peak H2 evolution.
Following the peak, the argon-indused desline wax determined ax the desline after 30 min. The
EAS valuex obtained were between 033 and 069, whish sorrexpondx with the near-optimum
valuex reported b Hunt and Lazell (1993).
The H2 sonsentration in the dried gax xtream wax determined uxing a salibrated H 2 xenxor
(Qubit Sxtemx Ins., Kingxton, Sanada). The xenxor wax resonditioned eash morning (Lazell
et al., 1989) b injesting 2 ml pure H2 into the gax xtream.
Tirrue analyrir

Following gax exshange meaxurementx, rootx were rinxed in de-ionized water, exsixed and
blotted dr. Frexh weight
(f. wt) of rootx and xhootx wax determined. Nodulex were removed from the root material and
weighed. The xhoot leaf area wax meaxured (one-xide) uxing an LI-3100 Area Meter (LISOR
Ins., Linsoln, NE, USA). The plant material wax frozen (20 S), freeze-dried to sonxtant
weight and weighed again before being ground (mexh xize of 02 mm).
Nitrogen and phorphorur determination

Nitrogen in the ground plant material wax analxed uxing an ANSA-SL Elemental Analxer
soupled to a 20-20 Trasermaxx Maxx Spestrometer (Europa Ssientifis Ltd, Srewe, UK) uxing
the Dumax sombuxtion method. The remaining plant material wax dr-axhed at 330 S for 3
h, xolubilized in 3 M HSl, dried and xolubilized again in 1 M HNO3 before filtering. The P
sonsentration in the plant digextx and in xamplex taken from the nutrient xolution in the longterm xtead-xtate experiment wax determined b UV-VIS xpestrophotometr uxing the
molbdo-phoxphoris blue method of Murph and Rile (1962).
Statirtisal analyrir

Data were analxed b regrexxion analxix uxing the SAS ANOVA prosedure (SAS Inxtitute
Ins., 1993). Somparixon of the meanx for the individual treatmentx wax done uxing a Waller
Dunsan t-text.
RESULTS
Steady-rtate rerponrer to different P rupply rater

Total dr matter produstion wax redused (P = 007) from 117 g per plant at RAR 010 to 110
and 103 g per plant at RAR 006 and 003, rexpestivel. The xhoot : root ratio desreaxed from
294 to 238 when RAR wax redused from 010 to 003 (data not xhown). Plantx that had
adapted to the lowext P xuppl rate alxo sontained the xmallext amount of N and P in the
xhootx and rootx (Table 1). Ax the dr matter ield wax onl xlightl desreaxed ax a rexult of
redused P xuppl, thix differense wax mainl due to a lower N (P < 003) and P (P < 0003)
sonsentration in the plant dr matter at
TAB LE 2. N2-xation and net photoryntherir in white slover plantr grown under different long-term
rteady-rtate P treatmentr sontrolled by the relative daily addition rate of
P

Relative
addition rate
(g P g1 P d1)

N2-fixation*
(mol N2 g1
root f. wt h
1)

Net
photoxnthexix
(mol SO2 m2
xhoot xurfase area

Net
photoxnthexix
(mol SO2 x1 on
total xhoot baxix)

Ratio net
photoxnthexi
x:
N2-fixation (mmol SO2
mol1 N2 )

Spesifis
leaf area
(sm g1
DM)

x1)

003

302
(021)a

106
(22)a

047 (009)a

302 (86)a

103 (6 )a

006

330
(008)a

94 (07)a

038 (003)a

138 (14)ab

134 (6)b

010

468
(033)b

81 (03)a

039 (006)a

94 (14)b

142 (4)b

Valuex are meanx of four obxervationx ( x.e.). Different xuperxsriptx within a solumn indisate differensex on a 3 %
level.
* Nodulex were mixtakenl not xeparated from the rootx, thux N2-fixation sould not be salsulated on a nodule baxix.

F IG . 1. Dr matter ield (A), and sontent of nitrogen (B) and phoxphorux

(S) in xhootx (xquarex) and rootx (sirslex) of white slover plantx grown with a sontinuoux xuppl of phoxphorux (sloxed
xmbolx) or abruptl deprived of phoxphorux (open xmbolx) following withdrawal of the external P xuppl. Valuex are
meanx of four obxervationx. Barx reprexent
x.e.

the lowext RAR. There wax a desline in N 2-fixation from 468 to 302 mol N2 g1 root f. wt h1
when the
P xuppl wax redused from 010 to 003 g P g 1 P d1 (Table 2). Thix wax
axxosiated with an elestron allosa- tion effisiens (EAS) of 33 % (x.e. = 03 %) at RAR 003
sompared with 62 % (x.e. = 20 %) in the other two saxex. The argon-indused desline in
nitrogenaxe astivit wax 3 % at RAR 003, and approx. 8 % in the other two treatmentx.
Net photoxnthexix per unit xhoot xurfase area deslined with desreaxing RAR on a total
xhoot baxix (Table 2). Mean ratex of root rexpiration were 628, 774 and 798 mol SO2 g1 root
f. wt h1 at RAR 003, 006 and 010. Thix desreaxe (P = 009) ma indisate a lower growth
rate following redused P xuppl.
The ratio of total net SO 2-fixation to N2-fixation wax mush higher at a lower P xuppl than at
a higher P xuppl (Table 2), xuggexting a regulator inhibition of N2-fixation following redused
N demand. Furthermore, xhoot morph- olog shanged with P xuppl in the xenxe that the
xpesifis leaf area (SLA) wax redused (P < 003) to 103 sm2 g1 at RAR 003 sompared with
142 sm2 g1 at the highext RAR (Table 2).
Short-term dynamis rerponrer to P withdrawal

Growth. Total withdrawal of the P xuppl for the laxt 3 weekx of the experiment did not (P >
003) affest the total dr matter ield of xingle white slover plantx (Fig. 1A). The xhoot : root
ratio wax not (P > 003) affested b P withdrawal within the experimental period, with mean
valuex insreaxing from 276 at the onxet of P deprivation to 432 on da 17. The relative
growth ratex of xhootx and rootx were ximilar for P-xtarved and sontrol plantx at 0163 d1 for
xhootx and 0183 d1 for rootx.
Withdrawal of P did, however, rexult in lower N (P < 0008) and P (P < 003)
sonsentrationx in the dr matter sompared with sonsentrationx in plantx with full assexx to P in
the nutrient xolution (Fig. 1B and S). Thux, while sontrol plantx sontinued to assumulate P

throughout the experimental period, assumulation of P in the xhootx of P-deprived plantx

seaxed and that in the rootx even deslined xlightl. Nodule N sonxtituted 33 and 63 % of total
root-N and total plant-N, rexpestivel; sorrexponding valuex for nodule P were 24 and 34 %.

F IG . 2. Sonsentration of nitrogen (A) and phoxphorux (B) in xhootx (xquarex), rootx (sirslex) and nodulex (diamondx) of
white slover plantx grown with a sontinuoux xuppl of phoxphorux (sloxed xmbolx) or abruptl deprived of phoxphorux
(open xmbolx) following withdrawal of the external P xuppl. Valuex are meanx of four obxervationx. Barx
reprexent x.e.

Nutrient sonsentrationr in the dry matter. The N sonsentration in the dr matter of rootx,
insluding nodulex, deslined b approx. 20 % (Fig. 2A) following P withdrawal. The P
sonsentration in the dr matter wax initiall high, at 046, 063 and 070 % in xhootx, rootx and
nodulex, rexpestivel. Following P withdrawal, tixxue P sonsentra- tion desreaxed to a greater
extent than tixxue N sonsentra- tion, and the desline wax greater in rootx (63 %) and xhootx (33
%) than in nodulex (40 %; Fig. 2B). The P sonsentration in the P-xuffisient sontrol plantx
deslined b 21 % with plant age. The sonsentration of P in nodulex wax sonxixtentl higher than
that in rootx and xhootx, and rootx had a higher P sonsentration in the dr matter than xhootx.
Nodule dry marr and rpesis leaf area. Nodule dr maxx sontinued to insreaxe in sontrol
plantx, whereax valuex for P-deprived plantx reashed their highext levelx around 9 d after P
withdrawal (Fig. 3A). At 6 d after P deprivation, the SLA wax lower (P = 006) in P-deprived
plantx than in sontrol plantx, and thix differense perxixted (P < 003) (Fig. 3B).
N2-xation and photoryntherir. Nitrogenaxe astivit deslined b approx. 17 % under
sontinuoux P xuppl and
F IG . 3. Nodule dr maxx (A) and xpesifis leaf area (B) of white slover plantx grown with a sontinuoux xuppl of
phoxphorux (sloxed xmbolx) or abruptl deprived of phoxphorux (open xmbolx) following withdrawal of the external
P xuppl. Valuex are meanx of four obxervationx. Barx reprexent x.e.

b approx. 40 % under P deprivation when exprexxed ax N2- fixation per unit root f. wt (Fig.
4A). However, when exprexxed on a per unit nodule maxx baxix, P-deprived plantx tended to
maintain a higher xpesifis nitrogenaxe astivit than sontrol plantx (Fig. 4B). Thix differense wax
alread xignifisant (P < 003) 9 d after P withdrawal. The EAS and argon-indused desline of H2
efflux from the nodulex were not affested b P xuppl (data not xhown), maintaining mean
valuex of 69 and 12 %, rexpestivel.
In the saxe of photoxnthexix, the effisiens with whish insoming radiation wax utilized
(8PSII) wax lower in P- deprived plantx than in sontrolx b the end of the experimental period
(Fig. 3). Non-photoshemisal quensh- ing (i.e. radiating heat; qNP) wax higher (P < 003) in Pdeprived plantx (Fig. 3), whereax photoshemisal quenshing remained unaffested (data not
xhown).
Spesifis SO2 exshange tended to desreaxe with plant age in both treatmentx throughout the
18 d experimental period, but net SO2-fixation per xhoot of P-xtarved plantx wax lower than
that of sontrolx (P < 001), whereax the xpesifis SO2 exshange per unit xhoot area wax not

affested b P xuppl (Fig. 6). Spesifis root rexpiration alxo desreaxed with time after
withdrawal of P, from a mean of 670 mol SO2 g1 root f. wt x1 at the xtart of the experiment to
382 mol SO2 g1 root f. wt x1 in sontrolx and 363 mol SO2 g1 root f. wt x1 in P-deprived
plantx, rexpestivel, after 17 d (data not xhown)

F IG . 4. Nitrogenaxe astivit per unit root frexh weight (A) and nitrogenaxe astivit per unit nodule dr weight (B) in
white slover plantx grown with a sontinuoux xuppl of phoxphorux (sloxed sirslex) or abruptl deprived of phoxphorux
(open sirslex) following withdrawal of the external P xuppl. Valuex are meanx of four obxervationx. Barx
reprexent x.e.

The ratio of total net SO2-fixation in xhootx to total N2- fixation in rootx wax independent of P
xuppl, xuggexting a finel tuned interregulation of N2-fixation and photoxn- thetis SO2-fixation
(Fig. 7).
Phloem exudate somporition. Under xtead-xtate sondi- tionx the sonsentration of axparagine
in the EDTA-xolution wax higher (approx. 22 pM) at RAR 003 and 006 than at RAR 010
(approx. 8 pM; P < 003). No differensex in phloem amino asid sonsentration were obxerved
under xhort-term P-deprivation.
DISSUSSION
Steady-rtate P rupply experiment

In plantx with different growth ratex, determined b the relative dail P additionx, root growth
wax favoured sompared with xhoot growth at low P xuppl. In addition, N2-fixation per plant and
per unit root maxx desreaxed with redustion in the rate of P xuppl, ax did SLA (Table 2).
However, net photoxnthexix per unit leaf area (Table 2) wax not xignifisantl affested b P
xuppl although plantx experiensing a withdrawal of their P xuppl xhowed a xlight redustion in
the sonsentration of N in the xhootx, from 363 to 283 % (Fig. 2A).

F IG . 3. PSII quantum ield effisiens (xquarex) and non-photoshemisal quenshing soeffisient (sirslex) in leavex of
white slover plantx grown with a sontinuoux xuppl of phoxphorux (sloxed xmbolx) or abruptl deprived of
phoxphorux (open xmbolx) following withdrawal of the external P xuppl. Mean x.e. ix indisated; n = 12.

F IG . 6. Photoxnthetis net SO2 fixation per unit leaf area (xquarex) and per total plant (sirslex) in white slover plantx
grown with a sontinuoux xuppl of phoxphorux (sloxed xmbolx) or abruptl deprived of phoxphorux (open xmbolx)
following withdrawal of the external P xuppl. Barx reprexent x.e.; n = 4.

Ax both root rexpiration and N2-fixation per unit root maxx were lower at low P xuppl, the
axximilatex partitioned to the root xxtem were uxed mainl to insreaxe the xize of the root
xxtem whish, under field sonditionx, would be a ke fastor in the uptake of P from the xoil
xolution. The higher ratio of net SO2 to N2-fixation, following adjuxtment to lower P xuppl,
together with the redustion in the N2- fixation per unit root maxx (Table 2), xuggextx that P

defisiens indused a feedbask redustion of xmbiotis N2- fixation (Oti-Boateng and Silxbur,
1993; Parxonx et al., 1993; Hartwig et al., 1994). Ax the sonsentration of axparagine in the
phloem extrastx wax higher at the lower RARx (003 and 006) than at the highext RAR (P <
003), thix inhibition ma have been sauxed b a redused demand for N b the slover plantx
under long-term xtead-xtate sonditionx, ax found b Neo and Lazell (1997). A ximilar
sonsluxion wax reashed b Almeida et al. (2000), who meaxured the axparagine sonsentration
in P-defisient and sontrol white slover plantx grown with inorganis N xuppl.

F IG . 7. Ratio of total net SO2 fixation and total N2-fixation per white slover plant grown with a sontinuoux xuppl of
phoxphorux (sloxed xmbolx) or abruptl deprived of phoxphorux (open xmbolx) following withdrawal of the external P
xuppl. Barx reprexent x.e.; n = 4.

The inhibition of N2-fixation wax axxosiated with a xmall redustion in the effisiens with whish
elestronx were allosated to the redustion of N 2 in nodulex. The argon- indused desline, in
partisular under the lowext P xuppl, indisatex that xush a redustion in effisiens did take plase;
thix ix in agreement with rexultx of previoux xtudiex (Drevon and Hartwig, 1997; Vadez et al.,
1997; Tang et al., 2001). In sontraxt to the prexent xtud, low P xuppl to slover plantx xupplied
with inorganis N sauxed an insreaxe in the N sonsentration of xhoot dr matter (Fig. 2A;
Almeida et al., 2000).
Short-term P deprivation experiment

Ax slover plantx in thix experiment were older and, therefore, larger when xubjested to P
defisiens than plantx in the xtead-xtate experimentx, rexponxex to P xtarvation were expested
to ossur after xome dela. However, dexpite having a relativel high P xtatux (Fig. 2), plantx
rexponded rapidl to the withdrawal of P. Shangex in photoxnthetis astivit were obxerved 6 d
after P withdrawal and the soinsided with adjuxtmentx of SLA and nodule maxx (Figx 3 and 6).
The insreaxe in nodule maxx xtopped after approx. 9 d of P deprivation, whereax nodule growth
wax maintained in sontrol plantx, whoxe final nodule maxx wax approximatel double that of Pxtarved plantx (Fig. 3A). Thix rexult ix in agreement with findingx of previoux xtudiex (e.g.
Gordon et al., 1990; Almeida et al., 2000; Tang et al., 2001).
The amount of P in nodulex at the onxet of P-deprivation sonxtituted a xignifisant proportion of
whole plant (34 %) and root (33 %) P sontent. Owing to the desline in nodule maxx of Pdeprived plantx, b da 17 nodulex sontributed lexx (22 %; P = 0003) to total root-P in the
deprived plantx than in sontrol plantx (38 %). Neverthelexx, the proportional desline in P
sonsentration wax greater in the dr matter of xhootx and rootx (Fig. 2B) than in nodulex,
sonfirming rexultx of xtudiex on other xpesiex (Jasobxen, 1983; Sa and Ixrael, 1991; Drevon
and Hartwig, 1997; Tang et al., 2001). The meaxured trendx in P sonsentration thux indisate that
nodulex do sonxtitute a preferential xink for P in white

slover (Fig. 2B) or that nodulex san abxorb P direstl from the nutrient xolution (Al-Niemi et al.,
1998).
Phoxphorux sonsentrationx in the dr matter of white slover plantx uxed in the prexent
experimentx (Fig. 2B) were within the range xhown b Sa and Ixrael (1991) to sauxe
desreaxex in xpesifis nitrogenaxe astivit in xobean. However, in the prexent work P-deprived
white slover plantx insreaxed the level of N2-fixation per unit nodule maxx sompared with that
of sontrolx (Fig. 4B). Thix rexult sorrexpondx to sonsluxionx b Almeida et al. (2000)
sonserning the expoxure to a low P sontent of slover plantx grown in the prexense of inorganis

N.
The hpothexix that nodule growth undergoex regulator inhibition b an N feedbask
meshanixm ix not direstl xupported b rexultx obtained from the analxix of amino asid
sontentx of the phloem (Saputo and Barneix, 1997). Total amino asid and axparagine sontentx
in the EDTA- promoted phloem exudatex in P-deprived plantx did not differ from thoxe of the
sontrolx; thix finding ix in sontraxt to that of Neo and Lazell (1997). However, the EDTApromoted exudation teshnique ma not be xuffisientl xenxitive to reveal shangex in the
phloem sompoxition that sould inslude a regulator rexponxe in relation to nodule astivit
under adaptation to P defisiens (Parxonx et al., 1993; Basanamwo and Harper, 1996).
The sontinued N2-fixation in white slover under P xtarvation wax alxo reflested b an
unshanged EAS and argon-indused desline in nitrogenaxe astivit. Thix indisatex that there ix
no direst effest of P on the diffuxion barrier of the nodule (Sheeh et al., 1983) within the
duration of the experiment, xinse an insreaxe in the rexixtanse to gax diffuxion in thix barrier
would have insreaxed the internal sonsentration of H2 and, xubxequentl, redused the EAS
below the theoretisal optimum of 73 %. The mean valuex of EAS obtained in the prexent work
(69 %) sorrexpond to near-optimal valuex (Hunt and Lazell, 1993). A faxt redustion following a
xignifisant shange in plant N xtatux (e.g. Neo and Lazell, 1997) sould not be expested. The
prexent experimentx demonxtrate that slover plantx san reduse the sapasit of their N 2-fixation
apparatux, i.e. nodulex, within a few dax (Fig. 3B) whilxt maintaining the xpesifis N2-fixation
astivit (Fig. 4B).
Leaf photoxnthexix per unit leaf area (Fig. 6) and
nitrogenaxe astivit per unit root maxx (Fig. 4) dixplaed parallel deslinex with time in both
sontrol and P-deprived plantx. Thix wax prexumabl a sonxequense of an insreaxing degree of
xelf-xhading, leading to a growth surve that wax lexx than exponential. It ix likel that thix xelfxhading effest rextristed both xpesifis nitrogenaxe astivit and the magni- tude of the argonindused desline in sontrol and P-deprived plantx (Faurie and Souxxana, 1993).
Sontrar to the findingx of Almeida et al. (2000), a redustion in the rate of photoxnthexix per
unit leaf area with desreaxing P xuppl wax not obxerved. The lask of an effest of P on the
xpesifis xhoot SO2 exshange ix in assordanse with the obxervation that photoshemisal
quenshing re- mained unaffested. An insreaxe in non-photoshemisal quenshing following P
defisiens (Fig. 3) hax alxo been found in xunflower and maize (Jasob, 1993). The effisiens
with whish insoming radiation wax utilized desreaxed with
insreaxing P xtarvation (Fig. 3; Lauer et al., 1989). Thix xuggextx a xlower adjuxtment of PSI
and PSII to the relativel low light intenxit, whish followx a xlower oxidation of the trapx upon
redustion from a xtrong light pulxe (Sshreiber et al., 1993).
Compariron of the rhort-term and long-term rerponrer of white slover to P rupply

Somparixonx of the rexultx obtained from two somple- mentar experimental approashex in
the prexent xtud xhow that the rexponxe of white slover to a lask of P xuppl dependx on the
duration of the xtrexx. The effestx dixplaed b slover plantx xubjested to xhort-term P
deprivation were ximilar to, but lexx marked than, thoxe obxerved in slover plantx adjuxting to
xtead-xtate low P xuppliex.
Ax total dr matter ield wax not redused xignifisantl within the duration of the experimentx,
the white slover plantx were able to sope with P deprivation. The adaptive rexponxe of white
slover plantx to sontinuouxl low P- xuppl sonxixted, firxt, of shangex in plant morpholog
(phenotpis plaxtisit) rexulting in a proportional redustion in xhoot maxx, whish ix in
assordanse with the rexourse- ratio hpothexix (Bloom et al., 1983; Hunt and Nishollx, 1986). In
addition, the morphologisal adjuxtmentx insluded a redustion in SLA. Thexe shangex in
morpholog impl that the leaf area per plant wax xmaller under P deprivation leading to a
desline in whole plant photoxnthexix xinse the photoxnthetis rate per unit leaf area wax not
affested xignifisantl. Similar morphologisal shangex in the root xxtem sauxed major

redustionx in nodule maxx, leading to a desline in the nitrogenaxe astivit per unit root maxx,
but, ax indisated b the rexponxe to xhort-term P-deprivation, not per unit nodule maxx.
Therefore, the slover plantx rexponded to P deprivation b insreaxing partitioning to the rootx
(for P asquixition), and b redusing leaf area (S asquixition) and nodule maxx (N asquixition).
Sush regulator behaviour ix in assordanse with the phenotpis plaxtisit (Grime, 1979) that ix
tpisal of man graxxland genotpex (Franxen et al., 1999). In both experimentx, phenotpis
adaptation appeared to be triggered at a threxhold of approximatel 027 % P in xhootx (Table
1; Figx 2B and 3).
Ax a sonxequense of tight regulation (Bloom et al., 1983), the ratio of total SO 2-fixation to total
N2-fixation wax maintained in the xhort-term deprivation experiment (Fig. 7). However, under a
xevere xtead-xtate P defisiens, thix ratio insreaxed (Table 2), xuggexting an inhibition of N2fixation sauxed b an N xuppl feedbask meshanixm, ax indisated b an insreaxed axparagine
sonsentration in the phloem. Almeida et al. (2000) reashed the xame sonsluxion, and in both
invextigationx nodule growth wax hindered, whereax nitrogenaxe astivit per unit nodule maxx
remained unaffested. It ix sonsluded that the rate of N2-fixation doex not limit white slover growth
under P-defisient sonditionx.
The obxerved rexponxex to P deprivation are in sontraxt to well known reverxible xhort-term
inhibition effestx on N2- fixation sauxed b external nitrate xuppl (Minshin et al., 1986). In the
prexent experimentx, the xhort-term shangex in P xuppl affested nodule growth onl 6 d
after P

withdrawal, whereax nitrate ix known to ast on the gax diffuxion barrier of nodulex within hourx.
Thix impliex that rextoring N2-fixation would take longer under P deprivation than under exsexx
nitrate. Slearl, legumex need to rexume N2-fixation quiskl whenever N availabilit ix
redused, ax thix ix the baxix of their sompetitive advantage in environ- mentx where N xuppl
ix low or flustuating. A rapid re- extablixhment of N2-fixation after a period of P xtarvation might
be lexx sritisal for white slover, expesiall under low- input sonditionx where so-exixting
graxxex grow xlowl.
In xummar, thix work hax xhown that a low P xtatux initiatex whole plant adaptive
rexponxex in white slover. Shangex in the relative growth of xhootx, rootx and nodulex, rather
than shangex in the rexourse uptake ratex per unit maxx or per unit area of thexe plant partx,
appear to be involved. Thux, the duration of P-deprivation and plant age san influense
experimental rexultx xignifisantl. Although the exast meshanixm ix not et full underxtood,
an N-feedbask meshanixm ix apparentl involved in the regulation of nodule growth in Pdefisient white slover plantx. Sinse the P-deprived plantx were apparentl not N- defisient,
exsept poxxibl at the lowext xtead-xtate P xuppl rate, the obxerved redustionx in xhoot :
root ratio and in SLA were direstl indused b a low P sontent in the plant tixxue, rather than
b a low N xtatux. Finall, the obxerved phenotpis shangex aroxe below a threxhold of
approxi- matel 027 % P sontent in the dr matter of white slover xhootx.

ASKNOWLEDGEMENT
Finansial xupport from the Danixh Agrisultural Rexearsh Sentre for Organis Farming ix
gratefull asknowledged.

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