Metabolite profiling reveals distinct changes in carbon and nitrogen metabolism in...

Metabolite Profiling Reveals Distinct Changes in Carbon and Nitrogen

Metabolism in Phosphate-Deficient Barley Plants (Hordeum vulgare L)

Chun Y Huang1 Ute Roessner

2 Ira Eickmeier

3 Yusuf Genc

4 Damien L Callahan

5

Neil Shirley1 Peter Langridge

1and Antony Bacic

2

1 Australian Centre for Plant Functional Genomics The University of Adelaide Waite Campus PMB1 Glen OsmondSouth Australia 5064 Australia2 Australian Centre for Plant Functional Genomics School of Botany The University of Melbourne Victoria 3010 Australia3 Max-Planck-Institut fur Molekulare Pflanzenphysiologie Am Muhlenberg 1 D-14476 Golm Germany4 Molecular Plant Breeding Cooperative Research Centre The University of Adelaide South Australia 5005 Australia5 Metabolomics Australia School of Botany The University of Melbourne Victoria 3010 Australia

Plants modify metabolic processes for adaptation to low

phosphate (P) conditions Whilst transcriptomic analyses

show that P deficiency changes hundreds of genes related to

various metabolic processes there is limited information

available for global metabolite changes of P-deficient plants

especially for cereals As changes in metabolites are the

ultimate lsquoreadoutrsquo of changes in gene expression we profiled

polar metabolites from both shoots and roots of P-deficient

barley (Hordeum vulgare) using gas chromatographyndashmass

spectrometry (GC-MS) The results showed that mildly

P-deficient plants accumulated di- and trisaccharides

(sucrose maltose raffinose and 6-kestose) especially in

shoots Severe P deficiency increased the levels of metabolites

related to ammonium metabolism in addition to di- and

trisaccharides but reduced the levels of phosphorylated

intermediates (glucose-6-P fructose-6-P inositol-1-P and

glycerol-3-P) and organic acids (a-ketoglutarate succinatefumarate and malate) The results revealed that P-deficient

plants modify carbohydrate metabolism initially to reduce

P consumption and salvage P from small P-containing

metabolites when P deficiency is severe which consequently

reduced levels of organic acids in the tricarboxylic acid (TCA)

cycle The extent of the effect of severe P deficiency

on ammonium metabolism was also revealed by liquid

chromatographyndashmass spectrometry (LC-MS) quantitative

analysis of free amino acids A sharp increase in the

concentrations of glutamine and asparagine was observed in

both shoots and roots of severely P-deficient plants Based on

these data a strategy for improving the ability of cereals to

adapt to low P environments is proposed that involves

alteration in partitioning of carbohydrates into organic

acids and amino acids to enable more efficient utilization of

carbon in P-deficient plants

Keywords Ammonium mdash Barley (Hordeum vulgare L) mdash

Carbohydrate mdash Metabolite profile mdash Phosphate

deficiency

Abbreviations GC-MS gas chromatographyndashmass spec-trometry LC-MS liquid chromatographyndashmass spectrometryTCA tricarboxylic acid

Introduction

Inorganic phosphorus (Pi) is an essential macronutrient

for plant growth and development It influences virtually all

biochemical processes in plants Pi is a component of many

cellular molecules such as ATP nucleic acids phospholipids

and phosphorylated sugars and thus plays a crucial role in

carbon and nitrogen metabolism Plant cells have to main-

tain Pi concentrations within a critical range (Schachtman

et al 1998) Although phosphorus (P) is abundant in

most soils P availability and mobility often are low

(Raghothama 1999) which limits crop growth and produc-

tivity Large amounts of Pi fertilizers are applied to achieve

high crop yields which has led to the rapid depletion of

non-renewable P resources and environmental pollution

(Vance et al 2003) Therefore improving the Pi acquisition

and utilization efficiency of crop species is important for

sustainable agriculture

Plants modify metabolic processes to reprioritize

utilization of internal Pi and maximize acquisition of

external Pi in low Pi environments (Vance et al 2003)

Thus plants increase root surface area and root shoot

ratios to enable exploration of more soil volume under low

Pi conditions Furthermore P-deficient plants optimize

P utilization through mobilization of P from different

subcellular compartments and organs and through the

modification of metabolic processes and hydrolysis of many

P-containing molecules such as nucleic acids phospholipids

and small phosphorylated metabolites (Mimura 1999

Plaxton 2004 Cruz-Ramirez et al 2006) Plant roots also

modify their rhizosphere by the secretion of organic acids

(Lopez-Bucio et al 2000) and enzymes to increase P

availability (Vance et al 2003) and simultaneously

Corresponding author E-mail chunyuanhuangadelaideeduau Fax thorn61-8-8303-7102

Plant Cell Physiol 49(5) 691ndash703 (2008)doi101093pcppcn044 available online at wwwpcpoxfordjournalsorg The Author 2008 Published by Oxford University Press on behalf of Japanese Society of Plant PhysiologistsAll rights reserved For permissions please email journalspermissionsoxfordjournalsorg

691

synthesize more Pi transporter proteins in the plasma

membrane of root epidermal cells for higher Pi uptake

capacity (Muchhal and Raghothama 1999) Transcriptomic

analyses in Arabidopsis rice and lupin have shown that

coordinated changes in the expression of several hundred

genes take place in P-deficient plants (Uhde-Stone et al

2003 Wasaki et al 2003a Wu et al 2003 Mission et al

2005 Morcuende et al 2007) These genes are involved in

various metabolic pathways such as photosynthesis carbon

metabolism nitrogen assimilation and synthesis of protein

and nucleic acids (Wu et al 2003 Misson et al 2005

Morcuende et al 2007) which could lead to adaptive

changes in metabolite profiles

Recent advances in analytical technologies such as gas

and liquid chromatography coupled to mass spectrometry

(GC-MS and LC-MS) have allowed the analysis of a large

number of compounds from a single plant sample (for a

review see Kopka 2006) Such metabolite profiles provide

not only a much broader view for a systematic adjustment

in metabolic processes than conventional biochemical

approaches but also an opportunity to reveal new insights

on metabolism Metabolite profiling with GC-MS has been

used for studies on nutrient deficiency including nitrogen

sulfate iron and P in a single-cell green alga (Bolling and

Fiehn 2005) and in higher plants such as Arabidopsis and

common beans (Hirai et al 2004 Nikiforova et al 2005

Hernandez et al 2007) A combination of transcriptomic

and metabolite analyses has also been made to increase

further our understanding of plant responses to various

environmental stresses such as sulfate and nitrogen (Hirai

et al 2004 Nikiforova et al 2004 Nikiforova et al 2005)

and P deficiency in Arabidopsis and common beans

(Hernandez et al 2007 Morcuende et al 2007) Despite

the agronomic importance of cereals such as wheat and

barley little information is available for metabolite changes

in response to P deficiency As Pi homeostasis is regulated

at the whole-plant level by translocating Pi from mature to

young tissues and from shoots to roots under Pi-limiting

conditions (Jeschke et al 1997 Mimura 1999) it is

necessary to determine alterations in both shoots and

roots to increase our understanding of the biochemical

mechanisms involved in whole-plant adaptation to the low

Pi environment

In this report barley (Hordeum vulgare L) was used as

a model plant for cereal crops We measured approximately

100 metabolites from both shoots and roots of mildly or

severely P-deficient barley plants using a recently developed

GC-MS method (Roessner et al 2006) Metabolite profiles

reveal distinct changes in carbon and nitrogen metabolism

in P-deficient barley plants leading to accumulation of

di- and trisaccharides and free amino acids The distinct

metabolite profiles suggested that the severely P-deficient

barley plants are confronted with an increased level of

ammonium Thus the effect of severe P deficiency on

ammonium metabolism was further investigated through

quantitative analysis of amino acid profiles using LC-MS

(Callahan et al 2007) Our results provide new insights

into the effects of P deficiency on carbon and nitrogen

metabolism in barley plants

Results

Plant responses to short- and long-term P starvation in

growth and P nutrition

Barley seedlings were grown from seeds for different

time periods in a nutrient solution either with no added

NH4H2PO4 (P) or with 01mM NH4H2PO4 (thornP control)

to obtain plants with different P status At the 10th day

after seed imbibition (day 10) shoot development was

similar in the two P treatments with both having a fully

expanded first leaf and a small second leaf There were no

apparent P deficiency symptoms in the shoots of P-treated

plants although a change in root appearance was evident

Shorter primary roots and more lateral roots were observed

in the P treatment Plant growth measured on a FW basis

was significantly different between P and thornP treatments

at day 10 (Fig 1A) but there was no difference in DW

between P and thornP treatments at day 10 (data not shown)

The P concentration of shoots in the P-treated plants was

26mg P gndash1 DW (Fig 1B) which was below the critical

P concentration of 4mg P gndash1 DW (Reuter and Robinson

1997) The concentration of P in the roots was 32mg gndash1

DW (Fig 1B) In contrast the P concentration in the

thornP-treated plants was adequate at day 10 (Fig 1B) The

P-treated plants at day 10 suffered from mild P deficiency

and henceforth were referred to as mildly P-deficient plants

Plant growth was severely retarded when the plants

were grown in the P treatment for an additional 7 d

(Fig 1A day 17) There were two small fully expanded

leaves with the third leaf emerging in the P-treated plants

whereas there were three large fully expanded leaves in the

thornP-treated plants The seminal roots of the P-treated

plants were much shorter than those in the thornP-treated

plants P concentrations in the P-treated plants further

declined to 12mg P gndash1 DW in shoots and 20mg P gndash1 DW

in roots at day 17 (Fig 1B) Therefore the P-treated

plants at day 17 were inferred to be severely P-deficient

plants in this study

Expression of HvIPS1 and HvPht16 in P-deficient plants

The TPSI1At4 genes are sensitive to P deficiency and

widely used as a molecular indicator for P deficiency

(Burleigh and Harrison 1997 Liu et al 1997 Martin et al

2000 Wasaki et al 2003b Hou et al 2005) HvIPS1 is a

barley homolog of the TPSI1At4 genes (Hou et al 2005)

and HvPht16 is a low-affinity Pi transporter of barley

692 Metabolite profiles in P-deficient barley plants

(Rae et al 2003) Both HvIPS1 and HvPht16 are sensitive

to P deficiency and are highly expressed in both shoots

and roots of Lofty Nijo barley plants The transcript levels

of HvIPS1 and HvPht16 therefore were determined by

quantitative real-time PCR to monitor the response of both

shoots and roots to P deficiency As shown in Fig 2A a low

level of HvIPS1 (12 106 normalized copies mg1 RNA)

was detected in the shoots of thornP-treated plants at day 10

but a 277-fold increase (343 106 normalized copies mg1

RNA) was observed in the shoots of the P-treated plants

The enhanced expression of HvPht16 (32-fold increase)

was also observed in the shoots of P-treated plants at day

10 compared with that in the shoots of thornP-treated plants

(Fig 2A) In contrast a 415-fold increase in the transcript

level of HvIPS1 was observed in the roots of P-treated

plants (Fig 2B) which was similar to the enhanced level

in the shoots (Fig 2A) However no difference in the

HvPht16 transcript level was found in the roots receiving

the two P treatments at day 10 (Fig 2B) suggesting that the

shoots of barley plants are more sensitive to P deficiency

than the roots Severe P deficiency sharply increased the

transcript levels of HvIPS1 and HvPht16 in both shoots

and roots at day 17 (Fig 2)

Metabolite profiles of shoots and roots in mildly P-deficient

plants

A GC-MS-based method developed by Roessner et al

(2006) was used for the analysis of polar metabolites from

barley plants More than 400 compounds were detected

in barley plants subjected to both thornP and P treatments

1800A

B

1600

1400

1200

1000

800

600

400

200

0

1800

1600

1400

1200

1000

800

600

400

200

0

No

rmal

ized

co

pie

s mg

minus1 R

NA

(times1

06 )N

orm

aliz

ed c

op

ies

mgminus1

RN

A (

times106 ) minus P

+ P

HvIPS1 HvPht16Day 10 Day 17

HvIPS1 HvPht16

Fig 2 Effect of P supply on transcript levels of HvIPS1 andHvPht16 in shoots (A) and roots (B) of barley seedlings Barleyseedlings were grown in a nutrient solution with 01mMNH4H2PO4

(thornP) or without NH4H2PO4 addition (P) The levels of transcriptsare presented as normalized copies per mg RNA Plants wereharvested at the 10th day after seed imbibition (day 10) and at the17th day (day 17) Standard errors (nfrac14 3) are shown as vertical bars

140A

B

120

100

080

060

040

020

0

10

8

6

4

2

0

Fre

sh w

eig

ht

(g p

lan

tminus1)

P c

on

cen

trat

ion

s (m

g g

minus1 D

W)

Shoots Roots

minusP

+P

minusP

+P

Criticallevel

Day 10 Day 17 Day 10 Day 17

Fig 1 Effect of P supply on plant growth and P nutrition (A) Freshweight of shoots and roots (B) P concentration in shoots and rootsBarley seedlings were grown in a nutrient solution with 01mMNH4H2PO4 (thornP) or without NH4H2PO4 addition (P) Plantswere harvested at the 10th day after seed imbibition (day 10)and at the 17th day (day 17) Standard errors (nfrac14 4) are shown asvertical bars

Metabolite profiles in P-deficient barley plants 693

of which approximately 130 compounds could be assigned a

chemical structure A total of 98 metabolites were identified

as known compounds including 23 amino acids 15 organic

acids 25 sugars and 35 non-polar compounds (Supplemen-

tary Tables S1 S2) Metabolite levels from both shoots and

roots and the ratios of PthornP are presented in Supplemen-

tary Table S1 for the mildly P-deficient plants at day 10

and in Supplementary Table S2 for the severely P-deficient

plants at day 17

The PthornP ratios for metabolite levels for mildly

P-deficient plants are summarized in Fig 3 The levels

of approximately half of the sugars analyzed were

significantly increased in the shoots of the mildly

P-deficient plants (PSD10 Supplementary Table S1)

A significant increase in the levels of di- and trisaccharides

(sucrose 11-fold maltose 27-fold raffinose 42-fold and

6-kestose 119-fold) and one aromatic compound (shiki-

mate 22-fold) was observed (Fig 3) Little change in

amino acids and organic acids was observed in the shoots

of the mildly P-deficient plants (PSD10 Supplementary

Table S1) In contrast only a few metabolites were

significantly changed in the roots of mildly P-deficient

plants compared with thornP-treated roots (PRD10

Supplementary Table S1 Fig 3) One sugar maltose rose

to 15-fold in the roots (PthornPRD10 Supplementary

Table S1) The Pi level in the polar fraction of mildly

P-deficient plants was not significantly affected either in

shoots or in roots (phosphoric acid in the polar fraction

of Supplementary Table S1) These results indicate that the

metabolites involved in sugar metabolism are more sensitive

di- and tri-saccharides

raffinose(419plusmn017)312plusmn041

sucrose(114plusmn008)159plusmn011

glc(144plusmn004)070plusmn011

shikimate(222plusmn014)161plusmn018

fru(120plusmn008)079plusmn011

glycerol(103plusmn006)115plusmn011

6-kestose(1186plusmn020)

292plusmn037

maltose(269plusmn016)149plusmn006

ribose(128plusmn012)052plusmn012

inositol-1-P(097plusmn010)103plusmn039

glycine(103plusmn006)091plusmn012

serine(089plusmn004)121plusmn016

gly-6-P(082plusmn009)112plusmn027

glycerol-3-P(030plusmn048)197plusmn052

tryptophan(117plusmn017)396plusmn075

fru-6-P(073plusmn008)127plusmn025

inositol(136plusmn005)117plusmn007

b-alanine(148plusmn007)105plusmn015

alanine(088plusmn006)193plusmn037

asparagine(115plusmn010)079plusmn012

succinate(125plusmn010)104plusmn003

fumarate(073plusmn009)086plusmn003

aspartate(084plusmn008)209plusmn017

malate (115plusmn005)275plusmn018

citrate (131plusmn023)173plusmn026

glutamate(486plusmn032)1258plusmn097

a-ketoglutarate(101plusmn009)118plusmn021

urea(106plusmn008)128plusmn008

lipids

fructanstarch

glycerate-3-P

PEP

pyruvate

oxaloacetate

aromatic compounds

Fig 3 Effect of P supply on the levels of primary polar metabolites from shoots and roots of 10-day-old plants Barley seedlings weregrown in a nutrient solution with 01mM NH4H2PO4 (thornP) or without NH4H2PO4 addition (P) and harvested at the 10th day after seedimbibition A simplified metabolic pathway is used to show the levels of the primary metabolites determined by GC-MS The metabolitesmeasured by GC-MS are shown in bold and undetermined metabolites in a regular font Relative ratios (PthornP) of metabolites in theshoots (in parentheses) and roots (no parentheses) are presented as means SE (nfrac14 4) Significant differences (P 005) in the ratiosbetween the P and thornP treatments are indicated in green for the increased ratios and in red for the reduced ratios


to mild P deficiency than others such as amino acids and

organic acids In addition it is obvious that shoots are more

responsive than roots to mild P deficiency

Metabolite profiles of shoots and roots in severely P-deficient

plants

The ratios (PthornP) of a wide spectrum of polar

metabolites were altered in 17-day-old P-deficient plants

(Supplementary Table S2) The elevated levels of di- and

trisaccharides remained in the shoots of severely P-deficient

plants (Fig 3) and more changes in di- and trisaccharides

including maltose (27-fold) sucrose (1374-fold) raffinose

(72-fold) and 6-kestose (70-fold) occurred in the roots

(Fig 4) Although the ratio of sucrose (PthornP) in the roots

of 17-day-old plants was extremely high the level of sucrose

(183) in the roots of the severely P-deficient plants

(Supplementary Table S2) was only slightly lower than

that (231) in the mildly P-deficient plants (Supplementary

Table S1) The extremely high ratio of sucrose in the roots

of the severely P-deficient plants is mainly due to the largely

reduced level of sucrose (01) in thornP-treated roots at day 17

(Fig 4 and Supplementary Table S2) In addition a large

increase in shikimate (120-fold) was observed in the roots

Ribose was the only monosaccharide which was reduced

03- to 06-fold in both shoots and roots of severely

P-deficient plants The levels of other monosaccharides

were either unaffected or increased (Fig 4) However Pi

levels in the polar fraction of the severely P-deficient plants

were sharply reduced to approximately 10 of those in

thornP-treated plants (phosphoric acid in the polar fraction of

di- and tri-saccharidesraffinose(335plusmn008)721plusmn012







glc-6-P(014plusmn012)007plusmn010


glyserol-3-P(015plusmn029)088plusmn008




tryptophan(5454plusmn042)

ndb-alanine(083plusmn005)038plusmn007










urea(nd)

3315plusmn009



695plusmn014


fructan

lipids

starch

glycerate-3-P

PEP

pyruvate

oxaloacetate

aromatic compounds

Fig 4 Effect of P supply on the levels of primary polar metabolites from shoots and roots of 17-day-old plants Barley seedlings weregrown in a nutrient solution with 01mM NH4H2PO4 (thornPi) or without NH4H2PO4 addition (P) harvested at day 17 after seed imbibitionand separated into shoots and roots A metabolic pathway is used to show the levels of the primary metabolites determined by GC-MS Themetabolites measured by GC-MS are shown in bold and undetermined metabolites in a regular font Relative ratios (PthornP) of metabolitein the shoots (in parentheses) and roots (no parentheses) are presented as means SE (nfrac14 4) Significant differences (P 005) in the ratiosbetween the P and thornP treatments are indicated in green for the increased ratios and in red for the reduced ratios


Supplementary Table S2) and the concomitant reduction in

phosphorylated sugars including glucose-6-P fructose-6-P

glycerol-3-P and inositol-1-P was also observed in both

shoots and roots of the severely P-deficient plants at day 17

(Fig 4 and Supplementary Table S2) The levels of seve-

ral organic acids of the TCA cycle were also reduced in

the severely P-deficient plants including a-ketoglutarate(04-fold) and malate (07-fold) in the shoots and succinic

acid (07-fold) and fumaric acid (05-fold) in the roots

(Fig 4)

There were hardly any changes in the levels of amino

acids in both shoots and roots of the mildly P-deficient

plants when compared with thornP-treated plants (Supplemen-

tary Table S1) but the levels of two-thirds of the amino

acids were increased in the shoots of the severely P-deficient

plants (PSD17 Supplementary Table S2) In addition

the polyamine putrescine was increased in shoots (PSD17

Supplementary Table S2) Similarly dramatic changes in

the levels of the amino acids and metabolites related to

ammonium metabolism were also observed in the roots of

severely P-deficient plants There was an increase in the

levels of asparagine (30-fold) glutamate (24-fold) and

putrescine (23-fold) in the roots of the severely P-deficient

plants as well as a sharp increase in the level of urea up to

332-fold in roots (Fig 4) This high ratio of the urea level

was due to the combination of an increase of urea (25-fold)

in the roots of the severely P-deficient plants (PRD17

Supplementary Table S2) compared with that of the mildly

P-deficient plants (PRD10 Supplementary Table S1)

and a reduction of the urea level from 08 in the roots of

thornP-treated plants at day 10 (thornPRD10 Supplementary

Table S1) to 01 at day 17 (thornPRD17 Supplementary

Table S2) These data should be viewed cautiously as the

quantification of urea by GC-MS can be unreliable so an

alternative approach would need to be utilized to explore

this effect further Furthermore the level of aspartate in the

roots of the severely P-deficient plants was reduced to

05-fold (Fig 4) The significant change in the metabolites

related to ammonium assimilation suggests that the severe

P deficiency increases levels of ammonium leading to

alterations in ammonium assimilation

Effect of severe P deficiency on the concentrations of free

amino acids

The observations described above led us to conduct

an additional experiment to determine the effect of severe P

deficiency on amino acid metabolism Absolute concentra-

tions of free amino acids were determined using LC-MS to

complement the data obtained by GC-MS In the additional

experiment the nutrient solutions were supplemented with

two levels of ammonium (01 and 04mM) The low

ammonium treatment (01mM) was the same as that used

for plant growth in the first experiment (Fig 1) The high

ammonium treatment (04mM) is designed to investigate

the effect of external ammonium supply on the accumula-

tion of free amino acids in P-deficient plants and to prevent

possible depletion of ammonium in the nutrient solution

when only low concentrations (01mM) were supplied

Barley is an ammonium-sensitive species (Britto and

Kronzucker 2002) We determined that the maximum

level of ammonium that could be added to the nutrient

solution without obvious adverse effect on plant growth

was 04mM with co-provision of ammonium and nitrate

In thornP treatments shoot fresh weights for the 01 and

04mM ammonium treatments were similar (Fig 5A) but

the fresh weight of roots in the 04mM ammonium

treatment was 072 g plantndash1 which was slightly lower

than that of 081 g plantndash1 in the 01mM treatment (Fig 5B)

In contrast plant growth in the P treatment supplied with

either 01 or 04mM ammonium was severely retarded

compared with that of thornP treatment (Fig 5A B) The

growth inhibition in both shoots and roots was similar to

that shown in Fig 1A P concentrations in the shoots

of thornP-treated plants supplemented with either 01 or

04mM ammonium were above the critical levels (Reuter

and Robinson 1997) whereas those in the shoots of

P-treated plants were 12mg P gndash1 DW for the 01mM

ammonium treatment which is equal to that observed

in the severely P-deficient plants in the first experiment

(Fig 1B) but slightly lower (10mg P gndash1 DW) in the

04mM ammonium treatment than that in the 01mM

ammonium treatment (Fig 5C)

The concentrations of all 20 amino acids and GABA

(g-aminobutyric acid) were obtained by LC-MS (Supple-

mentary Table S3) and the results were compared with

those determined by GC-MS (Supplementary Table S2)

The extent of the effect of severe P deficiency on the

concentrations of free amino acids was confirmed by the

LC-MS analysis (Supplementary Table S3) In the shoots

severe P deficiency led to an increase in the concentrations

of half of the amino acids especially glutamine and

asparagine (Supplementary Table S3) and a large reduction

in the concentrations of glutamate and aspartate regardless

of the ammonium concentration (01 or 04mM) (Fig 6A)

The concentrations of glutamine and asparagine also

increased in the roots (Fig 6B) but the concentrations

of some of the abundant amino acids such as alanine

aspartate glutamate and serine were decreased in the

roots irrespective of the rate of ammonium supply

(Supplementary Table S3) The changes in the levels

of amino acids generally agreed with those obtained

by GC-MS in the first experiment (PSD17 Supple-

mentary Table S2) but there were some differences in the

relative levels of some of the amino acids such as

asparagine glutamate proline and phenylalanine between

the two experiments using these two different technologies


(Supplementary Tables S2 S3) These differences in amino

acid levels reflect the differential stabilities of the respective

amino acids to the different derivatization and detection

methodologies

The concentrations of glutamine and asparagine in

both shoots and roots were sensitive to ammonium

supplied They increased from approximately 10 mmol g1

DW in the shoots of the thornP treatment with 01mM

ammonium to 40 mmol g1 DW in the shoots of the thornP

treatment with 04mM ammonium (Fig 6A) In the

severely P-deficient plants the concentrations of glutamine

and asparagine in the shoots rose above 60 mmol g1 DW in

01mM ammonium and above 90 mmol g1 DW in 04mM

ammonium (Fig 6A) A similar magnitude of increase in

the concentrations of glutamine and asparagine was also

observed in the roots of the severely P-deficient plants

(Fig 6B) These results indicate that severe P deficiency

further increases the concentrations of glutamine and

asparagine in both shoots and roots When PthornP ratios

of amino acids in both shoots and roots of the plants treated

with 04mM ammonium were compared with those treated

with 01mM ammonium the high ammonium supply had

little effect on the PthornP ratios of amino acids except for

a reduction in the ratios of glutamine and asparagine and

140

120

100

Fre

sh w

eig

ht

(g p

lan

tminus1)

Fre

sh w

eig

ht

(g p

lan

tminus1)

P c

on

cen

trat

ion

(m

g g

minus1 D

W)

080

060

040

020

0

140minusP

+P120

100

080

060

040

Critical level

020

0

10

8

6

4

2

001 mM 04 mM

A

B

C

Fig 5 Effect of P and ammonium supply on plant growth andP nutrition (A) Fresh weight of shoots (B) Fresh weight of roots(C) P concentration of shoots Barley seedlings grown in a nutrientsolution containing either no NH4H2PO4 (P) or 01mMNH4H2PO4 (thornP) with two rates of ammonium (01 and 04mM)Plants were harvested at the 16th day after seed imbibitionStandard errors (nfrac14 4) are shown as vertical bars

1200Glu

Gln

Asp

Asn

1000

800

600

400

200

0

1200

1000

800

600

400

200

001N+P 04N+P01NminusP 04NminusP

Co

nce

ntr

atio

n (

mg g

minus1 D

W)

Co

nce

ntr

atio

n (

mg g

minus1 D

W)

A

B

Fig 6 Effect of P and ammonium supply on concentrations of fouramino acids (A) Concentrations of four amino acids in shoots(B) Concentrations of four amino acids in roots Barley seedlingsgrown in a nutrient solution containing either no NH4H2PO4 (P)or 01mM NH4H2PO4 (thornP) with two rates of ammonium (01 and04mM) Plants were harvested at the 16th day after seedimbibition The concentrations of amino acids were determinedby LC-MS (see Supplementary Table S3) Standard errors (nfrac14 4) areshown as vertical bars


a couple of other amino acids (Supplementary Table S3)

These results indicate that the effect of severe P deficiency

on the concentrations of free amino acids in both shoots

and roots is largely independent of ammonium supply

In addition the concentrations of both glutamate

and aspartate precursors for synthesis of glutamine and

asparagine were affected by severe P deficiency The con-

centrations of glutamate and aspartate were reduced by

approximately 50 and 25 respectively in the shoots

of P treatments with either 01 or 04mM ammonium

relative to those of thornP treatments (Fig 6A and Supple-

mentary Table S3) The greater reductions in the concen-

trations of glutamate and aspartate were also observed in

the roots of P treatments with two rates of ammonium

supplied compared with those of thornP treatments (Fig 6B

and Supplementary Table S3) Interestingly the high

ammonium supplied did not reduce the concentrations of

glutamate and aspartate either in the shoots or in the roots

of thornP-treated plants (Fig 6A B Supplementary Table S3)

These results suggest that severely P-deficient plants utilize

amino acids as an alternative carbon resource especially in

the roots

Discussion

P deficiency in barley plants results in increases in di- and

trisaccharides and decreases in small P-containing

metabolites

P deficiency initially enhanced the accumulation of

di- and trisaccharides especially for maltose raffinose and

6-kestose in the shoots of the moderately P-deficient plants

(Fig 3) Only maltose increased in the roots (Fig 3

Supplementary Table S1) The transcript levels of HvPht16

(Fig 2) also indicate that roots are less sensitive to P

deficiency Severe P deficiency leads to a further increase in

di- and trisaccharides in both shoots and roots These data

are consistent with previous reports that P deficiency led

to an increase in the levels of starch sucrose and mono-

saccharides in common beans and Arabidopsis (Ciereszko

and Barbachowska 2000 Hernandez et al 2007 Nilsson

et al 2007) and a graminaceous plant Brachiaria hybrid

(Nanamori et al 2004) It also is consistent with the reduced

level of sucrose synthase in P-deficient maize roots (Li et al

2007) The increased levels of mono- di- and polysaccha-

rides suggest that glycolysis may be hindered in both shoots

and roots of P-deficient barley plants In contrast the

cDNA array analyses from lupin and rice suggest that P

deficiency stimulates glycolysis (Uhde-Stone et al 2003

Wasaki et al 2003a) It is conceivable that to maintain

a high level of organic acid secretion in the proteoid roots

of lupin a enhanced level of glycolysis is necessary under

P-deficient conditions (Pearse et al 2006) but it is not clear

why rice did not show enhanced accumulation of di- and

polysaccharides under P-deficient conditions (Nanamori

et al 2004) It appears that metabolic changes in glycolysis

in response to P deficiency differ among plant species

Severe P deficiency depleted inorganic Pi storage pools

as indicated by the sharp reduction of phosphoric acid

in the polar fractions of plant tissues to 510 of

the controls (PDS17thornPDS17 and PDR17thornPDS17

Supplementary Table S2) Under these conditions organic

P is an alternative resource of P for plant cells RNA

phospholipids and small phosphorylated metabolites are

three main classes of compounds containing phosphoesters

Degradation of RNA by increased RNase activity under

Pi-limiting conditions is well documented (Plaxton 2004)

The RNA content in the shoots of severely P-deficient

Arabidopsis plants was reduced to 13 of that in the

P-sufficient plants (Hewitt et al 2005) Considerable

amounts of organic P are present in small phosphorylated

metabolites which is approximately equivalent to that

in RNA or in phospholipids (Bieleski and Ferguson

1983 Doermann and Benning 2002) The levels of

small phosphorylated metabolites including glucose-6-P

fructose-6-P and inositol-1-P were largely reduced in both

shoots and roots of the severely P-deficient plants and the

level of glycerol-3-P in the polar fraction was also sharply

reduced in the shoots of the severely P-deficient plants

(Fig 4) The reduction of phosphorylated sugars was also

observed in bean roots and Arabidopsis under P-deficient

conditions (Rychter and Randall 1994 Morcuende et al

2007) These results indicate that severely P-deficient plants

salvage P from small phosphorylated metabolites for

essential cellular functions These small phosphorylated

metabolites are important intermediates in glycolysis and

synthesis of polysaccharides (glucose-6-P and fructose-6-P)

phospholipids (glycerol-3-P and inositol-1-P) amino acids

and nucleotides and in energy production Thus a depletion

of these phosphorylated metabolites would have a severe

impact not only on carbohydrate and nitrogen metabolism

but also on other metabolic processes

The enhanced accumulation of di- and tri-saccharides

in P-deficient plants can reduce the consumption of Pi in

phosphorylation of sugar metabolites and convert the small

phosphorylated metabolites to non-P-containing di- and

trisaccharides and other compounds such as aromatic

compounds (Figs 3 4) This could be a strategy to reduce

Pi consumption but may also have a benefit in osmotic

protection for stressed plants Increased accumulation of

di- and trisaccharides such as raffinose has been described

in plants exposed to heat stress (Panikulangara et al 2004)

and sulfate deficiency (Nikiforova et al 2005) maltose in

acute temperature stress (Kaplan and Guy 2004) and

6-kestose a precursor for synthesis of fructans under

drought conditions (Hendry 1993 Amiard et al 2003)

However carbohydrate formed as di- and trisaccharides


shown to accumulate in P-deficient plants cannot be utilized

readily for energy metabolism without involvement of Pi

to provide carbon metabolites for the TCA cycle An

alternative storage form of carbon such as organic acids

and amino acids would be preferred for survival of

P-deficient plants (Nanamori et al 2004)

P deficiency reduces organic acids in the TCA cycle

The reduced levels of several organic acids of the TCA

cycle including a-ketoglutarate succinate fumarate and

malate (Fig 4) and the reduction in the concentrations

of glutamate and aspartate especially in the roots of

the severely P-deficient plants (Fig 6B) suggest that the

shortage in carbohydrate supply is more apparent in the

roots than in the shoots of severely P-deficient plants

Reduced levels of fumarate were also observed in

P-deficient Arabidopsis (Misson et al 2005) In addition P

deficiency increases the secretion of organic acids into the

rhizosphere although cereals are weak secretors of organic

acids compared with legumes under low P conditions

(Neumann and Romheld 1999 Pearse et al 2006) The

secretion of organic acids could also contribute to

the reduction of the organic acids in the root tissues The

roots of severely P-deficient plants have to adjust metabolic

processes to utilize possible carbon metabolites such as

amino acids Some of the amino acids could be utilized as

an alternative carbon resource for energy production

reassimilation of the released ammonium and synthesis of

organic acids for secretion Interestingly the levels of several

organic acids in the TCA cycle were also reduced in

the roots of P-deficient common beans (Hernandez et al

2007) As legumes are efficient secretors of organic acids

(Neumann and Romheld 1999 Pearse et al 2006) secretion

could be a significant contributor to the reduced levels of

organic acids in the roots Nevertheless no reduction in

small P-containing metabolites nor organic acids in the

TCA cycle is observed in sulfate-deficient Arabidopsis plants

(Hirai et al 2004 Nikiforova et al 2005) indicating that

the reduction in the organic acids is unique to P-deficient

plant species

Severe P deficiency alters nitrogen metabolism

In P-deficient conditions transcript levels of the

genes coding for enzymes involved in protein degradation

were enhanced and the genes for protein synthesis were

suppressed in both Arabidopsis and common beans

(Wu et al 2003 Misson et al 2005 Hernandez et al

2007) The abundance of proteins related to protein

degradation through the ubiquitinndash26S proteasome path-

way was found to have increased in P-deficient roots of

maize (Li et al 2007) The reduced transcript levels of genes

related to nitrate reduction (Wu et al 2003) and reduced

activities for nitrate reduction have also been described in

P-deficient plants (Gniazdowska and Rychter 2000)

The increased concentrations of total free amino acids in

the shoots of severely P-deficient plants (Supplementary

Table S3) are likely to result from increased protein

degradation and repressed protein synthesis Various

metabolic reactions involved in either synthesis or degrada-

tion of amino acids produce ammonia such as aspara-

gine to aspartate glutamine to glutamate glutamate to

a-ketoglutarate and glycine to serine P deficiency enhances

the transcript levels of alanine aminotransferase genes

in both Arabidopsis and common beans (Wu et al 2003

Hernandez et al 2007) and the protein abundance of

glutamate dehydrogenase and phenylalanine ammonia-

lyase in maize roots (Li et al 2007) A high concentration

of ammonium is harmful to plants especially for barley an

ammonium-sensitive plant (Britto and Kronzucker 2002)

Thus it is critical to avoid excessive accumulation of

ammonium in P-deficient plants The levels of glutamine

and asparagine were elevated in both shoots and roots of

severely P-deficient plants and other metabolites related to

ammonium metabolism including putrescine and tyramine

were also enhanced in the severely P-deficient plants

especially in the roots (Supplementary Table S2) The

increased level of putrescine was also observed in P-deficient

rice cells (Shih and Kao 1996) These data suggest that

deamination of amino acids occurs in the severely

P-deficient plants for utilization in carbon metabolism

and a concomitant increase in assimilation of ammonium

takes place to ensure a reduction in levels of ammonium

The increased activities of glutamate dehydrogenase and

glutamine synthase in Arabidopsis plants (Morcuende et al

2007) and the enhanced protein abundance of root

glutamate dehydrogenase and glutamine synthase observed

in P-deficient maize plants (Li et al 2007) support this

interpretation It is interesting to note that the transcript

levels of glutamate synthase were down-regulated by P

deficiency in rice (Wasaki et al 2003a) Rice is an

ammonium-tolerant species which is in contrast to barley

that is an ammonium-sensitive species (Britto and

Kronzucker 2002) The difference in the metabolite levels

related to ammonium metabolism between P-deficient

barley and rice may be due to this different mechanism of

tolerance to ammonium

The synthesis of glutamine and asparagine requires

a carbon skeleton The enhanced levels of glutamine

and asparagine will require additional molecules of

a-ketoglutarate and oxaloacetate withdrawn from the

TCA cycle leading to a further reduction in the levels

of organic acids and restriction in energy production

in severely P-deficient plants The increased levels of

glutamine asparagine and other metabolites closely

related to ammonium metabolism are also observed in

other nutrient stresses such as nitrogen and sulfate


deficiency (Hirai et al 2004 Nikiforova et al 2005) but

the specific reduction in glutamate and aspartate is

only observed in P-deficient barley plants As synthesis

of glutamine and asparagine can incorporate two ammo-

nium molecules into one organic acid this is a highly

effective ammonium assimilation mechanism under the

carbohydrate-limiting conditions caused by P deficiency

Our metabolite analyses provide indirect evidence that

P-deficient plants are confronted with increased ammonium

To establish a direct link of P deficiency to the increased

accumulation of ammonium in barley tissues further

experiments are required by supplying low concentrations

of ammonium and monitoring ammonium levels in plant

tissues

The increased levels of glycine and serine in the shoots

of the severely P-deficient plants indicate that photorespira-

tion is enhanced in the shoots of the severely P-deficient

plants because glycine and serine are intermediates for

photorespiration (Wingler et al 2000) and the increased

levels of glycine and serine correlate well with the rate of

photorespiration in P-deficient bean plants (Kondracka and

Rychter 1997) Increases in the levels of glycine and serine

were also observed in sulfur-deficient Arabidopsis plants

(Nikiforava et al 2005) indicating that the increase in

photorespiration is a general stress response (Wingler et al

2000)

Carbohydrate partitioning in P-deficient barley differs

from a P-efficient Gramineae grass

P deficiency results in the enhanced accumulation of

di- and trisaccharides and reduced organic acids in barley

plants which generally is similar to rice a P-inefficient

plant (Nanamori et al 2004) A P-efficient grass (Brachiaria

hybrid) is very different from rice in carbohydrate par-

titioning under P-deficient conditions (Nanamori et al

2004) The Brachiaria hybrid can allocate more carbohy-

drates into organic acids and amino acids Maintaining high

activities of phosphoenolpyruvate carboxylase and phos-

phoenolpyruvate phosphatase under the low P condition

appears to be critical for allocating a large proportion of

carbohydrate into organic acids and amino acids Our data

suggest that barley behaves as a P-inefficient plant in

metabolic adaptation to low P environments Thus to

improve P efficiency in barley and perhaps other cereal

crops manipulation of metabolism through a shift in

carbohydrate partitioning could provide a logical strategy

In summary the metabolite profiles generated from

both shoots and roots provide an overview of the metabolic

adjustments that occur in P-deficient barley plants As

illustrated in Fig 7 P-deficient barley plants alter the levels

of many key metabolites which affect various metabolic

pathways including glycolysis photorespiration and

metabolism of nitrogen and phospholipids These adaptive

P inefficiency

P efficiency

ribose

inositol-1-P

glycine(shoots)

serine(shoots)

alanine(shoots)

aspartate

asparagine malate

fumarate

succinate

citrate

a-ketoglutarate

glutamine

glutamate

urea(roots)

glc

glc-6-P

fru

glycerol-3-P

aromaticcompounds

fru-6-P


Carbohydratepartitioning

glycerate-3-P

PEP

pyruvate

oxaloacetate

Fig 7 Schematic summary of metabolic adjustments in P-deficient plants and the proposed carbohydrate partitioning for improvementof P efficiency in cereals Thick arrows in green indicate an increase in metabolite levels and thin arrows in red indicate a decreasein metabolite levels The dashed line indicates carbohydrate partitioning for high P efficiency (below the line) and for low P efficiency(above the line)


adjustments reduce Pi consumption by increasing the

levels of di- and trisaccharides and salvage Pi from various

P-containing compounds including small P-containing

metabolites to maintain essential cellular functions An

immediate consequence which is unique to P deficiency is

greatlly reduced levels of the key metabolites in glycolysis

leading to the decrease in carbohydrate supply to the TCA

cycle especially in the roots In addition P-deficient plants

are likely to be confronted with the challenge of increased

ammonium derived from the degradation of protein The

assimilation of the increased ammonium requires additional

organic acids from the TCA cycle which depletes the

organic acid supply further It is noteworthy that although

there are di- and trisaccharides present in the P-deficient

plants they cannot be readily utilized Organic acids and

amino acids are the preferred storage metabolites in

P-deficient plants (Nanamori et al 2004) as they can be

readily utilized as the carbon resource by P-deficient plants

Therefore if barley (and other cereals) are to be engineered

to be better adapted to growth in P-deficient soils there

is a need to shift the partitioning of carbohydrate from

di- and tri-saccharides to organic acids and amino acids

Investigating the expression of genes (transcriptome) and

proteins (proteome) in barley under P-deficient conditions

would provide additional evidence to support this strategy

for enhancing the P efficiency

Materials and Methods

Plant materials

Barley (H vulgare L cv Lofty Nijo) seeds were surface-sterilized with 70 ethanol for 1min and 3 hypochlorite for5min rinsed with deionized water and incubated in Petri dishes for2 d at room temperature Two seeds with an emerged radicle wereput into a seedling cup which was placed in the lid of a black plasticcontainer Each container contained either four seedling cups fortwo harvests or two seedling cups for one harvest and were filledwith 1 liter of nutrient solution The basal nutrients were as follows(in mM) Ca(NO3)2 1000 KNO3 1000 MgSO4 250 KCl 50H3BO3 125 Fe-HEDTA 10 MnSO4 04 CuSO4 01 ZnSO405 NiSO4 01 and MoO3 01 with NH4H2PO4 100 (thornPtreatment) or without NH4H2PO4 (P treatment) To balanceNH4 ions added in the thornP treatment 100 mM NH4NO3 was addedinto the nutrient solution of the P treatment In the additionalexperiment with two levels of ammonium either 100 or 400 mMNH4NO3 was added in the thornP treatment and either nil or 300 mMNH4NO3 was added in the P treatment All treatments werereplicated four times Half-strength nutrient solution was useduntil the 10th day after seed imbibition for the plants harvested atday 10 and day 17 but the eighth day for the plants harvestedat day 16 Full strength was applied thereafter The full-strengthnutrient solution was replaced at day 14 for the plants harvested atday 17 but at day 12 for the plants harvested at day 16 The pH ofthe nutrient solution was adjusted to 60 with 1M KOH and thesolution was aerated continuously

Plants were grown in a controlled environment at 208Cday158C night with a photoperiod regime of 14 h day10 h nightat 300 mmolm2 sndash1 photon flux intensity at the plant level At each

harvest two seedling cups were removed from each containerPlants were collected in 5ndash6 h of the light period to eliminatediurnal changes in metabolite levels The roots of plants from oneseedling cup were rinsed briefly in deionized water excess waterwas blotted on fresh laboratory tissues and the roots and shootswere separated and oven-dried at 658C for 48 h The dry plantsamples were used for mineral element analysis by inductivelycoupled plasma spectrophotometry (Zarcinas et al 1987) Anothercup of plants was separated into roots and shoots frozenimmediately in liquid nitrogen and stored at 808C for metaboliteand transcript analyses

Real-time quantitative RTndashPCR

Shoots and roots of three replicates from each P treatmentwere used for transcript analysis Total RNA was prepared usingTrizol reagent according to the manufacturerrsquos instructions(Invitrogen Carlsbad CA USA) Total RNA was treated withDNase I using the DNA free kit (Ambion Austin TX USA) toremove any contaminating genomic DNA and then RNA integritywas checked on an agarose gel cDNA was prepared from 2mg oftotal RNA using SuperScriptTM III reverse transcriptase accord-ing to the manufacturerrsquos instructions (Invitrogen) The reactionwas incubated at 508C for 45min and at 708C for 15min toinactivate the enzyme

The transcript levels of HvIPS1 and HvPht16 weredetermined by real-time quantitative PCR essentially as describedby Burton et al (2004) The transcript levels of three control genes(barley a-tubulin heat shock protein 70 and cyclophilin) weredetermined and used for normalization Normalization was carriedout as described by Vandesompele et al (2002) and Burtonet al (2004) The normalized copies mg RNA1 are presentedThe primers 50 GCGGCGACTTCTCACCTCTACTAAG and50 CGAAGGATTCAAACAGGATCACACA (GenBank acces-sion No BI780234) were used for amplification of HvIPS1 and50 GGTCAAGAACTCGCTATTCATCC and 50 CCATACATCCATCCAAACCTG (GenBank accession No AF543198) foramplification of HvPht16

Analysis of metabolites with GC-MS

Metabolite analysis was carried out by a GC-MS-basedmethod modified from Roessner et al (2006) Fresh tissues of rootsand shoots from barley plants were homogenized using a mortarand pestle with liquid nitrogen Frozen tissue powder (100 10mgexact fresh weight was recorded) was extracted with methanol(350 ml) A polar internal standard (20 ml of 02mgmlndash1 ribitol inwater) and a non-polar internal standard (30 ml of 2mgmlndash1

nonadecanoic acid methylester in chloroform) were subsequentlyadded as quantification standards The mixture was extracted for15min at 708C and then mixed vigorously with 1 vol of waterIn order to separate polar and non-polar metabolites 300 ml ofchloroform was added to the mixture and centrifuged at 2200gfor 10min The upper methanolwater phase was collected andwashed with chloroform (300 ml) The polar phase of shoot extracts(100 ml) and root extracts (250 ml) was separately dried undervacuum Both lipid layers were combined and dried under vacuumIn order to methylate fatty acids the residues of the non-polarfractions were further incubated with chloroform (100 ml) andacidified methanol (300 ml of 125N HCl in methanol) for 24 h at508C and afterwards dried under vacuum The dry polar residuewas redissolved and derivatized for 120min at 378C in methox-yamine hydrochloride (40 ml of 20mgml1 in pyridine) followedby a 30min treatment with N-methyl-N-[trimethylsilyl] trifluoro-acetamide (MSTFA) (70 ml) at 378C The dry non-polar residue


was derivatized for 60min at 378C in pyridine (70 ml) and MSTFA(40 ml) To both a retention time standard mixture [10 ml of 0029(vv) n-dodecane n-pentadecane n-nonadecane n-docosanen-octacosane n-dotracontane n-hexatriacontane dissolved inpyridine] was added prior to trimethylsilylation Sample volumesof 1ml were then injected using the splitless mode onto a GCcolumn using a hot needle technique

The GC-MS system and conditions were as described inRoessner et al (2006) Acquired total ion chromatograms andmass spectra were evaluated using the Xcalibur program(ThermoFinnigan Manchester UK) and the resulting data areprepared normalized and presented as described by Roessner et al(2001) Mass spectra of eluting compounds were identified usingthe commercial mass spectra library NIST (httpwwwnistgov)and the public domain mass spectra library of the Max-Planck-Institute for Plant Physiology Golm Germany (httpcsbdbmpimp-golmmpgdecsbdbdbmamsrihtml) All matchingmass spectra were additionally co-verified by determination of theretention time and mass spectra by analysis of authentic standardsubstances

Analysis of free amino acids with LC-MS

Leaf material (15mg) was accurately weighed intoEppendorf tubes (2ml) and a solution of EDTA (400 ml 5mM)was added to each sample The samples were sonicated for15min then heated in a water bath (808C 15min) centrifuged(14500 rpm 5min) and the supernatant derivatized Thederivatization of the amino acids is based on a procedure outlinedby Cohen and Michaud (1993) and involved mixing the super-natant obtained above (20 ml) with borate buffer (200 ml 02MpH 88) followed by the addition of 10mM 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate solution (50 ml) in dry aceto-nitrile The reaction mixture was then heated at 558C in a waterbath for 10min and analyzed by LC-MS as described by Callahanet al (2007)

The LC-MS system comprised a Surveyor LC PumpSurveyor Auto Sampler and LCQ Deca XP Max ion trapMS (Thermo Finnigan USA) The ESI (electron spray ionization)source was optimized by infusing a derivatized amino acidstandard in line with the mobile phase then tuning the sourceconditions and ion optics to optimize the signals for the derivatizedamino acids This resulted in the following conditions sheath gas30 arbitrary units auxiliary gas 10 arbitrary units spray voltage30 kV capillary temperature 2508C capillary voltage 56V andtube lens 53V

Derivatized amino acids were eluted on an Altantis dC18150 21mm 3mm (Waters Melbourne Australia) HPLC columnusing a three-step gradient of deionized water (01 formic acid)and acetonitrile (01 formic acid) at a flow rate of 025mlminndash1

and a column temperature of 308C (Callahan et al 2007)

Statistical analysis

Students t-test at P 005 was applied for the means (nfrac14 4) ofmetabolites presented in Supplementary Tables S1ndashS3 Statisticalanalyses were carried using the Pirouette 311 software (InfometrixInc Woodinville WA USA)

Supplementary material

Supplementary material mentioned in the article is avail-able to online subscribers at the journal website wwwpcpoxfordjournalsorg

Funding

The Grains Research and Development Corporation

the Australian Research Council the South Australian

Government the University of Adelaide and the University

of Melbourne to the Australian Center for Plant Functional

Genomics (CYH UR NS PL AB) the Molecular

Plant Breeding CRC to YG Metabolomics Australia to

(UR DC AB)

Acknowledgments

We are grateful to M Tester for discussion and to T Fowlesand L Palmer for ICP analysis

References

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Bieleski RL and Ferguson IB (1983) Physiology and metabolism ofphosphate and its compounds In Encyclopedia of Plant PhysiologyEdited by Lauchli A and Bieleski RL Vol 15a pp 422ndash449 SpringerVerlag Berlin

Bolling C and Fiehn O (2005) Metabolite profiling of Chlamydomonasreinhardtii under nutrient deprivation Plant Physiol 139 1995ndash2005

Britto DT and Kronzucker HJ (2002) NH4thorn toxicity in higher plants

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Medicago truncatula roots is suppressed in response to colonization byvesicularndasharbuscular mycorrhizal (VAM) fungi and to phosphatenutrition Plant Mol Biol 34 199ndash208

Burton RA Shirley NJ King BJ Harvey AJ and Fincher GB(2004) The CesA gene family of barley Quantitative analysis oftranscripts reveals two groups of co-expressed genes Plant Physiol134 224ndash236

Callahan DL Kolev SD OrsquoHair RAJ Salt DE and Baker AJM(2007) Relationships between nicotianamine and other amino acids withnickel zinc and iron in Thlaspi hyperaccumulators New Phytol 176836ndash848

Ciereszko I and Barbachowska A (2000) Sucrose metabolism in leavesand roots of bean (Phaseolus vulgaris L) during phosphate deficiencyJ Plant Physiol 156 640ndash644

Cohen SA and Michaud DP (1993) Synthesis of a fluorescentderivatizing reagent 6-aminoquinolyl-N-hydroxysuccinimidyl carba-mate and its application for the analysis of hydrolysate amino acidsvia high-performance liquid chromatography Anal Biochem 211279ndash287

Cruz-Ramirez A Oropeza-Aburto A Razo-Hernandez F Ramirez-Chavez E and Herrera-Estrella L (2006) Phospholipase DZ2 playsan important role in extraplastidic galactolipid biosynthesis andphosphate recycling in Arabidopsis roots Proc Natl Acad Sci USA103 6765ndash6770

Doermann P and Benning C (2002) Galactolipids rule in seed plantsTrends Plant Sci 7 112ndash118

Gniazdowska A and Rychter AM (2000) Nitrate uptake by bean(Phaseolus vulgaris L) roots under phosphate deficiency Plant Soil 22679ndash85

Hendry GAF (1993) Evolutionary origins and natural functions offructansmdasha climatological biogeographic and mechanistic appraisalNew Phytol 123 3ndash14

Hernandez G Ramirez M Valdes-Lopez O Tesfaye MGraham MA et al (2007) Phosphorus stress in common bean roottranscript and metabolic responses Plant Physiol 144 752ndash767


Hewitt MM Carr JM Williamson CL and Slocum RD (2005)Effects of phosphate limitation on expression of genes involved in

pyrimidine synthesis and salvaging in Arabidopsis Plant Physiol

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Awazuhara M Arita M Fujiwara T and Saito K (2004) Integration

of transcriptomics and metabolomics for understanding of global

responses to nutritional stresses in Arabidopsis thaliana Proc Natl

Acad Sci USA 101 10205ndash10210Hou XL Wu P Jiao FC Jia QJ Chen HM Yu J Song XW

and Yi KK (2005) Regulation of the expression of OsIPS1 and OsIPS2in rice via systemic and local Pi signaling and hormones Plant Cell

Environ 28 353ndash364Jeschke WD Kirkby EA Peuke AD Pate JS and Hartung W

(1997) Effects of P deficiency on assimilation and transport of nitrate andphosphate in intact plants of castor bean (Ricinus communis L) J Exp

Bot 48 75ndash91Kaplan F and Guy CL (2004) Beta-amylase induction and the protective

role of maltose during temperature shock Plant Physiol 135 1674ndash1684Kondracka A and Rychter AM (1997) The role of Pi recycling processes

during photosynthesis in phosphate-deficient bean plants J Exp Bot 48

1461ndash1468Kopka J (2006) Current challenges and developments in GC-MS based

metabolite profiling technology J Biotechnol 124 312ndash322Li K Xu C Zhang K Yang A and Zhang J (2007) Proteomic

analysis of roots growth and metabolic changes under phosphorus deficitin maize (Zea mays L) plants Proteomics 7 1501ndash1512

Liu CM Muchhal US and Raghothama KG (1997) Differential

expression of TPS11 a phosphate starvation-induced gene in tomatoPlant Mol Biol 33 867ndash874

Martin AC del Pozo JC Inglesias J Rubio V Solano Rde la Pena A Leyva A and Paz-Ares J (2000) Influence of cytokinins

on the expression of phosphate starvation responsive genes inArabidopsis Plant J 24 559ndash567

Mimura T (1999) Regulation of phosphate transport and homeostasis inplant cells Int Rev Cytol Cell Biol 191 149ndash200

Misson J Raghothama KG Jain A Jouhet J Block MA et al

(2005) A genome-wide transcriptional analysis using Arabidopsis thaliana

Affymetrix gene chips determined plant responses to phosphate depriva-

tion Proc Natl Acad Sci USA 102 11934ndash11939Morcuende R Bari R Gibon Y Zheng WM Pant BD Blasing O

Usadel B Czechowski T Udvardi MK Stitt M and Scheible WR(2007) Genome-wide reprogramming of metabolism and regulatory

networks of Arabidopsis in response to phosphorus Plant Cell Environ

30 85ndash112Muchhal US and Raghothama KG (1999) Transcriptional regulation of

plant phosphate transporters Proc Natl Acad Sci USA 96 5868ndash72Nanamori M Shinano T Wasaki J Yamamura T Rao IM and

Osaki M (2004) Low phosphorus tolerance mechanisms phosphorus

recycling and photosynthate partitioning in the tropical forage grassBrachiaria hybrid cultivar mulato compared with rice Plant Cell Environ

45 460ndash469Neumann G and Romheld V (1999) Root excretion of carboxylic acids

and protons in phosphorus-deficient plants Plant Soil 211 121ndash130Nikiforova VJ Gakiere B Kempa S Adamik M Willmitzer L

Hesse H and Hoefgen R (2004) Towards dissecting nutrient

metabolism in plants a systems biology case study on sulphur

metabolism J Exp Bot 55 1861ndash1870Nikiforova VJ Kopka J Tolstikov V Fiehn O Hopkins L

Hawkesford MJ Hesse H and Hoefgen R (2005) Systems rebalan-

cing of metabolism in response to sulfur deprivation as revealed bymetabolome analysis of Arabidopsis plants Plant Physiol 138 304ndash318

Nilsson L Muller R and Nielsen TH (2007) Increased expression of theMYB-related transcription factor PHR1 leads to enhanced phosphate

uptake in Arabidopsis thaliana Plant Cell Environ 30 1499ndash1512

Panikulangara TJ Eggers-Schumacher G Wunderlich M Stransky Hand Schoffl F (2004) Galactinol synthase 1 A novel heat shock factortarget gene responsible for heat-induced synthesis of raffinose familyoligosaccharides in Arabidopsis Plant Physiol 136 3148ndash3158

Pearse SJ Veneklaas EJ Cawthray G Bolland MDA andLambers H (2006) Triticum aestivum shows a greater biomass responseto a supply of aluminium phosphate than Lupinus albus despite releasingfewer carboxylates into the rhizosphere New Phytol 169 515ndash524

Plaxton WC (2004) Plant response to stress biochemical adaptations tophosphate deficiency In Encyclopedia of Plant and Crop Science Editedby Goodman R pp 976ndash980 Marcel Dekker Inc New York

Plaxton WC and Carswell MC (1999) Metabolic aspects of thephosphate starvation response in plants In Plant Response toEnvironmental Stresses From Photohormones to GenomeReorganization Edited by Lerner HR pp 349ndash372 Marcel DekkerInc New York

Rae AL Cybinski DH Jarmey JM and Smith FW (2003)Characterization of two phosphate transporters from barley evidencefor diverse function and kinetic properties among members of the Pht1family Plant Mol Biol 53 27ndash36

Raghothama KG (1999) Phosphate acquisition Annu Rev Plant PhysiolPlant Mol Biol 50 665ndash693

Reuter DJ and Robinson JB (1997) Plant Analysis An InterpretationManual 2nd edn pp 86ndash92 CSIRO Australia Melbourne

Roessner U Luedemann A Brust D Fiehn O Linke TWillmitzer L and Fernie AR (2001) Metabolic profiling allowscomprehensive phenotyping of genetically or environmentally modifiedplant systems Plant Cell 13 11ndash29

Roessner U Patterson JH Forbes MG Fincher GB Langridge Pand Bacic A (2006) An investigation of boron toxicity in barley usingmetabolomics Plant Physiol 142 1087ndash1101

Rychter AM and Randall DD (1994) The effect of phosphate deficiencyon carbohydrate metabolism in bean roots Physiol Plant 91 383ndash388

Schachtman DP Reid RJ and Ayling SM (1998) Phosphorus uptakeby plants from soil to cell Plant Physiol 116 447ndash453

Shih CY and Kao CH (1996) Growth inhibition in suspension-culturedrice cells under phosphate deprivation is mediated through putrescineaccumulation Plant Physiol 111 721ndash724

Uhde-Stone C Zinn KE Ramirez-Yanez M Li AG Vance CP andAllan DL (2003) Nylon filter arrays reveal differential gene expressionin proteoid roots of white lupin in response to phosphorus deficiencyPlant Physiol 131 1064ndash1079

Vance CP Uhde-Stone C and Allan DL (2003) Phosphorus acquisitionand use critical adaptations by plants for securing a non renewableresource New Phytol 157 423ndash447

Vandesompele J De Preter K Pattyn F Poppe B Van Roy NDe Paepe A and Speleman F (2002) Accurate normalization of real-time quantitative RTPCR data by geometric averaging of multipleinternal control genes Genome Biol 3 1ndash11

Wasaki J Yonetani R Kuroda S Shinano T Yazaki J et al (2003a)Transcriptomic analysis of metabolic changes by phosphorus stress in riceplant roots Plant Cell Environ 26 1515ndash1523

Wasaki J Yonetani R Shinano T Kai M and Osaki M (2003b)Expression of the OsPI1 gene cloned from rice roots using cDNAmicroarray rapidly responds to phosphorus status New Phytol 158239ndash248

Wingler A Lea PJ Quick WP and Leegood RC (2000)Photorespiration metabolic pathways and their role in stress protectionPhilos Trans R Soc B Biol Sci 355 1517ndash1529

Wu P Ma L Hou X Wang M Wu Y Liu F and Deng XW (2003)Phosphate starvation triggers distinct alterations of genome expression inArabidopsis roots and leaves Plant Physiol 132 1260ndash1271

Zarcinas BA Cartwright B and Spouncer LR (1987) Nitric aciddigestion and multi-element analysis of plant material by induc-tively coupled plasma spectrometry Commun Soil Sci Plant Anal 18131ndash146

(Received February 21 2008 Accepted March 13 2008)


synthesize more Pi transporter proteins in the plasma

membrane of root epidermal cells for higher Pi uptake

capacity (Muchhal and Raghothama 1999) Transcriptomic

analyses in Arabidopsis rice and lupin have shown that

coordinated changes in the expression of several hundred

genes take place in P-deficient plants (Uhde-Stone et al

2003 Wasaki et al 2003a Wu et al 2003 Mission et al

2005 Morcuende et al 2007) These genes are involved in

various metabolic pathways such as photosynthesis carbon

metabolism nitrogen assimilation and synthesis of protein

and nucleic acids (Wu et al 2003 Misson et al 2005

Morcuende et al 2007) which could lead to adaptive

changes in metabolite profiles

Recent advances in analytical technologies such as gas

and liquid chromatography coupled to mass spectrometry

(GC-MS and LC-MS) have allowed the analysis of a large

number of compounds from a single plant sample (for a

review see Kopka 2006) Such metabolite profiles provide

not only a much broader view for a systematic adjustment

in metabolic processes than conventional biochemical

approaches but also an opportunity to reveal new insights

on metabolism Metabolite profiling with GC-MS has been

used for studies on nutrient deficiency including nitrogen

sulfate iron and P in a single-cell green alga (Bolling and

Fiehn 2005) and in higher plants such as Arabidopsis and

common beans (Hirai et al 2004 Nikiforova et al 2005

Hernandez et al 2007) A combination of transcriptomic

and metabolite analyses has also been made to increase

further our understanding of plant responses to various

environmental stresses such as sulfate and nitrogen (Hirai

et al 2004 Nikiforova et al 2004 Nikiforova et al 2005)

and P deficiency in Arabidopsis and common beans

(Hernandez et al 2007 Morcuende et al 2007) Despite

the agronomic importance of cereals such as wheat and

barley little information is available for metabolite changes

in response to P deficiency As Pi homeostasis is regulated

at the whole-plant level by translocating Pi from mature to

young tissues and from shoots to roots under Pi-limiting

conditions (Jeschke et al 1997 Mimura 1999) it is

necessary to determine alterations in both shoots and

roots to increase our understanding of the biochemical

mechanisms involved in whole-plant adaptation to the low

Pi environment

In this report barley (Hordeum vulgare L) was used as

a model plant for cereal crops We measured approximately

100 metabolites from both shoots and roots of mildly or

severely P-deficient barley plants using a recently developed

GC-MS method (Roessner et al 2006) Metabolite profiles

reveal distinct changes in carbon and nitrogen metabolism

in P-deficient barley plants leading to accumulation of

di- and trisaccharides and free amino acids The distinct

metabolite profiles suggested that the severely P-deficient

barley plants are confronted with an increased level of

ammonium Thus the effect of severe P deficiency on

ammonium metabolism was further investigated through

quantitative analysis of amino acid profiles using LC-MS

(Callahan et al 2007) Our results provide new insights

into the effects of P deficiency on carbon and nitrogen

metabolism in barley plants

Results

Plant responses to short- and long-term P starvation in

growth and P nutrition

Barley seedlings were grown from seeds for different

time periods in a nutrient solution either with no added

NH4H2PO4 (P) or with 01mM NH4H2PO4 (thornP control)

to obtain plants with different P status At the 10th day

after seed imbibition (day 10) shoot development was

similar in the two P treatments with both having a fully

expanded first leaf and a small second leaf There were no

apparent P deficiency symptoms in the shoots of P-treated

plants although a change in root appearance was evident

Shorter primary roots and more lateral roots were observed

in the P treatment Plant growth measured on a FW basis

was significantly different between P and thornP treatments

at day 10 (Fig 1A) but there was no difference in DW

between P and thornP treatments at day 10 (data not shown)

The P concentration of shoots in the P-treated plants was

26mg P gndash1 DW (Fig 1B) which was below the critical

P concentration of 4mg P gndash1 DW (Reuter and Robinson

1997) The concentration of P in the roots was 32mg gndash1

DW (Fig 1B) In contrast the P concentration in the

thornP-treated plants was adequate at day 10 (Fig 1B) The

P-treated plants at day 10 suffered from mild P deficiency

and henceforth were referred to as mildly P-deficient plants

Plant growth was severely retarded when the plants

were grown in the P treatment for an additional 7 d

(Fig 1A day 17) There were two small fully expanded

leaves with the third leaf emerging in the P-treated plants

whereas there were three large fully expanded leaves in the

thornP-treated plants The seminal roots of the P-treated

plants were much shorter than those in the thornP-treated

plants P concentrations in the P-treated plants further

declined to 12mg P gndash1 DW in shoots and 20mg P gndash1 DW

in roots at day 17 (Fig 1B) Therefore the P-treated

plants at day 17 were inferred to be severely P-deficient

plants in this study

Expression of HvIPS1 and HvPht16 in P-deficient plants

The TPSI1At4 genes are sensitive to P deficiency and

widely used as a molecular indicator for P deficiency

(Burleigh and Harrison 1997 Liu et al 1997 Martin et al

2000 Wasaki et al 2003b Hou et al 2005) HvIPS1 is a

barley homolog of the TPSI1At4 genes (Hou et al 2005)

and HvPht16 is a low-affinity Pi transporter of barley


























plants





1800A

B

1600

1400

1200

1000

800

600

400

200

0

1800

1600

1400

1200

1000

800

600

400

200

0

No

rmal

ized

co

pie

s mg

minus1 R

NA

(times1

06 )N

orm

aliz

ed c

op

ies

mgminus1

RN

A (

times106 ) minus P

+ P


HvIPS1 HvPht16



140A

B

120

100

080

060

040

020

0

10

8

6

4

2

0

Fre

sh w

eig

ht

(g p

lan

tminus1)

P c

on

cen

trat

ion

s (m

g g

minus1 D

W)

Shoots Roots

minusP

+P

minusP

+P

Criticallevel












plants at day 17






























292plusmn037






















lipids

fructanstarch

glycerate-3-P

PEP

pyruvate

oxaloacetate

aromatic compounds







plants

















































urea(nd)

3315plusmn009



695plusmn014


fructan

lipids

starch

glycerate-3-P

PEP

pyruvate

oxaloacetate

aromatic compounds











(Fig 4)

































amino acids



































































methodologies





















140

120

100

Fre

sh w

eig

ht

(g p

lan

tminus1)

Fre

sh w

eig

ht

(g p

lan

tminus1)

P c

on

cen

trat

ion

(m

g g

minus1 D

W)

080

060

040

020

0

140minusP

+P120

100

080

060

040

Critical level

020

0

10

8

6

4

2

001 mM 04 mM

A

B

C


1200Glu

Gln

Asp

Asn

1000

800

600

400

200

0

1200

1000

800

600

400

200


Co

nce

ntr

atio

n (

mg g

minus1 D

W)

Co

nce

ntr

atio

n (

mg g

minus1 D

W)

A

B
























the roots

Discussion



metabolites






















































































































plant species
















































































tissues












2000)



























P inefficiency

P efficiency

ribose

inositol-1-P

glycine(shoots)

serine(shoots)

alanine(shoots)

aspartate

asparagine malate

fumarate

succinate

citrate

a-ketoglutarate

glutamine

glutamate

urea(roots)

glc

glc-6-P

fru

glycerol-3-P

aromaticcompounds

fru-6-P



glycerate-3-P

PEP

pyruvate

oxaloacetate






























Plant materials






















Funding







(UR DC AB)

Acknowledgments


References







































































































plants





1800A

B

1600

1400

1200

1000

800

600

400

200

0

1800

1600

1400

1200

1000

800

600

400

200

0

No

rmal

ized

co

pie

s mg

minus1 R

NA

(times1

06 )N

orm

aliz

ed c

op

ies

mgminus1

RN

A (

times106 ) minus P

+ P


HvIPS1 HvPht16



140A

B

120

100

080

060

040

020

0

10

8

6

4

2

0

Fre

sh w

eig

ht

(g p

lan

tminus1)

P c

on

cen

trat

ion

s (m

g g

minus1 D

W)

Shoots Roots

minusP

+P

minusP

+P

Criticallevel












plants at day 17






























292plusmn037






















lipids

fructanstarch

glycerate-3-P

PEP

pyruvate

oxaloacetate

aromatic compounds







plants

















































urea(nd)

3315plusmn009



695plusmn014


fructan

lipids

starch

glycerate-3-P

PEP

pyruvate

oxaloacetate

aromatic compounds











(Fig 4)

































amino acids



































































methodologies





















140

120

100

Fre

sh w

eig

ht

(g p

lan

tminus1)

Fre

sh w

eig

ht

(g p

lan

tminus1)

P c

on

cen

trat

ion

(m

g g

minus1 D

W)

080

060

040

020

0

140minusP

+P120

100

080

060

040

Critical level

020

0

10

8

6

4

2

001 mM 04 mM

A

B

C


1200Glu

Gln

Asp

Asn

1000

800

600

400

200

0

1200

1000

800

600

400

200


Co

nce

ntr

atio

n (

mg g

minus1 D

W)

Co

nce

ntr

atio

n (

mg g

minus1 D

W)

A

B
























the roots

Discussion



metabolites






















































































































plant species
















































































tissues












2000)



























P inefficiency

P efficiency

ribose

inositol-1-P

glycine(shoots)

serine(shoots)

alanine(shoots)

aspartate

asparagine malate

fumarate

succinate

citrate

a-ketoglutarate

glutamine

glutamate

urea(roots)

glc

glc-6-P

fru

glycerol-3-P

aromaticcompounds

fru-6-P



glycerate-3-P

PEP

pyruvate

oxaloacetate






























Plant materials






















Funding







(UR DC AB)

Acknowledgments


References























































































plants at day 17






























292plusmn037






















lipids

fructanstarch

glycerate-3-P

PEP

pyruvate

oxaloacetate

aromatic compounds







plants

















































urea(nd)

3315plusmn009



695plusmn014


fructan

lipids

starch

glycerate-3-P

PEP

pyruvate

oxaloacetate

aromatic compounds











(Fig 4)

































amino acids



































































methodologies





















140

120

100

Fre

sh w

eig

ht

(g p

lan

tminus1)

Fre

sh w

eig

ht

(g p

lan

tminus1)

P c

on

cen

trat

ion

(m

g g

minus1 D

W)

080

060

040

020

0

140minusP

+P120

100

080

060

040

Critical level

020

0

10

8

6

4

2

001 mM 04 mM

A

B

C


1200Glu

Gln

Asp

Asn

1000

800

600

400

200

0

1200

1000

800

600

400

200


Co

nce

ntr

atio

n (

mg g

minus1 D

W)

Co

nce

ntr

atio

n (

mg g

minus1 D

W)

A

B
























the roots

Discussion



metabolites






















































































































plant species
















































































tissues












2000)



























P inefficiency

P efficiency

ribose

inositol-1-P

glycine(shoots)

serine(shoots)

alanine(shoots)

aspartate

asparagine malate

fumarate

succinate

citrate

a-ketoglutarate

glutamine

glutamate

urea(roots)

glc

glc-6-P

fru

glycerol-3-P

aromaticcompounds

fru-6-P



glycerate-3-P

PEP

pyruvate

oxaloacetate






























Plant materials






















Funding







(UR DC AB)

Acknowledgments


References



















































































plants

















































urea(nd)

3315plusmn009



695plusmn014


fructan

lipids

starch

glycerate-3-P

PEP

pyruvate

oxaloacetate

aromatic compounds











(Fig 4)

































amino acids



































































methodologies





















140

120

100

Fre

sh w

eig

ht

(g p

lan

tminus1)

Fre

sh w

eig

ht

(g p

lan

tminus1)

P c

on

cen

trat

ion

(m

g g

minus1 D

W)

080

060

040

020

0

140minusP

+P120

100

080

060

040

Critical level

020

0

10

8

6

4

2

001 mM 04 mM

A

B

C


1200Glu

Gln

Asp

Asn

1000

800

600

400

200

0

1200

1000

800

600

400

200


Co

nce

ntr

atio

n (

mg g

minus1 D

W)

Co

nce

ntr

atio

n (

mg g

minus1 D

W)

A

B
























the roots

Discussion



metabolites






















































































































plant species
















































































tissues












2000)



























P inefficiency

P efficiency

ribose

inositol-1-P

glycine(shoots)

serine(shoots)

alanine(shoots)

aspartate

asparagine malate

fumarate

succinate

citrate

a-ketoglutarate

glutamine

glutamate

urea(roots)

glc

glc-6-P

fru

glycerol-3-P

aromaticcompounds

fru-6-P



glycerate-3-P

PEP

pyruvate

oxaloacetate






























Plant materials






















Funding







(UR DC AB)

Acknowledgments


References























































































(Fig 4)

































amino acids



































































methodologies





















140

120

100

Fre

sh w

eig

ht

(g p

lan

tminus1)

Fre

sh w

eig

ht

(g p

lan

tminus1)

P c

on

cen

trat

ion

(m

g g

minus1 D

W)

080

060

040

020

0

140minusP

+P120

100

080

060

040

Critical level

020

0

10

8

6

4

2

001 mM 04 mM

A

B

C


1200Glu

Gln

Asp

Asn

1000

800

600

400

200

0

1200

1000

800

600

400

200


Co

nce

ntr

atio

n (

mg g

minus1 D

W)

Co

nce

ntr

atio

n (

mg g

minus1 D

W)

A

B
























the roots

Discussion



metabolites






















































































































plant species
















































































tissues












2000)



























P inefficiency

P efficiency

ribose

inositol-1-P

glycine(shoots)

serine(shoots)

alanine(shoots)

aspartate

asparagine malate

fumarate

succinate

citrate

a-ketoglutarate

glutamine

glutamate

urea(roots)

glc

glc-6-P

fru

glycerol-3-P

aromaticcompounds

fru-6-P



glycerate-3-P

PEP

pyruvate

oxaloacetate






























Plant materials






















Funding







(UR DC AB)

Acknowledgments


References


















































































methodologies





















140

120

100

Fre

sh w

eig

ht

(g p

lan

tminus1)

Fre

sh w

eig

ht

(g p

lan

tminus1)

P c

on

cen

trat

ion

(m

g g

minus1 D

W)

080

060

040

020

0

140minusP

+P120

100

080

060

040

Critical level

020

0

10

8

6

4

2

001 mM 04 mM

A

B

C


1200Glu

Gln

Asp

Asn

1000

800

600

400

200

0

1200

1000

800

600

400

200


Co

nce

ntr

atio

n (

mg g

minus1 D

W)

Co

nce

ntr

atio

n (

mg g

minus1 D

W)

A

B
























the roots

Discussion



metabolites






















































































































plant species
















































































tissues












2000)



























P inefficiency

P efficiency

ribose

inositol-1-P

glycine(shoots)

serine(shoots)

alanine(shoots)

aspartate

asparagine malate

fumarate

succinate

citrate

a-ketoglutarate

glutamine

glutamate

urea(roots)

glc

glc-6-P

fru

glycerol-3-P

aromaticcompounds

fru-6-P



glycerate-3-P

PEP

pyruvate

oxaloacetate






























Plant materials






















Funding







(UR DC AB)

Acknowledgments


References




































































































the roots

Discussion



metabolites






















































































































plant species
















































































tissues












2000)



























P inefficiency

P efficiency

ribose

inositol-1-P

glycine(shoots)

serine(shoots)

alanine(shoots)

aspartate

asparagine malate

fumarate

succinate

citrate

a-ketoglutarate

glutamine

glutamate

urea(roots)

glc

glc-6-P

fru

glycerol-3-P

aromaticcompounds

fru-6-P



glycerate-3-P

PEP

pyruvate

oxaloacetate






























Plant materials






















Funding







(UR DC AB)

Acknowledgments


References





















































































































plant species
















































































tissues












2000)



























P inefficiency

P efficiency

ribose

inositol-1-P

glycine(shoots)

serine(shoots)

alanine(shoots)

aspartate

asparagine malate

fumarate

succinate

citrate

a-ketoglutarate

glutamine

glutamate

urea(roots)

glc

glc-6-P

fru

glycerol-3-P

aromaticcompounds

fru-6-P



glycerate-3-P

PEP

pyruvate

oxaloacetate






























Plant materials






















Funding







(UR DC AB)

Acknowledgments


References




























































































tissues












2000)



























P inefficiency

P efficiency

ribose

inositol-1-P

glycine(shoots)

serine(shoots)

alanine(shoots)

aspartate

asparagine malate

fumarate

succinate

citrate

a-ketoglutarate

glutamine

glutamate

urea(roots)

glc

glc-6-P

fru

glycerol-3-P

aromaticcompounds

fru-6-P



glycerate-3-P

PEP

pyruvate

oxaloacetate






























Plant materials






















Funding







(UR DC AB)

Acknowledgments


References










































































































Plant materials






















Funding







(UR DC AB)

Acknowledgments


References


























































































Funding







(UR DC AB)

Acknowledgments


References















































































Metabolite profiling reveals distinct changes in carbon and nitrogen metabolism in...

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