DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS
Research and Development
CSG 15
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to [email protected]
Project title
Changes in Gene Expression Profiles under Nutrient Deficiency
DEFRA project code
HH3502SFV
Contractor organisation
and location
Horticulture Research International,
Wellesbourne,
Warks CV35 9EF
Total DEFRA project costs
Project start date
£ 44,624
10/01/01
Project end date
31/03/02
Executive summary (maximum 2 sides A4)

This project contributes to DEFRA's policy objectives: (a) to promote sustainable management and prudent
use of natural resources, and (b) to protect the environment and conserve and enhance biodiversity.

It addresses the problem of excessive P fertilisation of crops, which is both costly and can lead to
unnecessary pollution. Excessive P fertilisation occurs because chemical assays of both soil and plant P are
unreliable. Novel biosensor technologies, based on knowledge of the changes in plant gene expression
under P-starvation, could better inform P-fertiliser application.

Novel biosensor technologies might utilise transcripts from P-responsive genes to develop customised
DNA-array and antibody-based bioassays of P-stress. Alternatively, the promoters of P-responsive genes
could underpin the development of 'smart plants', in which P-responsive promoters control the expression of
genes encoding visible products whose abundance reflects plant P status.

Our aim was to identify genes, expressed in shoot tissues of the model plant Arabidopsis thaliana, whose
transcripts increased rapidly and specifically in response to P starvation. Expression profiling using
Affymetrix GeneChip technology allowed us to assay the abundance of transcripts from approximately
8,000 genes (33% genome coverage) simultaneously.

Our initial objectives were to determine the effects of withdrawing the essential mineral nutrients P, N or K
on shoot growth, mineral content and gene expression in Arabidopsis growing hydroponically. Withdrawing
P reduced shoot P concentration after about 24 h, and slowed shoot growth after about 100 h. Thus, gene
transcripts that increased in abundance between about 24 to 100 h following P withdrawal might be useful
CSG 15 (9/01)
1
Project
title
Changes in Gene Expression Profiles under Nutrient
Deficiency
DEFRA
project code
HH3502SFV
for biosensor development. Withdrawing P had no effect on shoot N or K concentration over this period.
Similarly, withdrawing N or K did not affect shoot P concentration.

Our ultimate objective was to identify genes whose expression was increased specifically by P-starvation
and before P-starvation affected plant growth. The expression of several hundred genes increased during P
starvation. Of these, 37 genes were identified as being potentially useful for biosensor technologies. The
expression of these 37 genes did not increase immediately (4 h) after P withdrawal, but increased at least
threefold 28 to 100 h after P withdrawal. However, the expression of 19 of these genes was also increased
by both N and K starvation, which suggests that they might be controlled by a common mineral-imbalance
stress-response system. The expression of a further 5 genes was increased by both N and P starvation, and
the expression of 8 genes was increased by both P and K starvation. The expression of the remaining four
genes appeared to be increased specifically by P starvation.

The four (provisional) marker-genes specific for P starvation were identified as encoding LEA M17
(which is related to proteins that confer tolerance of stresses associated with a water-deficit), a nodulin-like
protein (which is homologous to the MtN21 protein expressed during nodule organogenesis in Medicago
truncatula), strictosidine synthase (which catalyses the condensation of tryptamine and secologanin to form
strictosidine, a key intermediate in indole alkaloid production) and a protein with unknown function.

In conclusion: Transcripts and promoters from genes responding specifically to P-starvation can now be
used to develop biosensor technologies to assay plant P status. These technologies will help reduce the
application of P fertiliser. This will lower costs and reduce pollution, thereby delivering DEFRA's policy
objectives for the sustainable use of natural resources, protection of the environment and enhanced
biodiversity.
CSG 15 (9/01)
2
Project
title
Changes in Gene Expression Profiles under Nutrient
Deficiency
DEFRA
project code
HH3502SFV
Scientific report (maximum 20 sides A4)
1. INTRODUCTION
The UK horticultural and agricultural industries routinely apply large amounts of inorganic fertiliser-P to
maintain crop yields and quality. It is estimated that up to £50/ha is spent annually on P-fertilisers for
vegetables (equivalent to £7M nationally). More P is applied than is required because chemical assays of both
soil and plant P are unreliable. Analyses of soil P are unreliable because critical concentrations of extractable
soil P for maximum yield depend on soil type, and few farmers know the critical values for their fields.
Analyses of plant P are difficult to interpret not only because little information is available for the critical tissue
P concentration of vegetables, but also because these values depend on environmental, physiological and
developmental parameters.
Excessive P fertilisation is both costly and can lead to unnecessary pollution. Possible solutions to this
problem include in situ assays of plant-P (Bollons & Barraclough, 1997, 1999), or the use of novel biosensor
technologies based on knowledge of the changes in plant gene expression under P-starvation (White & Rahn,
1999), to inform fertiliser application. Several biosensor technologies can be envisaged. These include: (i)
'smart plants', in which P-responsive promoters control the expression of genes encoding visible products whose
abundance reflects plant P status, (ii) custom-designed DNA microarrays, to detect the transcripts of genes whose
expression is altered by P starvation, and (iii) traditional antibody-based assays that detect the presence of proteins
accumulating when plants lack P.
The aim of this project was to identify genes expressed in shoot tissues that are upregulated rapidly
and specifically in response to P starvation. It utilises the model plant, Arabidopsis thaliana, since gene
expression profiling can be performed readily in Arabidopsis using Affymetrix GeneChip technology, which
allows the experimenter to assay the expression of approximately 8,000 genes (33% genome coverage)
simultaneously. It is envisaged that transcripts of these genes will underpin the development of customised
DNA-array and antibody-based bioassays of P-stress, and that promoters of these genes will underpin the
development of 'smart plants'. In addition, knowledge of the changes in gene expression that occur when P
supply is compromised will improve our general understanding of the physiology of P nutrition.
Our approach was to combine knowledge of how the withdrawal of individual mineral nutrients (N, P,
K) affects shoot mineral content, growth and gene expression to realise two overall scientific objectives: (A)
to identify genes whose expression increases as the P concentration of the shoot declines, but before a shoot
growth is compromised, and (B) to determine the subset of these genes that are upregulated specifically by P
deficiency.
2. EXPERIMENTAL METHODS
Plant Material
Seeds of Arabidopsis thaliana (L.) Heynh. (Columbia Col–5, from Nottingham Arabidopsis Stock Centre,
NASC, #N1688) were washed in 70% (v/v) ethanol/water and surface sterilised using NaOCl (1% active chlorine).
Sterilised seeds were imbibed for 3-5 d in sterile distilled water at 4°C to break dormancy. Following
imbibation, seeds were sown in unvented, polycarbonate culture boxes (Sigma-Aldrich Company Ltd., Dorset
UK). Seedlings were grown for 21 d on perforated polycarbonate discs (diameter 91 mm) placed over 75 ml 0.8%
(w/v) agar containing 1% (w/v) sucrose and a basal salt mix (Murashige & Skoog, 1962). Boxes were placed in a
growth room set to 24°C, with 16 h light per day. Illumination was provided by a bank of 100W 84 fluorescent
tubes (Philips, Eindhoven, Netherlands) giving an intensity of 45 mol photons m-2 s-1 at plant height. Roots grew
into the agar, but shoots remained on the opposite side of the disc.
After 21 d, seedlings were transferred, still on polycarbonate discs, to a hydroponics system in a Saxcil
growth cabinet. Plants were illuminated for 16 h daily at a light intensity of approximately 75 µmol photons m-2
s-1 at plant height. Temperature was maintained at 24°C during the light period and 16°C during the dark. The
CSG 15 (9/01)
3
Project
title
Changes in Gene Expression Profiles under Nutrient
Deficiency
MAFF
project code
HH3502S
FV
relative humidity was approximately 80%. Each polycarbonate disc was placed in a light-proof 500 ml beaker
over 450 ml aerated complete nutrient solution containing 0.75 mM K+, 4.025 mM Ca2+, 0.75 mM Mg2+, 0.01 mM
Mn2+, 0.001 mM Zn2+, 0.003 mM Cu2+, 0.001 mM Na+, 0.25 mM H2PO42-, 8.0 mM NO3-, 0.764 mM SO42-, 0.05
mM Cl-, 0.03 mM H2BO3-, 0.0005 mM MoO42-, 0.1 mM FeNaEDTA. Nutrient solution was recirculated using a
peristaltic pump at a flow rate of 30 ml min-1 through four beakers and a central reservoir containing 6 l aerated
nutrient solution. Four such hydroponic units could be operated simultaneously. Nutrient solutions were replaced
twice a week. Plants were grown hydroponically for 7 d in complete nutrient solution prior to experimentation.
Experiments began 28 d after sowing. To determine the effects of P, N or K starvation on shoot growth,
mineral content and gene expression, the complete nutrient solution was replaced four hours into the light period
with nutrient solutions lacking these elements. Sulphate replaced either phosphate or nitrate in solutions lacking P
or N, respectively. Calcium replaced potassium in solutions lacking K.
Analysis of Plant Growth and Mineral Content
Plants were harvested at various intervals following P, N or K withdrawal. At each harvest the fresh weight of
the shoot was determined for individual plants. Shoot material was bulked, dried at 80°C for 48 hours and the
dry weight determined. Tissue P and K contents were determined following digestion of bulked and weighed
material from batches of 25 plants by inductively coupled plasma optical emission spectrophotometry
(JYHoriba Ultima 2 ICP-OES, Jobin Yvon Ltd, Middlesex, UK). Tissue N content was determined on a
subsample of dried material loaded directly into a Leco CN 2000 combustion analyser (Leco UK Ltd, Cheshire,
UK).
Gene Expression Studies
To avoid any complications resulting from light- or circadian-regulation of gene expression (Desprez et al.
1998; Kehoe et al. 1999; Harmer et al. 2000; Ma et al. 2001; Schaffer et al. 2001), all plants were harvested at
the same point in the light cycle. Gene expression was determined on shoot material harvested -20, 4, 28 and 100
h after transfer to experimental conditions. To control for biological variation, shoot material from four to eight
plants was bulked and snap frozen in liquid nitrogen. Thus, the gene expression recorded for each sample was the
common (average) response of biological replicates. Samples were stored at –70°C prior to total RNA extraction.
Total RNA was extracted from tissue samples following the addition of 1 ml TRIzol reagent, according to the
manufactures instructions (Invitrogen Life Technologies, Rockville, Maryland). To test biological
reproducibility, replicate experiments were performed (Table 1). To provide quality control of total RNA
samples, OD260/OD280 was determined and RNA gel pictures were scrutinised. Total RNA was sent to AROS
Applied Biotechnology (Aarhus, Denmark) for labelling and GeneChip™ analysis (Affymetrix, Santa Clara,
Ca, USA). Following the precedent of Chen et al. (2002), any average difference (expression level) below 5
was floored to 5. The fold-change for each gene was calculated by dividing the average difference of an
experimental sample by the average difference of an appropriate control sample. If, for any complete set of
comparisons, the Affymetrix software declared the gene transcripts "absent", these data were eliminated from
analyses. The database of Ghassemian et al. (2001) was used to map GeneChip™ ID to Arabidopsis Genome
Initiative (AGI) identifiers.
Note: The work relating to genechip analysis carried out as part of this Project was subject to the agreement of
certain licensing conditions with Affymetrix. The recipient(s) of this report therefore need(s) to be aware that
any commercial exploitation of the results of this Project may require the further negotiation of licence terms
with Affymetrix.
4
Changes in Gene Expression Profiles under Nutrient
Deficiency
Project
title
MAFF
project code
HH3502S
FV
3. THE EFFECT OF NUTRIENT STARVATION ON SHOOT GROWTH AND MINERAL CONTENT
Twenty-eight days after sowing, P, N or K were withdrawn individually from Arabidopsis plants growing
hydroponically and the timecourses of development of nutrient deficiencies were determined (Fig. 1). Shoot
mass was unaffected for at least 100 hours after nutrient withdrawal, but P starvation significantly reduced
shoot mass subsequently (Fig. 1A). For use as a diagnostic of P status, changes in gene expression upon P
starvation must be observed within the initial 100 h following P withdrawal. No change in shoot P
concentration was observed over the initial 24 h following P withdrawal, but shoot P concentration was reduced
significantly between 24 and 72 h after P withdrawal (Fig. 1B). Thus, for use as a diagnostic of P status, it is
likely that changes in gene expression upon P starvation need to be observed 24 to 72 h following P withdrawal,
as the shoot P concentration declines. The withdrawal of neither N nor K affected P status over this period (Fig.
1B), and P starvation did not affect shoot N or K concentrations (data not shown). It would appear that a
deficiency of one nutrient does not affect the tissue concentration of another. It is possible, therefore, to identify
genes regulated specifically by P starvation.
12
Shoot P content (mg/g DW)
35
Shoot mass (mg)
30
25
20
15
10
5
0
10
8
6
4
2
0
0
100
200
300
0
Time after nutrient withdrawal (h)
100
200
300
Time after nutrient withdrawal (h)
Figure 1. The effect of P, N or K withdrawal from the nutrient solution bathing roots of Arabidopsis plants on shoot mass
and shoot P concentration. Plants were grown in complete nutrient solution (●) and solutions lacking P (○), N (□) or K
(Δ). Data are expressed as mean ± SEM.
4. THE EFFECT OF NUTRIENT STARVATION ON GENE EXPRESSION IN ARABIDOPSIS SHOOTS
Total RNA was extracted from shoots of Arabidopsis grown with a variety of nutritional treatments for various
periods (Table 1). Timepoints were chosen at which (i) no nutrient deficiency was apparent (4 h), (ii) plants
approached the critical shoot nutrient concentration for deficiency (28 h) and (iii) the effects of nutrient
deficiency on shoot growth were imminent (100 h). About 5000 gene transcripts could be detected in each of
these experiments.
Treatment
Full nutrient
Minus N
Minus P
Minus K
Time of Harvest (h)
28
-20
4
1, 21
1, 21
1, 21
1, 21
9
11
2, 10, 22
12
3, 13, 23
5, 15, 25
6, 14, 24
4, 16, 26
100
7, 17, 27
19
8, 18, 28
20
Table 1. HRI numbering of 28 Affymetrix GeneChips™ used for gene expression profiling. These are divided into
individual experiments (colours) and include the experiments originally designated 'Genomic Arabidopsis Resource
Network (GARNet) Experiment 1' (red), 'GARNet experiment 2' (blue) and 'Objective Experiment' (black).
5
Changes in Gene Expression Profiles under Nutrient
Deficiency
Project
title
MAFF
project code
HH3502S
FV
This array of treatments yields several biological comparisons. First, it enables the changes in gene expression
during ontogeny in plants grown in a complete nutrient solution to be determined. These are appreciable (Fig.
2; Ruan et al. 1998; Zhu et al. 2001). Thus, comparison of the changes in gene expression following nutrient
withdrawal can only be determined relative to a control plant grown in complete nutrient solution harvested at a
comparable time. Second, by comparison with appropriate control plants, the temporal changes in the
expression of genes following nutrient withdrawal can be determined (Fig. 3). By comparing the changes in
expression profiles following P withdrawal, genes that are upregulated rapidly, but not transiently, by P
depletion (ie genes upregulated at both 28 and 100 h) can be identified (Overall Scientific Objective A;
Genomic Arabidopsis Resource Network, GARNet, Experiment 2). By comparing the gene expression profiles
in shoots of plants in which either N, P or K are withdrawn with appropriate control plants, subsets of genes
that are upregulated by lack of specific nutrients and those that are upregulated by a common mineralimbalance stress-response system can be identified (Overall Scientific Objective B; GARNet Experiment 1).
100000
Gene expression at 100 h
Gene expression at 28 h
100000
10000
1000
100
10
10000
1000
100
10
1
1
1
10
100
1000
10000
1
10
Gene expression at -20 h
100
1000
10000
Gene expression at -20 h
100000
100000
10000
10000
10000
1000
100
10
1
Gene expression -K
100000
Gene expression -P
Gene expression -N
Figure 2. Changes in gene expression during the development of Arabidopsis. Arabidopsis were grown in a complete
nutrient solution. GeneChip™ analysis was performed on RNA from shoots of plants harvested at three timepoints (-20,
28 and 100 h). Gene expression at 28 and 100 h was compared with gene expression at -20 h for all 5257 transcripts
declared present by the Affymetrix software. Lines indicate a two-fold difference in gene expression between timepoints.
1000
100
10
10
100
1000
Gene expression FNS
10000
100
10
1
1
1
1000
1
10
100
1000
10000
1
10
100
1000
10000
Gene expression FNS
Gene expression FNS
Figure 3. Changes in gene expression in shoots of Arabidopsis following the withdrawal of N, P or K from the nutrient
solution. Gene expression in plants from which N, P or K had been withdrawn for 28 hours was compared with gene
expression in plants grown in complete nutrient solution (FNS) for all 5500 transcripts declared present by the Affymetrix
software. Lines indicate a two-fold difference in gene expression.
Validation of the Microarray Data: Nitrate-regulated Gene Expression
A comparison of the genes regulated by nitrate identified in our microarray experiments with those reported in
the literature provided a simple validation of our data. We observed the downregulation of many genes when
nitrate was withdrawn (Fig. 3), which is consistent with the upregulation of many genes when nitrate is
6
Changes in Gene Expression Profiles under Nutrient
Deficiency
Project
title
MAFF
project code
HH3502S
FV
supplied (Wang et al. 2000). Further, several genes (although not all genes) classically found to be upregulated
in the presence of nitrate were duly downregulated upon nitrate starvation. Thus, the expression of ACH1, a
gene encoding a high-affinity nitrate transporter, was downregulated fourfold by N starvation for 28 h, but the
expression of genes for the nitrate transporters CHL1 and NTL1/NRT1 was unaffected. Similarly, among the
'novel' genes found to be upregulated in Arabidopsis in the presence of nitrate by Wang et al. (2000), we
observed that the expression of the gene encoding a non-symbiotic hemoglobin (AHB1) was halved by
withdrawing N for 28 h, but that the expression of neither the high affinity vacuolar calcium antiporter (CAX1)
nor the late embryogenesis abundant (LEA) protein homolog SAG21 was affected after 28 h N starvation.
Wang et al. (2000) also reported that several genes were repressed by nitrate in Arabidopsis. In our
experiments, removing nitrate had no effect on the expression of genes encoding AMT1 (an ammonium
transporter), ANR1 (a MADS box protein supposedly expressed specifically in roots), or an homeobox-leucine
zipper protein (Athb-12). However, the gene encoding phosphoglycerate dehydrogenase (AT4g20380;
BAA24440.1) was upregulated fivefold when nitrate was removed, which is consistent with the observations of
Wang et al. (2000). Our inability to detect changes in the expression of some genes following N withdrawal
may reflect the mildness of N-deficiency. In our experiments, a critical shoot N concentration had not been
reached 28 h after withdrawing N.
Genes Upregulated during P Starvation
Time after P
withdrawal (h)
Downregulated
>2
Upregulated
>2
Upregulated
>3
4
28
100
261
413
452
435
466
649
152
139
266
Table 2. The number of genes whose expression in Arabidopsis shoots was downregulated by at least twofold, or
upregulated by at least twofold or threefold at various times following the removal of P from the nutrient solution. In
these experiments 5770 transcripts were declared present by the Affymetrix software.
The conventional criterion for differential gene expression is a twofold difference, at which the Affymetrix
GeneChip™ has a rate of approximately 0.2% false-positives (Zhu & Wang, 2000). We observed that the
expression of many genes was significantly affected by removing P from the nutrient solution (Fig. 3B; Table
2). The expression of several hundred genes was upregulated by P starvation. Genes whose expression is
greatly increased upon P starvation will provide stronger molecular markers for physiological P deficiencies.
Thus, only the genes whose expression more than tripled following P starvation were selected for subsequent
analyses. The expression of 37 genes, which were not upregulated immediately (4 h) after P withdrawal, was
increased both 28 and 100 h after P withdrawal (Fig. 4).
4h
87
6
44
15
170
100 h
37
81
28 h
Figure 4. The number of genes whose expression was increased at least threefold upon the withdrawal of P from nutrient
solutions for 4, 28 or 100 h. The identities of the 37 genes that were upregulated after 28 and 100 h, but not after 4 h, are
given in Table 4.
7
Changes in Gene Expression Profiles under Nutrient
Deficiency
Project
title
MAFF
project code
HH3502S
FV
Genes Upregulated Specifically by P Starvation
Nutrient
Withdrawn
Upregulated
>2
Upregulated
>3
P
N
K
463
1191
722
135
467
260
Table 3. The number of genes whose expression in Arabidopsis shoots was increased at least twofold or at least threefold
following the removal of P, N or K from the nutrient solution for 28 h.
Many more genes were upregulated following the withdrawal of N or K from the nutrient medium than
following the withdrawal of P (Fig. 3; Table 3). Nineteen of the 37 candidate marker genes whose expression
was increased at least threefold as shoot P concentration declined, but before shoot growth was slowed, were
also upregulated at least twofold by both N and K starvation. These genes may be upregulated by a common
mineral-imbalance stress-response system. The expression of 5 candidate marker genes was upregulated by
both N and P starvation, and the expression of 8 more genes was upregulated by both P and K starvation. Only
four candidate marker genes were specifically upregulated by P starvation. These genes encoded LEA AtM17
(which is a member of the Late Embryogenesis Abundant proteins that are implicated in tolerance of several
stresses associated with a water-deficit, Raynal et al. 1999, Bray et al. 2000), a nodulin-like protein (which is
homologous to the MtN21 protein expressed during nodule organogenesis in Medicago truncatula, Gamas et al.
1996), strictosidine synthase (which catalyses the condensation of tryptamine and secologanin to form
strictosidine, a key intermediate in indole alkaloid production) and a protein with unknown function. The
expression of LEA AtM17 also appears to be induced by low temperature stress in young Arabidopsis seedlings
(Raynal et al. 1999). However, this is unsurprising, since interrelationships between plant P status and the
expression of genes impacting on low temperature acclimation have been reported previously (Hurry et al.
2000). It has also been suggested that the ABI3 transcription factor participates in regulating LEA AtM17
expression (Raynal et al. 1999). Because the expression of the four genes we have identified appears to be
regulated nutritionally solely by P status, their transcripts could immediately be used to develop customdesigned DNA-arrays to assay plant P status, or, following heterologous expression, could be used to produce
protein to develop antibody-based assays for plant P status. Promoters from these genes could be used in 'smart
plants' to control the expression of genes encoding visible products (such as the Green Fluorescent Protein) whose
abundance would then reflect plant P status. Indeed, it would be interesting to discover whether the promoters of
these genes share common regulatory elements.
8
Project
title
Changes in Gene Expression Profiles under Nutrient
Deficiency
P starved
For 4 h
GeneChip™ Expression
ID
Level
P starved
For 28 h
P starved
For 100 h
N starved
For 28 h
MAFF
project code
HH3502S
FV
K starved
For 28 h
Increased
Expression
Expression relative to full nutrient solution
Arabidopsis Genome Initiative (AGI) ID
16896_s_at
M
1.00
4.98
5.66
1.00
1.00
P
LEA M17 protein
13967_at
M
1.05
3.57
3.47
1.27
1.97
P
CAB36773.1
15622_s_at
H
2.84
3.23
5.73
1.87
1.35
P
Medicago nodulin MtN21-like AT4g28040
protein
Strictosidine synthase (STS1) At1g74020
20558_at
M
2.04
3.17
3.10
ND
ND
P
Putative protein
CAB16817.1
12571_s_at
M
1.18
7.91
4.11
15.66
0.21
NP
Ferrochelatase-I (SW:P42043) At5g26030
AAD40138.1
20489_at
M
1.42
4.64
11.72
3.57
1.95
NP
At2g44840
AAC31840.1
12002_at
M
2.10
4.45
5.46
9.96
0.54
NP
Putative ethylene response
element-binding protein
(EREBP)
Hypothetical protein
AT4g02940
AAC79112.1
13100_at
M
2.66
3.42
3.11
3.29
1.20
NP
Putative cytochrome P450
At2g45570
AAC06158.1
13219_at
M
1.70
3.00
3.73
3.25
0.44
NP
Class IV chitinase (CHIV)
AT3g54420
CAA74930.1
20641_at
M
0.04
11.76
4.08
1.00
9.28
PK
LEA76 homologue type1
At1g52690
CAA63012.1
At2g41270
AT4g36850
AAC78545.1
AAB40594.1
13949_at
H
0.52
7.31
7.06
1.74
4.05
PK
Thioesterase like protein
AT4g17470
CAB10528.1
15141_s_at
H
0.73
5.65
13.88
0.83
4.79
PK
AT5g24770
BAA22096.1
20096_at
H
1.91
5.03
5.13
1.34
2.21
PK
At2g34810
AAC12821.1
15389_at
H
1.33
4.32
3.15
1.42
4.76
PK
Vegetative storage protein
(D85191)
Putative berberine bridge
enzyme
Unknown protein
At2g22860
AAC32433.1
17187_at
H
0.94
4.05
3.98
1.79
5.47
PK
Similar to arginases
AT4g08870
AAD17371.1
17843_s_at
M
1.19
3.57
3.69
1.48
3.41
PK
Putative cytochrome P450
At2g23220
AAB87109.1
14573_at
M
0.52
3.34
3.24
0.72
2.61
PK
T14P8.17 gene product
AT4g02360
AAC19282.1
19672_at
M
0.22
20.52
7.62
10.12
33.56
NPK
RAP2.6
At1g43160
AAC36019.1
15531_i_at
M
0.79
13.52
10.98
27.16
20.04
NPK
Putative protein
AT4g24340
CAB45069.1
18946_at
M
0.96
12.88
4.85
16.46
18.58
NPK
Peroxidase ATP24a
AT5g39580
CAA72484.1
Putative transcription factor
(MYB75)
Putative protein
At1g56650
AAC83630.1
AT4g17950
CAA17138.1
AT5g24770
BAA22095.1
At1g54020
AAD25774.1
AT4g11320
CAB51416.1
AT3g50970
AAB00374.1
AT4g20010
CAA16609.1
16073_f_at
M
1.83
12.16
3.05
4.46
4.16
NPK
13009_i_at
M
2.98
11.46
3.66
14.82
8.82
NPK
15125_f_at
H
0.34
9.77
24.71
3.14
10.04
NPK
19150_at
M
0.51
7.91
6.56
7.25
8.30
NPK
12746/8
M
0.47
5.84
7.65
2.84
4.73
NPK
19186_s_at
M
0.43
4.85
14.76
2.30
11.35
NPK
Vegetative storage protein
(D85190)
PF|00657 type
Lipase/Acylhydrolase with
GDSL-motif
Drought-inducible cysteine
proteinase RD21A precursorlike protein
Dehydrin (Xero 2)
16654_at
M
1.15
4.44
3.59
5.97
4.50
NPK
Hypothetical protein
18909_s_at
M
2.80
3.83
4.95
3.39
4.56
NPK
Subtilisin-like protease (AIR3) Not found
AAC62611.1
15779_g_at
L
2.44
3.62
3.40
4.14
10.12
NPK
Zinc finger protein (Zat7)
AT3g46090
CAA67234.1
16232_s_at
M
0.79
3.46
9.74
6.12
6.76
NPK
Putative protein
At1g07160
CAB45796.1
20524_at
M
0.62
3.34
3.94
7.24
4.89
NPK
T3P18.12 gene product
At1g62560
AAD43613.1
16510_at
M
1.15
3.28
4.12
2.95
2.97
NPK
Putative protein
AT4g32480
CAA22575.1
15965_at
M
1.05
3.28
3.91
6.28
4.61
NPK
At4g39090
AAB63631.1
14614_at
H
2.13
3.17
3.02
3.25
3.68
NPK
Jasmonate inducible protein
isolog
Putative glucosyltransferase
At2g30140
AAC16958.1
19641_at
M
2.19
3.03
5.49
3.40
3.13
NPK
At2g29490
AAC95189.1
15005_s_at
L
1.08
3.02
3.62
3.26
7.18
NPK
Putative glutathione Stransferase
Putative protein kinase
At2g30040
AAC31848.1
Table 4. Identities of the 37 genes upregulated at least threefold 28 and 100 h after P withdrawal, as shoot P concentration
declined but before shoot growth was compromised, but not immediately following P withdrawal. Changes in gene
expression following N, P or K starvation are expressed relative to controls grown in a complete nutrient solution.
Qualitative estimates of absolute gene expression are classified as low (L), medium (M) and high (H), and the nutrient
specificity of increased gene expression is summarised. The database of Ghassemian et al. (2001) was used to map
Affymetrix GeneChip™ identifiers to Arabidopsis Genome Initiative (AGI) identifiers.
9
Project
title
Changes in Gene Expression Profiles under Nutrient
Deficiency
MAFF
project code
HH3502S
FV
5. CONCLUSIONS
A. Thirty-seven genes were identified whose expression increased at least threefold as shoot P concentration
declined, but before shoot growth was compromised, between 28 and 100 h after P was removed from the
nutrient solution.
B. Four genes were identified whose expression was increased at least threefold as shoot P concentration
declined, but was unaffected by N or K stress.
6. COMMUNICATION OF RESULTS
1. An abstract of the NASC transcriptome analyses will be published on the Genomic Arabidopsis Resource
Network (GARNet) website at http://www.york.ac.uk/res/garnet/garnet.htm.
2. Results will be presented in papers at national and international scientific conferences, including: [1] J
Hammond, PJ White, MJ Bennett, MR Broadley (2002) Abstract P7.18: Plants that make the most of
phosphate. Society for Experimental Biology Annual Meeting, Swansea, April 2002. [2] J Hammond, M
Bennett, M Broadley, P White (2002) Changes in gene expression upon nutrient removal and the
development of novel sensor technologies. GARNet, York, September 2002. [3] J Hammond, MJ Bennett,
MR Broadley, PJ White (2002) Gene expression and mineral nutrition. SEB Plant Transport Group, York,
September 2002.
3. A high-impact refereed publication on "Changes in gene expression in Arabidopsis shoots under nutrient
deficiency" is in preparation.
4. Illustrative data will be included in Dr White's lecture on "Mineral Nutrition of Higher Plants" in the HRI
Applied Biotechnology Course, October 2002, and other HRI technology transfer events.
5. Results will be incorporated into the PhD thesis of John Hammond (University of Nottingham).
7. FUTURE PROSPECTS
In this short-term Project we have taken the first, but most important, step towards the development of novel
biosensor technologies to help reduce P fertiliser application. Ultimately these will lower costs, reduce pollution
and enhance biodiversity. The identification of specific nutrient-responsive genes will underpin the
development of novel biosensor technologies for stress monitoring and crop improvement. These may include:
(i) custom-designed DNA-arrays, to detect the transcripts of genes whose expression is altered by P starvation, (ii)
traditional antibody-based assays that detect the presence of proteins accumulating when plants lack P, and (iii)
'smart plants', in which P-responsive promoters control the expression of genes encoding visible products whose
abundance reflects plant P status. However, further research and development are required before these
practical benefits can be realised.
DNA Arrays and Transcript Profiling
This Project has identified genes whose expression is altered following the withdrawal of the mineral nutrients
P, N and K (Fig. 3). Knowledge of these nutrient-sensitive genes, whether their appearance or disappearance is
specific or non-specific to the absence of a particular nutrient, can be used to design DNA-arrays for assaying
mineral nutrient status of plants through transcript profiling. By comparing the relative expression of a small
suite of genes that respond differently to plant mineral nutrient status, it is possible, theoretically, to diagnose
the identity of a deficient mineral element. Thus, the mineral nutrient status of a plant could be assayed
relatively rapidly in a small leaf sample. Such an assay is an exciting prospect, and immediately within our
technological grasp.
10
Project
title
Changes in Gene Expression Profiles under Nutrient
Deficiency
MAFF
project code
HH3502S
FV
It is important that transcript-profiling following the withdrawal of mineral nutrients is extended to crop
plants. Now that P-responsive genes have been identified in Arabidopsis, it would be informative to ascertain
whether homologous genes are also induced in Brassica upon P starvation. To effect this it may be possible to
probe Arabidopsis microarrays with mRNA from Brassica (Girke et al. 2000). Alternatively a customised
Brassica cDNA microarray could be produced to confirm the anticipated changes in gene expression in
Brassica leaves following P withdrawal. These experiments would enable suitable DNA-arrays for assaying the
mineral nutrient status of crop plants to be designed.
Immunoassays for Plant Mineral-Nutrient Status
In addition to their immediate utility in designing DNA-arrays for mRNA profiling, transcripts of any genes
whose expression is upregulated by nutrient starvation could be expressed in heterologous systems to produce
antigenic proteins. Antibodies to these proteins might form the basis of simple immunoassay kits for screening
for plant mineral-nutrient status.
Smart Plants, and Transgenic Plants Tolerating Reduced P Inputs
In a 'proof of concept' study initiated by DEFRA (HH0915SFV), and taken forward through an ongoing HRI
Browning Studentship to John Hammond (2000-2003), we have generated Arabidopsis lines expressing green
fluorescent protein (GFP) and -glucuronidase (GUS) marker genes under the control of a specific P-responsive
promoter (for the SQD1 gene). These plants show enhanced marker gene expression under P stress, and prove the
concept of smart plants (Hammond et al. 2002). However, we identified a need to obtain alternative 'hypersensitive' P-specific promoters that were upregulated earlier than SQD1, and prior to 100 h following P withdrawal.
The promoters of the four genes identified in this Project as being regulated specifically by P status would fulfil
this requirement.
Smart plants can be used as a basic research tool (e.g. in determining the role of root and/or soil structure
in the acquisition of P) as well as for more applied ends (e.g. by providing a bioassay for P availability within
soils). For example, smart plants could be used (i) in conjunction with a screen of mutants or ecotypes, to
identify plants with enhanced nutrient use efficiency, which would be useful in breeding programs, (ii)
following mutation of the transgenic lines to identify components of signaling cascades impacting on nutrientspecific gene regulation, (iii) as experimental material to assist and ratify computer predictions for the
application of fertiliser and (iv) as a model bioassay for rapid assessment of plant available P in situations
where existing chemical tests provide unclear results. Many downstream applications can be envisaged for such
smart plant technology when transferred to horticultural crops. Marker plants that indicate plant nutrient status
will allow efficient temporal and spatial fertiliser applications and further the development of decision-making
systems for precision farming. In addition to enabling us to identify four new, and specific, P-responsive
promoters, the data produced in this Project could presage the development of smart plants for both N and K
nutrition, pending further analysis and experimentation.
Transcription factors whose expression is regulated by plant nutrient status can be identified directly
from the microarray data collected in this Project (Table 3). In addition, a comparison of the promoter
sequences of genes expressed under specific conditions would enable the identification of potential cis-acting
DNA elements regulating gene expression, as well as confirming the target genes for known transcription
factors (Harmer et al. 2000; Maleck et al. 2000; Petersen et al. 2000; Ghassemian et al. 2001; Seki et al. 2001;
Reymond, 2001; Zhu et al. 2001; Chen et al. 2002, Wu et al., 2002). In the future, regulating the expression of
transcription factors and harnessing cis-acting DNA elements to particular beneficial genes could be used to
control a suite of genes that effect tolerance of low fertiliser inputs.
11
Project
title
Changes in Gene Expression Profiles under Nutrient
Deficiency
MAFF
project code
HH3502S
FV
8. REFERENCES
Bollons HM, Barraclough PB (1997) Inorganic orthophosphate for diagnosing the phosphorus status of wheat
plants. Journal of Plant Nutrition 20, 641-655.
Bollons HM, Barraclough PB (1999) Assessing the phosphorus status of winter wheat crops: inorganic
orthophosphate in whole shoots. Journal of Agricultural Science 133, 285-295.
Bray EA, Bailey-Serres J, Weretilnyk E (2000) Responses to abiotic stresses. In: Biochemistry & Molecular
Biology of Plants, pp. 1158-1203. B Buchanan, W Gruissem, R Jones, eds. American Society of Plant
Physiologists.
Chen W et al. (2002) Expression profile matrix of Arabidopsis transcription factor genes suggests their putative
functions in response to environmental stresses. Plant Cell 14, 559-574.
Desprez T, Amselem J, Caboche M, Höfte H (1998) Differential gene expression in Arabidopsis monitored
using cDNA arrays. Plant Journal 14, 643-652.
Gamas P, de Carvalho Niebel F, Lescure N, Cullimore J (1996) Use of a subtractive hybridization approach to
identify new Medicago truncatula genes induced during root nodule development. Molecular Plant
Microbe Interactions 9, 233-242.
Ghassemian M, Waner D, Tchieu J, Gribskov M, Schroeder JI (2001) An integrated Arabidopsis annotation
database for Affymetrix Genechip® data analysis, and tools for regulatory motif searches. Trends in
Plant Sciences 6, 448-449.
Girke T, Todd J, Ruuska S, White J, Benning C, Ohlrogge J (2000) Microarray analysis of developing
Arabidopsis seeds. Plant Physiology 124, 1570-1581.
Hammond J, White PJ, Bennett MJ, Broadley MR (2002) Plants that make the most of phosphate. Society for
Experimental Biology Annual Meeting, Swansea, April 2002. [Abstract P7.18]
Harmer SL, Hogenesch JB, Straume M, Chang H-S, Han B, Zhu T, Wang X, Kreps JA, Kay SA (2000)
Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science 290, 21102113.
Hurry V, Strand Ǻ, Furbank R, Stitt M (2000) The role of inorganic phosphate in the development of freezing
tolerance and the acclimatization of photosynthesis to low temperature is revealed by the pho mutants of
Arabidopsis thaliana. Plant Journal 24, 383-396.
Kehoe DM, Villand P, Somerville S (1999) DNA microarrays for studies of higher plants and other
photosynthetic organisms. Trends in Plant Science 4, 38-41.
Ma LG, Li JM, Qu LJ, Hager J, Chen ZL, Zhao HY, Deng XW (2001) Light control of Arabidopsis
development entails coordinated regulation of genome expression and cellular pathways. Plant Cell 13,
2589-2607.
Maleck K, Levine A, Eulgem T, Morgan A, Schmid J, Lawton KA, Dangl JL, Dietrich RA (2000) The
transcriptome of Arabidopsis thaliana during systemic acquired resistance. Nature Genetics 26, 403410.
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures.
Physiologia Plantarum 15, 473-497.
Petersen M, Brodersen P, Naested H, Andreasson E, Lindhart U, Johansen B, Nielsen HB, Lacy M, Austin MJ,
Parker JE, Sharma SB, Klessig DF, Martienssen R, Mattsson O, Jensen AB, Mundy J (2000)
Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance. Cell 103, 1111-1120.
Raynal M, Guilleminot J, Gueguen C, Cooke R, Delseny M, Gruber V (1999) Structure, organization and
expression of two closely related novel Lea (late-embryogenesis-abundant) genes in Arabidopsis
thaliana. Plant Molecular Biology 40, 153-163.
Reymond P (2001) DNA microarrays and plant defence. Plant Physiology & Biochemistry 39, 131-321.
Ruan Y, Gilmore J, Conner T (1998) Towards Arabidopsis genome analysis: monitoring expression profiles of
1400 genes using cDNA microarrays. Plant Journal 15, 821-833.
Schaffer R, Landgraf J, Accerbi M, Simon V, Larson M, Wisman E (2001) Microarray analysis of diurnal and
circadian-regulated genes in Arabidopsis. Plant Cell 13, 113-123.
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Project
title
Changes in Gene Expression Profiles under Nutrient
Deficiency
MAFF
project code
HH3502S
FV
Seki M, Narusaka M, Abe H, Kasuga M, Yamaguchi-Shinozaki K, Carninci P, Hayashizaki Y, Shinozaki K
(2001) Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold stresses by
using a full-length cDNA microarray. Plant Cell 13, 61-72.
Wang R, Guegler K, LaBrie ST, Crawford NM (2000) Genomic analysis of a nutrient response in Arabidopsis
reveals diverse expression patterns and novel metabolic and potential regulatory genes induced by
nitrate. Plant Cell 12, 1491-1509.
Wang YH, Garvin DF, Kochian LV (2001) Nitrate-induced genes in tomato roots. Array analysis reveals novel
genes that may play a role in nitrogen nutrition. Plant Physiology 127, 345-359.
White PJ, Rahn CR (1999) A first step to ‘smart’ plants: isolating nutrient responsive promoters. Final Report
on MAFF Project HH0915SFV, 6 pp.
Wu K, Tian L, Hollingworth J, Brown DCW, Miki B (2002) Functional analysis of tomato Pti4 in Arabidopsis.
Plant Physiology 128, 30-37.
Zhu T, Budworth P, Han B, Brown D, Chang HS, Zou GZ, Wang X (2001) Toward elucidating the global gene
expression patterns of developing Arabidopsis: Parallel analysis of 8 300 genes by a high-density
oligonucleotide probe array. Plant Physiology and Biochemistry 39, 221-242.
Zhu T, Wang X (2000) Large-scale profiling of the Arabidopsis transcriptome. Plant Physiology 124, 14721476.
13
Project
title
Changes in Gene Expression Profiles under Nutrient
Deficiency
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MAFF
project code
HH3502S
FV