Research and Development s13
title / Changes in Gene Expression Profiles under Nutrient Deficiency
/ DEFRA
project code / HH3502SFV
Department for Environment, Food and Rural Affairs CSG 15
Research and Development
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
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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 / £ 44,624
Project start date / 10/01/01 / Project end date / 31/03/02
Executive summary (maximum 2 sides A4)
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CSG 15 (9/01) 3
Projecttitle / Changes in Gene Expression Profiles under Nutrient Deficiency
/ DEFRA
project code / HH3502SFV
· 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 GeneChipä 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 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) 3
Projecttitle / Changes in Gene Expression Profiles under Nutrient Deficiency
/ DEFRA
project code / HH3502SFV
Scientific report (maximum 20 sides A4)
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CSG 15 (9/01) 3
Projecttitle / Changes in Gene Expression Profiles under Nutrient Deficiency
/ MAFF
project code / HH3502SFV
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 GeneChipä 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 mmol 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 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.
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.
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.