Higher biomass accumulation by increasing phosphoribosylpyrophosphate synthetase activity in Arabidopsis thaliana and Nicotiana tabacum

Plants are able to produce all the organic compounds required for development and growth. As developmental processes and metabolic pathways use a common resource pool, the tight regulation of the distribution of metabolites between growth, production of defence compounds and storage products can be assumed. A transgenic approach was used to investigate the importance of supplying the key intermediate phosphoribosylpyrophosphate (PRPP) for plant growth and biomass accumulation in the model plant Arabidopsis thaliana and in Nicotiana tabacum. For this purpose, the Ashbya gossypii genes coding for either PRPP synthetase (PRS) or a mutated variant of the same gene were over-expressed under the control of a constitutive promoter. It was shown that increased PRS activity in A. thaliana or N. tabacum leads to a substantial increase in biomass accumulation under different standardized growth conditions. Growth enhancement was accompanied by significant changes in the amount of sugars and other metabolites. This study provides evidence that the supply of PRPP co-limits growth rates, and has obvious implications for biotechnological strategies aiming to increase plant biomass as an alternative renewable energy source.


Introduction
Plants are photoautotrophic organisms that can produce all the organic compounds needed for development and growth. Over recent years, many factors that influence the cell and organ growth of plants have been identified, and the molecular functions of growth-related proteins have started to be elucidated (Anastasiou and Lenhard, 2007). As developmental processes and metabolic pathways use a common resource pool, and both processes respond to changes in environmental energy and resource supplies, it is evident that resource availability may have a direct influence on cell proliferation and growth. This close interrelation has recently been demonstrated by Baldet et al . (2006), who showed that fruit load reduction in tomato plants resulted in increased photoassimilate availability and increased growth rates in all other plant organs, including roots, stems, leaves, flowers and other fruits. Therefore, a tight regulation of the distribution of metabolites between growth, production of defence compounds and storage products can be assumed. Faster growing Arabidopsis accessions are characterized by lower levels of central metabolites, such as carbohydrates and amino acids, showing that faster growth draws on and depletes the levels of these central resources (Cross et al ., 2006). A detailed knowledge of the relations between metabolism and biomass accumulation can be expected to yield powerful novel tools to accelerate plant breeding programmes in the future. Furthermore, metabolic profiling in the model Arabidopsis using a recombinant inbred line (RIL) population has recently revealed a close link between Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation. et al ., 2007). This demonstrates the predictive power of metabolite composition as a biomarker for biomass production, opening up new opportunities for plant breeding.
Phosphoribosylpyrophosphate (PRPP) is a key intermediate in metabolism, essential for nucleotide production (Zrenner et al ., 2006) and the synthesis of the pyridine nucleotide cofactors NAD and NADP (Noctor et al ., 2006) and the amino acids histidine (Stepansky and Leustek, 2006) and tryptophan (Radwanski and Last, 1995). PRPP is needed in the respective de novo synthesis pathways, but is of equal importance in the salvaging processes of nucleobases and pyridines. Thus, the sufficient availability of PRPP is a crucial prerequisite for any metabolic process and, subsequently, for growth. Phosphoribosylpyrophosphate synthetase (ATP: D -ribose-5-phosphate pyrophosphotransferase; EC 2.7.6.1; PRS) catalyses the synthesis of PRPP from ribose 5-phosphate (R5P) and ATP. PRSs are a family of enzymes grouped into three classes on the basis of their dependence on phosphate ions for activity, their allosteric regulation and their specificity for diphosphoryl donors. Class I proteins are found in all organisms and require Mg 2+ and phosphate for activity, can be inhibited allosterically by ADP and prefer ATP or dATP as diphosphoryl group donor (Krath and Hove-Jensen, 2001a). Class II PRSs are found exclusively in plants, do not depend on phosphate for activity, lack the allosteric regulation of ADP and have a much broader specificity for the diphosphoryl donor Hove-Jensen, 1999, 2001b). Recently, a novel class III PRS has been identified from Methanocaldococcus jannaschii resembling class I properties, but also lacking an allosteric site for ADP (Kadziola et al ., 2005).
In previous experiments, it has been shown that, with decreased nucleotide de novo synthesis, the growth of potato and tobacco plants is reduced without further pleiotropic effects (Schröder et al ., 2005). In this study, our aims were to globally increase nucleotide availability by increasing the supply of the common precursor PRPP, and to investigate its influence on plant growth and biomass accumulation. For this purpose, a transgenic approach was used to increase the PRPP supply in the model plant Arabidopsis thaliana and in Nicotiana tabacum , representing a plant that achieves a larger vegetative biomass. An Ashbya gossypii gene coding for PRS ( PRS ) and a mutated variant of the same gene were expressed in transgenic plants using constitutive expression from the 35S promoter of the cauliflower mosaic virus. It was shown that increased PRS activity leads to a substantial increase in plant growth and biomass accumulation under different standardized growth conditions. This provides evidence that PRPP supply co-limits growth rates, and has obvious implications for biotechnological strategies aiming to increase plant biomass as an alternative energy source.

Results and discussion
Production and selection of transgenic plants with expression of a PRS gene from As. gossypii Two different PRS genes of fungal origin were used for expression in plants. One encodes the wild-type PRS class I activity of As. gossypii ATCC 10895 (AGR371Cp) and the other represents a mutant form. The mutant variant carries three point mutations leading to the exchange of leucine 133 by isoleucine and histidine 196 by glutamine; the exchange of these two amino acids results in an alteration of the allosteric mechanisms regulating both enzyme inhibition by purine nucleotides and activation by inorganic phosphate (Becker et al ., 1995). Therefore, this mutant form of the As. gossypii PRS protein resembles a protein of PRS class II activity. As. gossypii PRS genes were chosen because both variants of the gene were available that were highly heterologous to plant genes. All constructs used to transform either N. tabacum or A. thaliana plants were prepared with the binary vector pBinAR (Höfgen and Willmitzer, 1990), a derivative of the pBin19 vector containing the 35S promoter of the cauliflower mosaic virus for constitutive expression of the target gene and the octopine synthase polyadenylation signal. Primary transformants (T 1 ) were grown on selection medium containing 50 mg/L kanamycin. Kanamycin-resistant plantlets were transferred to soil and grown in growth chambers under standard cultivation conditions. For each transformation, about 30 plants that had survived the selection process were regenerated and analysed further. The leaves of these plants were analysed for the expression of the transgene 3-5 weeks after the transfer (Figure 1). Seeds were collected, and the T 2 offspring were grown on selection medium.
Resistant plants of the T 2 generation were selected when about one-quarter of the offspring were unable to survive the selection process. These plants were transferred to soil in growth chambers and further grown under optimum conditions, and the seeds were harvested (T 3 ). The T 3 seeds were again transferred to selection plates to identify the seed sets in which all plants were kanamycin resistant and could be regarded as homozygous for at least one functional insertion of the T-DNA. All experiments were carried out with seed from the T 3 or T 4 generation that had passed this selection process. Three to four lines originating from different individual primary transformants were chosen for each experiment. All experiments were carried out with control plants that had passed the same selection criteria after transformation with an empty vector.
PRS expression leads to a significant but not severe increase in extractable PRS activity It was confirmed that the expression of the PRS gene resulted in an increase in enzyme activity by assaying PRS activity using a standardized enzyme-coupled spectrophotometric determination procedure. This assay does not allow separate analysis of class I and class II PRS activities. As plant crude enzyme extracts contain both class I and class II PRS activities (Krath and Hove-Jensen, 1999), the plant enzyme activity cannot be distinguished from the additional Ashbya activities.
The total PRS activity was assayed in soluble protein extracts of A. thaliana and N. tabacum seedlings grown for 7 or 8 days in liquid culture in their respective growth media ( Figure 2). All data were calculated on a plant fresh weight basis as a percentage of the respective control transformants ( N. tabacum , 2.17 ± 0.31 μ mol/min/g fresh weight; A. thaliana , 2.83 ± 0.14 μ mol/min/g fresh weight). Data were also calcu- Our experiments were carried out on plants of the T 4 generation, complying with the selection criteria described above for plants that could be regarded as homozygous for at least one functional insertion of the T-DNA. Below a certain number of identical transgenes in the genome, the gene copy number and expression are positively correlated (Schubert et al ., 2004). Therefore, stable expression of the PRS gene over the analysed generations and a comparable level for the independent transformants are to be expected, and are reflected in the very low variation of increased PRS activity.

Increased PRS activity increases fresh weight accumulation of seedlings in liquid culture
To investigate the influence of increased PRS activity on overall plant performance and growth, the fresh weight accumulation of seedlings in the T 4 generation was measured in a highly controlled environment. Seedlings of A. thaliana and N. tabacum were grown in liquid cultures in their respective growth media, and plants were harvested and analysed after 7 or 8 days of growth. Expression of the native PRS gene and the mutant form in either A. thaliana or N. tabacum led to a significant increase in fresh weight accumulation in these young seedlings. Figure 3a shows that expression of the  Values are the means ± standard deviation of three to four lines originating from different individual primary transformants. Data are given as percentages of the respective control transformants, with N. tabacum at 2.17 ± 0.31 μmol/min/g fresh weight and A. thaliana at 2.83 ± 0.14 μmol/min/g fresh weight. Unpaired two-tailed t-tests were used. Significantly different values (P < 0.05) are labelled with an asterisk.

Figure 3
Correlation of phosphoribosylpyrophosphate synthetase (PRS) activity and fresh weight accumulation. Plants were grown in liquid seedling cultures for 7 or 8 days in their respective growth media as in Figure 2. Values are the means ± standard error of three to four lines originating from different individual primary transformants. Data are given as percentages of the respective control transformants, with N. tabacum at 49.6 ± 9.03 mg/seedling and A. thaliana at 2.7 ± 0.48 mg/seedling. Unpaired two-tailed t-tests were used. All fresh weight values are significantly different from the controls (P < 0.05), except for AtP-9. Linear correlation analysis was performed and the respective correlation coefficients are given. In all seeds analysed, the proportion of fatty acids of the individual seeds of A. thaliana was between 21% and 25%.
These values are lower than those reported in other studies (O'Neill et al., 2003), and are presumably a consequence of the growth conditions. It has been shown previously that abiotic factors, mainly light intensity, have a strong influence on lipid accumulation of A. thaliana seeds (Li et al., 2006), with low light intensities causing lower seed weight and lipid accumulation. All of our A. thaliana plants for seed production and seed analysis were grown in a long-day phytotron at light intensities of only 145 μmol/m 2 /s, which might explain the low proportion of fatty acids in the seeds. In addition, the values for oil accumulation in all N. tabacum transformants were lower than those in other studies (Tomlinson et al., 2004). It is assumed that N. tabacum plants normally grown Metabolite analysis reveals a negative correlation of sucrose content with biomass accumulation PRS is required for the synthesis of PRPP. This metabolite is a key intermediate, and is essential for nucleotide biosynthesis (Zrenner et al., 2006), the salvaging of nucleobases (Allen et al., 2002;Noctor et al., 2006) and tryptophan and histidine synthesis (see 'Introduction'). Sufficient availability of PRPP is a crucial prerequisite for any metabolic process and, in particular, in dividing tissues, where it is an important metabolite for the production of nucleotides needed for DNA replication.
To investigate whether increased fresh weight accumulation caused by increased PRS activity leads to changes in metabolic composition, extracts of the seedlings were analysed for carbohydrate, nucleotide and amino acid content and composition. Metabolite levels were calculated on a fresh weight basis to analyse differences in concentrations and on a total seedling basis to summarize productivity.
Total carbohydrates (summarized as glucose, fructose, sucrose and starch on a fresh weight basis) were not changed significantly in either A. thaliana or N. tabacum seedlings with increased PRS activity (data not shown). Significant changes were found for the individual sugar levels, however. Although hexose concentrations were increased, the sucrose content declined significantly (Figure 5a,d), leading to a highly significant increase in the hexose-to-sucrose ratio in A. thaliana (Figure 5b) and N. tabacum (Figure 5e) seedlings. The decreased sucrose content and the increased hexose-to-sucrose ratio correlated well with the increased PRS activity in both A. thaliana and N. tabacum expressing either the wild-type or mutant PRS gene. The starch content was also correlated with the increased PRS activity, although these changes were not significantly different from the controls. In A. thaliana, the starch content increased with increasing PRS activity ( Figure 5c); by contrast, the reverse was observed in N. tabacum (Figure 5f).
The carbon supply is highly co-ordinated with plant growth and, as a result of environmental changes, acute signalling and acclimatory responses can modulate plant growth (Smith and Stitt, 2007). However, the carbohydrate content of  Indeed, as already shown previously, nucleotide pools in fully grown organs are unaltered in transgenic plants with reduced pyrimidine de novo synthesis (Schröder et al., 2005).

Figure 6
Metabolite analyses of Arabidopsis thaliana seedlings. Plants were grown in liquid seedling culture for 7 days, as described in Figure 2. Values are given as the means and standard errors of three to four replicates. Unpaired two-tailed t-tests were used. Significantly different values (P < 0.05) are labelled with a red asterisk. Linear correlation analysis was performed and the respective correlation coefficients are given. Total nucleotides: adenosine monophosphate (AMP), adenosine diphosphate (ADP), uridine diphosphate (UDP), guanosine diphosphate (GDP), uridine triphosphate (UTP), adenosine triphosphate (ATP) and guanosine triphosphate (GTP). Total amino acids: all L-α-amino acids without proline and cysteine, including β-alanine, γ-aminobutyric acid, citrulline and ornithine. Minor amino acids: arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan, tyrosine and valine. BCAA, branched-chain amino acids: isoleucine, leucine and valine. In these cases, the reduced availability of nucleotides is compensated by reduced growth rates, without alterations in nucleotide pools. In this context, it should also be noted that the measurements of metabolites were made over the entire seedlings, and may not reflect the levels in dividing and growing cells. Assuming that the increase in growth is somehow accomplished by higher division rates, higher fluxes through nucleotide biosynthetic pathways are expected to provide adequate amounts of nucleotides for DNA and RNA synthesis.
The analysis of total amino acid concentrations revealed an increase in A. thaliana seedlings that correlated well with increasing PRS activity (Figure 6d), but only some of the values were significantly different from the controls. A more detailed analysis of amino acid composition revealed identical behaviour in A. thaliana and N. tabacum seedlings, but again only some results from A. thaliana were significant. Although the total amount of amino acids was increased in all seedlings with increased PRS activity, the proportion of minor amino acids decreased (Figure 6e). This decreased proportion of minor amino acids may indicate an increased demand for protein biosynthesis (Fritz et al., 2006;Osuna et al., 2007). The proportion of histidine, an amino acid whose biosynthesis requires PRPP, was not changed in any of the transformation lines (data not shown). The other amino acid that requires PRPP for its synthesis is tryptophan. The proportion of tryptophan in the total amino acid pool was increased in both A. thaliana and N. tabacum seedlings with increased PRS activity (data not shown). However, tryptophan biosynthesis occurs in plastids (Radwanski and Last, 1995), and an increase in PRS activity in the cytosol is not necessarily expected to increase PRPP availability in plastids. Further studies of the subcellular distribution of metabolites are needed to clarify this. Other changes included an increase in the major amino acids serine and glycine (data not shown), and a correlating decrease in the proportion of the branched-chain amino acids (BCAA), in A. thaliana seedlings with increased PRS activity (Figure 6f). At this point, it is assumed that these changes in the proportion of specific classes of amino acids in relation to the total amino acid pool are a result of their co-ordinated regulation within individual amino acid biosynthesis pathways.
To investigate whether increased PRS activity leads to an increase in the immediate product of catalysis, several attempts were made to extract and measure PRPP in these seedling cultures. Using standardized protocols for metabolite extraction, such as trichloroacetic acid (TCA)-ether or perchloric acid, no reliable amounts of PRPP were measurable from seedling cultures (Dancer and apRees, 1989; data not shown). Therefore, a rather indirect approach was used to indicate the better supply of PRPP for metabolism and growth.

Recycling of nucleobases is increased in A. thaliana seedlings with higher PRS activity
To investigate whether increased PRS activity has an influence on nucleobase recycling in the cytosol, seedling cultures were incubated with labelled uracil and the metabolism of the isotope was monitored in various fractions (Figure 7). The higher PRS activity in A. thaliana transformants had no influence on the total uptake of uracil, or the amount and proportion of uracil that was immediately degraded to CO 2 . When the label of soluble metabolites, including unprocessed uracil and nucleotides, was separated from the label of the insoluble fraction containing DNA, a shift of the flux of the label into the insoluble fraction was observed in transformed seedlings with increased PRS activity. Although the difference in flux in the insoluble fraction was not highly significant (P < 0.1), a clear increase in the amount of radioactivity distributed in the insoluble fraction (from 10% of the total uptake for control transformants to 15% of the total uptake for the seedlings) with increased PRS activity was observed. It is concluded that the increased activity of PRS enhances the availability of PRPP in the cytosol for salvaging processes.
Nucleotide de novo synthesis pathways are mainly located in plastids (Zrenner et al., 2006). So far, no evidence has been found for a transport system exchanging PRPP between plastids and the cytosol. Therefore, an increase in PRS activity in the cytosol, and the subsequent production of PRPP in this compartment, is not necessarily expected to increase PRPP availability in plastids for nucleotide de novo synthesis. In the cytosol, several PRPP-consuming reactions are present. These include salvage pathways for nucleobases using the respective phosphoribosyltransferases (Allen et al., 2002;Islam et al., 2007) and the recycling of pyridine nucleotides (Noctor et al., 2006).
In our uracil feeding experiment, a higher flux through the uracil phosphoribosyltransferase reaction in the cytosol is assumed to increase the recycling of the nucleobase uracil, finally leading to an increased incorporation of pyrimidine nucleotides into the insoluble fraction, namely into DNA or RNA.
The A. thaliana plant culture on soil was performed as follows. Seeds were surface sterilized and aseptically grown on medium containing half-strength Murashige and Skoog salts (micro-and macro-elements including vitamins), 0.25 mM MES at pH 5.8 (KOH), 50 mg/L kanamycin, 0.5% (w/v) sucrose and 0.8% (w/v) agar. Seeds were imbibed at 5 °C in complete darkness for 3 days and grown in a 12-h photoperiod (photon flux density, 150 μmol/m 2 /s; 22 °C light; 18 °C dark). After 2 weeks, the plants were transferred to soil in pots of 6 cm in diameter. For the 'adequate nutrients' condition, the pots were filled with a 2 : 1 (v/v) mix of GS90 soil (composition: peat, clay, coconut fibre, 2 g/L salt, 160 mg/L N, 190 mg/L P 2 O 5 , 230 mg/L K 2 O, pH 6; supplied by Werner Tantau GmbH & Co. KG, Hetersen, Germany) and vermiculite (Gebrüder Patzer, Sinntal-Jossa, Germany), and grown under short-day conditions (8 h light, 16 h dark) at a light intensity of 145 μmol/m 2 /s, relative humidity of 60% and temperatures of 20 °C (day) and 18 °C (night). For the 'limiting nutrients' condition, the GS90 soil was replaced by a 1 : 10 (v/v) mix of GS90 soil and 'Null-soil' (composition: peat, clay, coconut fibre, 0.8 g/L salt, For expression analysis and seed production and analysis, plants were grown in high-nitrogen conditions under a long-day (16 h light, 8 h dark) regime at a light intensity of 145 μmol/m 2 /s, relative humidity of 80% and temperatures of 20 °C (day) and 18 °C (night, 50% relative humidity).
The N. tabacum seedling culture was performed in an identical manner to the Arabidopsis seedling culture, but seedlings were harvested after 8 days and a different type of nutrient solution was applied. The tobacco full nutrition medium contained: Murashige and Skoog salts (micro-and macro-elements including vitamins), 0.25 mM MES at pH 5.8 (KOH), 50 mg/L kanamycin and 0.5% (w/v) sucrose.

Cloning procedures and plasmid construction
Escherichia coli strain XL-1 Blue and Agrobacterium tumefaciens strain C58C1 containing pGV2260 were cultivated using standard procedures (Sambrook and Russell, 2001). Sequenz primers AgPRSv (GGATCCAATATGTCGTCCAAT) and AgPRSh (GGATCCTACAT-GACAGCG) were used to amplify the wild-type PRS from plasmid pJRAgprs1486 and the mutant PRS from pJRAgprs1404 (mutant) using standard procedures. Subcloning into pCR Script (Stratagene Europe Biocrest BV, Amsterdam Zuidoost, The Netherlands) was in accordance with the protocol provided by the supplier. Clones were confirmed by sequence analysis. The 965-bp BamHI fragments encoding the full-length proteins were cloned into the BamHIrestricted binary vector pBinAR (Höfgen and Willmitzer, 1990). The sense direction of the insertion was checked by SalI digestion and polymerase chain reaction (PCR) analysis using the primers 35Shv (TATAGAGGAAGGGTCTTGCG) and AgPRSh. Definite plasmids to be transformed into plants were finally verified by sequence analysis.

Plant transformation and expression analysis
Agrobacterium-mediated gene transfer was performed according to Rosahl et al. (1989) for tobacco plants and to Bent and Clough (1998) for Arabidopsis. Expression of the transgene was analysed by Northern hybridization according to Giermann et al. (2002), using the full-length wild-type PRS as a probe.

Metabolite analysis
In liquid nitrogen, frozen plant material was ground to a powder using a ball mill (Retsch, Haan, Germany). Carbohydrates, amino acids and nucleotides were extracted and measured according to Schröder et al. (2005). Fatty acids were extracted according to the method of Bligh and Dyer (1959), and the lipid content was measured by gas chromatography of fatty acid methyl esters using pentadecanoic acid as internal standard (Benning and Somerville, 1992).

Uracil feeding
The A. thaliana seedling culture was performed in full nutrition medium. After 7 days of growth, the seedlings were fed with uracil for 24 h under the same growth conditions by adding 1 μCi of [2-14 C]uracil (7.4 GBq/mmol) at a final concentration of 2 mM to each flask. To measure the degradation of uracil, Whatman 3MM filter paper soaked with 3 M KOH was placed on top of each flask to capture [ 14 C]-CO 2 . The seedlings were harvested in liquid nitrogen and frozen plant material was ground to a powder using a ball mill. The radioactivity of the filter papers was measured after 24 h of equilibration in water. Metabolites were extracted using an adapted perchloric acid extraction procedure (Katahira and Ashihara, 2002). Frozen plant material (250 mg) was immediately mixed with cold 10% perchloric acid containing 10 mM ethyleneglycol-bis(βaminoethylether)-N,N′-tetraacetic acid (EGTA), and extracted for 30 min on ice. After centrifugation at 16 000 g for 5 min at 4 °C, the supernatant and pellet were separated. The pellet was washed with 250 μL of cold 80% ethanol and the supernatant, after centrifugation at 16 000 g for 5 min at 4 °C, was combined with the first supernatant and neutralized using 5 M KOH with 1 M triethanolamine. In this combined and neutralized supernatant fraction, radioactivity was measured for the soluble metabolites including nucleobases and nucleotides. In the pellet, the radioactivity of the insoluble fraction containing DNA and RNA was also determined.

Enzyme activity
Frozen plant material was ground to a powder in liquid nitrogen using a ball mill. Aliquots of 10-20 mg fresh weight were extracted by vigorous vortexing with 500-1000 μL of extraction buffer. The composition of the extraction buffer was 50 mM KH 2 PO 4 /K 2 HPO 4 at pH 7.5, 10% (v/v) glycerol, 0.1% (v/v) Triton X-100, 5 mM MgCl 2 , 1 mM ethylenediaminetetraacetic acid, 1 mM EGTA, 1 mM phenylmethylsulphonyl fluoride and 5 mM dithiothreitol. The extract was centrifuged at 16 000 g for 10 min at 4 °C. Measurements were performed in microplates by mixing 10 μL of the supernatant of the enzyme extract with 100-200 μL of measuring buffer (KH 2 PO 4 / K 2 HPO 4 at pH 7.5, 5 mM MgCl 2 , 3.75 mM R5P, 2 mM ATP, 3.75 mM phosphoenolpyruvate, 0.2 mM NADH, 1.5 U myokinase, 3 U pyruvate kinase and 1.5 U lactate dehydrogenase). The soluble protein content of the supernatant was determined using a dye-binding assay (Bradford, 1976).