Udo Moehren

Sarah Denayer

Michael Podvinec

Guy Verrijdt

Frank Claessens
email: frank.claessens@med.kuleuven.be

Identification of androgen-selective DNA response elements in the human Aquaporin 5 and RAD9 genes

Keywords: NUBIScan, androgen response element, androgen selectivity, Aquaporin5, RAD9, androgen receptor

published or not. The documents may come

Introduction

Being a ligand-induced transcription factor, the androgen receptor (AR) influences gene-expression by binding specific DNA motifs (called androgen response elements or AREs) in the regulatory regions of its target genes. The AR is known to functionally interact with two different sets of AREs [START_REF] Claessens | Selective DNA binding by the androgen receptor as a mechanism for hormone-specific gene regulation[END_REF]. One set is that of the classical steroid hormone response elements (cAREs), which are three-nucleotide-spaced partial palindromic repeats of the AR monomer consensus binding site 5'-TGTTCT-3'. The cAREs are also recognized by the other steroid hormone receptors that belong to the same subgroup of the nuclear receptor superfamily as does AR [START_REF] Verrijdt | Selective DNA recognition by the androgen receptor as a mechanism for hormone-specific regulation of gene expression[END_REF].

The AR is able to interact with another set of motifs that are essentially threenucleotide-spaced partial direct repeats of the same monomer binding element. In that respect, they resemble the binding elements for the VDR and RAR/RXR-type of nuclear receptors [START_REF] Germain | Overview of nomenclature of nuclear receptors[END_REF]. These motifs are called selective androgen response elements (sAREs).

They have so far been characterized in the rat Probasin promoter [4;5], the human Secretory Component gene upstream enhancer [START_REF] Verrijdt | Androgen specificity of a response unit upstream of the human secretory component gene is mediated by differential receptor binding to an essential androgen response element[END_REF], the mouse Sex-Limited Protein enhancer [START_REF] Adler | Multiple components of a complex androgen-dependent enhancer[END_REF][START_REF] Verrijdt | Functional interplay between two response elements with distinct binding characteristics dictates androgen specificity of the mouse sex-limited protein enhancer[END_REF][START_REF] Adler | Androgen-specific gene activation via a consensus glucocorticoid response element is determined by interaction with nonreceptor factors Proc[END_REF], the mouse Rhox5 promoter [START_REF] Barbulescu | New androgen response elements in the murine pem promoter mediate selective transactivation[END_REF] and the human Selective Androgen Responsive Gene [START_REF] Steketee | A bioinformatics-based functional analysis shows that the specifically androgen-regulated gene SARG contains an active direct repeat androgen response element in the first intron[END_REF]. In each of these genes, the sARE was identified as necessary to confer selective androgen versus glucocorticoid stimulation of gene expression to a heterologous promoter since its deletion or mutation results in a strong decrease of androgen responsiveness. These elements are also sufficient to mediate selectivity of androgen versus glucocorticoid stimulation to reporter gene expression, when taken out of the contexts of their original enhancers and tested individually in transient transfection assays [START_REF] Rennie | Characterization of two cis-acting DNA elements involved in the androgen regulation of the probasin gene[END_REF][START_REF] Claessens | The androgen-specific probasin response element 2 interacts differentially with androgen and glucocorticoid receptors[END_REF][START_REF] Verrijdt | Androgen specificity of a response unit upstream of the human secretory component gene is mediated by differential receptor binding to an essential androgen response element[END_REF][START_REF] Adler | Multiple components of a complex androgen-dependent enhancer[END_REF][START_REF] Verrijdt | Functional interplay between two response elements with distinct binding characteristics dictates androgen specificity of the mouse sex-limited protein enhancer[END_REF][START_REF] Adler | Androgen-specific gene activation via a consensus glucocorticoid response element is determined by interaction with nonreceptor factors Proc[END_REF][START_REF] Barbulescu | New androgen response elements in the murine pem promoter mediate selective transactivation[END_REF][START_REF] Steketee | A bioinformatics-based functional analysis shows that the specifically androgen-regulated gene SARG contains an active direct repeat androgen response element in the first intron[END_REF].

Sequence logos illustrating the conservation of bases in cognate sARE and cARE motifs are shown in Figure 1 [12;13]. In previous work, we have been able to demonstrate the in vivo relevance of at least one sARE (the Rhox5 ARE 1) in the socalled SPARKI mouse model in which the wild-type AR was exchanged for a mutant receptor that had lost its ability to interact with sAREs [START_REF] Schauwaers | Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model[END_REF]. We demonstrated that Rhox5 expression in mutant mice was more than 10-fold lower compared to wild-type littermates, whereas the expression levels of other known androgen responsive genes, driven by classical ARE motifs, were unchanged.

Here, we describe the application of a combined computational and experimental approach to discover previously unknown sAREs in the promoter regions of androgenresponsive genes. Using an extension of the NUBIScan algorithm [START_REF] Podvinec | NUBIScan, an in silico approach for prediction of nuclear receptor response elements[END_REF], we examined the promoter region of a collection of genes that had previously been described to respond to activation of the androgen receptor.

After computational analysis, we selected 7 motifs from 7 genes, which both according to the algorithm and visual inspection, are the best candidate androgenselective AREs. A first assessment of whether or not these motifs are functional sAREs was done by evaluating their roles in androgen regulation of reporter gene expression in transient transfection experiments using luciferase promoter constructs containing 4 copies of each element. Next we performed in vitro DNA binding assays using isolated DBD fragments of either AR or GR and comparing their affinities for each element.

Three elements that in both tests displayed significant androgen selectivity were tested in band-shift assays using the full-size receptors. Finally, the functionality of the RAD9 and ABCC1 motif was evaluated in the original genomic contexts by performing transfection assays using reporter constructs containing the enhancer region encompassing the ARE. We were able to confirm the androgen-specificity of the Aquaporin 5 ARE by assessing Aquaporin 5 expression in the SPARKI mouse model that expresses an AR mutant that is not able to bind sAREs.

We have thus succeeded in identifying and characterizing two new functional androgen-selective response elements and enhancers in two human genes that were previously known to respond to androgens but so far lacked more data on the DNA regions or motifs that are responsible for their androgen regulation.

Experimental

Reporter plasmids

Luciferase reporter constructs are all derived from the pGL3 basic reporter vector (Promega, Madison, WI). The empty promoter vector pTK-TATA-Luc construct was described previously [START_REF] Nelson | The human (PEDB) and mouse (mPEDB) Prostate Expression Databases[END_REF]. The empty pE1b-TATA-Luc vector was made by cutting out by PstI and BglII digestion the two copies of the TAT-GRE from the (ARE)2-E1b-Luc vector, which was described in [START_REF] Segawa | Androgen-induced expression of endoplasmic reticulum (ER) stress response genes in prostate cancer cells[END_REF]. Four copies of each ARE were inserted in pTK-TATA-Luc by cloning the corresponding oligonucleotides, having SacI-and HindIII-compatible overhangs, in the SacI-digested vector. Oligonucleotides having NheI-and XhoI compatible overhangs were inserted in the NheI-digested pTK-TATA-Luc vector. A genomic fragment containing the RAD9 putative ARE was amplified by PCR from genomic DNA from HeLa cells using the following primers (5'ccccggtaccgctggtgacgaggggagcag-3' and 5'-ccccctcgagtggcctctcaaagtggagtgag-3'). For the enhancer fragment encompassing the ABCC1 motif, in a similar PCR reaction, the primers (5'-ccccggtacccagtgcctggctcttgaataatg-3') and (5'ccccctcgagggaggaagaggctttcaaactga -3') were used. The resulting PCR products were inserted in the pE1b-TATA-Luc vector. All constructs were checked by sequencing.

The AR and GR mammalian expression vectors are a kind gift from Prof. Rusconi (Univ. of Fribourg, Switzerland).

Cell culture experiments

HeLa cells were plated on day 1 in 96-well microtiterplates in Dulbecco's modified Eagle's medium containing 1000 mg/ml glucose, penicillin (100 IU/ml), streptomycin (100 µg/ml) and 5% dextran coated charcoal-stripped fetal calf serum. The second day, cells were transfected with a DNA mixture of 100 ng reporter vector, 10 ng of an expression plasmid for β-galactosidase and 1 ng of the appropriate AR or GR expression vector, per well. Transfections were performed using the Genejuice transfection reagent (Novagen, Merck, Darmstadt, Germany) according to the manufacturer's instructions. On day three, medium was replaced with fresh medium, with or without the addition of the synthetic androgen methyltrienolone, R1881 (1 nM) or Dexamethasone, Dex, (10 nM), as appropriate. 24 hours after addition of the hormone, cells were harvested in 25 µl of Passive Lysis Buffer (Promega, Madison, WI) per well.

Luciferase light emission of 2 µl samples of each well were determined using the

Purification of receptor DBDs and full-size receptors

AR-and GR-DBD fragments were grown and purified as described previously [START_REF] Nelson | The human (PEDB) and mouse (mPEDB) Prostate Expression Databases[END_REF].

Full-size receptors for use in the band-shift assays were obtained by transfecting 15-cm plates of COS7 cells with 7 µg of the appropriate AR or GR expression plasmid (see higher). After 36 hrs and an additional 12 hr stimulation of the cells with the appropriate hormone (either 1 nM R1881, or 10 nM Dexamethasone), the cells were harvested as described previously [START_REF] Deprimo | Transcriptional programs activated by exposure of human prostate cancer cells to androgen[END_REF].

In vitro DNA-binding assays

For the calculation of the apparent dissociation constant (Ks) value for each motifreceptor DBD combination, an equal amount of radiolabeled oligonucleotide was incubated with increasing amounts (from 8 nM to 1.6 µM) of AR-or GR-DBD in 20 µl of binding buffer as described in [START_REF] Nelson | The human (PEDB) and mouse (mPEDB) Prostate Expression Databases[END_REF]. After non-denaturing PAGE electrophoresis, gels were dried and scanned in a STORM 840 PhosphorImager (GE Healthcare Biosciences, Uppsala, Sweden). Percentage of binding was calculated for each lane by calculating the ratio of the intensity of the bound probe, versus the total amount of radioactivity in that lane. Binding curves were made by plotting percentages of binding as a function of the concentration of AR-or GR-DBD that was used. For each curve, best fits to 4 parameter curves with allosteric Hill kinetics were calculated using the SigmaPlot software package (SPSS Inc. Chicago, IL) and Ks values were determined accordingly.

Band-shift assays detecting the binding of the full-size receptors were performed as described above, except that the radiolabeled probe was incubated with approximately 5 µg of total extract of COS7 cells transiently transfected with the appropriate AR or GR

Results and discussion

Selection of androgen responsive genes

Androgen regulated genes were compiled from a Virtual Expression Analysis Tool (VEAT) comparison of two datasets of expression profiles of androgen-starved and androgen-stimulated LNCaP cells in the Prostate Expressed DataBase (PEDB) at http://www.pedb.org/AR/microarray/ [19], as well as from two other comparative microarray analyses of androgen-starved and androgen-stimulated LNCaP cells [20;21]. This resulted in the compilation of a set of 85 known androgen-regulated genes in the promoter regions of which we searched for putative androgen-selective AREs. Using the SOURCE web site [START_REF] Sui | Specific androgen receptor activation by an artificial coactivator[END_REF], LocusLink (now: Entrez Gene) IDs were retrieved for all but 9 of the genes in the set. Using these identifiers, the first exon of each gene and 5 kb of 5' flanking region were retrieved from TRASER [http://genome-www6.stanford.edu/cgi-bin/Traser/traser] and saved for further analysis.

Analysis of promoter regions

We have previously published NUBIScan, an algorithm for the sensitive detection of nuclear receptor binding sites [START_REF] Podvinec | NUBIScan, an in silico approach for prediction of nuclear receptor response elements[END_REF]. In the present study, we have used a slightly modified version of the NUBIScan algorithm, which allows the definition of different positional weight matrices for each of the two hexamer half sites.

For each of the half-sites, we created positional weight matrices. Initially, a nucleotide distribution matrix was calculated based on the aligned known positive binding site sequences. Next, an arbitrarily scaled positional weight factor was calculated based on the degree of conservation at each position of the alignment. Due to this term, highly conserved positions contribute more to a site's scores than variable positions. Next, promoter sequences were scanned with these matrices, obtaining match quality scores at each sequence position. Finally, the scores from the relevant matrices, representative of the individual half-sites, were combined by multiplication for a final score.

A major challenge in this approach was that at the time of design of the study, only three confirmed naturally occurring and two synthetic selective ARE elements were described [23;24], and therefore, our matrices had to be composed using this small training set. We have used leave-one-out cross-validation to investigate the sensitivity of our algorithm. Here, one of the ARE elements is chosen as the validation data point, and the algorithm is trained with all other ARE elements. Next, the validation data point is classified using the algorithm. This procedure is then repeated with all data points and allows a rough estimate of the algorithm's predictive quality in spite of the small sample size. A preliminary study furthermore indicated that the algorithm was able to discriminate known positive sequences from randomized sequence and thus to identify binding sites within large sequence contexts. For each predicted binding site in the analyzed candidate promoter regions, expectation values were estimated by performing the same analysis on 100 randomly shuffled sequences of the same length and base composition. We are aware, however, that such a small and consistent training set may miss some of the less well conserved binding sites. Notably, elements organized as For 76 of the candidate AR-responsive genes collected from the literature, 5 kb of 5' flanking region sequence and the first exon were retrieved. All of these sequences were subjected to computational analysis, searching for the direct repeat-type arrangement of sAREs. After this search, motifs from seven genes from the top-ranked predictions by NUBIScan, ABCC1, AQP5, INPP5A, SREBF2, RAD9A, THRA, UCK2 (Table 1) were chosen for an initial analysis. The binding sites in this set all had a raw score > 0.75 and an expectation value < 1*10 -5 . Next, we set out to experimentally verify the ability of these elements to be activated by AR, and more specifically to ascertain their specificity towards AR.

Functional assays using the isolated ARE motifs

Four copies of each putative ARE were cloned in a luciferase reporter vector upstream of the TK minimal promoter. Theoretically they would thereby confer steroid responsiveness to luciferase expression in transient transfection experiments in cells cotransfected with the appropriate receptor.

The SREBF2, INPP5A, THRA and UCK2 motifs did not confer androgen-nor glucocorticoid stimulation to the minimal promoter (Fig. 2). Because of their intrinsic inability to confer androgen responsiveness, these motifs were excluded from subsequent experiments.

The ABCC1 motif displayed strong responsiveness to androgens (a 20-fold induction) but no selectivity towards glucocorticoids (20-fold induction). The slpHRE-2 control, the RAD9 and AQP5 motifs display a clear androgen inducibility (9.7, 14.1-and 14-fold induction, respectively), whereas glucocorticoid responsiveness was significantly lower (4.2, 1.2 and 4.4-fold induction, respectively). These motifs are therefore able to selectively confer androgen as opposed to glucocorticoid responsiveness to a heterologous promoter.

Band-shift assays using isolated DBDs

Oligonucleotides carrying the RAD9, AQP5 and ABCC1 motifs were radiolabeled and incubated with increasing amounts of recombinant AR-or GR-DBD and subjected to non-denaturing polyacrylamide gel electrophoresis (PAGE; Fig. 3). From the binding patterns for each element, binding curves were obtained and apparent dissociation constant (Ks) values were calculated as was described e.g. for the slp-HRE2 [START_REF] Schoenmakers | Differential DNA binding by the androgen and glucocorticoid receptors involves the second Zn-finger and a C-terminal extension of the DNA-binding domains[END_REF].

The RAD9 and AQP5 motifs displayed the most striking AR-versus GR-DBD selectivity: their dissociation constants (Ks) for AR-DBD interaction are 124 and 112 nM, respectively, while the Ks values for their interaction with the GR-DBD could not be calculated (> 1000 nM). This correlates well with their functional androgen versus glucocorticoid selecitivity in transfection assays.

The ABCC1 motif displayed a relatively high affinity for the AR-DBD (Ks value is 65 nM) and approximately 6-fold lower affinity for the GR-DBD (Ks = 405 nM).

Confirmation of selective receptor interaction to the RAD9, ABCC1 and AQP5 motifs

To confirm the results obtained in the two previous experiments, we performed bandshift experiments using the RAD9, ABCC1 and AQP5 motifs and full-size AR and GR (Fig. 4). As a positive control, band-shift experiments were performed using the nonselective C3(1)ARE [START_REF] Claessens | Functional characterization of an androgen response element in the first intron of the C3(1) gene of prostatic binding protein[END_REF] and the selective slp-HRE2 [START_REF] Adler | Multiple components of a complex androgen-dependent enhancer[END_REF]. All four motifs were bound by the AR as demonstrated by the super-shifted band upon the addition of an anti-AR antibody and the disappearance of this super-shifted band upon the addition of an excess of an unlabeled specific competitor oligonucleotide (the C3(1)ARE) but not of a non-specific competitor. When using cell extracts of cells not transfected with either receptor, no super-shifts appear upon the addition of anti-AR or anti-GR antibody. Only the C3 (1) ARE, but none of the putative selective AREs were significantly recognized by the GR, thereby confirming the binding assays using the isolated DBDs as well as the transfection assays. For ABCC1, a very faint super shift is visible when using cell extracts containing GR. Apparently, the GR is a stronger transcriptional activator than is the AR, as demonstrated for example for the non-specific TAT-GRE and the C3(1)ARE.

Despite having similar affinities for both DBDs, the fold-induction these elements and promoter regions confer upon glucocorticoid stimulation is always much higher compared to their androgen effects [23;24]. It is therefore not surprising that the ABCC1 motif displays low androgen versus glucocorticoid specificity in transient transfections.

On the other hand, the affinity of the GR-DBD for the RAD9 and AQP5 motifs is probably below the threshold for the GR to function as a transcriptional activator, which makes them genuine androgen-specific AREs.

Functional confirmation of androgen selectivity of the AREs in their original contexts

S t a g e 2 ( a ) P O S T -P R I N T

We subsequently cloned the enhancer regions, or androgen regulatory units (ARU), that surround the RAD9 and ABCC1 motifs in the E1b minimal promoter-driven luciferase reporter vector and performed transient transfection experiments in HeLa cells (Fig. 5). The RAD9 ARU shows a clear responsiveness to androgens (a 5.7-fold induction), while it does not respond to glucocorticoids (1.1-fold induction).

We also determined the expression levels of the AQP5 gene in the previously developed SPARKI mouse model containing a mutated AR that is able to interact with classical but not selective AREs [START_REF] Schauwaers | Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model[END_REF]. In a comparative microarray analysis on ventral prostate, the mouse AQP5 gene is approximately 3.6-fold down regulated in the SPARKI mice compared to the wild type mice, confirming that its expression is strongly dependent on an AR able to interact with (a) selective ARE(s). This difference in AQP5 expression level is not due to a generally lower abundance of transcripts in mutant versus wild type mice or a general lower activity of the androgen signaling pathway, since other known androgen-responsive genes (for example NKx3.1) display equal expression levels in the SPARKI versus wild type mice.

The AQP5 ARE located approximately 570 bp downstream of the AQP5 transcripition initiation site is well conserved between human (5'-tgttcgcagagttct-3') and mouse (5'-tgttcgcagagttcc-3'). The T to C at pos. +7 (underscored) most probably does not significantly affect AR interaction.

The RAD9 motif, however, is not conserved between human and mouse. Although it is located in a region with 60% sequence identity between human (at +/-1800 bp upstream of the transcription initiation site) and mouse (at +/-3800 bp upstream of the promoter), the element is changed (5'-ggctctggggctggg-3', underlined nucleotides are different from the human motif), so that it no longer corresponds to a consensus classical or selective ARE.

Androgen regulation of RAD9 and AQP5

RAD9 was initially found in fission yeast as a gene involved in resistance to ionizing radiation [START_REF] Lieberman | Rad9, an evolutionarily conserved gene with multiple functions for preserving genomic integrity[END_REF]. Apart from its function as a checkpoint control protein, it also plays a more direct role in DNA repair. As part of a complex with RAD1 and HUS1 (called 9-1-1 complex) it is thought to act as a DNA damage sensor [START_REF] Tomlins | Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer[END_REF]. Next to its functions in cell cycle control and DNA damage repair, it was also found to play a role in apoptosis and have dual functions in transcription. On the one hand, it acts as a transcriptional activator to stimulate the expression of genes that could be essential in cell cycle [START_REF] Parrilla-Castellar | Dial 9-1-1 for DNA damage: the Rad9-Hus1-Rad1 (9-1-1) clamp complex DNA Repair[END_REF][START_REF] Komatsu | Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xL and promotes apoptosis Nat[END_REF][29]. On the other hand, it has been proposed to be a co-repressor of ligand-activated AR [START_REF] Wang | Human checkpoint protein hRad9 functions as a negative coregulator to repress androgen receptor transactivation in prostate cancer cells[END_REF]. RAD9 could therefore theoretically play an important role in the onset and evolution of prostate cancer, protecting prostate epithelial cells from the influence of androgens on their proliferation. AQP5, one of 13 aquaporin transmembrane fluid channels, is expressed amongst others in salivary and lacrimal glands, sweat glands and lung and uterus epithelium. It functions as a channel through which fluids can flow from the inside of the cell to the external environment [START_REF] Delporte | Distribution and roles of aquaporins in salivary glands[END_REF]. AQP5 knockout mice show lower rates of saliva production with a higher osmolality and viscosity of saliva [START_REF] Ma | Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels[END_REF]. In the progesterone-primed uterus, its expression can be significantly upregulated by estrogens and a functional ERE was characterized in its proximal promoter [START_REF] Kobayashi | Chromatin immunoprecipitation-mediated target identification proved aquaporin 5 is regulated directly by estrogen in the uterus[END_REF]. In mouse lung epithelial cells, its expression can be regulated at the transcriptional level by all-trans retinoic acid [START_REF] Nomura | All-trans retinoic acid increases expression of aquaporin-5 and plasma membrane water permeability via transactivation of Sp1 in mouse lung epithelial cells[END_REF]. A conserved transcriptional enhancer region was identified in the first intron of the AQP5 gene [START_REF] Flodby | Conserved elements within first intron of aquaporin-5 (Aqp5) function as transcriptional enhancers[END_REF].

The motif we have discovered here resides in the first exon of the gene, which is well conserved between species. AQP5 also seems to be involved in the Sjögren's Syndrome pathology [START_REF] Konttinen | Immunohistopathology of Sjogren's syndrome[END_REF]. Since Sjögren's Syndrome affects mainly women (9:1 female -male ratio) and often displays a late onset of disease, the presumed androgen regulation of AQP5 expression could be an important factor in the etiology of Sjögren's Syndrome. In the acinar cells of normal individuals, AQP5 is expressed primarily at the apical membrane, while in cells of SS patients, AQP5 molecules are redistributed to the basal membrane, diverting a large portion of the fluid flow to the interstitium instead of to the acinar lumen [START_REF] Konttinen | Immunohistopathology of Sjogren's syndrome[END_REF].

Genome-wide searches for androgen response elements

Recent results from several genome-wide searches for AR interaction sites indicate that the AR is more promiscuous than thought before in the selection of motifs it interacts with to stimulate gene expression. Wang et al. [START_REF] Wang | A hierarchical network of transcription factors governs androgen receptor-dependent prostate cancer growth[END_REF] reported the results of a ChIP-on-chip analysis on human chromosomes 21 and 22. Only 10% of genomic regions interacting with the AR were found to contain a classical ARE, 22% did not contain an ARE-like motif, while the majority (68%) was found to contain a non-canonical ARE, including isolated half-sites and everted and direct repeat elements. One of the genomic regions in which a non-canonical ARE was found was the TMPRSS2 gene [START_REF] Tomlins | Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer[END_REF].

Using a similar technique, Bolton et al. [START_REF] Bolton | Cell-and gene-specific regulation of primary target genes by the androgen receptor[END_REF] found a number of AREs involved in the androgen regulation of a set of androgen responsive genes (ARGs). In this study, 69% However, the sequence logo presentation of the enriched sites in the AR-binding regions does not allow discrimination between the type of ARE. The relatively high abundance of non-canonical or half-site ARE motifs in genomic regions that were found to bind the AR, was confirmed by Massie et al. [START_REF] Massie | New androgen receptor genomic targets show an interaction with the ETS1 transcription factor[END_REF]. In ChIP-on-chip analysis of androgen-deprived and -stimulated LNCaP cells, this group found only a minority of 27% of the AR binding regions to be significantly enriched in 15 bp classical palindromic AREs. This study also claims that half sites are sufficient for the AR to bind and transactivate. It is important to consider, however, that not all half sites will be sufficient for AR binding and transactivation since the present study has shown that even motifs with a partial repeat structure not always bind AR or can confer androgen responsiveness to a promoter (Fig. 2). We have also shown previously that for example the HRE1 in the mouse Sex-Limited Protein ARU, which contains a perfect 5'-TGTTCT-3' without a proper upstream half-site does not bind the AR-DBD nor does it transactivate in reporter assays [START_REF] Verrijdt | Functional interplay between two response elements with distinct binding characteristics dictates androgen specificity of the mouse sex-limited protein enhancer[END_REF]. AR binding to DNA fragments containing 5'-TGTTCT-3' motifs was demonstrated by our group already in 1990 [START_REF] Claessens | Sequence-specific binding of androgen-receptor complexes to prostatic binding protein genes[END_REF] but most of the elements identified in that study were not functioning as AREs in transient transfection asays. The possibility for (near-)consensus ARE half-site to act as AREs, therefore needs to be investigated more thoroughly, and the putative modulatory role of flanking and spacer sequences should not be overlooked [START_REF] Haelens | DNA recognition by the androgen receptor: evidence for an alternative DNA-dependent dimerization, and an active role of sequences flanking the response element on transactivation[END_REF][START_REF] Nelson | Determinants of DNA sequence specificity of the androgen, progesterone, and glucocorticoid receptors: evidence for differential steroid receptor response elements[END_REF][START_REF] Ham | Characterization of response elements for androgens, glucocorticoids and progestins in mouse mammary tumour virus[END_REF].

Conclusions

From seven motifs found in the vicinity of androgen-regulated promoters using NUBIScan (Table I), we characterized three putative AREs by transactivation assays Fig. 2) and by band-shift (Fig. 3 and4). We have identified two new androgen-selective AREs, one in the human RAD9 and one in the AQP5 genes. For the RAD9 gene, we confirmed that its androgen regulation was impaired in the SPARKI mouse model. Our Table 1. Names and sequences of the putative selective AREs studied in this report. To the left are indicated the names and sequences of the motifs that were used to create the weblogo depictions (http://weblogo.berkeley.edu/). For references: PB2 [START_REF] Rennie | Characterization of two cis-acting DNA elements involved in the androgen regulation of the probasin gene[END_REF], SC [START_REF] Verrijdt | Androgen specificity of a response unit upstream of the human secretory component gene is mediated by differential receptor binding to an essential androgen response element[END_REF],

SLP2 [START_REF] Adler | Multiple components of a complex androgen-dependent enhancer[END_REF], SARG [START_REF] Steketee | A bioinformatics-based functional analysis shows that the specifically androgen-regulated gene SARG contains an active direct repeat androgen response element in the first intron[END_REF], MMTV [START_REF] Ham | Characterization of response elements for androgens, glucocorticoids and progestins in mouse mammary tumour virus[END_REF], C3(1) [START_REF] Claessens | Functional characterization of an androgen response element in the first intron of the C3(1) gene of prostatic binding protein[END_REF], PSA [START_REF] Cleutjens | Two androgen response regions cooperate in steroid hormone regulated activity of the prostate-specific antigen promoter[END_REF], SLP3 [START_REF] Adler | Multiple components of a complex androgen-dependent enhancer[END_REF], GUS [46], p21 [START_REF] Lu | Androgen regulation of the cyclin-dependent kinase inhibitor p21 gene through an androgen response element in the proximal promoter[END_REF],

MVDP [START_REF] Darne | Ubiquitous transcription factors NF1 and Sp1 are involved in the androgen activation of the mouse vas deferens protein promoter[END_REF], HK2 [49].

Figure 2.

Transient transfection assays in HeLa cells of TK-TATA-box-driven luciferase reporter constructs containing 4 copies of the motif as indicated (for sequences see Table 1). All

Biochemical

  Journal Immediate Publication. Published on 10 Jan 2008 as manuscript BJ20071352 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071352 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 10 Jan 2008 as manuscript BJ20071352 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071352 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 10 Jan 2008 as manuscript BJ20071352 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071352 S t a g e 2 ( a ) P O S T -P R I N T luciferase assay reagent (Promega, Madison, WI) in a Luminoskan Ascent Luminometer (Thermo Labsystems, Franklin, MA). To correct for transfection efficiency, luciferase values were always related to the β-galactosidase value as measured by the Galacto-Light Chemiluminscent Reporter Gene Assay System (Tropix, Bedford, MA). Results are expressed as corrected luciferase values relative to the luciferase value of the nonstimulated samples (which was arbitrarily set at 1). These values therefore also express folds induction, as a ratio of the stimulated samples versus the non-stimulated samples transfected with the same reporter construct. Experiments were performed in triplicate and repeated at least three times independently.

Biochemical

  Journal Immediate Publication. Published on 10 Jan 2008 as manuscript BJ20071352 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071352 S t a g e 2 ( a ) P O S T -P R I N T mammalian expression plasmid. The double-stranded oligonucleotides used as specific or non-specific competitors for AR or GR interaction had the following sequences (C3(1)ARE: 5'-agcttacatagtacgtgatgttctcaagctcga-3', and NFI: 5'attttggctacaagccaatatgat -3'). Anti-AR is an in-house antibody against the first 21 aminoacids of the AR, anti-GR was a kind gift of Dr. Wikström of the Karolinska Institute, Stockholm, Sweden. Gene array analysis Total RNA was extracted from ventral prostates of 11-week old wild type or SPARKI mice using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) with an additional oncolumn DNase treatment. RNA quality was checked in a NanoDrop spectrophotometer (NanoDrop Technologies, Centerville, DE, USA) and in an Agilent 2100 BioAnalyzer (Agilent, Palo Alto, CA, USA). Total RNA from ventral prostates of three wild type, or three SPARKI litter mates were pooled, creating paired control and SPARKI samples. These pools were used for hybridization with the Agilent Whole Mouse Genome 4x44K Dual-Mode Microarray. Two biological repeats were done in the dual-label mode, creating four data-sets. Normalized transcript signals in each sample were compared with background signals and scored either present or absent. A student's t-test was performed to filter all transcripts that were significantly different in SPARKI versus wild type (p<0.01). Transcripts that were more than two-fold up or down regulated in SPARKI were regarded as differentially expressed. Overall 364 signals were significantly down, and 344 signals were up regulated in SPARKI prostates.

Biochemical

  Journal Immediate Publication. Published on 10 Jan 2008 as manuscript BJ20071352 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071352 S t a g e 2 ( a ) P O S T -P R I N T

  Biochemical Journal Immediate Publication. Published on 10 Jan 2008 as manuscript BJ20071352 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071352 S t a g e 2 ( a ) P O S T -P R I N T direct versus inverted repeats are not easy to distinguish, as individual binding sites are degenerate, and share sequence overlap. For this reason, some of the sites detected by the algorithm may serve as binding sites in either of the two configurations.

Biochemical

  Journal Immediate Publication. Published on 10 Jan 2008 as manuscript BJ20071352 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071352 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 10 Jan 2008 as manuscript BJ20071352 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071352

Biochemical

  Journal Immediate Publication. Published on 10 Jan 2008 as manuscript BJ20071352 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071352 S t a g e 2 ( a ) P O S T -P R I N T regulation or DNA damage repair

Biochemical

  Journal Immediate Publication. Published on 10 Jan 2008 as manuscript BJ20071352 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071352 S t a g e 2 ( a ) P O S T -P R I N T of all AR-interacting DNA fragments contain sequence motifs resembling classical AREs.

  work also indicates that selective, non-canonical AREs are probably more general than was previously thought. What the precise criteria are for an ARE to be able to recruit AR and function as a transcriptional regulator in vivo warrants further investigation. Wetenschappelijk Onderzoek -Vlaanderen. This work was supported by grants from the 'Geconcerteerde Onderzoeks Actie' and the 'FWO-Vlaanderen'. Biochemical Journal Immediate Publication. Published on 10 Jan 2008 as manuscript BJ20071352 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071352 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical SocietyTables

For 3 '

 3 each gene (full name and abbreviation in the left column), the sequences of the putative ARE are depicted (right column). The halfsite hexamers are underlined. Biochemical Journal Immediate Publication. Published on 10 Jan 2008 as manuscript BJ20071352 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071352 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure legends Figure 1 .

 legends1 Figure legends

  hormone-stimulated activities are calculated relative to the activity of the same samples that were not stimulated with hormone (white bars). Black bars are the relative activities of each construct as indicated, co-transfected with the AR-expression plasmid and stimulated with 1 nM of the synthetic androgen Methyltrienolone (R1881). Striped bars are the relative activities of each construct as indicated, co-transfected with the GRexpression plasmid and stimulated with 10 nM of the synthetic glucocorticoid Dexamethasone (Dex). Values are the averages of at least 3 independent experiments performed in duplicate. Fold-induction upon hormone stimulation is indicated above each black or striped bar. The empty pTK-TATA-Luc and the same vector containing four copies of the TAT-GRE or the SLP-HRE2 were used as negative and positive controls for androgen and glucocorticoid function, and androgen selectivity, respectively. Error bars indicate standard deviations.

Figure 3 .

 3 Figure 3. Band-shift assays using AR-or GR-DBD and a selection of putative selective ARE motifs. A representative band-shift assay gel is shown for each (putative) ARE. Binding curves depict the percentage of binding of the AR-or GR-DBD to the radiolabeled oligonucleotide as a function of the concentration of the DBD that was used in the binding assay. % Binding values are the averages of at least three experiments +/-S.E.M. values. Ks values are apparent dissociation constants for the AR-or GR-DBD interaction with each motif. Solid line: AR-DBD binding, dotted line: GR-DBD binding.

Figure 4 .

 4 Figure 4. Binding of the full-size receptors to the putative AREs. The rat C3(1) ARE was used as a positive control for AR and GR interaction, the slpHRE2 as a control for AR-selectivity. In each panel, lanes 1-3 contains cell extracts from non-transfected COS7 cells; lanes 4-7 extracts from cells transfected with an AR expression plasmid; lanes 8-11 extracts from cells transfected with a GR expression plasmid. Anti-AR or GR antibody and specific or non-specific competitor was added as indicated at the top. The single asterisks indicate the AR-DNA complexes, the double asterisks indicate the super-shifted complexes. The accolades indicate the positions of non-specific binding to the probes.

Figure 5 .Fig. 2 .Fig

 52 Figure 5.Transient transfection assays in HeLa cells of E1b minimal promoter-driven luciferase reporter constructs containing the ABCC1 or RAD9 ARU. Activities are depicted as in Fig.2. The empty pE1b-TATA-Luc vector and a similar vector containing the SC upstream ARU[START_REF] Verrijdt | Androgen specificity of a response unit upstream of the human secretory component gene is mediated by differential receptor binding to an essential androgen response element[END_REF] were used as a negative and positive control, respectively. Error bars indicate S.E.M.

Fig. 2 .

 2 Fig. 2.

FigFig. 4 .Fig

 4 Fig. 3.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

We gratefully acknowledge the excellent technical assistance of R. Debruyn, R. Bollen and K. Bosmans. F.C. is the beneficiary of a grant of the Association for International Cancer Research (AICR). G.V. is a postdoctoral fellow of the Fonds voor Biochemical Journal Immediate Publication. Published on 10 Jan 2008 as manuscript BJ20071352 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071352 S t a g e 2 ( a ) P O S T -P R I N T


Sabine König

Sabine Freitag

Dr Ingo Heilmann

Till Ischebeck Christian Löfke

Alternative metabolic fates of phosphatidylinositol produced by PI-synthase isoforms in Arabidopsis thaliana

Keywords: PI-synthase, Phosphatidylinositol, Lipid-metabolism, Lipid-pools, PIS2 cytidinediphospho-diacylglycerol, CFP, cyan fluorescent protein, DAG, diacylglycerol, ER, endoplasmic reticulum, EYFP, enhanced yellow fluorescent protein, GC, gaschromatography, HFT, NFT, types of beam splitters, MBS, main beam splitter, PIS, phosphatidylinositol synthase, PLD, phospholipase D, PtdEtn, phosphatidylethanolamine, PtdIns, phosphatidylinositol, PtdIns4P, phosphatidylinositol-4phosphate, PtdIns

Alternative metabolic fates of phosphatidylinositol produced by PI-synthase isoforms in Arabidopsis thaliana

Christian Löfke, Till Ischebeck,

Introduction

The complex interplay of physiological processes in eukaryotic organisms, including plants, relies in part upon the regulation by inositol-containing lipids, such as polyphosphoinositides [START_REF] Balla | Phosphoinositide-derived messengers in endocrine signaling[END_REF][START_REF] Stevenson | Inositol signaling and plant growth[END_REF][START_REF] Meijer | Phospholipid-based signaling in plants[END_REF][START_REF] Drobak | Phosphoinositide kinases and the synthesis of polyphosphoinositides in higher plant cells[END_REF]. As regulatory functions of polyphosphoinositides require tight spatio-temporal coordination [START_REF] Santarius | Supervised membrane swimming: small G-protein lifeguards regulate PIPK signalling and monitor intracellular PtdIns(4,5)P2 pools[END_REF]6], it has been proposed for a range of eukaryotic model systems that alternative regulatory functions of inositol-containing lipids are performed by discrete lipid pools of different metabolic origin [START_REF] Santarius | Supervised membrane swimming: small G-protein lifeguards regulate PIPK signalling and monitor intracellular PtdIns(4,5)P2 pools[END_REF][6][START_REF] King | Multiple metabolic pools of phosphoinositides and phosphatidate in human erythrocytes incubated in a medium that permits rapid transmembrane exchange of phosphate[END_REF][START_REF] Heilmann | Changes in phosphoinositide metabolism with days in culture affect signal transduction pathways in Galdieria sulphuraria[END_REF][START_REF] Heilmann | Plasma membrane phosphatidylinositol 4,5-bisphosphate levels decrease with time in culture[END_REF][START_REF] Kost | Rac homologues and compartmentalized phosphatidylinositol 4, 5bisphosphate act in a common pathway to regulate polar pollen tube growth[END_REF][START_REF] König | Stress-inducible and constitutive phosphoinositide pools have distinct fatty acid patterns in Arabidopsis thaliana[END_REF]. For instance, we have previously demonstrated that stress-induced and constitutive polyphosphoinositides of Arabidopsis plants differ in the composition of their respective associated fatty acids, indicating an origin of these lipid pools from precursors differing in fatty composition [START_REF] König | Stress-inducible and constitutive phosphoinositide pools have distinct fatty acid patterns in Arabidopsis thaliana[END_REF]. So far, it is unresolved how independent pools of inositol-containing lipids can be established and maintained.

The key biosynthetic precursor of inositol-containing lipids is phosphatidylinositol (PtdIns), formed by phosphatidylinositol synthase (PI-synthase, EC 2.7.8.11) catalyzing the transfer of myo-inositol onto an activated cytidinediphospho-diacylglycerol (CDP-DAG) [START_REF] Klezovitch | Characterization of reactions catalysed by yeast phosphatidylinositol synthase[END_REF]. PI-synthases from different biological sources have previously been characterised, including examples from mammals [START_REF] Parries | Phosphatidylinositol synthase from canine pancreas: solubilization by n-octyl glucopyranoside and stabilization by manganese[END_REF][START_REF] Takenawa | CDP-diglyceride:inositol transferase from rat liver. Purification and properties[END_REF], fungi [START_REF] Fischl | Phosphatidylinositol biosynthesis in Saccharomyces cerevisiae: purification and properties of microsome-associated phosphatidylinositol synthase[END_REF] and plants [START_REF] Collin | Phosphatidylinositol synthesis and exchange of the inositol head are catalysed by the single phosphatidylinositol synthase 1 from Arabidopsis[END_REF][17][START_REF] Justin | Compared selectivities of the phosphatidylinositol-synthase from maize coleoptiles either in microsomal membranes or after solubilization[END_REF]. A characteristic property of reported PI-synthase sequences is a CDP-alcohol phosphatidyltransferase-domain and the possession of four hydrophobic membrane-spanning domains identifying PI-synthases as membrane-integral proteins. A blast-search (blastp) with the amino acid sequence of the Arabidopsis PI-synthase, PIS1, against publicly-accessible genomic databases indicates the presence of more than one PI-synthase isoform in most eukaryotic organisms, including plants, fungi and mammals, suggesting that distinct PI-synthase isoforms may contribute to the establishment of different physiological pools of inositol-lipids. Besides the known PIS1 gene locus (At1g68000), the Arabidopsis genome contains at least one more gene (At4g38570, PIS2) with similarity to PI-synthase-coding sequences, which has not been functionally characterised. Because mammalian and fungal PI-synthases have been reported to reside at the interface of the endoplasmic reticulum (ER) and Golgi-stacks [START_REF] Batenburg | Regulation and location of phosphatidylglycerol and phosphatidylinositol synthesis in type II cells isolated from fetal rat lung[END_REF]20], and plant PI-synthase activity has been found associated with microsomal fractions [START_REF] Justin | Compared selectivities of the phosphatidylinositol-synthase from maize coleoptiles either in microsomal membranes or after solubilization[END_REF], PtdInsproduction is thought to take place in the ER and/or the Golgi, although precise localisation studies have not been performed on plant enzymes. From multiple reports of polyphosphoinositide-formation in cellular compartments other than ER or Golgi [START_REF] Drobak | Phosphoinositide kinases and the synthesis of polyphosphoinositides in higher plant cells[END_REF][START_REF] Heilmann | Plasma membrane phosphatidylinositol 4,5-bisphosphate levels decrease with time in culture[END_REF][21][22][START_REF] Okpodu | Purification and Characterization of a Soluble Phosphatidylinositol 4-Kinase from Carrot Suspension Culture Cells[END_REF][START_REF] Stevenson-Paulik | Differential regulation of two Arabidopsis type III phosphatidylinositol 4-kinase isoforms. A regulatory role for the pleckstrin homology domain[END_REF] the question arises how PtdIns is channeled to those other cellular locations in order to provide PtdIns necessary for polyphosphoinositide production. Several mechanisms have been proposed by which PtdIns may be distributed throughout the cell, and possible explanations include the action of PtdIns-specific lipid transfer proteins [START_REF] Wirtz | Phospholipid transfer proteins revisited[END_REF][START_REF] Kearns | Novel developmentally regulated phosphoinositide binding proteins from soybean whose expression bypasses the requirement for an essential phosphatidylinositol transfer protein in yeast[END_REF], a distribution by targeted vesicle delivery, or a combination of both.

PtdIns can be converted into other lipid-intermediates by several alternative pathways.

The inositol-headgroup can be successively phosphorylated at the D3, D4, or D5-positions by phosphatidylinositol-kinases (PI-kinases) and phosphatidylinositolphosphate-kinases (PIPkinases), giving rise to a variety of polyphosphoinositides, many of which with reported regulatory effects on physiology [START_REF] Balla | Phosphoinositide-derived messengers in endocrine signaling[END_REF][START_REF] Meijer | Phospholipid-based signaling in plants[END_REF][START_REF] Santarius | Supervised membrane swimming: small G-protein lifeguards regulate PIPK signalling and monitor intracellular PtdIns(4,5)P2 pools[END_REF]. Alternatively, PtdIns can be degraded by phospholipase D (PLD) to phosphatidic acid (PtdOH), which is a central biosynthetic intermediate of phospholipid metabolism and can be transformed into any other glycerophospholipid [START_REF] Ohlrogge | Lipid biosynthesis[END_REF][28][29]. In addition to the pathways mentioned, a number of other lipidmodifying enzymes, including phospholipases C, A1 and A2, and inositolphosphoceramide synthase may convert PtdIns to a variety of other products [START_REF] Beisson | Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database[END_REF]. In this context we aimed to address the question whether PtdIns produced by different Arabidopsis PI-synthase isoforms may enter discernible alternative routes of further metabolic conversion.

Here we show that recombinant PIS2 protein is catalytically active as a PI-synthase in vitro and that Arabidopsis, thus, contains at least two PI-synthase isoforms. In the course of experiments for in vitro characterisation, subtle preferences of PIS1 and PIS2 for saturated and unsaturated CDP-DAG substrates, respectively, were observed. PIS1 and PIS2 are both localised in the ER with only very slight differences in localisation between the isoforms.

Increased PtdIns-levels with overexpression of PIS1 or of PIS2 in transgenic Arabidopsis plants were correlated to either increased DAG-and PtdEtn-levels (PIS1) or to increased levels of polyphosphoinositides (PIS2). The data suggest a role for PI-synthase isoforms in the channeling of PtdIns towards different pools of PtdIns-derived lipids.

Experimental cDNA-constructs

The cDNA sequences encoding Arabidopsis PIS1 and PIS2 were amplified from cDNA prepared from flowers and siliques using the primer combinations 5'-GATCGAGTCGACATGGCTAAAAAGGAGAGAC-3' (primer A)/5'-GATCGAGCGGCCGCTCAAGGCTTCTGCTGCTTCTCTATA-3' and 5'-GATCGAGTCGACATGGCTAAACAGAGACCGGCG-3' (primer B)/5'-GATCGAGCGGCCGCTCAAGGCTTCTTATGCTGTTTCTC-3', respectively. For constructs to be c-terminally tagged by enhanced yellow-fluorescent protein (EYFP), PIS1 and PIS2 cDNA fragments were amplified using the primer combinations primer A/5'-GATCGAGCGGCCGCTAGGCTTCTGCTGCTTCTCTATA-3' and primer B/5'-GATCGAGCGGCCGCTAGGCTTCTTATGCTGTTTCTC-3', respectively, leaving out the stop-codons and introducing one additional nucleotide base for in-frame-fusion with the tag.

The cDNAs encoding the enhanced yellow fluorescent protein (EYFP)-and cyan fluorescent protein (CFP)-tags were amplified from plasmids carrying the authentic clones provided by Dr. Martin Fulda (Göttingen), in both cases using the primer combination 5'-GATCGCGGCCGCCCATGGTGAGCAAGGGCGAG-3'/5'-GATCGATATCTTACTTGTACAGCTCGTCCATG-3'. All amplifications were carried out using Phusion High-Fidelity DNA polymerase (Finnzymes) and a PCR-protocol of 2 min denaturing at 95°C followed by 36 cycles of 30 s denaturing at 95°C, 30 s annealing at 60°C and 1 min elongation at 72°C, with a terminal elongation step for 5 min at 72°C. Amplicons were cloned into the pGEM-T-Easy vector (Promega) according to manufacturer's instructions and sequenced. PIS1-and PIS2-cDNA clones were moved as SalI/NotI-fragments into the bacterial expression vector pET28b (Novagen). For plant expression, the plasmid pUC18 (Fermentas) was modified by the addition of multiple cloning site and gateway cassette of the pENTR2b-vector (Invitrogen), yielding the plasmid pUC18-ENTR . The cDNAs encoding EYFP or CFP were introduced into pUC18-ENTR as NotI/EcoRVfragments, yielding the plasmids pUC18-ENTR-EYFP and pUC18-ENTR-CFP, respectively. PIS1-and PIS2-cDNA clones were moved as SalI/NotI-fragments first into pUC18-ENTR; PIS1-and PIS2-cDNA-fragments lacking the stop-codon were moved as SalI/NotI-fragments into pUC18-ENTR-EYFP or into pUC18-ENTR-CFP. Subsequently, cDNAs were moved from the pUC18-derived vectors into a pCAMBIA plasmid (pCAMBIA3300-0GC) modified to contain a Gateway cassette (Invitrogen), using Gateway technology (Invitrogen) according to manufacturer's instructions.

Heterologous expression in E. coli

PIS1-and PIS2-pET28b constructs were heterologously expressed in E. coli C43(DE3) cells optimised for the accumulation of integral membrane proteins [START_REF] Miroux | Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels[END_REF]. Cells were transformed by heat-shock-treatment [START_REF] Inoue | High efficiency transformation of Escherichia coli with plasmids[END_REF]. Expression cultures (400 mL) were grown in Luria-Bertani-medium [33] in 1-L Erlenmeyer flasks at 37°C shaking at 200 rpm. Protein expression was induced by addition of 1 mM isopropyl-ß-D-thiogalactoside at an optical density (600 nm) of 0.8. Cultures were harvested 4 h after induction, and cells were disrupted on ice by three 30 s-pulses of ultrasound using a Sonifier Cell disruptor B15 (Branson) at 50 % performance. The homogenate was centrifuged for 10 min at 840 x g at 4°C, and the cleared supernatants were subjected to ultracentrifugation at 100,000 x g for 60 min at 4°C. Membrane pellets were resuspended in 1 mL of 20 mM Tris-HCl, 1 mM EDTA, 8 mM ßmercaptoethanol, pH 8.0, containing 20 % (v/v) glycerol. Protein contents were determined by the Bradford method (BioRad) with BSA as a standard [34].

Synthesis of CDP-DAG substrates

CDP-DAG substrates not available commercially were synthesized from PtdOH by chemical modification via a lipid-morpholidate as previously described [START_REF] Van Wijk | Antiviral nucleoside diphosphate diglycerides: improved synthesis and facilitated purification[END_REF]. PtdOH-species used for chemical conversion were obtained from Avanti (1,2-dipalmitoyl-glycerin-3phosphate, 1,2-dioleoyl-glycerin-3-phosphate and 1,2-dilinoleoyl-glycerin-3-phosphate) or Sigma (1,2-diheptadecanoyl-glycerin-3-phosphate). In addition to synthetic lipids, PtdOH was extracted from 50 g of green leaf tissue [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF], purified by thin-layer-chromatography (TLC) and used for conversion to CDP-DAG. CDP-DAG species obtained comigrated with authentic CDP-DAG standards (Avanti) and were purified by TLC. Samples were dissolved in CHCl 3 , dried under streaming N 2 and diluted in CHCl 3 to a lipid content of approximately 0.3 mg mL - 1 .

PI-synthase activity tests

The activity of recombinant PIS1 and PIS2 proteins was detected in bacterial membrane pellets by monitoring the production of PtdIns from CDP-DAG (Avanti) and nonlabelled D-myo-inositol (Sigma). Before addition of soluble components, CDP-DAG was dissolved in ethanol and dried into glass reaction tubes under streaming nitrogen. The assay mixture contained 50 mM Tris-HCl, pH 8.0, 20 mM MgCl 2 , 1.5 mM D-myo-inositol, 0.9 mM CDP-DAG, and 1 % (v/v) Triton-X100 in a reaction volume of 200 µL, as described [START_REF] Justin | Synthetic capacity of Arabidopsis phosphatidylinositol synthase 1 expressed in Escherichia coli[END_REF]. In experiments to determine the fatty acid compositions of CDP-DAG substrates and PtdIns formed, CDP-DAG substrates were used at a concentration of 0.2 mM. Reactions were started by the addition of 20 µg of bacterial membranes and incubated for 30 min at room temperature. Lipid reaction products were extracted [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF], and the organic phase was washed with 1 % (w/v) NaCl solution and dried under streaming nitrogen. Samples were subjected to TLC using a developing solvent containing methylacetate, 2-propanol, CHCl 3 , CH 3 OH and 0.25 % (w/v) aqueous KCl-solution at volume ratios of 25:25:25:10:9 [START_REF] Christie | Thin-layer chromatography in Lipid analysis[END_REF]. Lipids were visualised by submerging dried chromatography plates in an aqueous solution of 10 % (w/v) CuSO 4 (Sigma) containing 8 % (v/v) H 3 PO 4 (Sigma) and subsequent heating to 180°C. Lipid products were identified according to comigration with authentic standards (Avanti).

Alternatively to destructive visualization, lipids were reisolated from TLC-plates and analyzed for their fatty acid composition as described below.

Lipid extraction and biochemical analysis

Plant material was ground under liquid nitrogen to a fine powder. Phospholipids were extracted from powdered plant material by using an acidic extraction protocol [START_REF] Cho | Separation and quantification of of [ 3 H]inositol phospholipids using thin-layer-chromatography and a computerized 3 H imaging scanner[END_REF]. Lipids were separated by TLC on silica gel plates (Merck) using developing solvents for optimal resolution: for phosphoinositidies and PtdOH, CHCl 3 :CH 3 OH:NH 4 OH:H 2 O (57:50:4:11(v/v/v/v)) [START_REF] Perera | S t a g e 2 ( a ) P O S T -P R I N T phosphate 5-kinase 10 reveals differences in Arabidopsis and human phosphatidylinositol phosphate kinases[END_REF]; for PtdEtn, acetone:toluol:H 2 O (91:30:7 (v/v/v)) [START_REF] Hartel | DGD1-independent biosynthesis of extraplastidic galactolipids after phosphate deprivation in Arabidopsis[END_REF]; for isolating phosphatidylinositol, the same developing solvent was used as described as part of the PIsynthase assay. Lanes with lipid standards (5 µg) run in parallel to biological samples or to lipids formed in in vitro-assays were cut and lipids visualised as described above. Unstained lipids were located on the remaining parts of the TLC plates according to standard migration, were scraped, redissolved in their respective developing solvents and dried under N 2 flow.

Lipids were transmethylated [START_REF] Hornung | Formation of conjugated Delta11Delta13-double bonds by Delta12-linoleic acid (1,4)-acyl-lipid-desaturase in pomegranate seeds[END_REF], fatty acid methyl esters dissolved in acetonitril and analysed using a GC6890 gas chromatograph with flame ionization detection (Agilent) fitted with a 30 m x 250 µm DB-23 capillary column (Agilent). Helium flowed as a carrier gas at 1 ml min -1 . Samples were injected at 220°C. After one min at 150°C, the oven temperature was raised to 200°C at a rate of 8°C min -1 , then to 250°C at 25°C min -1 , and then kept at 250°C for 6 min. Fatty acids were identified according to authentic standards and by their characteristic mass spectrometric fragmentation patterns (data not shown), and quantified according to internal tri-pentadecanoic acid standards of known concentration. Due to limiting material in samples representing isolated minor lipids, fatty acids of low abundance may be absent from fatty acid patterns.

Transient gene expression by particle bombardment

One-mm epidermal sections of quartered onion (Allium cepa) bulbs were placed on moist whatman paper and were transformed by bombardment with plasmid-coated 1 µm goldparticles using a helium-driven particle accelerator (PDS-1000/He; BIO-RAD) with 1350 psi rupture discs at a vacuum of 28 inches of mercury. Gold-particles (1.25 mg) were coated with 3-7 µg of plasmid DNA of the pCAMBIA-vectors also used for plant transformation. After bombardment onion sections were incubated in covered petri dishes for 24 h at room temperature in a closed moist styrofoam box. Images pf onion epidermal peels were recorded using a Zeiss LSM 510 meta confocal microscope. EYFP was excited at 514 nm and imaged using an HFT 405/514/633 nm major beam splitter (MBS) and a 530-600 nm band pass filter; CFP was excited at 405 nm and imaged using an HFT 405/514/633 nm MBS and a 470-500 nm band pass filter. In coexpression experiments, CFP and EYFP were synchronously excited at 405 nm and 514 nm, respectively, and imaged using an HFT 405/514/633 nm and NFT 515 nm MBSs and 470-500 nm and 530-600 nm band pass filters, respectively.

Plant culture and transformation

Arabidopsis thaliana (L.), ecotype Columbia-0 (col-0) plants were grown on soil in growth chambers (York) at 22-25 °C under a regime of 8 h exposure to 130 -150 µmol photons m -1 s -1 and 16 h darkness and at approximately 60 % humidity. Recombinant DNAconstructs were introduced into Arabidopsis plants by Agrobacterium tumefaciens-mediated transformation, using A. tumefaciens strain EHA105 [START_REF] Hood | New Agrobacterium helper plasmids for gene transfer to plants[END_REF] and the floral-dip method [44]. Independent transformants were selected according to resistance against aerosolic glufosinateammonium (Bayer). All analyses were carried out on six-week-old plants. Plant growth was estimated according to leaf area of plants of the same age. Leaf areas were determined using Blattfläche 1.0.4.6 software (DatInf GmBH) according to manufacturer's instructions.

Results

Arabidopsis contains two distinct PI-synthase isoforms capable of generating phosphatidylinositol

A search of publicly-accessible databases [START_REF] Beisson | Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database[END_REF][START_REF] Zimmermann | GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox[END_REF] suggested the gene locus At4g38570 to encode a previously-uncharacterised PI-synthase isoform in Arabidopsis in addition to the known PIS1 encoded by the locus At1g68000. The amino acid sequence of the deduced PIS2 protein indicates strong similarity to other PI-synthases from plants, fungi or mammals (Figure 1). According to published transcript-array data accessible through the Genevestigator portal [START_REF] Zimmermann | GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox[END_REF] the PIS1-and PIS2-genes are ubiquitously expressed at modest levels throughout the plant, indicating no tissue-specific functions for either isoenzyme. 1). As C148 is conserved in PI-synthases from plants and fungi, but not in mammalian enzymes (Figure 1), at present the relevance of the polymorphism is unclear. In order to test the functionality of the PIS2 gene product, the cDNA was recombinatly expressed in E. coli. Production of the correct recombinant proteins was verified by immunodetection of the poly-His-tagged proteins, and PIS1-and PIS2-proteins were present only in pellet-fractions representing insoluble membrane-proteins (data not shown). Protein levels of PIS1 and PIS2 preparations tested were adjusted according to immunodetection. Recombinant PIS2 (C144W) protein exhibited substantial activity, producing PtdIns from CDP-DAG and D-myo-inositol substrates (Figure 2). In side-by-side expression experiments, PIS2 activity was comparable to that of the previously reported PIS1 (Figure 2). Affinity-purification of poly-His-tagged recombinant enzymes was not successful, likely because of the inherent difficulties with reconstituting membrane-integral enzymes in an active form, and, therefore, no specific activity was calculated. Even so, the data presented establish PIS2 as an active PI-synthase isoform in Arabidopsis.

Sequencing of independently-cloned full-length PIS2-cDNAs consistently indicated a

PIS1 and PIS2 proteins have preferences for different CDP-DAG fatty acyl species

In oder to test the substrate preferences of PIS1 and PIS2, catalytic activity was assayed in the presence of CDP-DAG species differing in fatty acid composition (Figure 3). CDP-DAG substrates tested included 1,2-dipalmitoyl-CDP-DAG; 1,2-diheptadecanoyl-CDP-DAG; 1,2-dioleoyl-CDP-DAG and 1,2-dilinoleoyl-CDP-DAG. In a first experiment PIsynthase activity was assayed under identical conditions against substrates presented individually (Figure 3 A). Catalytic activity was consistently higher with substrates containing C18-fatty acids than with those containing medium-chain fatty acids. For PIS1, the highest activity was determined using 1,2-dioleoyl-CDP-DAG, whereas PIS2 was more active with 1,2-dilinoleoyl-CDP-DAG. In order to assess substrate preferences in a competition assay, activity tests were performed against a mixture of all four available synthetic CDP-DAG substrates (Figure 3 B). The preference of PIS1 and PIS2 for substrates containing C18-fatty acids was confirmed. In the competitive assay PIS1 preferentially converted 1,2-dioleoyl-CDP-DAG, whereas PIS2 preferred 1,2-dilinoleoyl-CDP-DAG. All CDP-DAG species tested so far were of synthetic origin and symmetrical with regard to the associated fatty acids. As biological lipids usually are not symmetrical and contain different fatty acids, CDP-DAG synthesized from PtdOH isolated from Arabidopsis leaves was also tested as a substrate for PIS1 and PIS2 (Figure 3 C). The pattern of fatty acids associated with the "biological" CDP-DAG input-mixture in these experiments resembled that of structural phospholipids of Arabidopsis leaves [START_REF] König | Stress-inducible and constitutive phosphoinositide pools have distinct fatty acid patterns in Arabidopsis thaliana[END_REF][START_REF] Mosblech | Phosphoinositide and inositolpolyphosphate-signaling in defense responses of Arabidopsis thaliana challenged by mechanical wounding[END_REF][START_REF] Welti | Lipid species profiling: a high-throughput approach to identify lipid compositional changes and determine the function of genes involved in lipid metabolism and signaling[END_REF]. After in vitro-conversion of the CDP-DAG by PIS1 or PIS2, the fatty acid composition of CDP-DAG not converted (CDP-DAG residue) and that of newly-formed PtdIns (PtdIns product) was determined. The biological CDP-DAG mixture was converted to PtdIns with high efficiency by PIS2, whereas conversion by PIS1 was only moderate (Figure 3 C). PtdIns formed by PIS1 contained mainly palmitic acid and, to a lesser degree, linoleic acid. The CDP-DAG residue after PIS1 conversion contained high proportions of linoleic acid and linolenic acids, indicating that CDP-DAG species containing these fatty acids were not effectively converted by PIS1. In contrast, PtdIns formed by PIS2 from biological CDP-DAG contained predominantly palmitic acid, linoleic acid and linolenic acid, concomitant with a severe depletion of these fatty acids in the corresponding CDP-DAG residue. Together the in vitro data suggest a preference of recombinant PIS2 for polyunsaturated CDP-DAG species.

PIS1 and PIS2 proteins localise in the ER of onion epidermal cells

In a previous study, plant PI-synthase activity was reported to associate with microsomal fractions [START_REF] Justin | Compared selectivities of the phosphatidylinositol-synthase from maize coleoptiles either in microsomal membranes or after solubilization[END_REF], and because mammalian and fungal PI-synthases have been reported to reside in both ER and Golgi-membranes [START_REF] Batenburg | Regulation and location of phosphatidylglycerol and phosphatidylinositol synthesis in type II cells isolated from fetal rat lung[END_REF]20], experiments were initiated to test the subcellular localisation of Arabidopsis PIS1 and PIS2 proteins. In-frame fusions of PIS1 or PIS2 with c-terminally-attached EYFP-CFP-tags were transiently expressed in onion epidermal cells, and the fluorescence distribution of the PIS1-EYFP and PIS2-CFP proteins synchronously monitored by laser-scanning confocal microscopy (Figure 4). PIS1-EYFP and PIS2-CFP both exhibited clear ER-localisation (Figure 4 A). Differences in the localisation of PIS1-EYFP and PIS2-CFP were restricted to small punctate structures of limited occurrence (Figure 4 A, arrowheads) where PIS1 was observed in the centre and PIS2 at the periphery (Figure 4 B). The distribution of PIS1 and PIS2 was evaluated in relation to that of the Golgiassociated signal peptide of the rat sialyl-S-transferase (Sial) [START_REF] Grebe | Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes[END_REF]. When PIS1-EYFP or PIS2-EYFP were coexpressed with CFP-Sial, a limited degree of overlap was observed, as shown for PIS1-EYFP in Figure 4 C. The combined localisation data indicate that PIS1 and PIS2 are present in the ER, including tubular domains in close proximity to Golgi-particles, and exhibit slight but reproducible differences in localisation patterns.

Constitutive ectopic overexpression of PIS1 or PIS2 in Arabidipsis affects plant growth

Increased formation of PtdIns and PtdOH with ectopic overexpression of PIS1 or PIS2 in Arabidopsis plants

Of the transgenic Arabidopsis lines expressing either PIS1 or PIS2, three lines each were chosen for detailed biochemical analysis of leaf lipids. The first question asked was whether PI-synthase overexpression was correlated with increased accumulation of PtdIns in the transgenic plants. GC-based quantification of fatty acids associated with PtdIns [START_REF] König | Stress-inducible and constitutive phosphoinositide pools have distinct fatty acid patterns in Arabidopsis thaliana[END_REF] indicates that PtdIns levels were significantly (p<0.01) increased in all PI-synthase overexpressing lines tested (Figure 6 A). This result contrasts somewhat with previous data indicating that the levels of PtdIns are tightly controlled and kept at a constant level with either metabolic disturbance or with applied stress-treatments [START_REF] Welti | Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis[END_REF]. When the levels of PtdOH as a potential breakdown-product of PtdIns were tested in the PI-synthase overexpressing lines, PtdOH-levels were found to also be significantly increased (p<0.01) over those detected in wild type plants (Figure 6 B), suggesting action of regulatory mechanisms to limit PtdInsaccumulation.

Alternative metabolic fates of PtdIns produced by PIS1 or PIS2

When other lipids were included in the analysis of PIS1-or PIS2-overexpressors, it was found that PIS1 and PIS2 exerted different effects on downstream metabolites (Figure 7).

Whereas overexpression of PIS1 resulted in a significant increase in the levels of DAG and phosphatidylethanolamine (PtdEtn) in all cases, no such increases were observed with expression of PIS2 (Figure 7 A and B). In contrast, the levels of the polyphosphoinositides, PtdIns4P and PtdIns(4,5)P 2 , were significantly (p<0.01) increased in plants overexpressing PIS2, but not in those overexpressing PIS1 (Figure 7

Lipid-backbone fatty acid composition indicates metabolic origin of downstream metabolites

In order to provide evidence for the metabolic origin of phospholipids exhibiting altered levels correlated with PIS1 or PIS2 overexpression (Figure 8), the flow of intermediates into alternative pathways must be traced. As [ 3 H]-myo-inositol headgrouplabeling is not feasible at a scale required for the detection of polyphosphoinositides in intact Arabidopsis tissue, and because the label would be lost from the part of the PtdIns-molecule that enters pathways for conversion to other phospholipids including PtdEtn, metabolites were traced according to the fatty acid composition of their glycerolipid-backbones, as previously described [START_REF] König | Stress-inducible and constitutive phosphoinositide pools have distinct fatty acid patterns in Arabidopsis thaliana[END_REF]. The fatty acid composition of relevant phospholipids isolated from wild type plants (Figure 8 A) indicates a high proportion (50-60 mol%) of polyunsaturated fatty acids associated with PtdIns, PtdOH and PtdEtn, whereas the polyphosphoinositides, PtdIns4P and PtdIns(4,5)P 2 , contained more saturated and monounsaturated fatty acids, as was reported previously [START_REF] König | Stress-inducible and constitutive phosphoinositide pools have distinct fatty acid patterns in Arabidopsis thaliana[END_REF]. With overexpression of PIS1 the fatty acid composition of PtdIns, PtdOH,

Discussion

The present study was initiated to explore effects of increased PtdIns-levels on plant lipid metabolism as a result of PI-synthase overexpression. In addition to a known Arabidopsis PI-synthase, PIS1, the previously uncharacterized PIS2-protein was included in this study and was first demonstrated to have PI-synthase activity in vitro (Figure 2). Activity tests were performed using crude bacterial extracts, because E. coli does not contain endogenous PI-synthase activity [START_REF] Collin | Phosphatidylinositol synthesis and exchange of the inositol head are catalysed by the single phosphatidylinositol synthase 1 from Arabidopsis[END_REF]. The lack of chaperones or posttranslational modifications required for correct functionality of many eukaryotic proteins expressed recombinantly in E. coli did not obviously affect the catalytic activity of PIS1 or PIS2 (Figure 2), indicating that chaperones or modifications are not essential for PI-synthase function. The presence of an unusual number of amino acid positions (>20 out of ~220 residues) conserved in PI-synthases either as T or S (Figure 1), some across organismic kingdoms, may indicate the presence of phosphorylation sites possibly regulating enzyme activity or localisation in vivo. A detailed investigation of catalytic activity of PIS1 and PIS2 in vitro against CDP-DAG species differing in associated fatty acyl moieties indicated that PIS1 and PIS2 differ in their substrate preferences (Figure 3). Whereas PIS1 preferred CDP-DAG substrates containing a combination of palmitic acid and linoleic acid, PIS2 was most active with CDP-DAG substrates containing the polyunsaturated linoleic acid and linolenic acid. The observation that PIS1 exhibited substantially less activity than PIS2 against the biological CDP-DAG suggests that the endogenous PIS1 substrate may not have been represented in the mixture of CDP-DAG-species tested.

By monitoring the subcellular distribution of fluorescence-tagged PIS1 and PIS2 proteins, ER-localisation is suggested for both plant enzymes (Figure 4). Both PIS1 and PIS2 associated with tubular ER-structures in close proximity to Golgi-particles (Figure 4 C) , consistent with reports of fungal and mammalian PI-synthases which localise to the interface of ER and Golgi [START_REF] Batenburg | Regulation and location of phosphatidylglycerol and phosphatidylinositol synthesis in type II cells isolated from fetal rat lung[END_REF]20]. While PIS1 and PIS2 generally colocalised in the ER, punctate ERpatterns were regularly observed to which both PIS1 and PIS2 associated, however with subtle differences in localisation (Figure 4 B). In this context it should be noted that algorithms for the prediction of subcellular targeting such as Predotar V1.03 [START_REF] Emanuelsson | Predicting subcellular localization of proteins based on their N-terminal amino acid sequence[END_REF] or iPSORT [START_REF] Nakai | Primary structure of beta-galactoside alpha 2,6-sialyltransferase. Conversion of membranebound enzyme to soluble forms by cleavage of the NH2-terminal signal anchor[END_REF] did not yield consistent targeting predictions for PIS1 and PIS2, despite of substantial similarity of primary sequences (Figure 1). The interpretation of the visual information obtained by confocal microscopy was aided by the use of the Golgi-marker constructs, CFP-Sial [52], which is an established organellar marker for use in plants and has been used in previous studies [START_REF] Nebenfuhr | Mobile factories: Golgi dynamics in plant cells[END_REF]. The results support a view of PtdIns biosynthesis at the interface of the Ectopic overexpression of PIS1 or the previously uncharacterised PIS2 resulted in significantly delayed growth of transgenic plants, suggesting that transgene expression was placing strain on metabolism or energy balance. It has previously been reported that the levels of PtdIns are not readily perturbed by stress-treatments affecting the levels of other phospholipids, including PtdIns-derived lipids, suggesting PtdIns-levels are tightly regulated [START_REF] Welti | Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis[END_REF]. Increases in PtdIns-levels in leaves of plants overexpressing PIS1 or PIS2 coincided with raised levels of PtdOH, the immediate PLD-breakdown product of PtdIns (Figure 6). The inhibitory effect of PI-synthase overexpression on plant growth (Figure 5) may, thus, be due to the establishment of a futile cycle of increased PtdIns-generation and breakdown and consequentially increased energy consumption. An alternative explanation are possible effects of altered polyphosphoinositide formation on signalling processes affecting plant growth or development [START_REF] Stevenson | Inositol signaling and plant growth[END_REF][START_REF] Meijer | Phospholipid-based signaling in plants[END_REF]. In order to monitor downstream metabolic effects of increased PtdInsturnover in the transgenic plants, a range of candidate sinks for PtdIns-derived lipid backbones was analysed (Figure 7). Overexpression of PIS1 resulted in elevated DAG and PtdEtn levels in leaves of all transgenic lines tested (Figure 7 A and B), whereas that of PIS2 consistently supported increased polyphosphoinositide production (Figure 7 C and D). The hypothesis that PtdIns produced by PIS1 or PIS2 was channeled towards different routes of metabolic conversion was tested by tracing the glycerolipid backbones of phospholipid intermediates according to their fatty acid composition (Figure 8). The observation that PtdIns and other phospholipids increasing with PIS1-overexpression contained fatty acids with a lower degree of unsaturation, whereas PtdIns and phospholipids increasing with PIS2overexpression contained fatty acids with a higher degree of unsaturation, suggests that the particular phospholipids may have their metabolic origin in distinct PtdIns-populations formed by PIS1 or PIS2, respectively. The data are consistent with the results of the in vitro characterisations, indicating differences in substrate preferences of PIS1 and PIS2 for CDP-DAG-species with particular fatty acid compositions. Influences of lipid-substrate fatty acid compositions on enzyme functionality have been previously demonstrated for other enzymes involved in the biosynthesis of inositol-lipids, including invertebrate PIP-kinases and phospholipase C [START_REF] Carricaburu | Phosphoinositide fatty acids regulate phosphatidylinositol 5-kinase, phospholipase C and protein kinase C activities[END_REF], and mammalian phosphoinositide phosphatases [START_REF] Schmid | Type II phosphoinositide 5-phosphatases have unique sensitivities towards fatty acid composition and head group phosphorylation[END_REF].

In contrast to the situation with PIS1, overexpression of PIS2 resulted in several-fold increased levels of PtdIns4P and PtdIns(4,5)P 2 (Figure 7 consequence of increased substrate availability for PI-kinases and PIP-kinases, respectively.

As the phosphorylation sequence leading to the formation of PtdIns(4,5)P 2 is likely not ERassociated in plants, PtdIns originating from PIS2-activity and entering this sequence must be delivered for further metabolic conversion by a different route than PtdIns produced by PIS1.

In summary, the data suggest that PIS1 and PIS2 may act in different metabolic contexts, resulting in PtdIns channeling towards different physiological pools. While it is clear that independent pools of inositol-lipids are present in plant cells [START_REF] Heilmann | Changes in phosphoinositide metabolism with days in culture affect signal transduction pathways in Galdieria sulphuraria[END_REF][START_REF] Heilmann | Plasma membrane phosphatidylinositol 4,5-bisphosphate levels decrease with time in culture[END_REF][START_REF] Kost | Rac homologues and compartmentalized phosphatidylinositol 4, 5bisphosphate act in a common pathway to regulate polar pollen tube growth[END_REF][START_REF] König | Stress-inducible and constitutive phosphoinositide pools have distinct fatty acid patterns in Arabidopsis thaliana[END_REF], the establishment of such pools may also be controlled by the distribution of lipids other than PtdIns. Future experiments will be aimed at elucidating the contribution of other phospholipid-biosynthetic enzymes in the formation of distinct lipid pools. error is approximately 15-20% and is not marked in the graphs for better clarity. Fatty acids are indicated by the bars: white, 16:0; light grey, 16:1? 9 ; diamonds, 18:0; dark grey, 18:1? 9 ; stripes, 18:2? 9,12 ; black, 18:3? 9,12,15 . For an estimate of fatty acid unsaturation in individual lipids in wild type and more transgenic lines, please refer to supplemental Table 1.

Biochemical

  Journal Immediate Publication. Published on 10 Apr 2008 as manuscript BJ20071371 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071371 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 10 Apr 2008 as manuscript BJ20071371 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071371 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 10 Apr 2008 as manuscript BJ20071371 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071371 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 10 Apr 2008 as manuscript BJ20071371 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071371 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 10 Apr 2008 as manuscript BJ20071371 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071371 S t a g e 2 ( a ) P O S T -P R I N T Fluorescence microscopy.

  Biochemical Journal Immediate Publication. Published on 10 Apr 2008 as manuscript BJ20071371 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071371 S t a g e 2 ( a ) P O S T -P R I N T mismatch against the published Arabidopsis genomic sequence at position 444 (G instead of C), resulting in the amino-acid exchange C148W, as indicated (Figure

Biochemical

  Journal Immediate Publication. Published on 10 Apr 2008 as manuscript BJ20071371 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071371 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 10 Apr 2008 as manuscript BJ20071371 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071371 S t a g e 2 ( a ) P O S T -P R I N T In order to test whether the metabolism of polyphosphoinositides or other phospholipids could be disturbed by overproduction of PtdIns in plants, PIS1 and PIS2 were individually overexpressed in transgenic Arabidopsis plants under the constitutive cauliflower mosaic virus 35S-promoter. Successful transformation was confirmed by PCR-based genotyping of glufosinate-ammonium-resistant transformants and detection of the 35S-PIS1 or 35S-PIS2 transgene (data not shown). The T2-progeny of resistant plants exhibited severely diminuished growth (Figure 4 A), indicating a substantial influence of transgene expression on physiology. Evaluation of plant size by automated leaf-area detection indicates a significant difference (p<0.01) in plant size compared to wild type plants grown in parallel (Figure 5 B).

  C and D). The lipid analyses performed Biochemical Journal Immediate Publication. Published on 10 Apr 2008 as manuscript BJ20071371 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071371 S t a g e 2 ( a ) P O S T -P R I N T indicate that despite of equivalent accumulation of PtdIns with overexpression of PIS1 or PIS2 (Figure 6 A) different lipid-metabolites were affected by PtdIns-overproduction through each PI-synthase isoform.

  DAG and PtdEtn shifted to contain less (30-45 mol%) polyunsaturated fatty acids and a greater proportion (55-65 mol%) of saturated and monounsaturated fatty acids (Figure 8 B), whereas the fatty acid composition of polyphosphoinositides was not obviously altered. In contrast, overexpression of PIS2 resulted in an increased proportion of 55-75 mol% polyunsaturated fatty acids associated with all lipids tested, most remarkably in PtdIns4P and PtdIns(4,5)P 2 (Figure 8 C). The proportion of polyunsaturated fatty acids associated with PtdIns4P and PtdIns(4,5)P 2 changed notably from only 25 and 15 mol% of the associated fatty acids in wild type plants, respectively, to 50 and 45 mol%, respectively, in plants overexpressing PIS2 (compare Figure 8 A with C). The data indicate that the degree of unsaturation of fatty acids associating with phospholipids affected by PI-synthase overexpression changes differentially for PIS1 or PIS2. Changes in the degree of unsaturation of fatty acids associated with phospholipids resulting from overexpression of PIS1 or PIS2 are summarized in supplemental Table 1. Biochemical Journal Immediate Publication. Published on 10 Apr 2008 as manuscript BJ20071371 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071371 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 10 Apr 2008 as manuscript BJ20071371 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071371 S t a g e 2 ( a ) P O S T -P R I N T lipid biosynthetic pathways in the ER and the secretory machinery of the Golgi. It is our working hypothesis that PI-synthases act in subtly-different metabolic contexts, and that the action of PIS1 and PIS2 may supply different pools of inositol-containing lipids with PtdIns.

  B and C), most likely as a Biochemical Journal Immediate Publication. Published on 10 Apr 2008 as manuscript BJ20071371 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071371 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 .

 1 Figure 1. Alignment of deduced amino acid-sequences of PI-synthases from different biological origins. Sequences were aligned using ClustalW and the alignment was optimised manually. Residues identical in five or more sequences are indicated in black, similar amino acids in grey. The dotted line indicates the conserved CDP-alcohol phosphatidyltransferasedomain. Black lines indicate hydrophobic domains suggested by analysis using TMpred [56]. The arrow indicates amino acid-position 148 of the deduced Arabidopsis PIS2-sequence affected (C148W) by the single-nucleotide-polymorphism (G444C) of the PIS2-allele isolated in this study. Origin of sequences: At, Arabidopsis thaliana; Os, Oryza sativa; Zm, Zea mays; Nc, Neurospora crassa; Mm, Mus musculus; Hs, Homo sapiens.

Figure 2 .

 2 Figure 2. PI-synthase activity of recombinant Arabidopsis PIS1 and PIS2 proteins in vitro. PIS1 and PIS2 proteins were heterologously expressed in E. coli C43(DE3) cells. Protein contents were adjusted according to immunodetection. Catalytic activity of recombinant PIS1 and PIS2 proteins was tested in total solubilised membrane extracts by monitoring the conversion of CDP-DAG and D-myo-inositol substrates to PtdIns. Lipids were visualised by charring and identified according to authentic standards, as indicated. Data shown are from two representative experiments. The experiment was performed three times with similar results.

Figure 3 .

 3 Figure 3. Substrate preferences of recombinant PIS1 and PIS2 for different CDP-DAG species. Recombinant PIS1 and PIS2 enzymes were tested in vitro against various species of CDP-DAG differing in the associated fatty acids. CDP-DAG substrate prior to conversion (white) and PtdIns formed by PIS1 (grey) or PIS2 (black) after conversion were reisolated from TLC-plates and quantified by GC-analysis of associated fatty acids. A, PtdIns formation from individual synthetic substrates. B, PtdIns formation from a mixture of synthetic substrates. C, A mixture of "biological" CDP-DAG species was synthesized from PtdOH isolated from Arabidopsis rosette leaves, and PtdIns formation from this substrate mixture was tested. First, the fatty acid composition of the CDP-DAG substrate was analysed (CDP-DAG input). After in vitro conversion by PIS1 or PIS2, the fatty acid compositions of nonconverted CDP-DAG (CDP-DAG residue) and of PtdIns produced (PtdIns product) was analysed, as indicated. Fatty acids are indicated at the bottom and are also distingushed by the

Figure 4 .Figure 5 .

 45 Figure 4. Subcellular distribution of Arabidopsis PIS1 and PIS2 transiently expressed in onion epidermal cells. PIS1 and PIS2 were transiently expressed in onion epidermal cells as fusion proteins to c-terminally-attached EYFP-or CFP-reporters. The fluorescence distribution was synchronously monitored for EYFP (left panels)and CFP (middle panels). The right panels show the merged image. Yellow colour indicates colocalisation. A, Coexpression of PIS1-EYFP and PIS2-CFP. Arrowheads, punctate structures with distinct patterns of EYFP and CFP-fluorescence. Inset, Magnified view of punctate structure. B, Gathering of punctate structures sometimes observed with coexpression of PIS1-EYFP and PIS2-CFP. C, Coexpression of PIS1-EYFP with the Golgi-marker, CFP-Sial. Bars, 10 µm (A, C); 5 µm (B)

Figure 6 .

 6 Figure 6. Increased levels of PtdIns and PtdOH in Arabidopsis plants constitutively overexpressing PIS1 or PIS2. The levels of isolated phospholipid classes were determined by GC-quantification of associated fatty acids relative to data obtained with total lipid extracts. A, PtdIns; B, PtdOH. Data are the means of 7-10 plants each for wild type (WT, white) and independent PIS1 (grey) or PIS2 (black) overexpressor lines, as indicated ± SD.Lipid levels in the transgenic lines are significantly increased over those of wild type plants according to a student's t-test (p<0.01).

Figure 7 .

 7 Figure 7. Differential effects of PIS1 or PIS2 overexpression on lipid metabolism. The levels of isolated phospholipid classes or DAG were determined by GC-quantification of

Figure 8 .

 8 Figure 8. Differential effects of PIS1 or PIS2 overexpression on phospholipid fatty acid composition. The fatty acid compositions of PtdIns, PtdOH, DAG, PtdEtn, PtdIns4P and PtdIns(4,5)P 2 were analysed by GC for wild type plants (left panel) and for plants overexpressing PIS1 (middle panel) or PIS2 (right panel). The proportion of each fatty acid is given as mol% of the associated fatty acids of each lipid. Fatty acids correspond to the bar segments, as indicated at the right. Data represent the means of 7-10 plants for wild type and 7-10 plants of the representative overexpressor lines, PIS1-1 and PIS2-1. The experimental

BiochemicalFigure 1 Biochemical 2 Biochemical

 12 Figure 1

Figure 4 PIS1

 4 Figure 3

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Acknowledgments

We would like to thank Dr. Ben Scheres for the Golgi-marker construct. We also thank the following individuals at Georg-August-University Göttingen: Dr. Martin Fulda for plasmid-DNA; Dr. Oliver Einsle for the E. coli C43 cells; Dr. Andreas Wodarz for access to and Dr. Michael Krahn for expert assistance with confocal microscopy; Susanne Mesters for expert plant culture; Dr. Ivo Feussner for helpful discussion. We gratefully acknowledge generous funding through an Emmy-Noether grant from the German Research Foundation (DFG, to I.H.).