Expression pattern and subcellular localization of Arabidopsis purple acid phosphatase AtPAP9

Gene Expression Patterns 14 (2014) 9–18

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Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep

Expression pattern and subcellular localization of Arabidopsis purple acid phosphatase AtPAP9 Katayoun Zamani a, Tahmineh Lohrasebi a, Mohammad S. Sabet b, Mohammad A. Malboobi a,⇑, Amir Mousavi a a b

Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, Islamic Republic of Iran Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Tarbiat Modares University, P. O. Box 14115-336, Tehran, Islamic Republic of Iran

a r t i c l e

i n f o

Article history: Received 27 November 2012 Received in revised form 9 August 2013 Accepted 12 August 2013 Available online 6 September 2013 Keywords: Arabidopsis AtPAP9 Cell adhesion Fungal infection Phosphate

a b s t r a c t Purple acid phosphatase (PAP; EC 3.1.3.2) enzymes are metallophosphoesterases that hydrolysis phosphate ester bonds in a wide range of substrates. Twenty-nine PAP-encoding loci have been identified in the Arabidopsis genome, many of which have multiple transcript variants expressed in response to diverse environmental conditions. Having analyzed T-DNA insertion mutants, we have provided strong pieces of evidence that AtPAP9 locus encodes at least two types of transcripts, designated as AtPAP9-1 and AtPAP9-2. These transcript variants expressed distinctly during the course of growth in medium containing sufficient phosphate or none. Further histochemical analysis by the use of AtPAP9-1 promoter fused to b-glucuronidase reporter gene indicated the expression of this gene is regulated in a tissue-specific manner. AtPAP9-1 was highly expressed in stipule and vascular tissue, particularly in response to fungal infection. Subcellular localization of AtPAP9-1:green fluorescent fusion protein showed that it must be involved in plasma membrane and cell wall adhesion. Ó 2014 Published by Elsevier B.V.

Purple acid phosphatase (PAP) enzymes catalyze the hydrolysis of a wide range of phosphoric acid mono- or di-esters and anhydrides at acidic or neutral pH (Cox et al., 2007). The characteristic pink or purple color of purified PAP proteins is related to charge transitions between a tyrosine residue and chromophoric ferric ion in the binuclear center (Plaxton and Tran, 2011). Multiple sequence alignments of eukaryotic and prokaryotic PAPs revealed seven invariant residues contained in five blocks of conserved amino acid sequences required for metal coordination, DXG/GDXXY/ GNH(D/E)/VXXH/GHXH (bold letters represent metal-ligating residues; Li et al., 2002). Structurally, plant PAP proteins are categorized into high molecular weight (HMW) and low molecular weight (LMW) phosphatases. The former is thought to be functional in homodimeric form while the latter is typically monomeric carrying only metallophosphoesterase motifs (Klabunde et al., 1996). As contrasted to functional monomeric LMW ones, the biological importance of a fibronectin motif within the N-terminus of HMW PAPs is still unknown. Our current knowledge suggests other functions for some plant PAPs as well. For example, AtPAP17 and AtPAP26 have both acid ⇑ Corresponding author. Tel./fax: +98 21 44580369. E-mail addresses: [email protected] (K. Zamani), [email protected] (T. Lohrasebi), [email protected] (M.S. Sabet), [email protected] (M.A. Malboobi), [email protected] (A. Mousavi). 1567-133X/$ - see front matter Ó 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.gep.2013.08.001

phosphatase and alkaline peroxidase activity. They could be involved in phosphate ion (Pi) scavenging and recycling as well as the metabolism of reactive oxygen species (del Pozo et al., 1999; Veljanovski et al., 2006). In comparison, AtPAP12 secreted by Pi-deficient Arabidopsis suspension cells and seedlings was highly active against several Pi-ester substrates over a broad range of pH range, making it ideally suited for scavenging Pi from the organic-Pi pools prevalent in many soils (Tran et al., 2010). The expression of several PAP-encoding genes in response to available Pi has been documented by several researchers (for example, see Lohrasebi et al., 2007; Misson et al., 2005; Morcuende et al., 2007; Wu et al., 2003). Increased expressions of PAP genes have also been reported in response to other environmental conditions including wounding, nematodes, insects, high NaCl, oxidative stresses and senescence (del Pozo et al., 1999; Feng et al., 2003; Jakobek and Lindgren, 2002; Liao et al., 2003; Liu et al., 2005; Lohrasebi et al., 2007; Williamson and Colwell, 1991). Recent studies have illustrated that PAP proteins are localized in various cellular components where they play different roles. For instance, AtPAP2 was localized in both plastids and mitochondria outer membrane (Sun et al., 2012). AtPAP12 and AtPAP10 were secreted out of the root cell (Tran et al., 2010; Wang et al., 2011) while AtPAP26 and AtPAP18 were dually targeted to vacuole and extracellular space (Tran et al., 2010; Zamani et al., 2012). In addition, it is well known that alternative-first-exon transcriptions as well as alternative splicing processes allow

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generation of large repertoire of proteins from a limited number of genes, enabling plants adaptation to changing environmental conditions (for a review see Reddy, 2007). Li et al. (2002) reported the existence of 29 loci within Arabidopsis genome encoding PAPs for which some preliminary expression data were presented. They also recorded two transcript variants for AtPAP9 gene located at At2g03450 locus at GenBank (Ac. Nos. AF492661 and AY090895). However, they did not provide any experimental evidence for the existence of these two transcripts and the expression level of each one. In the present work, we have shown the existence of two mRNA variants for AtPAP9 gene in Arabidopsis, named AtPAP9-1 and AtPAP9-2, by analyzing T-DNA knock-out lines. With this assumption, the expression patterns of the corresponding transcripts were compared in growing seedlings. As the variants differ for the Nterminal parts only, the importance of this sequence was investigated by further analysis of AtPAP9-1. Besides, the natures of cis-elements present in the AtPAP9-1 promoter leading to its responsiveness to biotic and abiotic stimuli were analyzed at tissue level. Having illustrated the subcellular location of AtPAP9-1 protein, this is the first report indicating a potential role in plasma membrane and cell wall adhesion for N-terminal domain of a HMW PAP protein.

1. Results and discussion 1.1. AtPAP9 mutation analysis Collections of T-DNA mutants of Arabidopsis were searched for disruptions in AtPAP9 locus. Two lines were identified with T-DNA insertion in the first and the second exons, named SALK_129905 and SALK_020806, respectively. Progenies of SALK_129905 line were genotyped by multiplex PCR method using a pair of AtPAP9 specific primers plus a primer derived from the T-DNA left boarder (Fig. 1A). Three homozygous plants were found for disruption in the first exon (Fig. 1B). However, no homozygous progeny for this line was found when over 130 progenies of self-pollinated heterozygote SALK_020806 parents were examined in the same way (Fig. 1C and D). Therefore, we proposed that SALK_020806 mutant possesses a recessive lethal trait. The segregation ratio close to 1:2 for wild type and the heterozygote progenies were in favor of this

hypothesis. This was further supported by the examination of immature siliques of the self-pollinated heterozygous plants in which there were a numbers of empty slots carrying aborted ovules and aborted shrunken brown seeds (Fig. 1E). The phenotypes of T-DNA insertion lines SALK_129905 versus wild type plants were compared on solid media containing sufficient or no Pi. Unlike SALK_020806 mutant, no detectable difference was noted in the growth rates or the appearance of shoots and roots of three-week old hetero/homozygote SALK_129905 mutants and wild type plants (data not shown). The above observations could only be explained by assuming the expression of an alternative transcript derived from the second exon in SALK_129905 helps plant survival. Its corresponding protein must have an important function that led to lethality if lacking. 1.2. Structural features of the AtPAP9 proteins Having a molecular weight of 74 kDa, AtPAP9-1 is classified as a HMW PAP with a noncatalytic domain at the N-terminal and a catalytic domain at C-terminal. As shown in Fig. 2, detailed analysis of AtPAP9-1 sequence revealed several important features including a signal peptide sequence at the N-terminus with 20 amino acid residues, an RSGD motif (position 33 to 36), a DLXXL motif (position 50 to 54) as recognition site of avb6 integrin (Kraft et al., 1999), two DGE motifs (position 165 to 167 and 568 to 570) as recognition site for a2b1 integrin (Coulson et al., 1997), a fibronectin type 3 (FNIII) domain (position 141 to 239), a metallophosphatase domain (position 254 to 487) and a transmembrane region (position 604 to 626). The T-DNA fragments were inserted upstream of the FNIII domain and transmembrane region in SALK-129905 line and SALK_020806, respectively (Fig. 2). 1.3. Expression patterns of AtPAP9 transcript variants Preliminarily, a comparative RT-PCR experiment was performed with cDNA molecules derived from wild type and the homozygous SALK_129905 seedlings using specific primer sets designed to amplify transcripts derived from each of the two exons (Fig. 3A). The amplification of two RT-PCR products with the expected sizes in wild type transcriptome and only one product corresponding to

Fig. 1. Characterization of T-DNA insertion line SALK_129905 and SALK_020806. (A and C) The structure of the AtPAP9 gene carrying T-DNA insert in the first and the second exons. Black boxes and line indicate exons and intron, respectively. The size of T-DNA is not drawn to scale. The locations of primers used for PCR genotyping (AP16F, 9-2F, 9MR, 9R and LBb1) are marked with arrows. (B) Illustration of homo/heterozygous T-DNA insertion loci by PCR on genomic DNA isolated from: (1) a wild-type plant; (2) a heterozygous mutant plant; and (3) a homozygous mutant plant. (D) Illustration of homo/heterozygous T-DNA insertion loci (SALK_020806) by PCR on genomic DNA isolated from: (1 and 2) heterozygous SALK_020806 plants; and (3) a wild type plant. M, 100-bp ladder DNA size marker. (E) Light microscopy of a wild-type immature siliques showing uniform seed development and heterozygous crossing SALK_020806 immature siliques containing aborted shrunken ovules (black arrowheads) and aborted brown shriveled seeds (white arrowheads).

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Fig. 2. AtPAP9-1 mRNA and protein sequences and known motifs. The signal peptide (1), fibronectin type III (2), metallophosphatase (3) and transmembrane domains (4) are boxed and numbered respectively. Integrin-ligand-related motifs (RSGD, DLXXL and DGE) are circled. All possible start sites (ATG and non-ATG) in the same open reading frame are underlined. Exon 1 and 2 boundary is shown by an open arrow head. The filled arrow heads indicated T-DNA insertion sites in the mutant lines.

the second exon in the mutant line reinforced the possibility of having two transcript variants, AtPAP9-1 and 2 (Fig. 3). Furthermore, this postulation was supported by illustrations of considerably different expression patterns for the presumed AtPAP9 transcripts in a series of time-coursed samples of plant grown in none or sufficient Pi (Fig. 4). Both transcripts were expressed in the examined organs and conditions, though the expression level of AtPAP9-2 was remarkably higher in the roots. Transcript levels of AtPAP9-1 increased within 3 days in the roots and shoots regardless of Pi availability. It returned to the initial level 7 days after growing in sufficient Pi while remained high in no-Pi roots for the duration of experiment (Fig. 4A). In contrast, the amounts of AtPAP9-1 transcripts gradually increased in Pi-fed shoots and decreased in no-Pi shoots for the samples taken after 7 and 14 days (Fig. 4B). Maximum AtPAP9–2 expression level was observed at day 3 followed by a sharp decrease to the basal level within 7 days in the well-fed roots. When exposed to Pi deprivation, AtPAP9-2 transcript level increased after 7 days and decreased slightly in the 14-day samples (Fig. 4C). In shoots, no significant changes were observed in AtPAP9-2 mRNA accumulation at the start and at the end of experiment period, while there were increases in its expression levels at days 7 and 3 for both Pi-fed and Pi-starved seedlings, respectively (Fig. 4D).

Interestingly, we observed that AtPAP9-2 expressed in response to high salt and fungi inoculation in roots and shoots of homozygous SALK_129905 plants (Data not shown). 1.4. Promoter sequence analysis of AtPAP9 A 1830-bp genomic DNA sequence upstream of AtPAP9-1 ATG codon in the first exon and the intronic sequence of it as AtPAP9-2 promoter were analyzed for the presence of known regulatory elements related to Pi availability (Supplementary Fig. S1). Scanning for Pi inducible cis-elements compiled by Muller et al. (2007), six AGTTTT elements, two NIT2 sequences, TATCTA and TATCTT, only one consensus Pho4 binding site (HLH), CA(T/ G)(A/C)TG, and four PHO-like, (G/T/A)(C/T/A)GTGG, sequences were found in AtPAP9-1 promoter. There are two PHO-like and one HLH elements in AtPAP9-2 promoter. Previously, Lohrasebi et al., 2007 indicated that AtPAP9-1 expression was induced by chitin and saline stress. Therefore, the promoter sequence was searched for known cis-element responsive to other environmental stresses too. The AtPAP9-1 promoter contains five WRKY box sequences, C/TTGACT/C two of which located within its 50 -UTR. AtPAP9-1 promoter also contains one AS-1 element, TGACG, at 1809 bp upstream of ATG codon. This element was earlier shown to be required for induced expression of

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pathogens as well as certain light treatments (Evrard et al., 2009). The AtPAP9-2 promoter contains one WRKY box sequence too.

A AtPAP9-1 AtPAP9-2

1.5. Localized expression of AtPAP9-1

B AtPAP9-1

AtPAP9-2

α-Tubulin Wild type

SALK_129905

Fig. 3. Expression of AtPAP9 transcript variants in wild type and mutant plants. (A) Illustrations of exons (thick lines), intron (thin line) and coding regions (solid lines) of AtPAP9 gene and primers used for expression analysis. (B) The expressions of AtPAP9 transcripts in wild type and in homozygous T-DNA insertion line SALK_129905. RT-PCR reactions were done for 28 cycles in shoots and roots with the specific primers for AtPAP9-1 (AP16F, R) and AtPAP9-2 (9-2F, 9MR) transcription variants as shown at the left sides. a-Tubulin transcript level was used as internal control (bottom panel).

the PR-1 gene by salicylic acid (Lebel et al., 1998). Another motif in this promoter is FORCA motif, (T/A)TGGGC, which is conserved in Arabidopsis clusters of co-expressed genes in response to fungal

Having in mind the presence of cis-elements in the promoter region, expression patterns were analyzed at tissue level in transgenic plants expressing GUS reporter gene fused to the AtPAP9-1 upstream sequence and grown in various environmental conditions. GUS staining patterns were recorded for at least ten independent T1 progenies of stably transformed Arabidopsis plants for which typical images are shown in Fig. 5. In the first set of experiments, seedlings grown in hydroponics culture were subjected to several abiotic stresses. When treated with jasmonic acid, abscisic acid, NaCl or H2O2, promoter activity was observed only at the root tips of 12-day old plant, though differed in color intensities (Fig. 5A and B). A very weak signal was found at the root tips in some of the plants grown under Pi starvation. In contrast, no GUS staining was detected when seedlings were treated with salicylic acid or grown in Pi-fed conditions. The strongest signal was detected for plants treated with jasmonic acid. If grown on solid medium, no staining was evident at the early stages of seedling establishment. A pale blue color was observed throughout the 4-week old Pi-fed plants. The signal intensity was stronger in vascular tissue and bract stipules (Fig. 5C–G). In contrast, relatively strong GUS staining was observed in the vascular cylinder of roots, petiole and the rosette leaves as scattered spots in 3-week old Pi-starved plants (Fig. 5H and I), particularly at trichoms bases (Supplemental Fig. S2).

Root

Shoot

B

C

D AtPAP9-2

Relative expression rates

AtPAP9-1

A

d 0

3

7

Days

14

0

3

7

14

Days

Fig. 4. Time-coursed expression of AtPAP9 transcript variants in wild type seedlings. Relative expression AtPAP9-1 (A and B) and AtPAP9-2 (C and D) transcript variants in roots (A and C) and shoots (B and D) are illustrated. Time 0 refers to when 14-day old seedlings were transferred into Pi sufficient (+P, black line) or Pi deficient (P, gray line) media and grown for indicated time periods when harvested for RNA extraction. The expression levels of AtPAP9-1 and 2 were distinguished by the use of specific primers in RT-PCR reactions (see Section 2). The band densities were quantified and standardized against a-tubulin gene as the internal control. Assuming similar amplification efficiencies, the value of RT-PCR products amplified with 9-2F and 9MR primers were subtracted from AtPAP9-1 expression value to calculate AtPAP9-2 expression level. Error bars represent standard errors among three replicates. Different letters show significant variations among the means as compared by Dunkan’s method at P < 0.05.

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Fig. 5. Tissue-specific expressions of AtPAP9-1 in various environmental conditions. The activity of AtPAP9-1 promoter fused to reporter gene was depicted by histochemical analyses of expressed GUS (see Section 2). (A–B) GUS activities in some epidermal cells at the root tips of 12-days old plants grown in liquid medium and treated with Jasmonic acid, abscisic acid, NaCl or H2O2. (C–G) Uniform staining 4-week old transgenic plants growth on Pi sufficient solid media. (C) whole plant, (D) petiole vascular tissue, (E) flower, (F) bract leaf and its stipule, (G) rosette leaf. (H–I) Rosette leaf and root of 3-weeks old transgenic plants grown in no Pi solid medium, blue color was seen in root vascular tissue. (J–M) GUS activity in 3-weeks old transgenic plants that inoculated with A. brassicicola. (J) the first and second internodes, (K) rosette leaf vascular tissue, and (L and M) bract stipules. (N–P) GUS activity in 3-weeks old transgenic plants that inoculated with H. arabidopsidis. (N) leaves and root junction, (O) rosette leaf, and (P) root. Arrows indicate: sp, stipule; st, stem; vs vascular tissue.

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When 18-day old Pi-fed transgenic plants were exposed to different fungi, blue color was only apparent if transgenic plants inoculated with Alternaria brassicicola or Hyaloperonospora arabidopsidis. Uniform staining of root tissues was observed in transgenic plants inoculated with A. brassicicola (Fig. 5J–M). Staining was much more intense in the junctions of roots and shoots, rosette leaves vascular tissues, predominantly in the first and the second internodes of stems and bract stipules. In comparison, high levels of reporter gene expression were detectable in the entire root and shoot tissues of the transgenic plants infected with pathogenic fungus H. arabidopsidis (Fig. 5N–P).

1.6. Subcellular localization of AtPAP9-1 Analysis by PSORT (Nakai and Horton, 1999) and Target P (Emanuelsson et al., 2000) revealed that AtPAP9-1 protein is very likely a transmembrane protein with N-terminal oriented towards outside. In order to find about the actual location of the AtPAP9-1 protein in plant cells experimentally, its signal peptide or the whole coding region were fused to GUS and GFP reporter genes (Supplementary Fig. S3). These constructs were used for both transient transformation onion (Allium cepa) epidermal cells and stable transformed of Arabidopsis plant under the control of the cauliflower mosaic virus 35S (CaMV-35S) promoter. As shown in Fig. 6, the signals for AtPAP9-1:GFP fusion proteins were detected in the region between plasma membrane and cell wall while the intact GFP protein was scattered throughout the cells. No blue color was observed in onion epidermal cells or Arabidopsis plants transformed with AtPAP9-1:GUS fusion construct. In order to recognize whether AtPAP9-1 was located on plasma membrane or cell wall, plasmolysis was performed by adding 30 percent sucrose solution. Interestingly, the plasma membrane did not detach from the cell wall in the cells overexpressing the transgene transiently while the collapse of neighboring cells was clearly evident (Supplementary Fig. S3). To investigate the importance of AtPAP9-1 signal sequence, a DNA fragment encoding for the first 49 amino acid residues at the N-terminus was fused to GUS protein under the control of the CaMV-35S promoter and transferred into Arabidopsis plants. Microscopy observation detected no specific targeting for the

fusion protein in the region between plasma membrane and cell wall (Supplementary Fig. S3).

1.7. AtPAP9 locus encodes both HMW and LMW PAP proteins Approximately forty-two percent of intron-containing genes in Arabidopsis have alternative mRNA species (Filichkin et al., 2010). It is believed that mRNA variants are involved in a range of functions including autoregulation of gene expression, signal transduction, disease resistance, biotic and abiotic stress responses, flowering time and the circadian clock (Simpson et al., 2008). Here, we report several pieces of evidence indicating the expressions of two types of transcripts derived from AtPAP9 locus by the use of T-DNA insertion mutants and expression analysis. Plant lacking AtPAP9-1 transcript variant showed no phenotypic change in comparison to wild type plants. Possibly, T-DNA insertion in the first exon affected only the long transcript with no detectable effect because a secondary promoter directs the expression of the alternative transcript. However, interruption in the second exon was lethal in homozygous progenies as it impairs both transcript variants. This is the first report of showing lethal effect for an acid phosphatase despite the fact that 29 PAP-encoding loci in Arabidopsis with several alternative transcripts could compensate for each other (Li et al., 2002; Lohrasebi and Malboobi, unpublished data). It is noteworthy that AtPAP9 putative first-exon alternative mRNA variants transcribed from a single locus encode both HMW and LMW PAPs. They share metallophosphatase and transmembrane domains while differ for having a signal peptide, some integrin-ligands motifs and a FNIII motif located at the N-terminal of AtPAP9-1. As already shown, alternative first exons can direct proteins localization to different cellular compartments (Chen et al., 2007). Such mRNA variants could be produced through alternative splicing process of the same transcript or – particularly in the case of large introns or distant start sites – through an alternative promoter to provide an extra level of gene expression regulation for the second transcript variant (Chen et al., 2007). The presented results showed that the former may not be true because the T-DNA insertion in the first exon (SALK_129905) did not affect the expression of the second transcript. Besides, the expression

Fig. 6. Subcellular localization of AtPAP9-1: GFP on onion epidermal cells. Intact GFP protein spread throughout the cytoplasm (A and B) while AtPAP9-1:GFP fusion protein was localized between plasma membrane and cell wall (C and D). Cells were observed by regular light (B and D) or UV-illuminated microscopy (A and C). Bars, 100 lm.

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pattern of the second mRNA variant shows that it must have its own regulatory sequences (Fig. 4). Such elaborations require further experimentations. 1.8. Various cis-regulatory elements direct AtPAP9-1 and AtPAP9-2 expression We have previously indicated constitutive AtPAP9-1 expression at low levels and its upregulation in response to Pi and sulfur starvation, cold and high-salt stresses as well as in the presence of chitin (Lohrasebi et al., 2007). Detailed analyses in this work showed that the expressions of AtPAP9 transcript variants were regulated differently during plant growth depending on the available Pi as in the most cases the resupply of Pi to starved plants led to a rapid return to the similar status as Pi-fed plants (Supplementary Fig. S4). For instance, the expression of AtPAP9-2 reduced to a very low level in the roots of Pi-starved plants and rapidly increased upon resupplying Pi such that the highest level occurred within 3 days. Up-regulation of AtPAP9-1 encoding gene in Pi-fed shoots in long term was consistent with the microarray data indicating its induced expression by developmental leaf senescence (Guo and Gan, 2012). Such differences in the expression levels must be due to the presence of various regulatory elements in the promoter regions of AtPAP9-1 and AtPAP9-2. As shown previously, the regulatory elements in the promoter of a Pi starvation-induced gene, psr3, interacted by different sets of specific transcription factors depend on the available Pi levels (Malboobi et al., 1998). The most frequent motif in AtPAP9-1 promoter is AGTTTT and then PHO-like element. AGTTTT found in the promoter of Pi-repressed genes in Arabidopsis leaf (Muller et al., 2007). This motif could be responsible for AtPAP9-1 down-regulation in the shoots when Pi starvation is continued for 7–14 days (Fig. 4B and Supplementary Fig. S4). Also, there are four PHO-like elements in the promoter of AtPAP9-1 while AtPAP9-2 promoter carries only two of it. PHO-like elements were found preferentially in promoter regions of early P-deficiency responsive genes (Hammond et al., 2003). Interestingly, none of the other five known Pi responsive cis-regulatory elements named PHR1binding site, P responsive element, TC–rich motif, HD-ZIP and GAATAT motifs (Muller et al., 2007) were found in AtPAP9-1 promoter. This analysis also revealed the existence of multiple pathogenesis, WRKY-box and abiotic responsive elements in AtPAP9-1 promoter regions. Microarray studies in Arabidopsis have revealed the roles of WRKY transcription factors in drought, cold, or high salinity stress (Seki et al., 2002). Also, it is known that WRKY75 positively regulates Pi starvation responses while negatively regulates lateral root and root hair growth (Devaiah et al., 2007). In addition WRKY factors act in a complex defense response network as both positive and negative regulators (Eulgem and Somssich, 2007). Consistently, AtPAP9-1 induction by chitin treatment was already reported (Lohrasebi et al., 2007). However, only inoculation with A. brassicicola (non-pathogenic) or H. arabidopsidis (pathogenic) out of four examined fungus species induced the promoter activity. Besides, the promoter:GUS staining observations indicated that AtPAP9-1 expression is distinctively regulated in certain cells and tissues. As such, the highest expression level was observed in the bract leaf stipules of seedlings infected with A. brassicicola (Fig. 5). It seems to us that AtPAP9-1 expression in response to fungus must be species specific and rather complex. 1.9. Possible roles of AtPAP9-1 protein AtPAP9 shares up to 72% sequence identity with AtPAP2 at amino acid sequence level which was shown to be targeted to both plastids and mitochondria via its C-terminal hydrophobic motif

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and to modulate carbon metabolism (Sun et al., 2012). However, a sequence comparison showed that AtPAP2 and AtPAP9 share only 54% identity at their C-terminals (data not shown). Now, it is well known that gene duplication is one of the primary source of new genes with novel or altered functions. In particular, alteration in protein subcellular localization is known to be a common mechanism for the functional diversification of duplicated genes (Marques et al., 2008). In agreement with this, we have illustrated the presence of AtPAP9-1:GFP between cell wall and plasma membrane (Fig. 6). To our knowledge, only a few cell wall or cell membrane-associated PAP proteins have already been identified, such as a glycosyl-phoshatidylinositol-anchored phosphatase isolated from Spirodela oligorhhiza cell membranes (Nakazato et al., 1998), PvPAP3 localized in plasma membrane of common bean cells (Liang et al., 2010), and four PAP isolated from tobacco cell wall that dephosphorylate wall proteins (Kaida et al., 2003). Protein sequence alignment and motif searches revealed the presence of FNIII- like domain in the N-terminal of all Arabidopsis HMW PAP proteins (Supplementary Fig. S5). FNIII domains are antiparallel b-structures composed of 90–100 amino acid residues interacting with integrins (Bork and Doolittle, 1992). In addition to FNIII, AtPAP9-1 possesses four integrin recognition motifs, one DLXXL, two DGE and one RSGD and a transmembrane domain (Fig. 2). These together resemble the structure of integrin ligands carrying interacting sites with integrin-like proteins in extracellular matrix (Coulson et al., 1997; Plow et al., 2000) that are identified as components of adhesome complex clamping plasma membrane to cell wall in plant cells. The integrin adhesome consist of over 150 distinct components including kinases, phosphatases and adaptor proteins which contribute to signaling events primarily (for a review see Kim et al., 2011). A number of integrin-like proteins have been identified in plants so far. For instance, Lu et al. (2012) have recently indicated that an integrin-like protein, AT14A, is a component of the cell wall-plasma membrane-cytoskeleton continuum. The at14a-1 mutant cells exhibit almost no cell clusters formation, while the AT14A over-expressed cells showed formation of large cell clusters, mostly over 10 cells per cluster. In line with the above, we found a tight association between cell wall and plasma membrane by transient expression of AtPAP9-1:GFP (Fig. 6 and Supplementary Fig. S3). Moreover, AtPAP9 promoter activity patterns resemble those of WAKL5 in Arabidopsis. For example, both of them expressed in bract leaf stipules and not in rosette leaf stipules (Verica et al., 2003). WAKL5 is a cell wall-associated kinase involved in signaling of plant defense response. The coexpression patterns of these two genes as well as co-localization of their gene products are considerably interesting, particularly with respect to coordinated reversible on/off switching of signaling components. In the same way, Knepper et al. (2011) identified an integrin-like protein NDR1, known as diseases resistance protein, with a broad role both in mediating cell wall-plasma membrane association and in signaling of biotic and abiotic stress responses. Such a postulated role for AtPAP9-1 needs to be investigated from several points of views.

2. Experimental procedures 2.1. Plant materials, culture conditions and treatments Seeds of Arabidopsis thaliana ecotype Col-1 were obtained from Arabidopsis Biological Resource Center (ABRC, Ohio State University, Ohio). Seeds of two selected T-DNA insertion lines of AtPAP9, SALK_129905 and SALK_020806, were obtained from SALK Institute Genomic Analysis Laboratory (http://signal.salk.edu/) and propagated on soil. The progenies were screened for T-DNA insertion by PCR using gene-specific primers, AP16F

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(50 -ATGATCGCCGCCGTTTACACTCTCTTC-30 ) and 9MR (50 -CCATAA AGCAACCGTCACATTGTTC-30 ) for SALK_129905 and 9-2F (50 -GAAATGAGAGCTCAGAAGAAACATTAG-30 ) and 9R (50 0 TTGATATACAAATCTGAGCTCCAAACAAACAA-3 ) for SALK_020806 and a primer anchored in the T-DNA left border, LBb1 (50 -GCGTGGACCGCTTGCTGCAACT-30 ). The correctness of PCR products were confirmed by sequencing. Interruption of the AtPAP9 gene was further confirmed by RT-PCR (see below) using gene-specific primers AP16F and AP16R (50 -ATTCCTCTCCGATTCACCCAC-30 ) for SALK_129905 and 9-2F and 9MR for SALK_020806. Seed sterilization and hydroponics growth conditions were as described by Lohrasebi et al. (2007). Prior to the treatments, 11day old seedlings grown on solid MS medium (Murashige and Skoog, 1962) were transferred into 15 ml of half-strength liquid MS medium supplemented with 1% sucrose and incubated for 3 days to achieve identical nutritional states. Then, the 14-day old seedlings were subjected to 5 mM Pi-fed and Pi-depleted treatments for 14 days. For Pi deprivation, 5 mM KCl replaced KH2PO4 in half-strength MS medium. To analysis AtPAP9-2 response to fungi and salt stresses, wild type and mutant plants cultured as above. Fungal inoculums A. brassicicola with spores were added to liquid medium in order to infect 20-day old Pi-fed plants. The infected plants were placed in a growth chamber for 24 h before harvesting. A group of Pi-fed plants were treated by 150 mM NaCl for 1 day before harvesting. In all cases, the culture medium was refreshed twice a week. Transgenic plants carrying AtPAP9-1 promoter:GUS construct (see below) were grown on solid MS medium for 7 days and then transferred into liquid MS medium lacking or containing 5 mM Pi for 5 days or grown in liquid MS medium containing 5 mM Pi and treated with either 250 mM NaCl, 4 mM H2O2, 100 mM abscisic acid, 50 mM Jasmonic acid or 100 mM salicylic acid for the last 24 h. For another set of treatments, 7-day old seedling of transgenic plants transferred to 18-cm Petri dishes containing solid MS medium with or without Pi for 12 days (2 seedlings per Petri dish). As a biotic stress, various fungi including A. brassicicola and H. arabidopsidis were used to inoculate Pi-fed plants and incubated for 2– 3 days as described (Hermanns et al., 2008; Schenk et al., 2003). All experiments were repeated two times with more than 10 plants. 2.2. RNA extraction and cDNA synthesis Shoots and roots were sampled from two separate set of hydroponically cultured seedlings at the four indicated time points for total RNA preparation (Fig. 4). Total RNA was extracted using a commercially prepared guanidine reagent RNX (Cinagen, Tehran, I.R. Iran) according to the manufacturer instruction. The RNA samples were further treated with the DNase free RNase A (Roche, Basel, Switzerland) according to the supplier instruction to eliminate genomic DNA contamination. For cDNA synthesis, 2 lg of each RNA sample was added to a reaction consisting of 1 ll of 100 lM oligo-dT(12–15) primer, 1 ll of 10 mM dNTP mix and sterile water up to a volume of 10 ll. The reaction was heated at 70 °C for 20 min and cooled on ice. 10 ll of master mix was added to each reaction which contained 2 ll 10xRT buffer, 0.1 M dithiothreitol, 50 mM MgCl2, 40 units of RNAse inhibitor (Roche Applied Sciences, Germany) and 50 units of Superscript II reverse transcriptase (Fermentas, Lithuania). The reaction was incubated at 42 °C for 1.5 h. The normalization of the cDNA contents of RT-PCR reactions were performed by comparing to the amplified transcripts of a-tubulin. 2.3. Semi-quantitative RT-PCR In order to evaluate the level of AtPAP9 transcript variants, a series of semi-quantitative RT-PCR were conducted. Two pairs of

AtPAP9 specific primers for exon 1, AP16F and AP16R, and exon 2, 9-2F and 9MR, and one pair for a-tubulin gene, Tub + 1 (50 -GCTTTCAACAACTTCTTCAG-30 ) and Tub-1(50 -CATCGTACCACCTTCAGACAC-30 ), were used in the PCR reactions. In all reactions, Master Mix PCR solution (CinnaGen, Tehran. I.R. Iran), 2 pmol of each primer and 0.1–1 ll cDNA were mixed in a volume of 20 ll and placed on a thermocycler (Peqlab, Germany). The PCR program was 1cycle of 4 min denaturation at 94 °C, 28–30 cycles of 1 min denaturation at 94 °C, 1 min annealing at 65 °C and 1 min extension at 72 °C and a final extension step at 72 °C for 5 min. The band intensities in ethidium bromide-stained gel images were quantified by TotalLab software (Phoretix International, New Castle, UK) and standardized against the level of transcripts of a-tubulin gene as the internal control. Assuming similar amplification efficiencies, the values of RT-PCR products amplified with 9-2F and 9MR primers were subtracted from AtPAP9-1 expression values to calculate AtPAP9-2 expression level. 2.4. AtPAP9-1 protein and promoter sequence analyses Conserved amino acid motifs were primarily recognized through searches in secondary database such as SMART (http:// smart.embl-heidelberg.de/; Letunic et al., 2009) and Pfam (http:// www.sanger.ac.uk/Software/Pfam/; Bateman et al., 2004). The AtPAP9-1 sequence was also searched manually for the integrinligand motifs described in Coulson et al. (1997) and Plow et al. (2000) using Editseq/DNASTAR software (DNASTAR, Madison, WI). For localization signals, detailed sequence analyses were done using tools in PSORT (Nakai and Horton, 1999) and TargetP sites (Emanuelsson et al., 2000). A1830-bp sequence upstream of the translation start codon of AtPAP9-1 gene was downloaded from Arabidopsis genome accessible through Map Viewer (http://www.ncbi.nlm.nih.gov/projects/ mapview/) was scanned with PLACE search tool (http:// www.dna.affrc.go.jp/htdocs/PLACE;). In addition, the AtPAP9-1 promoter region was scanned manually for all known Pi related ciselements described by Muller et al. (2007). 2.5. AtPAP9-1 promoter:GUS construct and transformation Genomic sequence located 1979-bp upstream of the AtPAP9-1 translation start site was amplified by PCR and introduced in T/A cloning vector (Fermentas, Lithuania). The utilized oligonucleotides were 50 -CGGTGGAGGAGTGAGAGTGGGAAGT-30 and 50 -TGTAAACGG CGGGGATCCTGATTTA-30 . The amplified fragment was digested with HindIII (at position 1830 of the promoter sequence from ATG) and BamHI (within the second primer) enzymes and ligated upstream of b-glucoronidase (GUS) gene in pAM194 binary vector (kindly provided by Plant Breeding Institute of Kiel University, Germany). The resulting construct, AtPAP9-1 promoter:GUS was transferred into Agrobacterium tumefaciens strain GV3101 via freeze and thaw method (Holsters et al., 1978) and then into A. thaliana by vacuum infiltration method (Bechtold and Pelletier, 1998). Having screened transgenic plants by PCR, one homozygous line was chosen to produce T3 generation which was used for the gene expression analysis in various biotic and abiotic conditions as explained above. GUS activity was detected in situ by incubating the plant materials in 5-bromo-4-chloro-3-indolyl b-Dglucuronide solution as described by Jefferson et al. (1987). 2.6. Constructs for subcellular localization of AtPAP9-1 To make the CaMV35S:AtPAP9-1-GUS and CaMV35S:AtPAP91-GFP fusion constructs, the open reading frame of AtPAP9-1 was PCR amplified with a pair of primers, 9F (50 -AAC CAC CAT CCT CCG CCT CTA GAG TAA ATC AT-30 ) and PAP9SPR (50 -CAA ATC AGT

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TGA ATC TAG ACT CCT CG-30 ). The PCR products were cloned into pTZ57R/T vector prior to subcloning into pBI121 (Clontech, Palo Alto, CA, USA) and pBIGFP at XbaI site (Mousavi et al., 1999). To make the CaMV35S:SP9-GUS, a DNA fragment (198 bp from the start codon of AtPAP9-1 coding region) was amplified by 9F and SP9R 5‘- ATT CCT AAC CAG TCG AGT CTA GAC GGT GAT TC-3‘ primers and subcloned into XbaI Sites of the pBI121 as an N-terminal translational fusion. The correctness of all cloning products was verified by restriction enzyme digestion and DNA sequencing. The CaMV35S:AtPAP9-1-GUS, CaMV35S:AtPAP9-1-GFP and also the intact GUS and GFP genes were transferred into the onion (Allium cepa L.) epidermal cells by particle bombardment using a Biolistic PDS/1000 Helium System (Bio-Rad, USA). Bombardment parameters were as follows: 1100 psi bombardment pressure, 1.0-lm gold particles and a target distance of 9 cm. The transformed epidermal cells were observed after 24 h by a confocal scanning microscopy system (Model TCS SP2; Leica). For plasmolysis, the transformed cells were treated with 30 percent sucrose solution before inspections. The CaMV35S:AtPAP9-1-GUS and CaMV35S:SP9-GUS constructs were transferred into A. thaliana as above. 2.7. Statistical analysis Expression data were statistically analyzed in a completely randomized design with at least three replications. Dunkan’s method was used to determine significance differences between the means at P < 0.05 using SPSS software V.16 (Statistical Package for the Social Science). Acknowledgments We gratefully acknowledge National Institute of Genetic Engineering and Biotechnology for funding through grant Nos. 113, 205 and 294 as well as the Salk Institute Genomic Analysis Laboratory for providing the sequence indexed Arabidopsis T-DNA insertion mutants. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.gep. 2013.08.001. References Bateman, A., Coin, L., Durbin, R., Finn, R.D., Hollich, V., Griffiths-Jones, S., Khanna, A., Marshall, M., Moxon, S., Sonnhammer, E.L.L., Studholme, D.J., Corin Yeats, C., Eddy, S.R., 2004. The Pfam protein families database. Nucleic Acids Res. 32, D138–D141. Bechtold, N., Pelletier, G., 1998. In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol. Biol. 82, 259–266. Bork, P., Doolittle, R.F., 1992. Proposed acquisition of an animal protein domain by bacteria. Proc. Natl. Acad. Sci. U.S.A. 89, 8990–8994. Chen, W.H., Lv, G., Lv, C., Zeng, C., Hu, S., 2007. Systematic analysis of alternative first exons in plant genomes. BMC Plant Biol. 7, 55. Coulson, B.S., Londrigan, S.L., Lee, D.J., 1997. Rotavirus contains integrin ligand sequences and a disintegrin-like domain that are implicated in virus entry into cells. Proc. Natl. Acad. Sci. U.S.A. 94, 5389–5394. Cox, R.S., Schenk, G., Mitic, N., Gahan, L.R., Hengge, A.C., 2007. Diesterase activity and substrate binding in purple acid phosphatases. J. Am. Chem. Soc. 129, 9550– 9551. del Pozo, J.C., Allona, I., Rubio, V., Leyva, A., de la Pena, A., Aragoncillo, C., Paz-Ares, J., 1999. A type 5 acid phosphatase gene from Arabidopsis thaliana is induced by phosphate starvation and by some other types of phosphate mobilising/ oxidative stress conditions. Plant J. 19, 579–589. Devaiah, B.N., Karthikeyan, A.S., Raghothama, K.G., 2007. WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis. Plant Physiol. 143, 1789–1801. Emanuelsson, O., Nielsen, H., Brunak, S., von Heijne, G., 2000. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J. Mol. Biol. 300, 1005–1016. Eulgem, T., Somssich, I.E., 2007. Networks of WRKY transcription factors in defense signaling. Curr. Opin. Plant Biol. 10, 366–371.

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