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Molecular characterization and expression of PsPK2, a PINOID-like gene from pea (Pisum sativum)
Plant Science 168 (2005) 1281–1291 www.elsevier.com/locate/plantsci
Molecular characterization and expression of PsPK2, a PINOID-like gene from pea ( Pisum sativum) Fang Bai a, John C. Watson b, Jason Walling c, Norman Weeden c, Aaron A. Santner b, Darleen A. DeMason a,* b
a Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA Department of Biology, Indiana University–Purdue University Indianapolis, 723 West Michigan Street, Indianapolis, IN 46202, USA c Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59717, USA
Received 26 December 2004 Available online 22 January 2005
Abstract The plant hormone auxin acts as a signal for cell division, extension and differentiation during the course of plant growth and development. With the aim of elucidating the molecular mechanism of auxin responses in pea leaf development, we have isolated a putative pea PINOID ( PID) homolog, PsPK2, and characterized it. The deduced protein sequence of PsPK2 had 445 amino acids and possessed 12 conserved catalytic subdomains that characterize protein kinases. The PsPK2 sequence was most closely related to PID, and together they defined a PINOID subfamily within the AGC-VIII protein kinases. PsPK2 mapped to the lower portion of linkage group VI between Pl and Gsp. By genome walking, 2.3 kb of the PsPK2 promoter was obtained and was predicted to possess auxin and gibberellin response elements. PsPK2 was strongly expressed in developing organs of wildtype (WT) pea, such as shoot tips, immature adult leaves, developing embryos and flower buds. Comparing the PsPK2 mRNA levels from tendrils and leaflets of WT as well as the other leaf form mutants, we found that irrespective of genotype, PsPK2 mRNA was much more abundant in tendrils than in leaflets. Furthermore, differential expression occurred in shoot tip RNAs of the different leaf form genotypes. Expression was slightly higher in the afila genotypes, and lower in acacia/tendrilless and tendrilledacacia compared to WT. In an auxin induction experiment, PsPK2 mRNA levels increased most after methyl-IAA treatment compared to IAA, 4-Cl-IAA, IBA and NAA treatments. Time course and concentration dependent experiments showed that PsPK2 mRNA levels were the highest after 30 min of methyl-IAA treatment compared to control and other treatments, and peaked at 10 mM methyl-IAA. In addition, PsPK2 mRNA levels peaked after 4 h of GA3 treatment and were highest in response to 75 mM GA3. Therefore, auxin and gibberellin positively regulate PsPK2 expression and it may play a role in the control of auxin transport and leaf form generation in pea. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Auxin; Gibberellin; Pea; PINOID; PsPK2; Ser/Thr protein kinases
1. Introduction Auxin is perceived by plant cells, and depending on their type and status, is rapidly transduced into a wide variety of responses in growth and development. Some of these responses include changes in direction of growth, in shoot and root branching and in vascular tissue differentiation. Auxin is transported in a strictly polar manner in plants. PIN1, the putative auxin efflux carrier, controls polar auxin * Corresponding author. Tel.: +1 951 827 3580; fax: +1 951 827 4437. E-mail address: [email protected].
transport via its position and function [1–3]. Recently, Benkova´ et al. [4] showed that lateral organ initiation and/or outgrowth requires local accumulation of auxin and this process requires correct targeting of PIN1. A recent model predicts that many of the components that mediate glucose transporter targeting in mammalian cells responsive to insulin have orthologs in auxin efflux carrier targeting [5]. These authors propose that protein phosphorylation by a protein kinase plays an important role in PIN1 targeting. DeLong et al. proposed that protein phosphorylation plays important regulatory roles in auxin signaling and response [6]. Generally, plant protein kinases can be placed
0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.01.005
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in one of three groups based on their ability to phosphorylate serine and threonine or tyrosine or histine residues. They are the serine/threonine (Ser/Thr) kinases, the tyrosine (Tyr) kinases, and the histine (His) kinases [7]. Protein kinases share many conserved structural features including sequence and three-dimension arrangements of the catalytic domain [7,8]. The functions of plant protein kinases are poorly known, although many have been identified in the past few years. The Arabidopsis Ser/Thr kinase predicted by Muday and Murphy [5] to participate in targeting of the auxin efflux carrier is PINOID (PID). The pid mutant was first described by Bennett et al. [9]. pid mutants share many characteristics with pin1 mutants, such as reduced auxin transport, naked inflorescences and defects in form and position of lateral organs, including cotyledons, leaves and floral organs. Christensen et al. [10] identified PID as a unique Ser/Thr kinase and characterized various aspects of PID expression. They found that it is expressed in developing embryos, the shoot apical meristem, leaf primordia and in the initial stages of floral organ development. They concluded that PID plays roles in positioning and/or outgrowth of lateral organs [6,10]. Benjamins et al. [11] further characterized expression and function of PID in Arabidopsis. They found that it is auxinregulated, because the PID mRNA level is upregulated in roots, vascular tissue and leaf primordia after 4 h of 5 mM IAA treatment. They also found a single auxin responsive element, TGTCTC, indicating that PID transcription is probably regulated by an auxin response factor (ARF) and Aux/IAA transcription co-factors [11]. Further, these authors showed that 35S::PID seedlings had enhanced auxin transport and therefore PID is a positive regulator of polar auxin transport. Recently, Furutani et al. [12] reported that PID and PIN1 redundantly promote cotyledon initiation and establishment of the bilateral symmetry in developing Arabidopsis embryos by establishing auxin maxima. Also, Friml et al. [13] showed that the Muday and Murphy model [5] is correct and PID controls PIN targeting. Characterization of PID orthologs and their patterns of expression in other plant species have not yet been done. Pea ( Pisum sativum) is among those plant species with mutations known to affect leaf form and development. This species has complex, pinnately compound leaves and mutants that span an extensive range of leaf form. The leaf of pea has large, foliaceous stipules at the base, a distinct petiole, pairs of lateral pinnae and a terminal pinna. On normal or wildtype (WT) leaves, the basal or proximal pinnae are leaflets and the distal and terminal pinnae are simple tendrils. The best known pea leaf mutants are afila (af) and acacia/tendrilless (tl). Leaflets on af plants are replaced by branched tendrils creating leaves with only tendrils; and tendrils on tl plants are replaced by leaflets creating leaves with only leaflets [14–16]. The af tl double mutant possesses leaves with branched pinnae, each segment of which ends in a miniaturized leaflet [14,17]. The tendrilled-acacia (unitac) mutant has a terminal leaflet and a reduced number of
pinna pairs with the missing ones being the distal, lateral tendrils [18,19]. The molecular controls resulting in these distinct leaf morphologies are not yet known. Developing leaf primordia have long been thought to be the source of auxin production in shoot tips [20,21]. Generally, it is thought that auxin plays several roles in leaf development, including leaf initiation on the apical meristem [4,22–24], growth of the lamina and development of the venation [25–27]. Recently, DeMason and Chawla [28] hypothesized that auxin and an auxin gradient play unique and fundamental roles in development of the compound leaves of pea. They proposed that auxin concentrated in the tip of leaf primordia is the signal controlling leaf tip growth and pinna initiation. Further, they proposed that a gradient in auxin concentration controls pinna determination. Pinna primordia become tendrils in the distal half of the developing leaf, where auxin levels are higher and they become leaflets in the proximal half, where auxin levels are lower. These conclusions were based on the effects of growing plantlets in culture media containing several different auxin response or transport inhibitors. The observed effects included reductions in the number of pinna pairs produced, with the distal forms being eliminated before proximal ones, and conversions from compound to simple, including tri- and bilobed, leaves. In the most extreme situations, the entire leaf blade was aborted or converted into a median stipule or scale leaf. Also, terminal tendrils on WT leaves were converted into leaflets. Recently, the possible PIN1 ortholog, PsPIN1, was cloned from pea [29]. PsPIN1 mRNA is ubiquitously expressed throughout pea plants, but is especially abundant in growing tissues. It is regulated by both indole-3-acetic acid (IAA) and another active auxin in pea, 4-chloro-indole acetic acid (4-Cl-IAA). PsPIN1 expression is also regulated by gibberellic acid (GA). This regulation explains its abundance in growing tissues. Finally, DeMason and Chawla [28,30] tested the direction of auxin transport in small, expanding pea leaves using [14C]IAA. They found that NPA-sensitive IAA transport was strictly basipetal in tendrils, rachis segments and petioles, but it occurred from the margins inward in leaflets. On a weight basis, tendrils transported auxin much more efficiently than other leaf parts. Further, several results from the study on the af mutants, including the fact that their petioles transport more auxin than those of WT leaves, support a hypothesis that the leaves of af genotypes produce more auxin during development [30]. In order to pursue our investigation of the role of auxin in pea leaf development we began looking for additional auxinregulated genes that are expressed in leaf primordia and might play a role in auxin transport in leaves. An initial BLAST search with PID revealed that a partial cDNA of gene called PsPK2 [31] from pea aligned well. We decided to clone this gene and investigate its general expression patterns in pea. The questions we addressed in this study are: (1) How similar is PsPK2 to PID? (2) Does PsPK2 map to a
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location associated with any known morphological mutants of pea? (3) What is the pattern of PsPK2 expression in the different leaf genotypes and different leaf and plant parts? (4) Is PsPK2 regulated by auxin and/or gibberellin, and if so, what are the characteristics of this regulation?
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BLAST/). The overall similarity of these three proteins, with Gap and Bestfit were compared. EMOTIF (http:// motif.stanford.edu/cgi-bin/emotif) and ScanProsite (http:// us.expasy.org/cgi-bin/scanprosite) were used to search coding domains of PsPK2. 2.3. Promoter sequence determination and analysis
2. Materials and methods 2.1. Plant materials and growth conditions P. sativum genotypes used for this study were obtained from the Marx collection, which resides at the USDA-ARS Pacific West Area germplasm collection in Pullman, Washington. The tendrilled-acacia (uni-tac) line was W615272. The other lines used were from a set of near isogenic lines constructed by G. Marx and designated: wildtype (WT) W622593, acacia/tendrilless (tl) W622594, afila (af) W622597, and the double mutant, af tl W622598. Plants were grown in a standard greenhouse at 20 8C in the day and 15 8C at night. For purposes of propagation, two seeds of each line were sown into 1-gal pots filled with UC soil mix [32] supplemented with slow-release fertilizer (Osmocote 14-14-14) and seeds were collected from drying pods of selfed plants. 2.2. Cloning and sequence analysis of the PsPK2 gene A cDNA of a partial PsPK2 clone (253 bp) (GenBank accession no. M69031) [31] was PCR-amplified and used to screen a WT shoot tip Zap Express cDNA library (Stratagene). A 717 bp clone was obtained that contained the poly(A) tail. To obtain the 50 portion of the gene sequence, we used the SMART RACE cDNA Amplification kit (Clontech). Gene specific RACE-ready cDNAs were generated using the following primer: (50 -TAT ACG TCA CCC GCC ATT TT-30 ) R. 50 RACE was performed according to the manufacturer’s instructions using the following primer: (50 -TAT CCC AAA CGA CCA CCA GTC CAC-30 ) R. This produced a 962 bp fragment that contained the translation start site. The complete coding sequence was determined and a pair of PsPK2-specific primers were designed that would amplify a 1.4 kb fragment that included 76 bp upstream of ATG through +1412 bp. This pair of primers was: (50 -TGA TCG TGA TTC TGG GAT GA-30 ) F and (50 -TAT ACG TCA CCC GCC ATT TT-30 ) R. These were used to verify the genomic and coding sequences of PsPK2. Also, these primers were used to map the gene and to perform RT-PCR. Analysis of the PsPK2 cDNA sequence (GenBank accession no. AY505304) and alignment of the putative amino acid sequence of PsPK2, PINOID ( PID) (GenBank accession no. NM_129019) from Arabidopsis and BcPK1 (GenBank accession no. U93559) from Brassica were performed on ClustalW (http://www.ebi.ac.uk/clustalw/) and BLAST at NCBI (http://www.ncbi.nlm.nih.gov/
The PsPK2 promoter sequence (2.3 kb) (GenBank accession no. AY785781) was obtained using PCR and adaptor-ligated genomic pea DNA restriction enzyme fragment libraries generated using the Universal GenomeWalker Kit (Clontech). An initial 1.2 kb fragment was amplified by two rounds of nested PCR using the library made with EcoRV. The two gene specific primers (GSPs) used were: PsPK2GSP1 (50 -GAG TTC GAA AGA GAG ACG GCT GAA ACTT-30 ) R and PsPK2GSP2 (50 -GGC GCT GCT TAA AGA AGT TCT CTG TGT AT-30 ) R. An additional 2.4 kb upstream sequence was amplified by another two rounds of nested PCR from the library made with SmaI. The two GSPs used in this case were: PsPK2GSP3: (50 -GGG TAC AGG GAA CCG TTT TTG AAG TGG-30 ) R and PsPK2GSP4: (50 -GAG TTG AAA ACA TCT TTA CCT AAG GCA TGG AC-30 ) R. The first round PCR was 2 s at 94 8C and 3 min at 72 8C for 7 cycles, followed by 2 s at 94 8C and 3 min at 67 8C for 36 cycles, and ended at 67 8C for 4 min. The second round PCR was 2 s at 94 8C and 3 min at 72 8C for 5 cycles, followed by 2 s at 94 8C and 3 min at 67 8C for 20 cycles, and ended at 67 8C for 4 min. Finally, using genomic DNA from pea as a template, a 2.3 kb PsPK2 promoter fragment was amplified using Ex Taq (TaKaRa). This pair of primers was PsPK2P4F (50 -CCC TTT TTG GGG GTT CC-30 ) and PsPK2P4R (50 AAA ACC GTG CTG AGG AGA GA-30 ). PCR was carried out at 94 8C for 5 min followed by 44 cycles of 94 8C for 1 min, gradient temperatures 53.5, 55.8, 58.5, 61.4, 64.2 8C for 1 min, and 72 8C for 5 min preceded a final extension at 72 8C for 10 min. Amplified products were fractionated on 1.0% agarose gels and purified for further sequencing. PsPK2 promoter sequence was analyzed using PlantCARE (http://oberon.rug.ac.be:8080/PlantCARE/index/ html) and PLACE (http://www.dna.affrc.go.jp/htdocs/ PLACE/fasta.html). 2.4. Phylogenetic analysis To build a phylogenetic tree from the sequences of catalytic domain AGC-VIII groups, multiple alignments were performed with ClustalW 1.74. The phylogenetic tree was generated by using 1000 bootstrap trials and visualized using NJplot. 2.5. Mapping The location of PsPK2 was determined by joint segregation analysis in two populations of F2-derived
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recombinant inbred lines (RILs). The first population consisted of 53 F10 RILs derived from the cross JI1794 slow and constituted the population on which the consensus linkage map for pea was based [33]. The second population consisted of 51 F8 RILs derived from the cross MN313 JI1794. Using the PsPK2 specific primers PCR amplification products were generated from P. sativum parental lines and RILs. Restriction digestion of these products was performed by taking a 10 ml aliquot of the PCR products and adding to 20 ml solution of 10 times restriction buffer, water and restriction enzyme and incubating for 60 min as per instructions from the supplier of the restriction enzymes. Products of the restriction digest were separated on 2% agarose gels. The AgeI restriction pattern was compared to each of the segregation patterns of the approximately 1000 markers previously mapped in the JI1794 slow RIL population and to each of the approximately 300 markers previously mapped in the MN313 JI1794 population. The location of PsPK2 was determined by minimizing the number of recombinants between it and flanking markers using the EXCEL macro QUIKMAP [34]. 2.6. RNA extraction and gene expression patterns Total cellular RNAs were isolated from different plant parts of WT and mutant plants using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocols. RNA samples were DNase I (DNA-free kit; Ambion, Austin, TX) treated on the mini spin column according to the manufacturer’s instructions to eliminate any contaminating genomic DNA. The RNA was carefully quantified and run on a gel with ethidium bromide staining to verify equal loading. The plant parts used were mature leaflets, mature tendrils, seedling roots, shoot tips, root tips, flower buds, immature adult leaves and internodes from WT plants, mature leaflets from tl and uni-tac plants, and mature tendrils from af plants. Developing pods of WT plants were collected and four samples of three stages of embryo development were obtained: young (embryo linear, occupied less than half the ovule’s volume and without cotyledon swelling), medium (embryo occupied approximately half the ovule’s volume and with swelling cotyledons), mature (embryo filled the ovule’s volume but had not yet started drying) and mature embryo axis. In addition, shoot tips were dissected from 2week-old seedling plants of WT, tl, af, uni-tac and af tl. All tissues were immediately frozen in liquid nitrogen and stored in a 80 8C freezer until RNA was isolated. cDNAs were synthesized separately from each RNA preparation in a 20 ml reaction mixture containing 1 mg total RNA, 2 ml 10 times RT buffer, 2 ml dNTPs mixture (each at 5 mM), 1 ml random hexamer primer mixture, 0.25 ml RNase inhibitor, 1 ml Ominiscript Reverse Transcriptase (Qiagen). Synthesis time was 1 h at 37 8C. PCR was performed in a 50 ml volume containing 2.5 U Taq DNA polymerase (Qiagen), 1 ml cDNAs, 200 mM
nucleotides, 40 pM each specific primer, and 5 ml 10 times reaction buffer. The PCR conditions were 3 min at 92 8C, followed by 44 cycles of 1 min at 92 8C, 1 min at annealing temperature 62 8C of primers and 2 min at 72 8C, followed by a final extension of 10 min at 72 8C. The PCR products were separated on 1% agarose gels, stained with ethidium bromide and photographed with a Gel Doc 2000 (Bio-Rad). Control PCR reactions using primers specific to pea b-actin (GenBank accession no. X90378) (0.6 kb) were done to confirm equivalent RNA inputs into the reactions. The actin primers were ActinF (50 -GTT GGA ATG GGA CAA AAA GA-30 ) and ActinR (50 -CGA TGG CTG GAA CAG AAC30 ). These primers flanked an intron in the pea b-actin gene and the product size verified the lack of contaminating genomic DNA. 2.7. Auxin and GA induction experiments WT seeds were surface sterilized in 10% bleach for 10 min, washed in four changes of deionized water, and imbibed overnight in 2 l of deionized water. They were then sown in sterile vermiculite, saturated with sterile deionized water, and grown for 7 days at room temperature in complete darkness. The shoot tips were dissected from seedlings under a dim green safelight and treated with hormones. The hormones used were: indole-3-acetic acid (IAA), 4-chloroindole acetic acid (4-Cl-IAA), indole-3-butyric acid (IBA), indole-3-acetic acid methylester (methyl-IAA), a-naphthalene-acetic acid (NAA) and gibberellin A3 (GA3). All hormones were obtained from Sigma except 4-Cl-IAA, which was a gift from Jerry Cohen. In the first experiment, eight shoot tips were incubated with water or 10 mM IAA, 4-Cl-IAA, IBA, methyl-IAA or NAA for 30 min. All auxin stock solutions were made in 100% ethanol and the appropriate amount of hormone solution was added to the deionized water used as the bathing medium. Shoot tips were immediately frozen in liquid nitrogen after treatment and stored at 80 8C until RNA purification. IAA, 4-Cl-IAA and IBA were used because they are naturally occurring auxins in pea. MethylIAA was used because it is transported through tissues and cell membranes more efficiently than other auxins, and when methyl-IAA enters a cell, the methyl group is cleaved off producing IAA (Dr. Jerry Cohen, personal communication). In the second experiment, the shoot tips were separated into two groups. One group was placed in sterile deionized water or 10 mM methyl-IAA (Sigma, I-9770) in the dark for 10 min, 30 min, 1 h, 2 h or 4 h, and in the other group, shoot tips were treated with 0, l, 10, 25, 50, or 75 mM methyl-IAA for 30 min. In each treatment, eight shoot tips were used. The methyl-IAA stock solution was made in 100% ethanol and the appropriate amount of methyl-IAA solution was added to the deionized water used as the bathing medium. Shoot tips were immediately frozen in liquid nitrogen after treatment and stored at 80 8C until RNA purification.
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In the third experiment, the shoot tips were separated into two groups. One group was placed in sterile deionized water or 75 mM GA3 in the dark for 10 min, 30 min, 1 h, 2 h or 4 h, and in the other group, shoot tips were treated with 0, 50, 75 or 100 mM GA3 for 4 h. In each treatment, eight shoot tips were used. The GA3 stock solution was made in ethanol and the appropriate amount of GA3 solution was added to the deionized water used as the bathing medium. Shoot tips were immediately frozen in liquid nitrogen after treatment and stored at 80 8C until RNA purification. 2.8. Semi-quantitative RT-PCR cDNAs were synthesized separately from each RNA preparation in a 20 ml reaction mixture containing 1 mg total RNA, 2 ml 10 times RT buffer, 2 ml dNTPs mixture (each at 5 mM), 1 ml random hexamer primer mixture, 0.25 ml RNase inhibitor, 1 ml Ominiscript Reverse Transcriptase (Qiagen). Synthesis time was 1 h at 37 8C. PCR was performed in a 30 ml containing 2.5 unit HotStarTaq DNA polymerase (Qiagen), 6 ml cDNAs, 200 mM dNTP, 40 pM each specific primer, and 3 ml 10 times reaction buffer. The PCR conditions were 15 min at 95 8C, followed by 19 cycles of 1 min at 94 8C, 1 min at annealing temperature 60 8C of primers and 2 min at 72 8C, followed by a final extension of 10 min at 72 8C. The PCR products were separated on 1% agarose gel. The following primer sets were used: DeadboxF (50 -TTC TCG TCA TCA ACC TCA CC-30 ) and DeadboxR (50 -TTC CTA CCA AAC CTT CCA CTA C-30 ) for control gene, DEAD box (GenBank accession no. AY167670), and the primers for the PsPK2 gene. The gels were transferred to nylon membranes, hybridized with a 32P-labeled probes PsPK2 and DEAD box separately, and quantification of the blots were done using Typhoon 9410 (Amersham Pharmacia Biotech). The densitometry ratio of PsPK2 value/DEAD box value was calculated and graphed using SigmaPlot 2000. The sample size was two separate sets of RNA extractions from each treatment. Means and standard errors were determined using Microsoft Excel xp.
3. Results and discussion 3.1. Cloning and structure analysis of PsPK2 The complete transcribed sequence of PsPK2 (GenBank accession no. AY505304) was obtained by screening a WT shoot tip cDNA library with a partial cDNA followed by 50 RACE-PCR. The PsPK2 full cDNA was 1573 bp. The deduced PsPK2 amino acid sequence consisted of 445 amino acid residues (Fig. 1A) and had a calculated pI of 9.16 and molecular mass of 49,869. The PsPK2 gene was also sequenced and found to contain a small 44 bp intron between amino acid R and F at 181 and 182, respectively.
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PsPK2 had the conserved catalytic core typical of eukaryotic protein kinases at positions 68–394, including the well characterized 12 conserved subdomains (Fig. 1A) [8]. A serine/threonine protein kinase active-site signature at 199–211 (LiYrDLKpeNVLV) (Fig. 1A) was characteristic of amino acid substrate specificity and predicted PsPK2 to be a protein serine/threonine kinase. The short amino acid sequence KKKS in subdomain III was predicted to be a cAMP- and cGMP-dependent protein kinase phosphorylation site using ScanProsite (Fig. 1A). The N-terminal domain of PsPK2 is rich in serines and contains several potential phosphorylation sites. The predicted PsPK2 polypeptide shared 71.7% identity and 77.6% similarity with PID from Arabidopsis thaliana, and had 72.9% identity and 78.9% similarity with the polypeptide encoded by a Brassica rapa gene, Bcpk1 (Fig. 1B). A highly conserved amino acid motif DFD occurred at positions 221–223 located in subdomain VII of the PsPK2 sequence and was also present in PID and Bcpk1 (Fig. 1B). This motif is characteristic of the AGC-VIII group of protein kinases. Between conserved domains I and II we found a stretch of approximately eight amino acids in PsPK2 that were not present in PID or Bcpk1 (Fig. 1B). These amino acids occur in a region between subdomains I and II that folds away from the ATP binding site in protein kinase PKA, and there are several kinases with extra amino acids here [8]. Also, a segment of about 50 amino acids is present between conserved domains VII and VIII (open bar, Fig. 1B), which has been suggested to be a regulatory domain [10] or to have an affect on catalytic activity [35] that PsPK2, PID and Bcpk1 all share. This ‘‘insert’’ between subdomains VII and VIII is another unique feature of the AGC-VIII kinases, but there was little conservation of amino acid sequence in this region for these three kinases. The Nterminal region of the PsPK2, PID and Bcpk1, shares low identity as well as does the C-terminal end beyond the catalytic domain. To determine the relationship of PsPK2 to these kinases, a phylogenetic analysis was performed to develop a tree of related kinases using the sequences of the catalytic domain (Fig. 2). The PINOID, phototropin, PVPK-1 and PsPK3 subfamilies belong to the AGC-VIII group, which are characterized by a DFD motif instead of DFG in conserved subdomain VII of the catalytic site. We propose that PsPK2, Bcpk1 and PID group together in a unique PINOID subfamily because they share high sequence conservation in the catalytic domain. Finally, Bcpk1 and PINOID are probably more similar to one another than to PsPK2 because Brassica and Arabidopsis are both members of the Brassicaceae. 3.2. PsPK2 promoter sequence We obtained 2.3 kb of PsPK2 promoter sequence (GenBank accession no. AY785781) by genome walking
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Fig. 1. The PsPK2 amino acid sequence. (A) The predicted PsPK2 protein contains the conserved catalytic core of protein kinases at amino acids 68-394. Portions of the 12 conserved subdomains are in boldface. The Ser/Thr protein kinase active-site signature is underlined. The cAMP and cGMP-dependent protein kinase phosphorylation site KKKS is enclosed by a box. (B) Alignment of the PsPK2 sequence with PINOID and Bcpk1 using ClustalW (http:// www.ebi.ac.uk/clustalw/). Identical amino acids present in all three proteins are shaded. The highly conserved amino acid sequence DFD motif is underlined. A segment of eight amino acids between subdomains I and II (dots) and a stretch of about 50 amino acids between subdomains VII and VIII (open bar) are marked.
(Fig. 3) and analyzed this sequence using software PlantCARE and PLACE. An AuxRE containing the consensus sequence TGTCTC, which is common to early response genes and is recognized by the transcription factor known as ARF was identified at 1695 to 1690. This element is thought to confer auxin inducibility of promoters [36–38] by a mechanism involving ARF and Aux/IAA proteins that is well characterized [39–44]. An AuxRR-core,
which is another regulatory element involved in auxin responsiveness, was found at seven locations, 815 to 809 (GGTCCAT), 1321 to 1315 (GGTCAAT), 1958 to 1952 (GGTCCTT), 2063–2057 (GGTCCCT), 2128 to 2122 (GGTCCCT), 2162 to 2156 (GGTCCCT), and 2207 to 2201 (GGTCCAA). AuxRRs were found in the promoters of PsPIN1 and Ps-IAA4/5 from pea [29,45], parB [46] and parC from tobacco [47], and SAUR from
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1 cM of these latter two markers, the limit of precision for our analysis. There are no known leaf morphology mutants of pea that map to this position, and so we must also conclude that PsPK2 cannot be considered a candidate gene for the various leaf mutations that map to other positions on the pea linkage map. Note that recombination values might be slightly reduced compared to some linkage maps of pea because JI1794 is a P. sativum ssp. elatius (Steven ex M. Bieb) Asch. & Graebner accession, and the two alternative parents are P. sativum ssp. sativum and P. sativum ssp. elatius. Wide crosses in Pisum tend to give lower map distances between standard markers. 3.4. Expression of PsPK2
Fig. 2. Phylogenetic tree of the relationships among Ser/Thr protein kinases. PsPK2, BcPK1 and PINOID are in the PINOID subfamily within the AGC-VIII group. Multiple alignments were performed with ClustalW 1.74 and visualized using NJplot.
soybean [48] and have been shown to be involved in auxin response. Many putative light responsive elements such as Gbox, AAAC-motif, AE-box, ATC motif, I-box, and others were present on the PsPK2 promoter suggesting that PsPK2 is possibly regulated by light. Lin et al. [31] studied light regulation of five protein kinases, PsPK1– PsPK5 of pea and found that transcripts were differentially regulated during deetiolation. PsPK2 mRNA levels did not respond to light significantly during this process. Because the putative gibberellin (GA) responsive elements, 16 P-boxes and a TATC-box were also identified in the promoter sequence, we tested the possibility that PsPK2 is GA regulated. 3.3. Mapping of PsPK2 The PsPK2 locus was positioned between Pl and Gsp in both RIL populations (Fig. 4). More precisely, PsPK2 mapped halfway between these two markers and cosegregated with the RAPDs B192420 and B162a, placing it within
In WT plants, PsPK2 mRNA was differentially expressed in different plant parts. PsPK2 was abundant in immature adult leaves, shoot tips and flower buds. It was also present in seedlings, roots, and stems. No expression was found in mature leaflets but it was abundant in mature tendrils (Fig. 5A). This pattern of expression is very similar to that described for PID in Arabidopsis. Both PsPK2 and PID mRNAs are most abundant in growing and developing regions such as flower buds, leaf primordia, the shoot apical meristem and seedlings [10,11]. We compared PsPK2 expression in pinna types from WT leaves and those of the leaf mutants, af, tl and uni-tac (Fig. 5B). PsPK2 was expressed in mature tendrils from leaves of af and WT, although no significant expression was detected in mature leaflets of WT, tl or uni-tac. Basically, PsPK2 mRNA was abundant in tendrils irrespective of genotype, but not in leaflets. Tendrils are thigmotropic, a process that involves auxin signaling, whereas leaflets are not. Therefore, the greater expression of PsPK2 may be associated with greater auxin production and transport in tendrils. Tendrils do transport more auxin than other leaf parts in pea [28]. Overexpression of 35S::PID in Arabidopsis resulted in a reduced gravitropic response in the primary root suggesting that PID may play an important role in control of gravitropism, which is also regulated by auxin [10,11]. Furthermore, differential expression occurred in shoot tips of the different pea leaf mutants. PsPK2 mRNA level was slightly more abundant in af and af tl, and less abundant in tl and uni-tac compared to WT (Fig. 5C). This was verified using semi-quantitative RT-PCR (data not shown). The expression of PsPK2 in shoot tips from different genotypes is similar to that of the Uni gene, which is known to play a
Fig. 3. Diagram of the PsPK2 promoter. The CAAT-box (67) and TATA-box (59) are designated by open rectangles. Auxin response elements: AuxRE (1695) and seven AuxRRs (815, 1321, 1958, 2063, 2128, 2162 and 2207) are designated by black rectangles. GA response elements: P-boxes (442, 1080, 1103, 1328, 1498, 1620, 1911, 1919, 1946, 1955, 1972, 1987, 2027, 2108, 2202 and 2259) and a TATC-box (94) are designated by grey rectangles.
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Fig. 4. Mapping of the PsPK2 gene. (A) Position of PsPK2 on Linkage Group VI on the map developed for the JI1794 slow RILs. (B) Position of PsPK2 on linkage group VI on the map developed for the MN313 JI1794 RILs. Distances between adjacent markers are given as recombination percentages and were calculated using the equation: r = R/(2 2R), where r is the frequency of recombination and R is the proportion of RILs displaying a recombinant phenotype [54]. Other markers used to position PsPK2 include Pl (black hilum), Gsp (plastid specific glutamine synthetase), Hop1 (homeodomain protein-1) and Prx (peroxidase-3) and RBIP-95x2 (a retrotransposon-derived marker obtained courtesy of Dr. Andy Flavell). The remaining markers are RAPDs, with priming sequences that can be obtained from the authors upon request.
central role in pea leaf development [19,49]. Uni mRNA is more abundant in tl, af and af tl and less abundant in uni-tac compared to WT [28]. Since both PID and PsPK2 are regulated by auxin (see below), this expression pattern might indicate that developing leaves of the af genotypes produce more auxin than those of WT, a hypothesis that was proposed in an earlier study [30]. In any case, this differential
expression of PsPK2 in the different leaf form mutants suggests that it might play an important role in leaf development in pea. Observations of its in situ expression patterns in developing leaves of these mutants may help us determine what this role might be. PsPK2 was also expressed differentially during embryo development (Fig. 5D). PsPK2 expression was weak in young embryos, higher in medium embryos, and weak in mature, whole embryos. However, expression was high in the axis of mature embryos. Expression was low in the whole, mature embryo due to the presence of the large, storage cotyledons where the mRNA level of PsPK2 was presumably low. The expression pattern is similar to that shown for PsPIN1 [29], and PID is known to be expressed during Arabidopsis embryogenesis. In addition to being present in globular stage embryos and on the adaxial side of cotyledon primordia in the late heart to torpedo stages, PID mRNA is mainly expressed at the boundary between cotyledon primordia [12]. 3.5. Hormone regulation of PsPK2 expression
Fig. 5. PsPK2 expression in different plant parts of WT and leaf form mutants: af, tl, uni-tac and af tl. (A) Expression in different parts of WT plants. (B) Expression in leaflets and tendrils of WT, tl, af and uni-tac. (C) Expression in shoot tips of WT, af, tl, uni-tac and af tl. (D) Expression in three stages of WT embryos, young, medium, mature whole and mature axis only.
We examined PsPK2 mRNA expression in response to treatment by different auxins, both natural and synthetic. Semi-quantitative analysis showed higher PsPK2 mRNA levels in response to all auxins used compared to controls. Methyl-IAA treatment produced the strongest response followed by IAA, 4-Cl-IAA, NAA, and IBA had the least (Fig. 6A). Therefore, among the naturally occurring auxin in pea, PsPK2 expression is most sensitive to IAA regulation. PsPK2 mRNA increased approximately 1.5-fold at 30 min after methyl-IAA treatment and then declined to near control levels after 4 h of treatment (Fig. 6B). In the dose response experiment, PsPK2 mRNA levels were most abundant at 10 mM methyl-IAA, less abundant at other concentrations, but higher than control at all concentrations except at 1 mM
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Fig. 6. PsPK2 mRNA expression relative to DEAD box mRNA levels in response to auxin treatment. (A) PsPK2 expression induced by 10 mM IAA, 4-Cl-IAA, IBA, methyl-IAA and NAA for 30 min. (B) PsPK2 expression induced by methyl-IAA for different time periods. (C) PsPK2 expression in different concentrations of methyl-IAA for 30 min. Control 1 = time zero control before methyl-IAA treatment, control 2 = control at 30 min without methyl-IAA treatment. The error bars are S.E. which are average from two repeats of each treatment.
(Fig. 6C). PID expression has also been shown to be regulated by auxin in Arabidopsis. Benjamins et al. [11] showed by Northern blotting that PID mRNA increased at 4 h after 5 mM IAA treatment, but declined by 20 h. Also, the PID promoter was found to possess a TGTCTC AuxRE. Rapid upregulation followed by a decline, and the presence of an ARF binding site in both PID and PsPK2, suggest that both are primary auxin response genes. PsPIN1 is also regulated by auxin, however, it is more responsive to 4-ClIAA than IAA, requires higher levels of auxin for induction, and responds more slowly than PsPK2 (2–4 h). Reinecke et al. [50] showed that 4-Cl-IAA was more active than IBA and IAA in stimulating both stem and pericarp growth in pea. To verify the GA responsiveness predicted by promoter sequence, we examined the kinetics and concentration dependence of PsPK2 expression in response to exogenous
GA3. PsPK2 mRNA increased approximately 1.8-fold at 4 h after GA3 treatment compared to the control (Fig. 7A). In the dose response experiment, PsPK2 mRNA levels were most abundant at 75 mM GA3, and were less abundant in the other treatments, although they were higher than control in all treatment concentrations (Fig. 7B). The promoter was found to possess several GA responsive elements, 16 P-boxes and a TATC-box and we verified that GA regulates the PsPK2 expression in pea. We do not know whether this is a unique feature of this gene in pea or whether GA also regulates PID. However, since GA was also found to enhance PsPIN1 mRNA levels in pea [29], it seems that auxin transport in pea may be regulated both by auxin and GA. Auxin is known to regulate GA levels via its control of biosynthetic and catalytic enzymes. It upregulates GA 3-oxidase transcription and down regulates GA 2-oxidase transcription [51]. GA 3oxidase converts inactive GA20 into active GA1 and GA 2-
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leaflets of pea leaves, irrespective of genotype, suggesting that it might play a role in the thigmotropic response. PsPK2 is not only abundantly expressed in developing pea leaves, but it is differentially expressed in shoot tips of the different leaf form mutants suggesting that PsPK2 plays an important role in leaf form generation. PsPK2 and PID have been shown to be early auxin response genes and PsPK2 is also gibberellin-regulated. Our goal is to determine the roles of PsPK2 in auxin and gibberellin regulation, which may control pea leaf development.
Acknowledgements We thank Janet Giles for technical assistance with library screening, cloning the full sequence of the PsPK2 gene and preparing four genome walker libraries. We thank Dr. Rekha Chawla for preparing four genome walker libraries, and for comments on the manuscript. We thank Dr. Jerry Cohen for providing the 4-Cl-IAA. The pea genotypes used in this study were obtained from the Marx collection, which currently resides at the USDA-ARS, Pacific West Area, Pullman, Washington. Sequencing was done by the UCR Genomics Institute. This work was partially supported by USDA/NRI grant no. 2001-35304-10958 to Dr. Darleen A. DeMason.
References
Fig. 7. PsPK2 mRNA expression relative to DEAD box mRNA levels in response to GA3 treatment. (A) PsPK2 expression induced by GA3 for different time periods. (B) PsPK2 expression in different concentrations of GA3 for 4 h. Control 1 = time zero control before GA3 treatment, control 2 = control at 30 min without GA3 treatment. The error bars are S.E. which are average from two repeats of each treatment.
oxidase converts GA1 into inactive GA29. Further, 4-Cl-IAA significantly increases expression of another GA biosynthetic enzyme (GA 20-oxidase) in pea pericarp [52,53]. The simplest explanation for our results is that auxin regulates PsPK2 both directly and indirectly through regulation of GA levels.
4. Conclusion PsPK2 is the second PID-like gene to be characterized in the plant kingdom. Its sequence is very similar to that of PID and another PID-like gene, Bcpk1, suggesting the existence of a PINOID subfamily within the AGC-VIII protein kinases. PsPK2 mRNA is more abundant in tendrils than in
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Molecular characterization and expression of PsPK2, a PINOID-like gene from pea ( Pisum sativum) Fang Bai a, John C. Watson b, Jason Walling c, Norman Weeden c, Aaron A. Santner b, Darleen A. DeMason a,* b
a Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA Department of Biology, Indiana University–Purdue University Indianapolis, 723 West Michigan Street, Indianapolis, IN 46202, USA c Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59717, USA
Received 26 December 2004 Available online 22 January 2005
Abstract The plant hormone auxin acts as a signal for cell division, extension and differentiation during the course of plant growth and development. With the aim of elucidating the molecular mechanism of auxin responses in pea leaf development, we have isolated a putative pea PINOID ( PID) homolog, PsPK2, and characterized it. The deduced protein sequence of PsPK2 had 445 amino acids and possessed 12 conserved catalytic subdomains that characterize protein kinases. The PsPK2 sequence was most closely related to PID, and together they defined a PINOID subfamily within the AGC-VIII protein kinases. PsPK2 mapped to the lower portion of linkage group VI between Pl and Gsp. By genome walking, 2.3 kb of the PsPK2 promoter was obtained and was predicted to possess auxin and gibberellin response elements. PsPK2 was strongly expressed in developing organs of wildtype (WT) pea, such as shoot tips, immature adult leaves, developing embryos and flower buds. Comparing the PsPK2 mRNA levels from tendrils and leaflets of WT as well as the other leaf form mutants, we found that irrespective of genotype, PsPK2 mRNA was much more abundant in tendrils than in leaflets. Furthermore, differential expression occurred in shoot tip RNAs of the different leaf form genotypes. Expression was slightly higher in the afila genotypes, and lower in acacia/tendrilless and tendrilledacacia compared to WT. In an auxin induction experiment, PsPK2 mRNA levels increased most after methyl-IAA treatment compared to IAA, 4-Cl-IAA, IBA and NAA treatments. Time course and concentration dependent experiments showed that PsPK2 mRNA levels were the highest after 30 min of methyl-IAA treatment compared to control and other treatments, and peaked at 10 mM methyl-IAA. In addition, PsPK2 mRNA levels peaked after 4 h of GA3 treatment and were highest in response to 75 mM GA3. Therefore, auxin and gibberellin positively regulate PsPK2 expression and it may play a role in the control of auxin transport and leaf form generation in pea. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Auxin; Gibberellin; Pea; PINOID; PsPK2; Ser/Thr protein kinases
1. Introduction Auxin is perceived by plant cells, and depending on their type and status, is rapidly transduced into a wide variety of responses in growth and development. Some of these responses include changes in direction of growth, in shoot and root branching and in vascular tissue differentiation. Auxin is transported in a strictly polar manner in plants. PIN1, the putative auxin efflux carrier, controls polar auxin * Corresponding author. Tel.: +1 951 827 3580; fax: +1 951 827 4437. E-mail address: [email protected].
transport via its position and function [1–3]. Recently, Benkova´ et al. [4] showed that lateral organ initiation and/or outgrowth requires local accumulation of auxin and this process requires correct targeting of PIN1. A recent model predicts that many of the components that mediate glucose transporter targeting in mammalian cells responsive to insulin have orthologs in auxin efflux carrier targeting [5]. These authors propose that protein phosphorylation by a protein kinase plays an important role in PIN1 targeting. DeLong et al. proposed that protein phosphorylation plays important regulatory roles in auxin signaling and response [6]. Generally, plant protein kinases can be placed
0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.01.005
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in one of three groups based on their ability to phosphorylate serine and threonine or tyrosine or histine residues. They are the serine/threonine (Ser/Thr) kinases, the tyrosine (Tyr) kinases, and the histine (His) kinases [7]. Protein kinases share many conserved structural features including sequence and three-dimension arrangements of the catalytic domain [7,8]. The functions of plant protein kinases are poorly known, although many have been identified in the past few years. The Arabidopsis Ser/Thr kinase predicted by Muday and Murphy [5] to participate in targeting of the auxin efflux carrier is PINOID (PID). The pid mutant was first described by Bennett et al. [9]. pid mutants share many characteristics with pin1 mutants, such as reduced auxin transport, naked inflorescences and defects in form and position of lateral organs, including cotyledons, leaves and floral organs. Christensen et al. [10] identified PID as a unique Ser/Thr kinase and characterized various aspects of PID expression. They found that it is expressed in developing embryos, the shoot apical meristem, leaf primordia and in the initial stages of floral organ development. They concluded that PID plays roles in positioning and/or outgrowth of lateral organs [6,10]. Benjamins et al. [11] further characterized expression and function of PID in Arabidopsis. They found that it is auxinregulated, because the PID mRNA level is upregulated in roots, vascular tissue and leaf primordia after 4 h of 5 mM IAA treatment. They also found a single auxin responsive element, TGTCTC, indicating that PID transcription is probably regulated by an auxin response factor (ARF) and Aux/IAA transcription co-factors [11]. Further, these authors showed that 35S::PID seedlings had enhanced auxin transport and therefore PID is a positive regulator of polar auxin transport. Recently, Furutani et al. [12] reported that PID and PIN1 redundantly promote cotyledon initiation and establishment of the bilateral symmetry in developing Arabidopsis embryos by establishing auxin maxima. Also, Friml et al. [13] showed that the Muday and Murphy model [5] is correct and PID controls PIN targeting. Characterization of PID orthologs and their patterns of expression in other plant species have not yet been done. Pea ( Pisum sativum) is among those plant species with mutations known to affect leaf form and development. This species has complex, pinnately compound leaves and mutants that span an extensive range of leaf form. The leaf of pea has large, foliaceous stipules at the base, a distinct petiole, pairs of lateral pinnae and a terminal pinna. On normal or wildtype (WT) leaves, the basal or proximal pinnae are leaflets and the distal and terminal pinnae are simple tendrils. The best known pea leaf mutants are afila (af) and acacia/tendrilless (tl). Leaflets on af plants are replaced by branched tendrils creating leaves with only tendrils; and tendrils on tl plants are replaced by leaflets creating leaves with only leaflets [14–16]. The af tl double mutant possesses leaves with branched pinnae, each segment of which ends in a miniaturized leaflet [14,17]. The tendrilled-acacia (unitac) mutant has a terminal leaflet and a reduced number of
pinna pairs with the missing ones being the distal, lateral tendrils [18,19]. The molecular controls resulting in these distinct leaf morphologies are not yet known. Developing leaf primordia have long been thought to be the source of auxin production in shoot tips [20,21]. Generally, it is thought that auxin plays several roles in leaf development, including leaf initiation on the apical meristem [4,22–24], growth of the lamina and development of the venation [25–27]. Recently, DeMason and Chawla [28] hypothesized that auxin and an auxin gradient play unique and fundamental roles in development of the compound leaves of pea. They proposed that auxin concentrated in the tip of leaf primordia is the signal controlling leaf tip growth and pinna initiation. Further, they proposed that a gradient in auxin concentration controls pinna determination. Pinna primordia become tendrils in the distal half of the developing leaf, where auxin levels are higher and they become leaflets in the proximal half, where auxin levels are lower. These conclusions were based on the effects of growing plantlets in culture media containing several different auxin response or transport inhibitors. The observed effects included reductions in the number of pinna pairs produced, with the distal forms being eliminated before proximal ones, and conversions from compound to simple, including tri- and bilobed, leaves. In the most extreme situations, the entire leaf blade was aborted or converted into a median stipule or scale leaf. Also, terminal tendrils on WT leaves were converted into leaflets. Recently, the possible PIN1 ortholog, PsPIN1, was cloned from pea [29]. PsPIN1 mRNA is ubiquitously expressed throughout pea plants, but is especially abundant in growing tissues. It is regulated by both indole-3-acetic acid (IAA) and another active auxin in pea, 4-chloro-indole acetic acid (4-Cl-IAA). PsPIN1 expression is also regulated by gibberellic acid (GA). This regulation explains its abundance in growing tissues. Finally, DeMason and Chawla [28,30] tested the direction of auxin transport in small, expanding pea leaves using [14C]IAA. They found that NPA-sensitive IAA transport was strictly basipetal in tendrils, rachis segments and petioles, but it occurred from the margins inward in leaflets. On a weight basis, tendrils transported auxin much more efficiently than other leaf parts. Further, several results from the study on the af mutants, including the fact that their petioles transport more auxin than those of WT leaves, support a hypothesis that the leaves of af genotypes produce more auxin during development [30]. In order to pursue our investigation of the role of auxin in pea leaf development we began looking for additional auxinregulated genes that are expressed in leaf primordia and might play a role in auxin transport in leaves. An initial BLAST search with PID revealed that a partial cDNA of gene called PsPK2 [31] from pea aligned well. We decided to clone this gene and investigate its general expression patterns in pea. The questions we addressed in this study are: (1) How similar is PsPK2 to PID? (2) Does PsPK2 map to a
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location associated with any known morphological mutants of pea? (3) What is the pattern of PsPK2 expression in the different leaf genotypes and different leaf and plant parts? (4) Is PsPK2 regulated by auxin and/or gibberellin, and if so, what are the characteristics of this regulation?
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BLAST/). The overall similarity of these three proteins, with Gap and Bestfit were compared. EMOTIF (http:// motif.stanford.edu/cgi-bin/emotif) and ScanProsite (http:// us.expasy.org/cgi-bin/scanprosite) were used to search coding domains of PsPK2. 2.3. Promoter sequence determination and analysis
2. Materials and methods 2.1. Plant materials and growth conditions P. sativum genotypes used for this study were obtained from the Marx collection, which resides at the USDA-ARS Pacific West Area germplasm collection in Pullman, Washington. The tendrilled-acacia (uni-tac) line was W615272. The other lines used were from a set of near isogenic lines constructed by G. Marx and designated: wildtype (WT) W622593, acacia/tendrilless (tl) W622594, afila (af) W622597, and the double mutant, af tl W622598. Plants were grown in a standard greenhouse at 20 8C in the day and 15 8C at night. For purposes of propagation, two seeds of each line were sown into 1-gal pots filled with UC soil mix [32] supplemented with slow-release fertilizer (Osmocote 14-14-14) and seeds were collected from drying pods of selfed plants. 2.2. Cloning and sequence analysis of the PsPK2 gene A cDNA of a partial PsPK2 clone (253 bp) (GenBank accession no. M69031) [31] was PCR-amplified and used to screen a WT shoot tip Zap Express cDNA library (Stratagene). A 717 bp clone was obtained that contained the poly(A) tail. To obtain the 50 portion of the gene sequence, we used the SMART RACE cDNA Amplification kit (Clontech). Gene specific RACE-ready cDNAs were generated using the following primer: (50 -TAT ACG TCA CCC GCC ATT TT-30 ) R. 50 RACE was performed according to the manufacturer’s instructions using the following primer: (50 -TAT CCC AAA CGA CCA CCA GTC CAC-30 ) R. This produced a 962 bp fragment that contained the translation start site. The complete coding sequence was determined and a pair of PsPK2-specific primers were designed that would amplify a 1.4 kb fragment that included 76 bp upstream of ATG through +1412 bp. This pair of primers was: (50 -TGA TCG TGA TTC TGG GAT GA-30 ) F and (50 -TAT ACG TCA CCC GCC ATT TT-30 ) R. These were used to verify the genomic and coding sequences of PsPK2. Also, these primers were used to map the gene and to perform RT-PCR. Analysis of the PsPK2 cDNA sequence (GenBank accession no. AY505304) and alignment of the putative amino acid sequence of PsPK2, PINOID ( PID) (GenBank accession no. NM_129019) from Arabidopsis and BcPK1 (GenBank accession no. U93559) from Brassica were performed on ClustalW (http://www.ebi.ac.uk/clustalw/) and BLAST at NCBI (http://www.ncbi.nlm.nih.gov/
The PsPK2 promoter sequence (2.3 kb) (GenBank accession no. AY785781) was obtained using PCR and adaptor-ligated genomic pea DNA restriction enzyme fragment libraries generated using the Universal GenomeWalker Kit (Clontech). An initial 1.2 kb fragment was amplified by two rounds of nested PCR using the library made with EcoRV. The two gene specific primers (GSPs) used were: PsPK2GSP1 (50 -GAG TTC GAA AGA GAG ACG GCT GAA ACTT-30 ) R and PsPK2GSP2 (50 -GGC GCT GCT TAA AGA AGT TCT CTG TGT AT-30 ) R. An additional 2.4 kb upstream sequence was amplified by another two rounds of nested PCR from the library made with SmaI. The two GSPs used in this case were: PsPK2GSP3: (50 -GGG TAC AGG GAA CCG TTT TTG AAG TGG-30 ) R and PsPK2GSP4: (50 -GAG TTG AAA ACA TCT TTA CCT AAG GCA TGG AC-30 ) R. The first round PCR was 2 s at 94 8C and 3 min at 72 8C for 7 cycles, followed by 2 s at 94 8C and 3 min at 67 8C for 36 cycles, and ended at 67 8C for 4 min. The second round PCR was 2 s at 94 8C and 3 min at 72 8C for 5 cycles, followed by 2 s at 94 8C and 3 min at 67 8C for 20 cycles, and ended at 67 8C for 4 min. Finally, using genomic DNA from pea as a template, a 2.3 kb PsPK2 promoter fragment was amplified using Ex Taq (TaKaRa). This pair of primers was PsPK2P4F (50 -CCC TTT TTG GGG GTT CC-30 ) and PsPK2P4R (50 AAA ACC GTG CTG AGG AGA GA-30 ). PCR was carried out at 94 8C for 5 min followed by 44 cycles of 94 8C for 1 min, gradient temperatures 53.5, 55.8, 58.5, 61.4, 64.2 8C for 1 min, and 72 8C for 5 min preceded a final extension at 72 8C for 10 min. Amplified products were fractionated on 1.0% agarose gels and purified for further sequencing. PsPK2 promoter sequence was analyzed using PlantCARE (http://oberon.rug.ac.be:8080/PlantCARE/index/ html) and PLACE (http://www.dna.affrc.go.jp/htdocs/ PLACE/fasta.html). 2.4. Phylogenetic analysis To build a phylogenetic tree from the sequences of catalytic domain AGC-VIII groups, multiple alignments were performed with ClustalW 1.74. The phylogenetic tree was generated by using 1000 bootstrap trials and visualized using NJplot. 2.5. Mapping The location of PsPK2 was determined by joint segregation analysis in two populations of F2-derived
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recombinant inbred lines (RILs). The first population consisted of 53 F10 RILs derived from the cross JI1794 slow and constituted the population on which the consensus linkage map for pea was based [33]. The second population consisted of 51 F8 RILs derived from the cross MN313 JI1794. Using the PsPK2 specific primers PCR amplification products were generated from P. sativum parental lines and RILs. Restriction digestion of these products was performed by taking a 10 ml aliquot of the PCR products and adding to 20 ml solution of 10 times restriction buffer, water and restriction enzyme and incubating for 60 min as per instructions from the supplier of the restriction enzymes. Products of the restriction digest were separated on 2% agarose gels. The AgeI restriction pattern was compared to each of the segregation patterns of the approximately 1000 markers previously mapped in the JI1794 slow RIL population and to each of the approximately 300 markers previously mapped in the MN313 JI1794 population. The location of PsPK2 was determined by minimizing the number of recombinants between it and flanking markers using the EXCEL macro QUIKMAP [34]. 2.6. RNA extraction and gene expression patterns Total cellular RNAs were isolated from different plant parts of WT and mutant plants using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocols. RNA samples were DNase I (DNA-free kit; Ambion, Austin, TX) treated on the mini spin column according to the manufacturer’s instructions to eliminate any contaminating genomic DNA. The RNA was carefully quantified and run on a gel with ethidium bromide staining to verify equal loading. The plant parts used were mature leaflets, mature tendrils, seedling roots, shoot tips, root tips, flower buds, immature adult leaves and internodes from WT plants, mature leaflets from tl and uni-tac plants, and mature tendrils from af plants. Developing pods of WT plants were collected and four samples of three stages of embryo development were obtained: young (embryo linear, occupied less than half the ovule’s volume and without cotyledon swelling), medium (embryo occupied approximately half the ovule’s volume and with swelling cotyledons), mature (embryo filled the ovule’s volume but had not yet started drying) and mature embryo axis. In addition, shoot tips were dissected from 2week-old seedling plants of WT, tl, af, uni-tac and af tl. All tissues were immediately frozen in liquid nitrogen and stored in a 80 8C freezer until RNA was isolated. cDNAs were synthesized separately from each RNA preparation in a 20 ml reaction mixture containing 1 mg total RNA, 2 ml 10 times RT buffer, 2 ml dNTPs mixture (each at 5 mM), 1 ml random hexamer primer mixture, 0.25 ml RNase inhibitor, 1 ml Ominiscript Reverse Transcriptase (Qiagen). Synthesis time was 1 h at 37 8C. PCR was performed in a 50 ml volume containing 2.5 U Taq DNA polymerase (Qiagen), 1 ml cDNAs, 200 mM
nucleotides, 40 pM each specific primer, and 5 ml 10 times reaction buffer. The PCR conditions were 3 min at 92 8C, followed by 44 cycles of 1 min at 92 8C, 1 min at annealing temperature 62 8C of primers and 2 min at 72 8C, followed by a final extension of 10 min at 72 8C. The PCR products were separated on 1% agarose gels, stained with ethidium bromide and photographed with a Gel Doc 2000 (Bio-Rad). Control PCR reactions using primers specific to pea b-actin (GenBank accession no. X90378) (0.6 kb) were done to confirm equivalent RNA inputs into the reactions. The actin primers were ActinF (50 -GTT GGA ATG GGA CAA AAA GA-30 ) and ActinR (50 -CGA TGG CTG GAA CAG AAC30 ). These primers flanked an intron in the pea b-actin gene and the product size verified the lack of contaminating genomic DNA. 2.7. Auxin and GA induction experiments WT seeds were surface sterilized in 10% bleach for 10 min, washed in four changes of deionized water, and imbibed overnight in 2 l of deionized water. They were then sown in sterile vermiculite, saturated with sterile deionized water, and grown for 7 days at room temperature in complete darkness. The shoot tips were dissected from seedlings under a dim green safelight and treated with hormones. The hormones used were: indole-3-acetic acid (IAA), 4-chloroindole acetic acid (4-Cl-IAA), indole-3-butyric acid (IBA), indole-3-acetic acid methylester (methyl-IAA), a-naphthalene-acetic acid (NAA) and gibberellin A3 (GA3). All hormones were obtained from Sigma except 4-Cl-IAA, which was a gift from Jerry Cohen. In the first experiment, eight shoot tips were incubated with water or 10 mM IAA, 4-Cl-IAA, IBA, methyl-IAA or NAA for 30 min. All auxin stock solutions were made in 100% ethanol and the appropriate amount of hormone solution was added to the deionized water used as the bathing medium. Shoot tips were immediately frozen in liquid nitrogen after treatment and stored at 80 8C until RNA purification. IAA, 4-Cl-IAA and IBA were used because they are naturally occurring auxins in pea. MethylIAA was used because it is transported through tissues and cell membranes more efficiently than other auxins, and when methyl-IAA enters a cell, the methyl group is cleaved off producing IAA (Dr. Jerry Cohen, personal communication). In the second experiment, the shoot tips were separated into two groups. One group was placed in sterile deionized water or 10 mM methyl-IAA (Sigma, I-9770) in the dark for 10 min, 30 min, 1 h, 2 h or 4 h, and in the other group, shoot tips were treated with 0, l, 10, 25, 50, or 75 mM methyl-IAA for 30 min. In each treatment, eight shoot tips were used. The methyl-IAA stock solution was made in 100% ethanol and the appropriate amount of methyl-IAA solution was added to the deionized water used as the bathing medium. Shoot tips were immediately frozen in liquid nitrogen after treatment and stored at 80 8C until RNA purification.
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In the third experiment, the shoot tips were separated into two groups. One group was placed in sterile deionized water or 75 mM GA3 in the dark for 10 min, 30 min, 1 h, 2 h or 4 h, and in the other group, shoot tips were treated with 0, 50, 75 or 100 mM GA3 for 4 h. In each treatment, eight shoot tips were used. The GA3 stock solution was made in ethanol and the appropriate amount of GA3 solution was added to the deionized water used as the bathing medium. Shoot tips were immediately frozen in liquid nitrogen after treatment and stored at 80 8C until RNA purification. 2.8. Semi-quantitative RT-PCR cDNAs were synthesized separately from each RNA preparation in a 20 ml reaction mixture containing 1 mg total RNA, 2 ml 10 times RT buffer, 2 ml dNTPs mixture (each at 5 mM), 1 ml random hexamer primer mixture, 0.25 ml RNase inhibitor, 1 ml Ominiscript Reverse Transcriptase (Qiagen). Synthesis time was 1 h at 37 8C. PCR was performed in a 30 ml containing 2.5 unit HotStarTaq DNA polymerase (Qiagen), 6 ml cDNAs, 200 mM dNTP, 40 pM each specific primer, and 3 ml 10 times reaction buffer. The PCR conditions were 15 min at 95 8C, followed by 19 cycles of 1 min at 94 8C, 1 min at annealing temperature 60 8C of primers and 2 min at 72 8C, followed by a final extension of 10 min at 72 8C. The PCR products were separated on 1% agarose gel. The following primer sets were used: DeadboxF (50 -TTC TCG TCA TCA ACC TCA CC-30 ) and DeadboxR (50 -TTC CTA CCA AAC CTT CCA CTA C-30 ) for control gene, DEAD box (GenBank accession no. AY167670), and the primers for the PsPK2 gene. The gels were transferred to nylon membranes, hybridized with a 32P-labeled probes PsPK2 and DEAD box separately, and quantification of the blots were done using Typhoon 9410 (Amersham Pharmacia Biotech). The densitometry ratio of PsPK2 value/DEAD box value was calculated and graphed using SigmaPlot 2000. The sample size was two separate sets of RNA extractions from each treatment. Means and standard errors were determined using Microsoft Excel xp.
3. Results and discussion 3.1. Cloning and structure analysis of PsPK2 The complete transcribed sequence of PsPK2 (GenBank accession no. AY505304) was obtained by screening a WT shoot tip cDNA library with a partial cDNA followed by 50 RACE-PCR. The PsPK2 full cDNA was 1573 bp. The deduced PsPK2 amino acid sequence consisted of 445 amino acid residues (Fig. 1A) and had a calculated pI of 9.16 and molecular mass of 49,869. The PsPK2 gene was also sequenced and found to contain a small 44 bp intron between amino acid R and F at 181 and 182, respectively.
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PsPK2 had the conserved catalytic core typical of eukaryotic protein kinases at positions 68–394, including the well characterized 12 conserved subdomains (Fig. 1A) [8]. A serine/threonine protein kinase active-site signature at 199–211 (LiYrDLKpeNVLV) (Fig. 1A) was characteristic of amino acid substrate specificity and predicted PsPK2 to be a protein serine/threonine kinase. The short amino acid sequence KKKS in subdomain III was predicted to be a cAMP- and cGMP-dependent protein kinase phosphorylation site using ScanProsite (Fig. 1A). The N-terminal domain of PsPK2 is rich in serines and contains several potential phosphorylation sites. The predicted PsPK2 polypeptide shared 71.7% identity and 77.6% similarity with PID from Arabidopsis thaliana, and had 72.9% identity and 78.9% similarity with the polypeptide encoded by a Brassica rapa gene, Bcpk1 (Fig. 1B). A highly conserved amino acid motif DFD occurred at positions 221–223 located in subdomain VII of the PsPK2 sequence and was also present in PID and Bcpk1 (Fig. 1B). This motif is characteristic of the AGC-VIII group of protein kinases. Between conserved domains I and II we found a stretch of approximately eight amino acids in PsPK2 that were not present in PID or Bcpk1 (Fig. 1B). These amino acids occur in a region between subdomains I and II that folds away from the ATP binding site in protein kinase PKA, and there are several kinases with extra amino acids here [8]. Also, a segment of about 50 amino acids is present between conserved domains VII and VIII (open bar, Fig. 1B), which has been suggested to be a regulatory domain [10] or to have an affect on catalytic activity [35] that PsPK2, PID and Bcpk1 all share. This ‘‘insert’’ between subdomains VII and VIII is another unique feature of the AGC-VIII kinases, but there was little conservation of amino acid sequence in this region for these three kinases. The Nterminal region of the PsPK2, PID and Bcpk1, shares low identity as well as does the C-terminal end beyond the catalytic domain. To determine the relationship of PsPK2 to these kinases, a phylogenetic analysis was performed to develop a tree of related kinases using the sequences of the catalytic domain (Fig. 2). The PINOID, phototropin, PVPK-1 and PsPK3 subfamilies belong to the AGC-VIII group, which are characterized by a DFD motif instead of DFG in conserved subdomain VII of the catalytic site. We propose that PsPK2, Bcpk1 and PID group together in a unique PINOID subfamily because they share high sequence conservation in the catalytic domain. Finally, Bcpk1 and PINOID are probably more similar to one another than to PsPK2 because Brassica and Arabidopsis are both members of the Brassicaceae. 3.2. PsPK2 promoter sequence We obtained 2.3 kb of PsPK2 promoter sequence (GenBank accession no. AY785781) by genome walking
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Fig. 1. The PsPK2 amino acid sequence. (A) The predicted PsPK2 protein contains the conserved catalytic core of protein kinases at amino acids 68-394. Portions of the 12 conserved subdomains are in boldface. The Ser/Thr protein kinase active-site signature is underlined. The cAMP and cGMP-dependent protein kinase phosphorylation site KKKS is enclosed by a box. (B) Alignment of the PsPK2 sequence with PINOID and Bcpk1 using ClustalW (http:// www.ebi.ac.uk/clustalw/). Identical amino acids present in all three proteins are shaded. The highly conserved amino acid sequence DFD motif is underlined. A segment of eight amino acids between subdomains I and II (dots) and a stretch of about 50 amino acids between subdomains VII and VIII (open bar) are marked.
(Fig. 3) and analyzed this sequence using software PlantCARE and PLACE. An AuxRE containing the consensus sequence TGTCTC, which is common to early response genes and is recognized by the transcription factor known as ARF was identified at 1695 to 1690. This element is thought to confer auxin inducibility of promoters [36–38] by a mechanism involving ARF and Aux/IAA proteins that is well characterized [39–44]. An AuxRR-core,
which is another regulatory element involved in auxin responsiveness, was found at seven locations, 815 to 809 (GGTCCAT), 1321 to 1315 (GGTCAAT), 1958 to 1952 (GGTCCTT), 2063–2057 (GGTCCCT), 2128 to 2122 (GGTCCCT), 2162 to 2156 (GGTCCCT), and 2207 to 2201 (GGTCCAA). AuxRRs were found in the promoters of PsPIN1 and Ps-IAA4/5 from pea [29,45], parB [46] and parC from tobacco [47], and SAUR from
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1 cM of these latter two markers, the limit of precision for our analysis. There are no known leaf morphology mutants of pea that map to this position, and so we must also conclude that PsPK2 cannot be considered a candidate gene for the various leaf mutations that map to other positions on the pea linkage map. Note that recombination values might be slightly reduced compared to some linkage maps of pea because JI1794 is a P. sativum ssp. elatius (Steven ex M. Bieb) Asch. & Graebner accession, and the two alternative parents are P. sativum ssp. sativum and P. sativum ssp. elatius. Wide crosses in Pisum tend to give lower map distances between standard markers. 3.4. Expression of PsPK2
Fig. 2. Phylogenetic tree of the relationships among Ser/Thr protein kinases. PsPK2, BcPK1 and PINOID are in the PINOID subfamily within the AGC-VIII group. Multiple alignments were performed with ClustalW 1.74 and visualized using NJplot.
soybean [48] and have been shown to be involved in auxin response. Many putative light responsive elements such as Gbox, AAAC-motif, AE-box, ATC motif, I-box, and others were present on the PsPK2 promoter suggesting that PsPK2 is possibly regulated by light. Lin et al. [31] studied light regulation of five protein kinases, PsPK1– PsPK5 of pea and found that transcripts were differentially regulated during deetiolation. PsPK2 mRNA levels did not respond to light significantly during this process. Because the putative gibberellin (GA) responsive elements, 16 P-boxes and a TATC-box were also identified in the promoter sequence, we tested the possibility that PsPK2 is GA regulated. 3.3. Mapping of PsPK2 The PsPK2 locus was positioned between Pl and Gsp in both RIL populations (Fig. 4). More precisely, PsPK2 mapped halfway between these two markers and cosegregated with the RAPDs B192420 and B162a, placing it within
In WT plants, PsPK2 mRNA was differentially expressed in different plant parts. PsPK2 was abundant in immature adult leaves, shoot tips and flower buds. It was also present in seedlings, roots, and stems. No expression was found in mature leaflets but it was abundant in mature tendrils (Fig. 5A). This pattern of expression is very similar to that described for PID in Arabidopsis. Both PsPK2 and PID mRNAs are most abundant in growing and developing regions such as flower buds, leaf primordia, the shoot apical meristem and seedlings [10,11]. We compared PsPK2 expression in pinna types from WT leaves and those of the leaf mutants, af, tl and uni-tac (Fig. 5B). PsPK2 was expressed in mature tendrils from leaves of af and WT, although no significant expression was detected in mature leaflets of WT, tl or uni-tac. Basically, PsPK2 mRNA was abundant in tendrils irrespective of genotype, but not in leaflets. Tendrils are thigmotropic, a process that involves auxin signaling, whereas leaflets are not. Therefore, the greater expression of PsPK2 may be associated with greater auxin production and transport in tendrils. Tendrils do transport more auxin than other leaf parts in pea [28]. Overexpression of 35S::PID in Arabidopsis resulted in a reduced gravitropic response in the primary root suggesting that PID may play an important role in control of gravitropism, which is also regulated by auxin [10,11]. Furthermore, differential expression occurred in shoot tips of the different pea leaf mutants. PsPK2 mRNA level was slightly more abundant in af and af tl, and less abundant in tl and uni-tac compared to WT (Fig. 5C). This was verified using semi-quantitative RT-PCR (data not shown). The expression of PsPK2 in shoot tips from different genotypes is similar to that of the Uni gene, which is known to play a
Fig. 3. Diagram of the PsPK2 promoter. The CAAT-box (67) and TATA-box (59) are designated by open rectangles. Auxin response elements: AuxRE (1695) and seven AuxRRs (815, 1321, 1958, 2063, 2128, 2162 and 2207) are designated by black rectangles. GA response elements: P-boxes (442, 1080, 1103, 1328, 1498, 1620, 1911, 1919, 1946, 1955, 1972, 1987, 2027, 2108, 2202 and 2259) and a TATC-box (94) are designated by grey rectangles.
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Fig. 4. Mapping of the PsPK2 gene. (A) Position of PsPK2 on Linkage Group VI on the map developed for the JI1794 slow RILs. (B) Position of PsPK2 on linkage group VI on the map developed for the MN313 JI1794 RILs. Distances between adjacent markers are given as recombination percentages and were calculated using the equation: r = R/(2 2R), where r is the frequency of recombination and R is the proportion of RILs displaying a recombinant phenotype [54]. Other markers used to position PsPK2 include Pl (black hilum), Gsp (plastid specific glutamine synthetase), Hop1 (homeodomain protein-1) and Prx (peroxidase-3) and RBIP-95x2 (a retrotransposon-derived marker obtained courtesy of Dr. Andy Flavell). The remaining markers are RAPDs, with priming sequences that can be obtained from the authors upon request.
central role in pea leaf development [19,49]. Uni mRNA is more abundant in tl, af and af tl and less abundant in uni-tac compared to WT [28]. Since both PID and PsPK2 are regulated by auxin (see below), this expression pattern might indicate that developing leaves of the af genotypes produce more auxin than those of WT, a hypothesis that was proposed in an earlier study [30]. In any case, this differential
expression of PsPK2 in the different leaf form mutants suggests that it might play an important role in leaf development in pea. Observations of its in situ expression patterns in developing leaves of these mutants may help us determine what this role might be. PsPK2 was also expressed differentially during embryo development (Fig. 5D). PsPK2 expression was weak in young embryos, higher in medium embryos, and weak in mature, whole embryos. However, expression was high in the axis of mature embryos. Expression was low in the whole, mature embryo due to the presence of the large, storage cotyledons where the mRNA level of PsPK2 was presumably low. The expression pattern is similar to that shown for PsPIN1 [29], and PID is known to be expressed during Arabidopsis embryogenesis. In addition to being present in globular stage embryos and on the adaxial side of cotyledon primordia in the late heart to torpedo stages, PID mRNA is mainly expressed at the boundary between cotyledon primordia [12]. 3.5. Hormone regulation of PsPK2 expression
Fig. 5. PsPK2 expression in different plant parts of WT and leaf form mutants: af, tl, uni-tac and af tl. (A) Expression in different parts of WT plants. (B) Expression in leaflets and tendrils of WT, tl, af and uni-tac. (C) Expression in shoot tips of WT, af, tl, uni-tac and af tl. (D) Expression in three stages of WT embryos, young, medium, mature whole and mature axis only.
We examined PsPK2 mRNA expression in response to treatment by different auxins, both natural and synthetic. Semi-quantitative analysis showed higher PsPK2 mRNA levels in response to all auxins used compared to controls. Methyl-IAA treatment produced the strongest response followed by IAA, 4-Cl-IAA, NAA, and IBA had the least (Fig. 6A). Therefore, among the naturally occurring auxin in pea, PsPK2 expression is most sensitive to IAA regulation. PsPK2 mRNA increased approximately 1.5-fold at 30 min after methyl-IAA treatment and then declined to near control levels after 4 h of treatment (Fig. 6B). In the dose response experiment, PsPK2 mRNA levels were most abundant at 10 mM methyl-IAA, less abundant at other concentrations, but higher than control at all concentrations except at 1 mM
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Fig. 6. PsPK2 mRNA expression relative to DEAD box mRNA levels in response to auxin treatment. (A) PsPK2 expression induced by 10 mM IAA, 4-Cl-IAA, IBA, methyl-IAA and NAA for 30 min. (B) PsPK2 expression induced by methyl-IAA for different time periods. (C) PsPK2 expression in different concentrations of methyl-IAA for 30 min. Control 1 = time zero control before methyl-IAA treatment, control 2 = control at 30 min without methyl-IAA treatment. The error bars are S.E. which are average from two repeats of each treatment.
(Fig. 6C). PID expression has also been shown to be regulated by auxin in Arabidopsis. Benjamins et al. [11] showed by Northern blotting that PID mRNA increased at 4 h after 5 mM IAA treatment, but declined by 20 h. Also, the PID promoter was found to possess a TGTCTC AuxRE. Rapid upregulation followed by a decline, and the presence of an ARF binding site in both PID and PsPK2, suggest that both are primary auxin response genes. PsPIN1 is also regulated by auxin, however, it is more responsive to 4-ClIAA than IAA, requires higher levels of auxin for induction, and responds more slowly than PsPK2 (2–4 h). Reinecke et al. [50] showed that 4-Cl-IAA was more active than IBA and IAA in stimulating both stem and pericarp growth in pea. To verify the GA responsiveness predicted by promoter sequence, we examined the kinetics and concentration dependence of PsPK2 expression in response to exogenous
GA3. PsPK2 mRNA increased approximately 1.8-fold at 4 h after GA3 treatment compared to the control (Fig. 7A). In the dose response experiment, PsPK2 mRNA levels were most abundant at 75 mM GA3, and were less abundant in the other treatments, although they were higher than control in all treatment concentrations (Fig. 7B). The promoter was found to possess several GA responsive elements, 16 P-boxes and a TATC-box and we verified that GA regulates the PsPK2 expression in pea. We do not know whether this is a unique feature of this gene in pea or whether GA also regulates PID. However, since GA was also found to enhance PsPIN1 mRNA levels in pea [29], it seems that auxin transport in pea may be regulated both by auxin and GA. Auxin is known to regulate GA levels via its control of biosynthetic and catalytic enzymes. It upregulates GA 3-oxidase transcription and down regulates GA 2-oxidase transcription [51]. GA 3oxidase converts inactive GA20 into active GA1 and GA 2-
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leaflets of pea leaves, irrespective of genotype, suggesting that it might play a role in the thigmotropic response. PsPK2 is not only abundantly expressed in developing pea leaves, but it is differentially expressed in shoot tips of the different leaf form mutants suggesting that PsPK2 plays an important role in leaf form generation. PsPK2 and PID have been shown to be early auxin response genes and PsPK2 is also gibberellin-regulated. Our goal is to determine the roles of PsPK2 in auxin and gibberellin regulation, which may control pea leaf development.
Acknowledgements We thank Janet Giles for technical assistance with library screening, cloning the full sequence of the PsPK2 gene and preparing four genome walker libraries. We thank Dr. Rekha Chawla for preparing four genome walker libraries, and for comments on the manuscript. We thank Dr. Jerry Cohen for providing the 4-Cl-IAA. The pea genotypes used in this study were obtained from the Marx collection, which currently resides at the USDA-ARS, Pacific West Area, Pullman, Washington. Sequencing was done by the UCR Genomics Institute. This work was partially supported by USDA/NRI grant no. 2001-35304-10958 to Dr. Darleen A. DeMason.
References
Fig. 7. PsPK2 mRNA expression relative to DEAD box mRNA levels in response to GA3 treatment. (A) PsPK2 expression induced by GA3 for different time periods. (B) PsPK2 expression in different concentrations of GA3 for 4 h. Control 1 = time zero control before GA3 treatment, control 2 = control at 30 min without GA3 treatment. The error bars are S.E. which are average from two repeats of each treatment.
oxidase converts GA1 into inactive GA29. Further, 4-Cl-IAA significantly increases expression of another GA biosynthetic enzyme (GA 20-oxidase) in pea pericarp [52,53]. The simplest explanation for our results is that auxin regulates PsPK2 both directly and indirectly through regulation of GA levels.
4. Conclusion PsPK2 is the second PID-like gene to be characterized in the plant kingdom. Its sequence is very similar to that of PID and another PID-like gene, Bcpk1, suggesting the existence of a PINOID subfamily within the AGC-VIII protein kinases. PsPK2 mRNA is more abundant in tendrils than in
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