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Developmental Biology and Developmental Genetics |
Department of Botany and Plant Sciences, University of California, Riverside, California 92521 USA
Received for publication 16 May 2007. Accepted for publication 6 December 2007.
ABSTRACT
The putative pea PINOID homolog, PsPK2, is expressed in all growing plant parts and is positively regulated by auxin, gibberellin, and cytokinin. Here, we studied hormonal regulation of PsPK2::GUS expression compared with DR5::GUS and PID::GUS in Arabidopsis. PsPK2::GUS, DR5::GUS, and PID::GUS expression in Arabidopsis shoots is mainly localized in the stipules, hydathodes, veins, developing leaves, and cotyledons. Unlike DR5::GUS, PsPK2::GUS, and PID::GUS are weakly expressed in root tips. Both DR5::GUS and PsPK2::GUS are induced by different auxins and are more sensitive to methyl indole acetic acid, 4-chloro-indole acetic acid, and 
-naphthalene acetic acid than others. GA3 has no significant effect on GUS activity in DR5::GUS-transformed seedlings compared to the control, but induction by auxin and gibberellin in combination is synergistic. Cytokinin increases auxin transport in Arabidopsis seedlings. Auxin, gibberellin, and cytokinin all increase GUS activity in shoots of PsPK2::GUS transformed plants compared to the control. However, only auxin and gibberellin increase GUS activity in PID::GUS shoots. In conclusion, auxin, gibberellin, and cytokinin positively regulate PsPK2 expression in shoots, but not in roots. Auxin and gibberellin also upregulate AtPIN1 and LEAFY expression, which is similar to PsPIN1 and Uni in pea. With minor exceptions, the orthologous genes from both species are regulated similarly.
Key Words: AtPIN1 • auxin • cytokinin • DR5::GUS • gibberellin • LFY • PID::GUS • PsPK2::GUS
Gibberellin (GA), auxin, and cytokinin are three classic plant hormones known to regulate plant growth and development. Cross-talk and signal integration among growth-regulating hormones is an active area of research. GA has long been recognized to play roles in seed germination, stem and petiole elongation, induction of flowering, fruit growth, and root development. GA20ox, GA3ox, and GA2ox are three genes involved in GA synthesis and deactivation. Most of the GA20ox and GA3ox genes are downregulated by applied GA (Yamaguchi and Kamiya, 2000
; Olszewski et al., 2002), but the genes encoding GA 2-oxidase (GA2ox), which convert active GA1 to inactive GA29 are upregulated by GA treatment (Thomas et al., 1999; Elliott et al., 2001). Auxin is known to regulate GA levels by enhancing transcription of GA20ox and GA3ox and reducing that of GA2ox (Ross et al., 2000). DELLA proteins are plant growth repressors that act as key integrators of plant growth from multiple hormonal signals (Achard et al., 2003; Fu and Harberd, 2003; Nemhauser et al., 2006). They are degraded by GA and are regulated by auxin (Fu and Harberd, 2003; Cao et al., 2006).
Cytokinins play roles in the regulation of cell division, development of the shoot and root, delay of senescence, and transduction of nutritional signals. Cytokinins interact with auxin to control many central developmental processes in plants, particularly apical dominance, shoot development, and root development. In their classic tissue culture experiments, Skoog and Miller (1957) showed that callus cultures grown in high cytokinin to auxin ratios form shoots, while low ratios stimulate root formation. The IPT genes are thought to be key regulatory genes in cytokinin biosynthesis and homeostasis (Sakakibara, 2006). In Arabidopsis roots, transcription of AtIPT5 and AtIPT7 is promoted by auxin and that of AtIPT1, AtIPT3, AtIPT5, and AtIPT7 is repressed by cytokinin (Miyawaki et al., 2004). In pea, the expression of two IPT genes, PsIPT1 and PsIPT2, which are expressed in nodes, is negatively regulated by auxin (Tanaka et al., 2006). Cytokinin upregulates the GA biosynthetic gene, GA3ox (LE) in pea (Bai and DeMason, 2006).
Auxin is an essential hormone that also provides directional and positional information for plant growth and development. PINFORMED (PIN) proteins, which are the auxin efflux carriers, control polar indole acetic acid (IAA) transport via their position and function (Gälweiler et al., 1998; Palme and Gälweiler, 1999; Friml and Palme, 2002; Friml et al., 2004). Lateral organ initiation and/or outgrowth require local accumulation of auxin, and this process requires correct targeting of PIN1 (Benková et al., 2003; Reinhardt et al., 2003; Heisler et al., 2005). The Arabidopsis Ser/Thr kinase, PINOID (PID), targets PIN by phosphorylating its central, hydrophilic domain (Friml et al., 2004; Furutani et al., 2004; Lee and Cho, 2006; Michniewicz et al., 2007). Arabidopsis pid mutants resemble pin1 mutants and plants that are grown on polar auxin transport inhibitors, such as naphthylphthalamic acid (NPA). These plants have similar defects in leaf initiation, phyllotaxy, lamina shape, cotyledons, vascular patterning, and floral organ form (Bennett et al., 1995; Palme and Gälweiler, 1999; Christensen et al., 2000; Aloni et al., 2003; Benková et al., 2003; Furutani et al., 2004; Heisler et al., 2005). PID is expressed in developing embryos, shoot tips, leaf primordia, floral organ primordia, and procambial strands in developing organs (Christensen et al., 2000; Benjamins et al., 2001; Furutani et al., 2004). IAA induces PID::GUS expression in vascular tissue and leaf primordia of Arabidopsis seedlings (Benjamins et al., 2001). Over-expression of PID under the control of the constitutive CaMV 35S promoter (35S::PID) causes a phenotype that is similar to that of auxin-insensitive plants with a collapsed primary root, reduced root elongation, and reduced expression of auxin-responsive DR5::GUS (Christensen et al., 2000; Benjamins et al., 2001).
An important tool to localize regions of auxin responsiveness is the synthetic auxin response reporter construct known as DR5::GUS. This construct consists of seven-copy tandem direct repeats of the ARF-binding site from the soybean G3 promoter placed upstream of a minimal cauliflower mosaic virus (CaMV) 35S promoter and the β-glucuronidase (GUS)-coding sequence (Ulmasov et al., 1997). Promoter response was tested in transient expression assays of carrot protoplasts and in transformed Arabidopsis thaliana (L.) Heyhn. (Brassicaceae) seedlings using the synthetic auxin naphthalene acetic acid (NAA) (Ulmasov et al., 1997). Although both Sabatini et al. (1999) and Mattsson et al. (2003) used 2,4-dichlorophenoxy acetic acid (2,4-D) treatments as controls for DR5 responsiveness in root tips and leaf primordia, and Nakamura et al. (2003) used IAA treatments of seedlings, no careful evaluation of this promoter’s responsiveness to a range of auxins in different plant parts has been done. DR5 is also responsive to brassinolide (Nakamura et al., 2003), but because these responses are distinguishable from those of auxin treatment, DR5::GUS is still considered to be an important indicator of auxin response.
We cloned the PID ortholog, PsPK2, from pea. PsPK2 mRNA is ubiquitously expressed throughout pea plants, but it is especially abundant in growing parts, such as shoot tips, immature adult leaves, developing embryos, and flower buds (Bai et al., 2005). PsPK2 mRNA levels are positively regulated by GA and auxin and are induced to higher levels by simultaneous application of auxin and GA (Bai et al., 2005; Bai and DeMason, 2006). PsPK2 is differentially responsive to different auxins, and auxin induction of PsPK2 does not require de novo protein synthesis (Bai and DeMason, 2006). PsPK2 is marginally upregulated by cytokinin (Bai and DeMason, 2006). In addition, PsPIN1 is also positively regulated by both auxin and GA and shows differential responses to different auxins (Chawla and DeMason, 2004; Bai and DeMason, 2006). Although PsPK2 is differentially expressed in the different leaf form mutants of pea (Bai et al., 2005), little is known about its role in pea leaf development. Nothing is known about cytokinin or GA regulation of PID in Arabidopsis.
To learn more about the regulation of PsPK2, we focused in this study on auxin, GA, and cytokinin interactions and regulation of transgenic PsPK2::GUS compared with DR5::GUS and PID::GUS in Arabidopsis. The comparison to DR5::GUS is necessary because cross talk between hormones affects the regulation of naturally occurring genes, but DR5 allows the isolation of strict auxin responsiveness as an important control for interpretation of the PsPK2 promoter responses. The questions we addressed in this study were the following: (1) What is the GUS expression pattern of PsPK2::GUS during Arabidopsis seedling development compared with DR5::GUS? (2) How does PsPK2::GUS respond to different auxins compared with DR5::GUS (3) Does PsPK2::GUS or PID::GUS respond to auxin, GA, cytokinin, and to pairwise combinations of these hormones? (4) What are the characteristics of this regulation compared with that of DR5::GUS (5) How do the functionally related genes AtPIN1 and LFY respond to these same hormone treatments in Arabidops is shoot tips?
MATERIALS AND METHODS
Plant materials and growth conditions
Arabidopsis thaliana DR5::GUS (Ulmasov et al., 1997), PsPK2::GUS, and PID::GUS (Benjamins et al., 2001) transgenic plants in Col-0 background were used for this study.
The PID::GUS construct contained a 3.6-kb SphI-MspAI genomic fragment of the 5' untranslated sequence and the complete PID gene, excluding the last six codons, in frame with the GUS gene in pCAMBIA1381Xb (Benjamins et al., 2001). Seeds were surface sterilized with 70% ethanol for 2 min and 10% bleach for 10 min and washed four times with sterile water. Sterile seeds were cold treated for 4 d at 4°C and germinated in pots or on Murashige and Skoog medium (MS) (Caisson Laboratories, Logan, Utah, USA) with 0.8% (w/v) agar (PhytoTechnology Laboratories, Shawnee Mission, Kansas, USA), 3% (w/v) sucrose. Seedlings were grown at 22°C with a light regime of 8 h darkness and 16 h light (33 µmolm·-2·s-1).
Construction of PsPK2::GUS
To study the transcriptional regulation of the PsPK2 gene (accession AY505304), the 2.2-kb promoter of PsPK2 (accession AY785781) including the 5' untranslated region (UTR) was fused to the GUS reporter gene to monitor the expression of PsPK2. The reporter construct was created by cloning GUS and OCS from the SLJ4D4 plasmid (Jones et al., 1992) and inserting it into the SstI and HindIII sites of pCAMBIA3300 T-DNA (pCAMBIA) close to the right border. The PsPK2 promoter was then cloned into the EcoRI site upstream of the GUS reporter gene. This fusion sequence, designed as PsPK2::GUS was confirmed by sequencing. This binary T-DNA vector was introduced into Agrobacterium tumefaciens strain GV3101 by electroporation and transformed cells were selected from LB-agar plate that contained 50 µg/mL kanamycin. The selected transformed cells with binary T-DNA vectors were used to transform wild-type Col ecotype of Arabidopsis by the floral dip method (Clough and Bent, 1998). T1 seeds were harvested and selected on the basis of resistance to phosphinothricin (PPT) by spraying with the commercial herbicide Basta. Thirty-six Basta-resistant Arabidopsis seedlings were grown to get T2 seeds. T2 seedlings were confirmed by GUS staining, and selection on LB-agar plates that contained 50 µg/mL kanamycin. Sixteen lines of T2 with one insertion were grown to harvest T3 seeds. T3 seeds were pooled for further experiments. The blue color observed in T3 transgenic Arabidopsis plants indicated that active expression of the GUS gene was driven by the PsPK2 promoter.
Hormone response assays
The hormones used were indole-3-acetic acid (IAA), 4-chloro-indole-3-acetic acid (4-Cl-IAA), indole-3-butyric acid (IBA), indole-3-acetic acid methylester (MeIAA), -naphthalene acetic acid (NAA), gibberellin A3 (GA3), and N6-benzyladenine (BAP). All hormones were obtained from Sigma (St. Louis, Missouri, USA) except 4-Cl-IAA, which was a gift from Dr. Jerry Cohen, University of Minnesota. All hormone stock solutions were made with 100% ethanol except BAP, which was dissolved in 1N HCl. The polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) (90% purity) was a gift from Uniroyal Chemical (Middlebury, Connecticut, USA). The NPA stock solution (50 mM) was made in warm water. The appropriate amount of hormone or NPA solution was added to the MS media or to the deionized water used as the bathing media.
Two methods were used to treat seedlings with different hormones or NPA. For the first method, seedlings of DR5::GUS, PsPK2::GUS, or PID::GUS were grown on MS medium for 5 d after germination and then transferred for another 2 d to MS medium containing the following chemicals: 1 µM MeIAA/NAA, 1 µM GA3, 1 µM BAP, 1 µM MeIAA/NAA + 1 µM GA3, 1 µM BAP + 1 µM GA3, 1 µM BAP + 1 µM MeIAA/NAA, 1 µM BAP + 2 µM NPA, or 2 µM NPA. For the second method, seedlings of DR5::GUS and PsPK2::GUS were grown on MS medium for 10 d and then incubated for 2 h with water or 10 µM 4-Cl-IAA, 10 µM IAA, 10 µM IBA, 10 µM MeIAA, 10 µM NAA, 15 µM GA3, or 10 µM MeIAA + 15 µM GA3 combination solution. A hundred seedlings were used for each treatment.
Histochemical analysis of GUS activity
DR5::GUS, PsPK2::GUS, and PID::GUS seedlings treated with or without hormones were submerged in GUS staining buffer (Jefferson et al., 1987) containing 1 mM 5-bromo-4-chloro-3-indolyl β-D-glucuronidase (Gold BioTechnology, St. Louis, Missouri, USA), 100 mM sodium phosphate (pH 7.5), 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 10 mM EDTA, and 0.1% (v/v) Triton X-100. Seedlings for all treatments were incubated at 37°C for 4 h and cleared with 70% (v/v) ethanol. Fifteen to 20 seedlings were observed for each treatment. Images were taken with a dissecting microscope (Leica MEFLIII) with a digital camera (Diagnostic Instruments, Sterling Heights, Michigan, USA).
Quantitative GUS activity assay
GUS activity was assayed with the substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) (Sigma) (Jefferson, 1987). First, seedlings were frozen in liquid nitrogen and ground in MUG extraction buffer (30 mg tissue/300 µL) composed of 50 mM sodium phosphate (pH 7.0), 10 mM β-mercaptoethanol, 10 mM EDTA (pH 8.0), 0.1% (w/V) N-lauroyl sarcosine (SLS) (Sigma), and 0.1% (v/v) Triton X-100. The extract was spun in a microcentrifuged for 15 min at 4°C at 13 000 rpm. The supernatant was transferred to new tubes and kept at –80°C. Next, 10 µL of the protein extract were mixed with 390 µL GUS assay buffer (22 mg MUG in 50 ml GUS extraction buffer) and incubated at 37°C for 1 h. Samples (100 µL) were taken from each tube at 0, 30, and 60 min incubation time, and reactions were stopped with 4.9 mL of 0.2 M Na2CO3. Fluorescence was determined with a fluorometer (DyNA Quant 200). Protein concentration was determined according to Peterson’s modification of the Lowry method (Peterson, 1977). GUS activity was expressed as nmol MUG/min/mg protein. The sample size was 40 seedlings for each treatment. Means ± SE of three replicates were calculated. A paired t test in Microsoft (Redmond, Washington, USA) Office Excel was used to check the P value of treatments vs. control. If P > 0.05, there was considered to be no significant difference between the two samples.
Promoter analysis
The promoter sequences of AtPIN1 (At1g73590.1) (2904 bp), AtPID (At2g34650.1) (3600 bp) and LEAFY (At5g61850.1) (2386 bp) including the 5' UTRs were analyzed using PlantCARE (http://oberon.rug.ac.be:8080/PlantCARE/index/html) and PLACE (http://www.dna.affrc.go.jp/htdocs/PLACE/fasta.html).
RNA extraction and cDNA synthesis
Total cellular RNAs were isolated from 7-d-old Arabidopsis seedlings using the RNeasy Mini Kit (Qiagen, Maryland, USA) according to the manufacturer’s protocols. RNA samples were DNase I (DNA-free kit, Ambion, Austin Texas, USA) treated on minispin columns 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 cDNAs were synthesized separately from each RNA preparation in a 20-µL reaction mixture containing 0.5 µg denatured RNA, 1 µL random primers (Promega, Madison, Michigan, USA), 10 mM dNTPs mixture (Promega), 4 µl 5x first strand buffer, 2 µL 0.1 M dithiothreitol, and 0.2 µL reverse transcriptase (Invitrogen, Carlsbad, California, USA). Synthesis time was 10 min at 42°C followed by 60 min at 50°C and 15 min at 70°C.
Semiquantitative RT-PCR
PCR was performed in 25 µL containing 1 unit HotMaster Taq DNA polymerase (Eppendorf, Westbury, New York, USA), 5 µL cDNAs, 100 µM dNTP (Promega), 5 pM each primer, and 2.5 µL 10x reaction buffer. The PCR conditions were 15 min at 95°C; followed by 19 cycles of 1 min at 94°C, 1 min at the appropriate annealing temperature for the primers, and 2 min at 72°C; and a final extension of 10 min at 72°C. The annealing temperature differed for each gene: ACTIN-2 was 57°C, LFY was 62°C, AtGA20ox4 was 63°C, and AtPIN1 was 61°C. The PCR products were separated on a 1% agarose gel. The following primer sets were used: ACT2F (5'-CTCATGAAGATTCTCACTGAG-3') and ACT2R (5'-ACAACAGATAGTTCAATTCCCA-3') for the control gene ACTIN-2 (ATU37281); AtPIN1F (5'-GCTTTTGATCTCCGAGCAGTTT-3') and AtPIN1R (5'-GTCTTGTCTTTTCCCACCAACC-3') for the AtPIN1 gene (AT1G73590); AtGA20ox4F (5'-GGAATGCATCATAAAGCTCCCTCAAAG-3') and AtGA20ox4R (5'-CAGAAACTTCCTTTGTTCTTGAGCC-3') for the AtGA20ox4 gene (AT1G60980); LFY-F (5'-TGAAGGACGAGGAGCTTGAAGAG-3') and LFY-R (5'-TTGCCACGTGCCACTTC C-3') for the LFY gene (AT5G61850). The PCR products were transferred to nylon membranes, hybridized separately with a 32P-labeled probe for the ACTIN-2, AtPIN1, AtGA20ox4, or LFY genes. Quantification of the blots was done using a Typhoon 9410 Phosphoimager (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA). The densitometry ratios for AtPIN1/ACTIN-2, AtGA20ox4/ACTIN-2, and LFY/ACTIN-2 were 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. The experiment was repeated five times, and representative results are presented.
RESULTS
Seedling expression of DR5::GUS
In 2- and 3-d-old (equivalent to days after germination) DR5::GUS seedlings, GUS activity was detected in both shoots and roots (Fig. 1A, B). Staining occurred at the tips of cotyledons and at the root tips. In 4-d-old seedlings, the first pair of developing leaves stained dark blue, and GUS activity was high at the tips of cotyledons, in the central cylinder of roots and in root tips (Fig. 1C). In 7-d-old seedlings, GUS activity was strong in the stipules, tips, and veins of cotyledons and leaves, root procambial strands, root apices, and lateral root primordia (Fig. 1D). At 2 wk, expanding leaves had higher GUS activity than mature leaves (Fig. 1E). In both young and mature leaves, GUS staining was typically seen in the veins, hydathodes, and leaf tips.
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GUS expression in shoots of PID::GUS seedlings did not respond to MeIAA or NAA (data not shown), so we used IAA for individual and combination auxin treatments. IAA and GA3 were the only individual treatments that produced an increase in GUS activity of PID::GUS seedling shoots (Fig. 10C). As for the other seedlings, the combination of IAA and GA3 produced the greatest upregulation, but in this case it was neither additive or synergistic compared to each hormone treatment used alone. Again the combination of IAA + BAP produced an intermediate effect between that of BAP and IAA used alone.
In general, these results were consistent with our computer analysis results since all three promoters have auxin responsive elements and that of PsPK2 has GA responsive elements. An unexpected but consistent result was the response of all promoters to auxin plus cytokinin, which was intermediate between that of each hormone alone. We had expected an additive or synergistic effect because cytokinin increases auxin transport and auxin increases GA homeostatsis. Our results could be explained by the downregulation of GA by cytokinin as demonstrated by Jasinski et al. (2005).
Auxin, gibberellin, and cytokinin interactions in Arabidopsis shoot tips
Many genes are known to be expressed in shoot tips and in response to auxin, GA, or cytokinin. Among them are the AtPIN1, LFY, and AtGA20ox genes. We evaluated the responsiveness of AtPIN1 and LFY to these hormones both empirically and by computational analysis of known regulatory sequences in their promoters. The promoter and 5' upstream noncoding regions of the PsPIN1 possess one AuxRE and one AuxRR-core, and the same GA response elements, (i.e., P-boxes and TATC-boxes) that are also present in the promoter of PsPK2 (Chawla and DeMason, 2004; Bai et al., 2005). No classical cytokinin response elements were found. The AtPIN1 promoter has six AuxRE sites, but no typical GA or cytokinin response elements. The Uni promoter possesses four AuxRR-cores, but no AuxRE sites for auxin response, and 21 P-boxes and 4 TATC-boxes, for GA response (Bai and DeMason, 2006), whereas the LFY promoter has one AuxRE and three GA-MYB binding sites, which also suggest auxin and GA regulation, but by different mechanisms. From these data, we would predict that only the LFY/Uni orthologs would have similar hormonal responses in both species, i.e., upregulated by both auxin and GA.
We treated 7-d-old Arabidopsis seedlings with different hormones individually and in pairwise combinations and evaluated the mRNA levels of AtPIN1 and LFY. MeIAA, GA3, MeIAA + GA3, and MeIAA + BAP treatments increased AtPIN1 mRNA levels compared with the control (Fig. 11). Based on a t test, neither BAP nor BAP + GA3 had any significant effect on AtPIN1 mRNA levels (t > 0.05). LFY mRNA levels were highest after MeIAA and MeIAA + GA3 treatments followed by the MeIAA + BAP treatment and GA3 treatment alone. The combination of auxin and GA resulted in the highest expression levels measured for both the AtPIN1 and LFY genes. However, the enhancement in comparison with auxin or GA alone was neither synergistic nor even additive. These results suggest that auxin and GA regulation mainly occurs in the same signaling pathway and not separately. BAP treatments increased LFY mRNA levels over the control, but less than the other treatments. BAP + GA3 had no significant effect on LFY mRNA levels (P > 0.05). NPA treatment increased AtPIN1 mRNA levels, but had no obvious affect on LFY mRNA levels compared to the seedlings grown on control media (P > 0.05).
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showed that these enzymes are downregulated by GA, whereas auxin differentially upregulates the expression of various AtGA20OX genes (Frigerio et al., 2006). Auxin increases AtGA20ox1 (GA5) and AtGA20ox2 mRNA levels, but has less effect on expression of AtGA20ox3 and AtGA20ox4. Our results verified that AtGA20ox4 is marginally upregulated by auxin and is downregulated by GA, and therefore our treatment regimes and experimental methods were effective (data not shown). DISCUSSION
One of the fundamental characteristics of plants is their polar organization and polar growth. This polarity is established very early in the plant life cycle of plants. All plant organs and assemblages of organs (shoots, flowers, and inflorescences) have distinct polarity or asymmetry in their organization and growth patterns. An auxin gradient has been implicated in directing patterns of differentiation in the cambial zone of Pinus (Uggla et al., 1996), root tip (Sabatini et al., 1999, Friml et al., 2004), the embryo (Hamann et al., 1999, Hamann et al., 2002; Jürgens, 2001), vascular system of leaves (Scarpella et al., 2006) and the gynoecium (Nemhauser et al., 2000) of Arabidopsis. These results have lead Doerner (2000) to conclude that an auxin gradient provides the positional framework for all patterning processes in plants. When new organs such as axillary buds, secondary roots, and leaf or floral primordia are initiated, a new axis of growth is formed, and a new auxin gradient is established. Control of auxin transport is, therefore, a critical component of controlling plant morphogenesis (Scarpella et al., 2006).
Auxin efflux carriers, PINs, present only in specific cell membranes, drive polar auxin transport (Gälweiler et al., 1998; Palme and Gälweiler, 1999). The PID protein kinase in Arabidopsis regulates transport by phosphorylating PIN proteins, which leads to correct targeting (Friml et al., 2004; Michniewicz et al., 2007). If this gene plays the same role in all plant species, it must be among the most important genes that control morphological diversity. In spite of this, PID orthologs have only been identified in four other plant species including Bcpk1 in Brassica rapa, OsPID in rice, bif2 in maize, and PsPK2 in pea. Many aspects of the expression patterns of PID, PsPK2, OsPID, and bif2 have been described. As expected, these genes are expressed in all developing plant parts, including embryos, leaf primordia, and flower buds (Christensen et al., 2000; Benjamins et al., 2001; Bai et al., 2005; McSteen et al., 2007; Morita and Kyozuka, 2007). Both PID and OsPID have low expression in roots (Benjamins et al., 2001; Morita and Kyozuka, 2007). PsPK2 is differentially expressed in shoot tips of the different leaf form mutants in pea, suggesting that it plays a role in leaf form generation (Bai et al., 2005). In the present study, we described the regulation of PsPK2 via its promoter in transgenic Arabidopsis plants to look at its (1) positional expression in seedlings, (2) auxin responsiveness in comparison to DR5::GUS and PID::GUS, (3) responsiveness to treatments with combinations of hormones, and (4) hormone expression responses compared to AtPIN1 and LFY.
Expression patterns of PsPK2::GUS compared with DR5::GUS and PID::GUS in transgenic Arabidopsis seedlings
Although DR5 has been used in many studies to evaluate specific sites and patterns of auxin distribution and response, few general descriptions of expression patterns in Arabidopsis have been done to provide basic information about locations of auxin activity during all stages of the plant’s life cycle. Such studies can provide a basis of comparison for other species as this auxin-responsive construct is placed in other plants. Some more general studies of DR5::GUS expression patterns include those of Aloni et al. (2003, Aloni et al. 2006a) in which the authors visualized changing patterns of GUS expression during leaf and flower development. We observed the patterns of DR5::GUS expression in seedling development as a basis to compare those of PsPK2::GUS because we know that PsPK2 is auxin-regulated in peas. GUS expression in shoots (e.g., developing leaves, procambium), due to expression of PsPK2::GUS, spatially correlates with that of DR5::GUS verifying that auxin regulates the pattern of PsPK2 transcript distribution in Arabidosis shoots as we predicted from RT-PCR experiments in pea shoot tips (Bai and DeMason, 2006). However, PsPK2::GUS expression does not match DR5::GUS expression patterns in roots. No staining occurs in the root tip, and only weak staining occurs in the procambium, unlike the strong staining in root tips and vasculature of DR5::GUS plants (Sabatini et al., 1999). PID::GUS plants have weak staining in the tips, procambium, and endodermis of roots (Benjamins et al., 2001). This difference in response between PsPK2::GUS and PID::GUS might be due to differences in the size of the DNA fragments used (2.2 vs. 3.6 kb) or to Arabidopsis transcriptional proteins not recognizing the pea promoter. However, using Northern blot analysis, McSteen et al. (2007) were unable to detect bif2 expression in corn roots. These results indicate that some transacting elements in roots suppress auxin upregulation of the promoters of these genes and that root regulation of these genes may differ in different plant species.
Auxin responsiveness of PsPK2 compared with DR5 in Arabidopsis
Although DR5 is generally thought to be sensitive to auxin in a dosage-dependent manner and its activity is thought to reflect endogenous auxin levels in plant parts, especially roots (Ulmasov et al., 1997; Sabatini et al., 1999; Casimiro et al., 2001; Benkova et al., 2003; Aloni et al., 2006b), there have been no careful studies testing its responsiveness to a range of auxins. We found that 4-Cl-IAA, MeIAA, and NAA, followed by IAA, produced the strongest staining response in DR5::GUS Arabidopsis plants. Only the weak auxin IBA had little effect compared with the controls. Because Mattsson et al. (2003) showed that DR5::GUS is responsive to 2,4-D, it is clear that it is responsive to all strong auxins. That 4-Cl-IAA, MeIAA, and NAA travel through cell membranes more efficiently than IAA probably explains their stronger effect compared with IAA.
The staining response of PsPK2::GUS in Arabidopsis to 4-Cl-IAA, MeIAA, NAA, and IAA treatments were similar to that of DR5::GUS, indicating that these responses are probably due to ARF-Aux/IAA transcriptional regulation at the AuxRE site in the PsPK2 promoter. However, PsPK2::GUS had a slightly stronger response to IBA than DR5::GUS does. In a previous study (Bai et al., 2005) using RT-PCR, we compared the mRNA levels of PsPK2 with auxin treatments in pea shoot tips and found that PsPK2 is responsive to MeIAA, IAA, 4-Cl-IAA, NAA, and IBA, in that order. Although lowest in response to the IBA treatment, PsPK2 expression was significantly higher than in the controls. This slight difference in response between PsPK2 and DR5 might result from elements other than the AuxRE sites in the gene’s promoter.
Hormone interactions and the regulation of PsPK2::GUS compared with responses in DR5::GUS and PID::GUS transgenic plants
Previous studies have shown that DR5 is stimulated by brassinosteroids, in addition to auxin, but not by a 24 h treatment of 10 µM ABA, ACC, jasmonic acid, zeatin, or GA (Nakamura et al., 2003). These authors did not look for hormone interactions. Interactions between auxin, GA, and cytokinin play many roles in plant development and regulate PsPK2 in pea shoot tips (Bai and DeMason, 2006). In whole seedlings, DR5 did not respond to GA treatment alone, but there was a slight response to the cytokinin BAP and to the auxin transport inhibitor NPA. There was an eightfold higher response in the auxin treatment compared with the control, as one would anticipate. The slight increase in GUS produced in response to BAP was due to increased GUS activity in the root procambium, whereas the slight increase in GUS produced in response to NPA was due to increases in GUS activity in root tips and developing leaf tips and margins. When DR5::GUS seedlings were treated with BAP and NPA simultaneously, the root procambium staining disappeared, but blue staining in leaf tips and margins was maintained. This suggests that the BAP treatment increases auxin transport into roots, which is antagonized by NPA. Treating seedlings with auxin and cytokinin simultaneously produced an even higher response in roots and increased the staining response in leaves, especially in the veins. Aloni et al. (2006b) discussed the interactive role that cytokinin and auxin plays in vascular differentiation, especially in roots.
The only hormone combination that resulted in a higher GUS response than either hormone applied alone to DR5::GUS plants is that of auxin and GA. This response is synergistic because it was greater than the individual responses summed. Positions of staining in the seedlings are the same as with auxin alone, but the stain intensity was greater at those positions. Because GA applied alone did not affect DR5, this suggests that there is a strong interaction between these hormones that synergistically increases the concentration of auxin or increases the concentration of brassinolid. Auxin and brassinolid measurements are needed to confirm this hypothesis.
The lack of GUS staining in roots of PsPK2::GUS plants was not reversed even after auxin or cytokinin treatments, which induced higher GUS activity in roots of DR5::GUS plants. PID::GUS was only weakly responsive to auxin treatment in roots, even though the auxin levels used caused callusing. It would be interesting to determine the reason for the promoter’s relative lack of auxin responsiveness in roots.
PsPK2’s auxin response was not as dramatic as that of DR5, suggesting that the PsPK2 promoter is somewhat less sensitive to auxin. This difference might be due to the fact that DR5 has a 7x repeat of the AuxRE core, whereas the PsPK2 promoter has only three AuxRE cores. Ulmasov et al. (1997) quantified the effect of the AuxRE copy number on auxin response. They found that the increases in auxin responsiveness correlated with increases of AuxRE copies up to seven, and the 7x construct has much higher auxin response than the 3x construct.
In contrast to DR5::GUS, PsPK2::GUS had a strong response to GA3, BAP, and NPA in transgenic seedlings. PsPK2’s response to GA in transgenic Arabidopsis was expected since the promoter has numerous P-boxes and several TATC-boxes and has been demonstrated to be GA-regulated in pea (Bai et al., 2005; Bai and DeMason, 2006). This regulation is independent of the AuxRE cores because it did not occur in DR5::GUS plants. NPA application resulted in a greater response of PsPK2 than of DR5 as well. NPA results in auxin accumulation adjacent to sites of synthesis as a result of short exposures (Ljung et al., 2001), and increases in GA levels due transcriptional regulation of GA3ox, GA20ox, and GA2ox may explain the modest response. Cytokinin increased GUS activity in shoots of PsPK2::GUS transgenic plants and did not enhance GUS activity in combination with auxin or GA compared with either of those hormones used alone. However, cytokinin had no effect on shoots of PID::GUS plants. These results indicate that these orthologous genes differ in their response to cytokinin.
PID::GUS was strongly responsive to IAA and GA, but not to cytokinin in shoots of transgenic seedlings. Benjamins et al. (2001) showed that PID is upregulated by IAA in the shoot. We found that PID::GUS was not very responsive to MeIAA or NAA. MeIAA and NAA induced PID expression slightly in hypocotyls and mature roots (data not shown). These results indicate that PID::GUS is selectively responsive to different auxins in a pattern that is not similar to DR5::GUS or PsPK2::GUS. The PID promoter is also less sensitive to auxin than DR5, perhaps because the PID promoter (At2g34650.1) has only four AuxRE cores for auxin response and a limited spatial response. PID::GUS was responsive to GA and was more responsive to the combination of GA and auxin than either alone, but this response was not additive or synergistic. Because there are no GA response elements of any known type in PID promoter, we predict that GA upregulates PID indirectly. Nothing is known about the GA response of PID orthologs in other species. PID::GUS has no response to cytokinin, and no cytokinin response elements are present in the PID promoter.
The most significant interaction between the possible pairs of hormones used to treat PID::GUS seedlings was that of IAA and GA, just as it was for DR5::GUS and PsPK2::GUS. However, this response was synergistic for PsPK2::GUS and DR5::GUS, but not for PID::GUS. It is not clear whether this synergism is due to the same transcriptional interactions at the AuxRE cores alone or due to a combination of responses including the GA response on its own.
Regulation of AtPIN1 and LFY in Arabidopsis compared with their orthologs in pea
A group of genes—PsPK2, PsPIN1, and Unifoliata (=LFY)—are all regulated by auxin and GA in pea shoot tips (Chawla and DeMason, 2004; Bai et al., 2005; Bai and DeMason, 2006). AtPIN1 is known to be regulated by auxin (Gälweiler et al., 1998; Heisler et al., 2005), but no one had tested its sensitivity to GA. LFY is known to be regulated by GA, via a GA-MYB site, but its response to auxin had not been specifically tested. To compare the responses of the PIN1 and LFY/Uni genes in these two eudicot species, we used semiquantitative PCR to determine expression in Arabidopsis shoot tips in response to treatments by this same set of hormones, and we analyzed the promoter regions to look for putative hormone regulatory elements.
We found that both LFY and AtPIN1 mRNA levels increased after treatment with auxin. LFY mRNA levels increased about twofold after auxin treatment. Previously, only indirect evidence suggested that LFY is regulated by auxin. Vernoux et al. (2000) showed that pin1 mutants have reduced LFY expression and reduced expression of its downstream targets. Also Heisler et al. (2005) showed that LFY expression correlates with pPIN::PIN1–green fluorescent protein (GFP) expression and suggested that auxin transport routes directly or indirectly regulate LFY expression patterns. Consistent with this, there is an AuxRE in LFY’s promoter.
The mRNA levels of both LFY and AtPIN1 also increase after exogenous GA application. GA is known to regulate LFY transcription and floral initiation in Arabidopsis (Blazquez and Weigel, 2000; Eriksson et al., 2006). This regulation occurs by a GA-MYB transcription factor (Blazquez and Weigel, 2000; Gocal et al., 2001). The mRNA levels of AtPIN1 had an obvious response to exogenous GA treatment that is consistent with its ortholog in pea, PsPIN1. The mechanism for this is not known because no GA response elements are present. BAP had little affect on AtPIN1 and weakly increased LFY mRNA levels. Therefore PID, AtPIN1, and LFY are all upregulated by auxin and GA in Arabidopsis as are their orthologs in pea.
Angiosperms have a great diversity in body plans, which required dramatic evolutionary changes in genes that control development. Evolutionary developmental changes often involve three possible processes: changes in (1) cis-regulatory elements and/or (2) protein function, or (3) the expression domain of key developmental regulators, with conserved molecular interactions of these regulators, within their new expression domains. In addition, changes in expression domain might result from changes in position of hormone synthesis and/or direction of transport. The first two processes have been documented in two evolutionary studies of LFY (Maizel et al., 2005; Sliwinski et al., 2006). We are attempting to understand the developmental regulation of pea leaf morphogenesis for which Uni plays a significant role, even though LFY does not do so in Arabidopsis. Like Uni, PsPK2 and PsPIN1 are expressed in pea shoot tips and in response to auxin and GA (Chawla and DeMason, 2004; Bai and DeMason, 2006). In an initial attempt to determine the evolutionary-developmental differences between the species, we have compared hormonal regulation of these three genes, their cis-regulatory elements and positional expression patterns of PID/PsPK2 in Arabidopsis seedlings compared to pea. Because auxin and GA regulate all pairs of orthologs in the two species and PID/PsPK2 promoter expression in Arabidopsis is so similar, our hypothesis is that a change in expression domain is the major difference. We will test this by extending our positional observations to LFY/Uni and PIN.
Conclusion
In pea and Arabidopsis, PsPK2/PID, PsPIN1/AtPIN1, and LFY/Uni are all upregulated by auxin and GA. PsPK2::GUS and PID::GUS expression is spatially similar in that GUS activity is present mainly in the shoots and hypocotyls, but low in root tips. PsPK2::GUS responds to different auxins like DR5::GUS, but PID::GUS ’s auxin responses are complex. Cytokinin enhances auxin transport and upregulates expression of PsPK2::GUS, but not that of PID::GUS. In conclusion, auxin and GA positively regulate PsPK2 in pea and PID in Arabidopsis in shoots, but root expression is reduced.
FOOTNOTES
1 The authors thank the Nottingham Arabidopsis Stock Centre (NASC) for providing PID::GUS seeds, and Dr. W.-C. Lin for providing the GUS reporter construct derived from pCAMBIA. Monetary support was provided by the Institute for Integrative Genomics to D.A.D. and from the Graduate Division to F.B. This work is a part of the Ph.D. dissertation of F.B. 
2 Author for correspondence (e-mail: demason{at}ucr.edu)
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