Introduction
SQUAMOSA PROMOTER-BINDING PROTEIN (SBP) and SBP-LIKE (SPLs) play multiple roles in plant development (Klein et al., Reference Klein, Saedler and Huijser1996; Cardon et al., Reference Cardon, Hohmann, Nettesheim, Saedler and Huijser1997; Unte et al., Reference Unte, Sorensen, Pesaresi, Gandikota, Leister, Saedler and Huijser2003; Zhang et al., Reference Zhang, Schwarz, Saedler and Huijser2006; see also the accompanying paper Martin et al., Reference Martin, Asahina, Liu, Kristof, Coppersmith, Pluskota, Bassel, Goloviznina, Nguyen, Martínez-Andújar, Kumar, Pupel and Nonogaki2010). Information concerning the regulatory mechanisms of SPL expression is emerging. Through bioinformatic analyses, it is known that 11 out of 17 Arabidopsis SPLs contain miR156/miR157 complementary sequences (Rhoades et al., Reference Rhoades, Reinhart, Lim, Burge, Bartel and Bartel2002) (miR156 and miR157 are nearly identical, so the term ‘miR156’ is used hereafter). Targeted cleavage of SPL3, SPL4 and SPL5 by miR156 has been demonstrated (Chen et al., Reference Chen, Li, Xie, Peng and Ding2004; Wu and Poethig, Reference Wu and Poethig2006). Constitutive expression of MIR156b reduces SPL3 expression, supporting the idea of SPL regulation by miRNA (Schwab et al., Reference Schwab, Palatnik, Riester, Schommer, Schmid and Weigel2005).
In addition to mRNA cleavage, translational repression of SPL3 has also been reported (Gandikota et al., Reference Gandikota, Birkenbihl, Hohmann, Cardon, Saedler and Huijser2007). While mRNA cleavage was thought to be the predominant mechanism of miRNA-mediated gene repression in plants, translational repression also appears to be widespread (Brodersen et al., Reference Brodersen, Sakvarelidze-Achard, Bruun-Rasmussen, Dunoyer, Yamamoto, Sieburth and Voinnet2008). The AP2 family genes are regulated by miR172 through translational repression (Aukerman and Sakai, Reference Aukerman and Sakai2003; Chen, Reference Chen2004), although cleavage of miR172 targets has also been observed (Schwab et al., Reference Schwab, Palatnik, Riester, Schommer, Schmid and Weigel2005). Analysis of SPL3 regulation by miR156 provides another example of the regulation of plant miRNA targets by both mRNA cleavage and translational repression. miR156 complementary sequences have been found in SPL orthologues in the moss Physcomitrella patens, suggesting an ancient origin of miRNA-dependent regulation of SPLs (Arazi et al., Reference Arazi, Talmor-Neiman, Stav, Riese, Huijser and Baulcombe2005; Riese et al., Reference Riese, Hohmann, Saedler, Munster and Huijser2007).
While the regulation of SPLs by miRNA seems to play a fundamental role in plant growth and development, information on other SPL family members targeted by miRNA156 is limited. In this study, we focused on the function of SPL13, which is expressed following seed germination. The analysis of the molecular mechanisms of SPL13 involvement in post-germinative events revealed that miRNA gene regulation cascades function during these stages. Potential interaction between the miR156 and miR172 pathways through SPL13 function is discussed.
Materials and methods
Generation of miR156-resistant SPL13 mutants
The SPL13 (At5g50570) gene including the 1.3 kb upstream regulatory region was amplified from genomic DNA of Arabidopsis thaliana ecotype Col-0 using SPL13 forward (SPL13 F: 5′-ACCTACTCCTGCCAACACAATGTTCTTACA-3′) and reverse (SPL13 R: 5′-ATCCTACAAGATGGCTCATCTCAACAAGGT-3′) primers. The intact gene was used to generate non-mutated SPL13 transgenic plants. Mutations at the miR156 target site were generated by site overlap extension polymerase chain reaction (PCR) mutagenesis (Ho et al., Reference Ho, Hunt, Horton, Pullen and Pease1989) using SPL13 mutant forward (SPL13mutF: 5′-CTGATTGTGCTCTCTCACTACTATCTTCCT-3′) and reverse (SPL13mutR: 5′-AGGAAGATAGTAGTGAGAGAGCACAATCAG-3′) primers. PCR products were cloned into pCAMBIA1301. Arabidopsis thaliana ecotype Col-0 plants were transformed by floral dip (Clough and Bent, Reference Clough and Bent1998) using Agrobacterium tumefaciens carrying pCAMBIA1301 with intact SPL13 (SPL13) or mutant SPL13 (mSPL13) constructs. Wild-type and transgenic plants were grown at 22°C under 12-h light/12-h dark conditions until rosette stages and then flowers were induced by transferring plants to 16-h light/8-h dark conditions.
Differential interference contrast microscopy
Seedlings were cleared with chloral hydrate solution [3.5 ml water, 0.5 g glycerol, 10 g chloral hydrate (Sigma, St. Louis, Missouri, USA)] for 16–18 h. Samples were mounted with Hoyer's solution (7.5 ml water, 1.3 g glycerol, 1.9 g gum arabic and 25 g chloral hydrate) and observed with an Axioskop 2 plus microscope (Zeiss, Jena, Germany). The differential interference contrast (DIC) optics coupled to a Pixera camera Model #PVC 100C (Pixera Corporation, Los Gatos, California, USA) were used to capture images, which were then processed with Pixera Visual Communication Suite software.
mRNA and small RNA extraction
High molecular weight (HMW) RNA for mRNA expression analysis was extracted using a standard phenol–SDS extraction protocol. Briefly, 100 Arabidopsis seedlings were homogenized in 2 ml RNA extraction buffer [45.5% (v/v) phenol, 9% (v/v) chloroform, 0.45% (w/v) SDS, 41 mM LiCl, 2 mM EDTA, 5.9 mM β-mercaptoethanol, 82 mM Tris–HCl, pH 8.2] with a mortar and pestle. The extract was centrifuged at 10,000 g for 2 min. The supernatant was extracted with one volume of phenol–chloroform–isoamyl alcohol [25:24:1 (v/v/v)] solution and then with one volume of chloroform. LiCl was added to the supernatant (2 M final concentration) and the sample was mixed thoroughly and kept at − 20°C overnight. The sample was thawed, mixed and centrifuged at 10,000 g for 5 min. The pellet was washed with 1 ml 80% (v/v) ethanol, dried, dissolved in water and used for mRNA expression analysis. To obtain low molecular weight (LMW) RNA for miRNA detection, the supernatant from the 2 M LiCl precipitation step in the total RNA isolation protocol was fractionated by isopropanol (Martin et al., Reference Martin, Liu and Nonogaki2005). The pellet from the 35–50% isopropanol fraction was washed with 1 ml 80% (v/v) ethanol, dried, dissolved in water and used for miRNA expression analysis.
Microarray analysis
Three different lots of wild type and three independent transgenic lines of SPL13 and mSPL13 were analysed using Arabidopsis ATH1 Genome GeneChips (Affymetrix, Santa Clara, California, USA). Seedlings (100) were grown at 22°C under 12-h light/12-h dark conditions and total RNA was extracted from them 3 DAI (days after the start of imbibition). RNA integrity was checked with Agilent Bioanalyzer 2100 (Agilent Technologies Inc., Santa Clara, California, USA). Four micrograms of total RNA from individual pools was used to produce double-stranded cDNAs with Affymetrix One-Cycle Target Labeling Kit, according to the GeneChip Expression Analysis Technical Manual. Biotinylated cRNAs (complementary RNAs) were synthesized from the double-stranded cDNA using T7 RNA polymerase and nucleotide mixture containing biotin-conjugated pseudouridine provided in the IVT Labeling Kit (Affymetrix). cRNA (25 μg) was purified and fragmented prior to hybridization in the Affymetrix GeneChip® Hybridization Oven 640. The arrays were washed in the Affymetrix GeneChip® Fluidics Station 450 and then stained with biotinylated anti-streptavidin (Vector Laboratories, Burlingame, California, USA) at the Center for Genome Research and Biocomputing Core Laboratories at Oregon State University, Corvallis, Oregon, USA. The arrays were scanned with an Affymetrix GeneChip® Scanner 3000 at 570 nm and signal values were obtained using the statistical algorithms on Affymetrix GeneChip® Operating (GCOS) software. The presence or absence of a reliable hybridization signal for each gene was determined by the detection call on GCOS and imported into GENESPRING GX 7.2 (Agilent Technologies Inc.). The sum of signal values from all probe sets was used for normalization across the different samples. Up- or down-regulated genes were selected when the signal values deviated twofold or more. The microarray data will be available at the Gene Expression Omnibus (GSE10414, http://www.ncbi.nlm.nih.gov/geo).
Quantitative PCR
First-strand cDNA was synthesized from total RNA (1 μg) with QuantiTect Reverse Transcription Kit according to the manufacturer's instructions (Qiagen, Valencia, California, USA). Quantitative reverse transcription (QRT)-PCR with Taq-Man technology (Holland et al., Reference Holland, Abramson, Watson and Gelfand1991) or SYBR Green I RT-PCR reagents (Qiagen) was performed using the first-strand cDNA as a template on a sequence detector system (ABI PRISM 7000; Applied Biosystems, Foster City, California, USA) as described in a previous report (Yamauchi et al., Reference Yamauchi, Ogawa, Kuwahara, Hanada, Kamiya and Yamaguchi2004) with several modifications. Results were normalized using 18S rRNA as the internal control. Duplicate experiments were performed using independent plant materials. Primers used for quantitative PCR were:
SNZ-forward (5′-AGCCTACACAGCCGCAAGA-3′)/reverse (5′-TGGAGTCCCCGGAATCTGA-3′);
MIR172a-forward (5′-TGGCTTCCAAGATCTGGTAATATG-3′)/reverse (5′-ACGAGACAAACCCACAAATTTCTAT-3′);
MIR172b-forward (5′-TGACACGTCAGCCCTTGGA-3′)/reverse (5′-GGGATATGAGGAAAAGTAGATAGGTGAA-3′);
MIR172c-forward (5′-GTCTACATCTATCTCTTTCTAGGTCACTAGCT-3′)/reverse (5′-GCACCATTTTGCTGGAAACA-3′); and
SPL13-qRT-forward (5′-CCTCGTCGTCAGTCCCTCAT-3′)/reverse (5′-TCAACTGCTTCTTGGGACAAAG-3′).
miRNA gel blot
The LMW RNA pellet was dissolved in 2 μl water followed by 4 μl formamide and 2 μl 4 × loading buffer [50% (v/v) glycerol, 0.03% (w/v) bromophenol blue (BPB), 50 mM Tris–HCl, pH 7.7, 5 mM EDTA] and applied to a 17% (w/v) denaturing polyacrylamide gel (85 mm wide, 80 mm long, 1.5 mm thick) containing 7 M urea, 0.5 × Tris–borate–EDTA (TBE) buffer, pH 8.0. The gel was pre-run at 180 V for 30 min. The samples were loaded and the gel was run at 180 V until the BPB line reached the bottom of the gel, stained with ethidium bromide and photographed to visualize the 5S rRNA and tRNA bands. After rinsing the gels with 0.5 × TBE buffer, the separated LMW RNAs were transferred to positively charged Hybond-N+membrane (GE Healthcare Bio-Sciences Corp./Amersham, Piscataway, New Jersey, USA) using a semi-dry transfer unit (Bio-Rad Laboratories, Hercules, California, USA). The transferred RNA was UV cross-linked and the membranes were dried and used for hybridization. miRNA probe synthesis was performed following the instruction manual of the mirVana™ miRNA Probe Construction Kit (Applied Biosystems/Ambion, Austin, Texas, USA). For miRNA probe synthesis, DNA templates were designed based on the miRNA sequence with the addition of part of the T7 promoter sequence (5′-cctgtctc-3′) at the 3′ end of the oligonucleotide. The DNA oligomer and the T7 promoter primer were mixed, heated at 70°C for 5 min and hybridized at room temperature for 5 min. Exo-Klenow DNA polymerase provided with the kit was added to the mix, which was then incubated at 37°C for 30 min to produce a double-stranded DNA template for transcription. Antisense miRNA probes were synthesized at 37°C for 30 min using T7 RNA polymerase (provided with the kit) and a digoxigenin (DIG) RNA-labelling mix (Roche Applied Science, Hague Road, Indianapolis, Indiana, USA). Prehybridization was performed in a hybridization buffer PerfectHyb™ Plus (Sigma-Aldrich) at 42°C for 30 min. Probe (4 μl) was added to 2 ml hybridization solution, heated at 95°C for 5 min and cooled to 42°C. The prehybridization solution was removed and replaced with hybridization solution. Hybridization was allowed to proceed for 16–18 h. The membrane was washed three times at 65°C for 20 min each time with 2 × saline–sodium citrate (SSC), 0.2% (w/v) SDS. Membranes were blocked for 30 min with 5% (w/v) non-fat milk in 0.1 M maleic acid buffer, pH 7.5, containing 0.15 M NaCl, and 0.3% (v/v) Tween 20 (buffer A) and were then incubated with alkaline phosphatase-conjugated anti-DIG antibody for 1 h at 25°C. After washing with buffer A, the membranes were subjected to chemiluminescence detection. The signal was detected on X-ray film after exposure.
Results
SPL13 represses primordium development post-germination
Silent mutations created in the SPL13 sequence that is complementary to the miR156 sequence cause the deregulation of mSPL13 from miR156. Transgenic seedlings that overaccumulate the miRNA-resistant mutant SPL13 (mSPL13) exhibited mutant phenotypes visible at post-germinative stages: the development of vegetative leaves was delayed in mSPL13 mutants (Fig. 1A). In transgenic plants expressing non-mutated SPL13, vegetative leaves emerged normally, indicating that the phenotype in mSPL13 was due specifically to the deregulation of mSPL13 mRNA from miRNA156. Vegetative leaves became visible only 4–5 DAI even in wild-type seedlings; however, differentiation of leaf primordia seemed to be initiated in very small seedlings right after germination (example of a 2-DAI seedling in Fig. 1A, bottom right). We examined the shoot apical meristems (SAMs) of seedlings at post-germinative stages using a DIC microscope. DIC examination revealed noticeable SAMs in the embryos excised from imbibed seeds. Apparent differentiation of leaf primordia was observed at the SAM around 2 DAI (Fig. 1B). No visible differences were detected between wild-type and mSPL13 seedlings at this stage. Slight differences in primordia development in the wild type and mSPL13 were detected 3 DAI (Fig. 1B). These results suggested that the over-accumulation of miR156-resistant mSPL13 affected the development of the leaf primordia. Based on the information from morphological analysis, seedlings 3 DAI were used for gene expression analysis (see below).
Post-germination phenotypes of miRNA-resistant mSPL13 mutants. (A) Arabidopsis 6-DAI seedlings of wild-type (WT, top left), control transgenic expressing non-mutated SPL13 (SPL13, top right) and mutant plants over-accumulating miR156-resistant SPL13 (mSPL13, bottom left). Note that there was a delay in the emergence of the first pair of vegetative leaves in mSPL13. An image of a 2-DAI seedling in which leaf primordia start to differentiate at the shoot apical meristem (see panel B) is also shown (bottom right). (B) Differential interference contrast microscope images of leaf primordia at the shoot apical meristem (SAM) are shown. A slight difference in the size of leaf primordia in WT and mSPL13 appeared in 3-DAI seedlings. Arrows indicate the position of SAM in a 1-DAI embryo.
Downstream genes affected by the over-accumulation of mSPL13
The effects of the deregulation of mSPL13 from miR156 at the molecular level were examined by microarray analysis using RNA extracted from seedlings 3 DAI, when slight morphological differences were visible between wild-type and mSPL13 seedlings (Fig. 1B). By comparing gene expression in three independent lines of wild-type, SPL13 and mSPL13 seedlings, genes up- or down-regulated (twofold or greater) in mSPL13 mutants were identified. SPL13 was one of the up-regulated genes which confirmed the deregulation of mSPL13 from miR156. Other up-regulated genes included plant hormone associated genes, such as 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ethylene biosynthesis) and NPH3 (auxin response) (Motchoulski and Liscum, Reference Motchoulski and Liscum1999), protein inhibitors, a membrane transporter and a dehydrogenase (Table 1). Down-regulated genes also included plant hormone associated genes, such as gibberellin responsive protein GAST1-LIKE and an auxin-induced IAA34 protein. Expansin, a cell wall modifying protein, was also down-regulated in mSPL13 (Table 2). (Detailed microarray data will be available at the Gene Expression Omnibus, GSE10414, http://www.ncbi.nlm.nih.gov/geo).
Genes up-regulated in mSPL13 mutant seedlings
ACC, 1-aminocyclopropane-1-carboxylate.
Genes down-regulated in mSPL13 mutant seedlings
miR156–miR172 gene regulation cascades
One of the genes most significantly down-regulated based on the microarray analysis (nine- to twelvefold decrease) was SCHNARCHZAPFEN (SNZ), an AP2-like gene (Table 2). Quantitative RT-PCR confirmed that SNZ was down-regulated specifically in mSPL13 mutants (Fig. 2A). The AP2 gene family contains well-known flower-patterning genes, but AP2 is also involved in SAM development in Arabidopsis seedlings (Wurschum et al., Reference Wurschum, Gross-Hardt and Laux2006). SNZ, another AP2-like gene, appears to play a critical role in SAM development.
Downregulation of SCHNARCHZAPFEN (SNZ) and overexpression of MIR172 genes in mSPL13 mutants. (A), (B) and (C) Quantitative RT-PCR of SNZ, MIR172a and MIR172b, respectively. Relative gene expression levels in three independent lines of wild type (WT; 1, 2 and 3), control transgenic (SPL13; 2B, 4C and 5B) and mutant (mSPL13; 2D, 7B and 12C) are shown. Bars indicate SE (n = 3). (D) miRNA gel blot for miR172 expression in wild-type (WT), control transgenic (SPL13) and mutant (mSPL13) seedlings (3 DAI). Results were normalized using 18S rRNA as the internal control.
Interestingly, SNZ is a target of miR172 (Schmid et al., Reference Schmid, Uhlenhaut, Godard, Demar, Bressan, Weigel and Lohmann2003) (Fig. 3). Therefore, the microarray data indicated that the deregulation of SPL13 from miR156 affected a target of miR172. This suggested a potential interaction between miR156 and miR172 pathways. We hypothesized that the down-regulation of SNZ in mSPL13 plants was caused by the up-regulation of miR172 (Fig. 4). We examined the expression of MIR172a, MIR172b and MIR172c predicted gene transcripts (Xie et al., Reference Xie, Allen, Fahlgren, Calamar, Givan and Carrington2005) in three independent lines of wild-type, SPL13 and mSPL13 seedlings. While MIR172c was barely detectable, MIR172a and MIR172b were specifically up-regulated in mSPL13 seedlings (Fig. 2B and C). Based on RNA gel blot of total small RNAs from these lines, mature miR172 over-accumulated in mSPL13 seedlings (Fig. 2D). These results indicated that over-accumulation of SPL13 caused over-expression of at least two MIR172 genes, which resulted in over-accumulation of mature miR172. This also suggested that the down-regulation of SNZ in mSPL13 was caused by this change in miR172 levels.
Schematic representation of SCHNARCHZAPFEN (SNZ) targeted by miR172. The miRNA target sequence in SNZ is aligned with the miR172a sequence according to the Arabidopsis Small RNA Project (http://asrp.cgrb.oregonstate.edu/). Characters in red indicate mismatch with the SNZ sequence.
Schematic representation of the miR156–miR172 regulation cascade model in Arabidopsis seedlings. Deregulation of mSPL13 from miR156, due to silent mutations in the miRNA complementary site, causes over-accumulation of functional SPL13, which promotes the expression of MIR172 genes directly or indirectly through the function of unknown factor X. miR172 then down-regulates SNZ which is involved in seedling development. The scheme presents a possible mechanism. A causal connection between SNZ down-regulation and the observed phenotype still needs to be verified by further experiments.
Discussion
Deregulation of SPL13 from miR156 caused a delay in the emergence of vegetative leaves at the post-germination stages. The effects of over-accumulation of SPL13 were partially exerted through the down-regulation of SNZ, an AP2-like transcription factor (Table 2). The involvement of AP2, a floral-patterning gene, in the SAM in Arabidopsis seedlings was shown in a previous study (Wurschum et al., Reference Wurschum, Gross-Hardt and Laux2006). The stem cell niche in the SAM of Arabidopsis seedlings is maintained primarily by the activity of WUSCHEL (WUS), a positive regulator, and CLAVATA3 (CLV3), a negative regulator. AP2 enhances WUS activity and reduces CLV3 activity, both events positively affecting SAM activity (Wurschum et al., Reference Wurschum, Gross-Hardt and Laux2006). SNZ also appears to be a positive regulator of SAM activity. The phenotype of mSPL13 seedlings where SNZ levels were reduced was similar to the phenotype found in wus loss-of-function mutant and CLV3 over-expressers, although the latter mutants have more severe phenotypes than those observed in mSPL13 seedlings. Therefore, SNZ seems to have a role similar to that of AP2. It is possible that SNZ functions through pathway(s) separate from the WUS–CLV3 pathway, since microarray data did not indicate a differential expression of these genes between wild-type or control SPL13 and mSPL13 seedlings. Microarray data also indicated that multiple genes other than SNZ were up- or down-regulated by the over-accumulation of mSPL13 (Tables 1 and 2). Functional analysis of genes differentially expressed in control and mSPL13 plants in the present study will provide further information on mechanisms of vegetative leaf development.
In terms of mechanistic analysis, the down-regulation of SNZ, a target of miR172, in mSPL13 mutants suggests the possibility that the miR156 pathway acts upstream of the miR172 pathway in Arabidopsis. This possibility was initially predicted by Wu and Poethig (Reference Wu and Poethig2006) based on complementary patterns of expression of miR156 and miR172 and their function. A recent study demonstrated that SPL9 mediates the interaction between the miR156 and miR172 pathways (Wu et al., Reference Wu, Park, Conway, Wang, Weigel and Poethig2009). Our findings provide evidence for the involvement of SPL13 in the miR156 and miR172 cascades at the post-germinative stages. A similar possibility has been suggested in maize, although the mechanisms are not known. glossy15 (gl15), an AP2 transcription factor loss-of-function mutant in maize (Zea mays), exhibits precocious adult cell characteristics in juvenile leaves (Evans et al., Reference Evans, Passas and Poethig1994; Moose and Sisco, Reference Moose and Sisco1994, Reference Moose and Sisco1996) suggesting that AP2 family proteins play critical roles in the juvenile-to-adult transition in monocotyledonous species. Corngrass1 (Cg1), a dominant mutant in maize, exhibits prolonged juvenile development. cg1 encodes two tandem miR156 genes (zma-MIR156b and zma-MIR156c) that are over-expressed in the meristem and lateral organs in the mutant maize (Chuck et al., Reference Chuck, Cigan, Saeteurn and Hake2007). A target of cg1/zma-MIR156 is teosinte glume architecture1 (tga1), a gene important for the domestication of maize from teosinte (Wang et al., Reference Wang, Nussbaum-Wagler, Li, Zhao, Vigouroux, Faller, Bomblies, Lukens and Doebley2005). The expression of tga1 is reduced in the Cg1 mutant (Chuck et al., Reference Chuck, Cigan, Saeteurn and Hake2007) providing evidence for tga1 repression by miR156. The tga1 protein contains an SBP domain (data not shown). SPL13 is a tga1 orthologue in Arabidopsis. Interestingly, miR172 levels are reduced in Cg1 mutants (Chuck et al., Reference Chuck, Cigan, Saeteurn and Hake2007), supporting the idea that miR172 expression is downstream of the miR156 pathway. These results, together with the results presented here, suggest that SBP-domain-containing transcription factors (SPL13 in Arabidopsis or tga1 in maize), which are negatively regulated by miR156, do promote the expression of MIR172. miR172 then targets AP2 transcription factors (SNZ in Arabidopsis or gl15 in maize) involved in seedling development. Thus, similar miRNA regulation cascades (miR156 ⊣ SBP-like → miR172 ⊣ AP2-like) appear to be conserved between monocotyledonous and dicotyledonous species.
The mechanisms involved in the induction of MIR172a and MIR172b by SPL13 over-accumulation are not known at this time. SPLs are transcription factors that bind conserved DNA motifs in promoter regions of target genes. SPL13 contains an SBP domain that is conserved among other SPLs (Klein et al., Reference Klein, Saedler and Huijser1996). The C-terminal end of this domain contains the bipartite nuclear localization signal (KR… RRRK) which is also found in other SPLs. The conserved DNA motif recognized by Antirrhinum SBP and SBP2 and Arabidopsis AP1 was first identified as GTCCGTACAA (Klein et al., Reference Klein, Saedler and Huijser1996). Through a more detailed analysis of the binding capacity of the SPL SBP domains, the essential palindromic GTAC core in the motif was identified (Birkenbihl et al., Reference Birkenbihl, Jach, Saedler and Huijser2005). This motif was found in the 5′ upstream regulatory region ( − 619/ − 604) of MIR172a (data not shown). However, this motif was not found in the promoter region of MIR172b which was also upregulated in mSPL13 mutants. Therefore, the regulation of the MIR172 genes by SPL13 may not be direct, although direct control mediated by other motifs is possible.
Acknowledgements
We are grateful to Tony Chen, Department of Horticulture, Oregon State University, for providing us with the transformation vector. This work was supported by NSF grant IBN-0237562 (to H.N.) and American Seed Research Foundation grant (to H.N. and R.C.M.).
