Abstract

Polar auxin transport, mediated by the PIN-FORMED (PIN) class of auxin efflux carriers, controls organ initiation in plants. In maize, BARREN INFLORESCENCE2 (BIF2) encodes a serine/threonine protein kinase co-orthologous to PINOID (PID), which regulates the subcellular localization of AtPIN1 in Arabidopsis. We show that BIF2 phosphorylates ZmPIN1a, a maize homolog of AtPIN1, in vitro and regulates ZmPIN1a subcellular localization in vivo, similar to the role of PID in Arabidopsis. In addition, bif2 mutant inflorescences have lower auxin levels later in development. We propose that BIF2 regulates auxin transport through direct regulation of ZmPIN1a during maize inflorescence development.

Plant architecture is shaped by the activity of axillary meristems which develop in the axils of leaves (McSteen and Leyser 2005). The initiation of axillary meristems and lateral organs is controlled by the hormone auxin, which is transported toward the site of primordia initiation (Delker et al. 2008). In Arabidopsis (Arabidopsis thaliana), the polarity of auxin transport is directed by the PIN-FORMED (PIN) family of auxin efflux carriers (Galweiler et al. 1998, Petrasek et al. 2006, Wisniewska et al. 2006). The subcellular localization of AtPIN proteins is regulated by PINOID (PID), a serine/threonine protein kinase (Christensen et al. 2000, Benjamins et al. 2001, Friml et al. 2004). pid mutants produce a pin-like inflorescence similar to pin mutants, with defects in the initiation of floral meristems (Bennett et al. 1995). PID phosphorylates AtPIN1, demonstrating a possible direct mechanism by which PID controls the subcellular localization of AtPIN proteins (Michniewicz et al. 2007).

The role of auxin transport appears to be conserved in maize (Zea mays) inflorescence development despite the divergence in plant morphology (Wu and McSteen 2007, Gallavotti et al. 2008). In maize, there are two types of inflorescences: the tassel, which produces male florets, and the ear, which produces female florets (Fig. 1A, Supplementary Fig. S1A). Within the inflorescence, four types of axillary meristems give rise to the highly branched tassel (Barazesh and McSteen 2008). First, branch meristems (BMs) grow out to produce the long branches at the base of the tassel (Fig. 1A). Next, spikelet pair meristems (SPMs) form on the main spike and the branches (Fig. 1B). The SPMs transition to produce spikelet meristems (SMs) which then give rise to the floral meristems (FMs). This process results in the production of a structure called a spikelet pair, with each spikelet containing two florets. The ear develops in an analogous manner, except that BMs are not produced and the lower floret aborts, resulting in paired rows of kernels (Supplementary Fig. S1A, B).

Fig. 1

ZmPIN1 immunolocalization in normal and bif2 inflorescences. (A) Normal and bif2 tassels. Normal tassels have long branches (br) at the base, and spikelet pairs (sp) covering the branches and main spike. bif2 tassels have a reduced number of branches and spikelets. SEM of 4–6 mm developing (B) normal and (C) bif2 tassels showing defects in the initiation of spikelet pair meristems (SPMs) and spikelet meristems (SMs) in bif2 mutants. (D–I) Immunolocalization of developing maize inflorescences using an Arabidopsis AtPIN1 antibody. The apex of (D) normal and (E) bif2 developing ears showing localization of ZmPIN1 proteins in the apical inflorescence meristem. Note: bif2 ears are often fasciated with multiple growing points (see Supplementary Fig. S1). The growing point on the right is sectioned through the center of the meristem, but the section through the growing point on the left is glancing. (F) Normal and (G) bif2 ears showing ZmPIN1 protein localization in the SPMs that form in the axils of bract primordia (bp). (H) Normal and (I) bif2 developing tassel spikelet showing localization of ZmPIN1 proteins in the vasculature (arrow) and in SMs in the process of producing FMs. bp, bract primordium; br, branch; fm, floral meristem; ig, inner glume; og, outer glume; p, palea; sm, spikelet meristem; sp, spikelet pair; spm, spikelet pair meristem. Scale bars = 2 cm (A) and 100 μm (B–I).

Fig. 1

ZmPIN1 immunolocalization in normal and bif2 inflorescences. (A) Normal and bif2 tassels. Normal tassels have long branches (br) at the base, and spikelet pairs (sp) covering the branches and main spike. bif2 tassels have a reduced number of branches and spikelets. SEM of 4–6 mm developing (B) normal and (C) bif2 tassels showing defects in the initiation of spikelet pair meristems (SPMs) and spikelet meristems (SMs) in bif2 mutants. (D–I) Immunolocalization of developing maize inflorescences using an Arabidopsis AtPIN1 antibody. The apex of (D) normal and (E) bif2 developing ears showing localization of ZmPIN1 proteins in the apical inflorescence meristem. Note: bif2 ears are often fasciated with multiple growing points (see Supplementary Fig. S1). The growing point on the right is sectioned through the center of the meristem, but the section through the growing point on the left is glancing. (F) Normal and (G) bif2 ears showing ZmPIN1 protein localization in the SPMs that form in the axils of bract primordia (bp). (H) Normal and (I) bif2 developing tassel spikelet showing localization of ZmPIN1 proteins in the vasculature (arrow) and in SMs in the process of producing FMs. bp, bract primordium; br, branch; fm, floral meristem; ig, inner glume; og, outer glume; p, palea; sm, spikelet meristem; sp, spikelet pair; spm, spikelet pair meristem. Scale bars = 2 cm (A) and 100 μm (B–I).

In maize, barren inflorescence2 (bif2) encodes a serine/threonine protein kinase co-orthologous to PINOID (McSteen et al., 2007). bif2 mutants have defects in the initiation of axillary meristems (Fig. 1C); consequently, bif2 mutants produce a reduced number of tassel branches and spikelets (Fig. 1A) (McSteen and Hake, 2001). bif2 mutants also produce a reduced number of ears with fewer kernels (Supplementary Fig. S1A). In addition, the apical meristem is often fasciated (Supplementary Fig. S1A, C). BIF2 is localized both at the cell periphery and in the nucleus (Skirpan et al., 2008). BIF2 interacts with and phosphorylates BARREN STALK1 (BA1), a basic helix–loop–helix (bHLH) transcription factor required for axillary meristem initiation, suggesting that BA1 is a target of BIF2 (Gallavotti et al. 2004, Skirpan et al. 2008).

Maize has three genes similar to AtPIN1, designated ZmPIN1a, ZmPIN1b and ZmPIN1c (Carraro et al. 2006, Gallavotti et al. 2008). Using two different methods, ZmPIN1 proteins were shown to be up-regulated as axillary meristems and lateral organ primordia initiate, similar to Arabidopsis (Reinhardt et al. 2003, Heisler et al. 2005, Carraro et al. 2006, Gallavotti et al. 2008). However, there are also differences between the two methods as the antibody against the Arabidopsis AtPIN1 protein (which cross-reacts with maize PIN1 proteins) does not detect any ZmPIN1 proteins in the epidermis of the inflorescence apex (Carraro et al. 2006), while the translational fusion of ZmPIN1a to yellow fluorescent protein (YFP) expressed under the native promoter detects ZmPIN1a expression in the epidermis similar to Arabidopsis (Gallavotti et al. 2008). Using immunolocalization, Carraro et al (2006) did not detect ZmPIN1 proteins at the apex of bif2 inflorescences, and proposed that there was a difference in the interaction between BIF2 and ZmPIN1 in maize compared with Arabidopsis, which is surprising given the similarity in phenotype of the bif2 and pid mutants.

In this study, we reinvestigated the localization of ZmPIN1 proteins in bif2 mutants using the same antibody used in Carraro et al. (2006) and the ZmPIN1a–YFP line described by Gallavotti et al. (2008). Although Carraro et al. (2006) did not detect ZmPIN1 in bif2 tassels, the tassels were described as ‘fully developed’, which would be past the time point at which axillary meristems would still be developing in normal inflorescences. In addition, ZmPIN1 was not detected in the apical inflorescence meristem of bif2 ears when the phenotype was severe (Carraro et al. 2006). Therefore, we tested the localization of ZmPIN1 in young (still developing) inflorescence meristems of bif2 mutants by immunolocalization with the Arabidopsis AtPIN1 antibody. In normal tassels and ears, ZmPIN1 proteins were localized in the inflorescence meristem (Fig. 1D), in SPMs, which form in the axils of bract leaf primordia (Fig. 1F), in SMs and FMs (Fig. 1H), and in the vasculature (Fig. 1H), as reported (Carraro et al. 2006). However, ZmPIN1 proteins were also detected in the apical inflorescence meristem of bif2 mutants (Fig. 1E). Furthermore, ZmPIN1 proteins were localized in bif2 mutants in SPMs when they formed (Fig. 1G), in SMs and FMs (Fig. 1I), and in the vasculature (Fig. 1I), similar to normal inflorescences. When axillary meristems failed to form in bif2 mutants, ZmPIN1 proteins were not detected on the flanks of the inflorescence (Fig. 1G).

To determine the subcellular localization of the ZmPIN1a protein we used the ZmPIN1a–YFP fusion protein described by Gallavotti et al. (2008). In normal tassel inflorescences, ZmPIN1a–YFP was observed in the apical inflorescence meristem and was up-regulated in a regular phyllotactic pattern on the flanks of the inflorescence, as reported (Fig. 2A) (Gallavotti et al. 2008). This up-regulation marks the position of the bract primordium and the subsequent site of axillary meristem initiation and outgrowth. In bif2 mutants, ZmPIN1a–YFP was expressed in the apical inflorescence meristem; however, ZmPIN1a up-regulation was not observed on the flanks of the inflorescence (Fig. 2B). In normal inflorescences, ZmPIN1a appeared to be apolarly localized in cells in the incipient primordium (Fig. 2C). However, in bif2 mutants, ZmPIN1a was often localized at the basal side of cells, as reported in the pid mutant (Fig. 2D, Supplementary Fig. S2) (Friml et al. 2004). When the SPM/bract primordium was visible in normal plants, the orientation of ZmPIN1a–YFP appeared to be towards the tip of the primordium (asterisk in Fig. 2E), while in bif2 plants basal orientation of ZmPIN1–YFP was often detected (Fig. 2F, Supplementary Fig. S2). Therefore, we propose that the BIF2/PID-dependent localization of PIN1 proteins is conserved in maize as in Arabidopsis.

Fig. 2

ZmPIN1a–YFP localization in normal and bif2 inflorescences. Confocal microscopy of 4 mm normal and bif2 tassels. (A–F) ZmPIN1a–YFP localization in (A, C, E) normal and (B, D, F) bif2 developing tassels. (C, E) Close-up of the surface of normal inflorescence as the SPM/bract primordium is initiating (C) and after the primordium is visible (E). The asterisk indicates the tip of the SPM/bract primordium. (D, F) Close-up of the surface of a bif2 inflorescence in the regions corresponding to normal, showing altered expression and subcellular localization of ZmPIN1a, early (D) and later (F) in development. Arrowheads indicate basal localization of ZmPIN1a in bif2. im, inflorescence meristem; bp, bract primordium. Scale bars = 50 μm (A, B) and 10 μm (C–F).

Fig. 2

ZmPIN1a–YFP localization in normal and bif2 inflorescences. Confocal microscopy of 4 mm normal and bif2 tassels. (A–F) ZmPIN1a–YFP localization in (A, C, E) normal and (B, D, F) bif2 developing tassels. (C, E) Close-up of the surface of normal inflorescence as the SPM/bract primordium is initiating (C) and after the primordium is visible (E). The asterisk indicates the tip of the SPM/bract primordium. (D, F) Close-up of the surface of a bif2 inflorescence in the regions corresponding to normal, showing altered expression and subcellular localization of ZmPIN1a, early (D) and later (F) in development. Arrowheads indicate basal localization of ZmPIN1a in bif2. im, inflorescence meristem; bp, bract primordium. Scale bars = 50 μm (A, B) and 10 μm (C–F).

To test whether there was a direct interaction between BIF2 and ZmPIN1a, we performed kinase assays. The structure of AtPIN1 consists of multiple predicted membrane-spanning domains flanking either side of a central cytoplasmic hydrophilic region, depicted for ZmPIN1a in Fig. 3A (Galweiler et al. 1998, Carraro et al. 2006). PID has been reported to phosphorylate the hydrophilic loop of AtPIN1 (Michniewicz et al. 2007). We noted that there was divergence in the hydrophilic loop of ZmPIN1a, b and c proteins compared with AtPIN1 and that the residues shown to be phosphorylated in AtPIN1 in vivo were not conserved in maize (arrows in Fig. 3B) (Michniewicz et al. 2007). Overall, the three ZmPIN1 genes are 65–67% identical (75–77% similar) to AtPIN1; however, in the hydrophilic loop, the ZmPIN1 proteins are only 48–50% identical (57–60% similar). Thus, we tested if BIF2 could phosphorylate ZmPIN1a in vitro. Purified proteins were co-incubated in kinase buffer in the presence of [γ-32P]ATP. Autophosphorylation of BIF2 (Fig. 3C, lanes 1 and 2) and strong transphosphorylation of ZmPIN1a were observed (Fig. 3C, lane 2). BIF2 autophosphorylation and ZmPIN1a transphosphorylation were not observed when the two different mutated kinase-dead versions of BIF2 were substituted for active BIF2 (Fig. 3C, lanes 3 and 4) (Skirpan et al. 2008). Finally, there was no transphosphorylation of ZmPIN1a when it was incubated with glutathione S-transferase (GST) alone (data not shown). A co-purifying bacterial kinase was observed in all lanes and served as an internal control for activity and specificity, as it did not phosphorylate BIF2, GST or ZmPIN1a. Thus, BIF2 strongly and specifically phosphorylated ZmPIN1a in vitro, despite the divergence in the hydrophilic loop, indicating that there must be multiple sites of phosphorylation. Interestingly, additional sites of phosphorylation were detected in AtPIN1 upon treatment with flg22 elicitor protein (Benschop et al. 2007), and these residues are conserved between maize and Arabidopsis PIN1 proteins (asterisks in Fig. 3B), suggesting that phosphorylation may occur at the conserved 5′ end of the hydrophilic loop.

Fig. 3

BIF2 phosphorylates ZmPIN1a in vitro. (A) Schematic representation of ZmPIN1a protein. The transmembrane domains (TMs) are indicated by black boxes. The hydrophilic loop encoding amino acids 183–440, indicated in gray, was fused to GST for the phosphorylation assay. (B) Alignment of the hydrophilic loop from the maize ZmPIN1a, b and c proteins with Arabidopsis AtPIN1. ZmPIN1a, amino acids 183–440; ZmPIN1b, amino acids 183–434; ZmPIN1c, amino acids 188–436; AtPIN1, amino acids 183–461. Arrows indicate the two sites in AtPIN1 proposed to be phosphorylated in vivo (Michniewicz et al. 2007), which are not conserved in maize. Asterisks indicate additional sites phosphorylated in AtPIN1 in response to flg22 (Benschop et al. 2007). The line indicates the peptide used to make the Arabidopsis antibody which is conserved with maize (Boutte et al. 2006, Carraro et al. 2006). (C) Coomassie-stained gel (left) and autoradiograph (right) showing BIF2 autophosphorylation and transphosphorylation of ZmPIN1a. Lane 1, GST–BIF2 co-incubated with GST. Lane 2, GST–BIF2 co-incubated with GST–ZmPIN1a. Lane 3, GST–BIF2K161R (Skirpan et al. 2008) co-incubated with GST–ZmPIN1a. Lane 4, GST–BIF2D258A co-incubated with GST–ZmPIN1a. An asterisk indicates a co-purifying bacterial kinase observed in all lanes.

Fig. 3

BIF2 phosphorylates ZmPIN1a in vitro. (A) Schematic representation of ZmPIN1a protein. The transmembrane domains (TMs) are indicated by black boxes. The hydrophilic loop encoding amino acids 183–440, indicated in gray, was fused to GST for the phosphorylation assay. (B) Alignment of the hydrophilic loop from the maize ZmPIN1a, b and c proteins with Arabidopsis AtPIN1. ZmPIN1a, amino acids 183–440; ZmPIN1b, amino acids 183–434; ZmPIN1c, amino acids 188–436; AtPIN1, amino acids 183–461. Arrows indicate the two sites in AtPIN1 proposed to be phosphorylated in vivo (Michniewicz et al. 2007), which are not conserved in maize. Asterisks indicate additional sites phosphorylated in AtPIN1 in response to flg22 (Benschop et al. 2007). The line indicates the peptide used to make the Arabidopsis antibody which is conserved with maize (Boutte et al. 2006, Carraro et al. 2006). (C) Coomassie-stained gel (left) and autoradiograph (right) showing BIF2 autophosphorylation and transphosphorylation of ZmPIN1a. Lane 1, GST–BIF2 co-incubated with GST. Lane 2, GST–BIF2 co-incubated with GST–ZmPIN1a. Lane 3, GST–BIF2K161R (Skirpan et al. 2008) co-incubated with GST–ZmPIN1a. Lane 4, GST–BIF2D258A co-incubated with GST–ZmPIN1a. An asterisk indicates a co-purifying bacterial kinase observed in all lanes.

To determine whether the altered localization of ZmPIN1 in bif2 mutants led to a difference in auxin levels, we measured free IAA levels in developing bif2 tassels compared with normal tassels. These experiments had not previously been performed on developing maize inflorescences and a protocol was adapted for this purpose (Barkawi et al. 2008). In both 4–6 mm and 7–9 mm developing tassels, no significant difference was observed in the concentration of free IAA between normal and bif2 tassels (Table 1). However, we noted that bif2 tassels were approximately half the weight of normal due to the developmental defects. Therefore, we calculated the amount of IAA per tassel, rather than per g FW, and discovered that there was a statistically significant difference in IAA levels in the 7–9 mm tassels (Table 1). We propose that early in development, auxin levels are normal in bif2 mutants because auxin is synthesized in the inflorescence but is not transported towards the site of primordia initiation. Instead, the basal localization of ZmPIN1a in bif2 mutants would lead to auxin being transported down the inflorescence, leading to the reduction in auxin levels detected later in development. Alternatively, the reduction in auxin levels detected could be a consequence of the developmental defects in bif2 mutants.

Table 1

 Measurement of free IAA levels in normal and bif2 tassel inflorescences

Phenotype IAA ng g–1 FW
 
IAA ng per tassel
 
 4–6 mm tassels 7–9 mm tassels 4–6 mm tassels 7–9 mm tassels 
Normal 83.61 ± 11.86 55.26 ± 8.77 0.58 ± 0.04 1.01 ± 0.05 
bif2 102.07 ± 7.84 42.02 ± 3.98 0.50 ± 0.03 0.57 ± 0.02a 
Phenotype IAA ng g–1 FW
 
IAA ng per tassel
 
 4–6 mm tassels 7–9 mm tassels 4–6 mm tassels 7–9 mm tassels 
Normal 83.61 ± 11.86 55.26 ± 8.77 0.58 ± 0.04 1.01 ± 0.05 
bif2 102.07 ± 7.84 42.02 ± 3.98 0.50 ± 0.03 0.57 ± 0.02a 

aA significant difference from normal (t-test, P < 0.05).

Values are the mean ± SE (n = 8, except bif2 first column where n = 7).

In conclusion, we show that BIF2, which was previously shown to be localized at the cell periphery in heterologous systems (Skirpan et al. 2008), phosphorylates ZmPIN1a and affects ZmPIN1a localization similar to the interaction between PID and AtPIN1 in Arabidopsis (Friml et al. 2004, Michniewicz et al. 2007). Therefore, BIF2 may regulate auxin transport through direct regulation of ZmPIN1a in maize inflorescence development. We propose that in bif2 mutants, altered ZmPIN1 localization results in auxin flowing out of the inflorescence meristem, failure to initiate axillary meristems and a reduction in free auxin levels. In addition, BIF2 is localized in the nucleus and it interacts with and phosphorylates BA1, which is required for axillary meristem initiation in the maize inflorescence (Skirpan et al. 2008). Therefore, the BIF2 kinase has multiple putative targets in the regulation of axillary meristem initiation during maize inflorescence development. An intriguing speculation is that the bifunctional role of the BIF2 kinase may provide a mechanism to induce genes required for axillary meristem initiation in response to auxin transport.

Materials and Methods

For scanning electron microscopy (SEM), 4–6 mm tassels were harvested from 5-week-old greenhouse-grown families segregating for bif2-77 in B73 (McSteen et al. 2007). The 5–7 mm ears were harvested from 8-week-old field-grown plants (Rocksprings, PA, USA). Fixation, critical point drying and SEM were performed as described (Wu and McSteen 2007). For confocal microscopy, plants homozygous for bif2-77 and heterozygous for ZmPIN1a–YFP (Gallavotti et al. 2008) were identified by genotyping for bif2 with primers bif2-57 and bif2-250 as described (Skirpan et al. 2008), and for YFP with the primers given in Supplementary Methods. Tassels 4 mm in size were dissected from 5-week-old greenhouse-grown plants and mounted in 5% glycerol. Fifteen normal and eight bif2 tassels were imaged using a Zeiss LSM510 Meta confocal microscope as described in Supplementary Methods.

Immunolocalization was performed as described in Carraro et al. (2006), except that samples (4–7 mm tassels and 2–12 mm ears) were fixed in FAA (3.7% formaldeyde, 5% glacial acetic acid, 50% ethanol) overnight. The secondary antibody was a 1 : 1,000 dilution of goat anti-rabbit polyclonal antibody conjugated to alkaline phosphatase (Novagen, EMD Chemicals, Madison, WI, USA), and detection was with 0.35 mg ml–1 4-nitroblue tetrazolium chloride and 0.175 mg ml–1 5-bromo-4-chloro-3-indoyl-phosphate (Roche Applied Science, Indianapolis, IN, USA) in 0.1 M Tris pH 9.5, 0.1 M NaCl, 50 mM MgCl2. Signal was not detected in control sections in which the primary or secondary antibody was omitted (data not shown).

To determine the levels of free auxin, normal and bif2-77 tassels, 4–9 mm in size, were harvested from 5-week-old field-grown plants (Rocksprings) and immediately frozen in liquid nitrogen. IAA extraction was performed by adapting the procedures of Chen et al. (1988) and Barkawi et al. (2008), as described in the Supplementary Methods.

For phosphorylation assays, the hydrophilic loop of ZmPIN1a was amplified from tassel cDNA (primers given in the Supplementary Methods) with the addition of 1 M betaine and 10% dimethylsulfoxide. The product was cloned into pGEX-5X-1 (GE Life Sciences, Piscataway, NJ, USA) in-frame with GST, and sequenced. Fusion proteins were induced and purified as described (Skirpan et al. 2008). Phosphorylation assays were performed as described (Skirpan et al. 2001). Multiple sequence alignments were performed using Clustal W (www.ebi.ac.uk/Tools/clustalw2) and shaded using boxshade (www.ch.embnet.org/software/BOX_form.html).

Supplementary data

Supplementary data are available at PCP online.

Funding

The National Science Foundation (IBN-0416616 to P.M., DBI-0501862 to D.J., MCB-0643845, MCB-0725149 and DBI-0606666 to J.D.C.); a Cold Spring Harbor Laboratory Association Fellowship (to A.G.)

Acknowledgments

We thank Missy Hazen for SEM training, Richard Cyr and Gloria Muday for advice on confocal microscopy, Tony Omeis for plant care, and members of the Braun and McSteen labs for discussions and comments on the manuscript.

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Abbreviations:

    Abbreviations:
  • BM

    branch meristem

  • FM

    floral meristem

  • GST

    glutathione S-transferase

  • IM

    inflorescence mer-istem

  • SEM

    scanning electron microscopy

  • SM

    spikelet meristem

  • SPM

    spikelet pair meristem

  • YFP

    yellow fluorescent protein.

Author notes

4Present address: Monsanto Company, 800 N. Lindbergh Blvd., Saint Louis, MO 63167, USA.
5Present address: Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.