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Developmental Biology and Developmental Genetics |
Department of Biology, The Pennsylvania State University, 208 Mueller Lab, University Park, Pennsylvania 16802 USA
Received for publication June 30, 2007. Accepted for publication September 20, 2007.
ABSTRACT
Axillary meristems play a fundamental role in inflorescence architecture. Maize (Zea mays) inflorescences are highly branched panicles because of the production of multiple types of axillary meristems. We used auxin transport inhibitors to show that auxin transport is required for axillary meristem initiation in the maize inflorescence. The phenotype of plants treated with auxin transport inhibitors is very similar to that of barren inflorescence2 (bif2) and barren stalk1 (ba1) mutants, suggesting that these genes function in the same auxin transport pathway. To dissect this pathway, we performed RNA in situ hybridization on plants treated with auxin transport inhibitors. We determined that bif2 is expressed upstream and that ba1 is expressed downstream of auxin transport, enabling us to integrate the genetic and hormonal control of axillary meristem initiation. In addition, treatment of maize inflorescences with auxin transport inhibitors later in development results in the production of single instead of paired spikelets. Paired spikelets are a key feature of the Andropogoneae, a group of over 1000 grasses that includes maize, sorghum, and sugarcane. Because all other grasses bear spikelets singly, these results implicate auxin transport in the evolution of inflorescence architecture. Furthermore, our results provide insight into mechanisms of inflorescence branching that are relevant to all plants.
Key Words: auxin transport • axillary meristem • barren inflorescence • branch • maize • Poaceae • spikelet • Zea mays
Development of higher plants is regulated by meristems (Steeves and Sussex, 1989
). Meristems have two main functions; organogenesis, whereby organs are produced in the peripheral zone, and maintenance, whereby further growth is supported by the central zone (McSteen and Hake, 1998
; Williams and Fletcher, 2005
). The shoot apical meristem reiteratively produces modular units called phytomers, consisting of a leaf, axillary meristem, node, and internode (Steeves and Sussex, 1989
; McSteen and Leyser, 2005
). During vegetative development in many plants such as Arabidopsis thaliana, the leaf is prominent, while the axillary meristem either remains dormant or grows out to become a branch. After the transition to reproductive growth, the axillary meristem enlarges and produces flowers, while the subtending leaf, called a bract, is often reduced (Long and Barton, 2000
; Grbic, 2005
). The use of polar auxin transport inhibitors, such as N-1-naphthylphthalamic acid (NPA), has shown that auxin transport is required for initiation of leaves during vegetative development and flowers during reproductive development (Okada et al., 1991
; Reinhardt et al., 2000
, 2003
; Scanlon, 2003
).
The role of auxin transport in organogenesis has also been shown by modeling and genetic studies. Auxin is transported towards primordia as they initiate, causing the primordia to become auxin sinks and preventing other organs from forming close by (Benkova et al., 2003
; Reinhardt et al., 2003
; Swarup et al., 2005
; de Reuille et al., 2006
; Jonsson et al., 2006
; Smith et al., 2006
). Arabidopsis thaliana plants with mutations in the auxin efflux carrier, PINFORMED1 (PIN1), fail to initiate floral meristems in the inflorescence (Okada et al., 1991
; Galweiler et al., 1998
; Petrasek et al., 2006
). Arabidopsis thaliana plants with mutations in PINOID (PID), a serine/threonine protein kinase proposed to regulate auxin transport, have a phenotype very similar to the pin1 mutants (Bennett et al., 1995
; Christensen et al., 2000
; Benjamins et al., 2001
; Friml et al., 2004
; Lee and Cho, 2006
). Therefore, regulated auxin transport is required for floral meristem initiation in A. thaliana (Cheng and Zhao, 2007
).
Unlike A. thaliana, which has a raceme type of inflorescence, maize (Zea mays) produces inflorescences that are branched panicles. Maize inflorescences are highly branched because of the production of multiple types of axillary meristems (Fig. 1) (Irish, 1997
; McSteen et al., 2000
; Bommert et al., 2005
; Kellogg, 2007
). The male inflorescence, the tassel, forms at the apex of the plant, and the female inflorescence, the ear, is produced from an axillary meristem a few nodes below the tassel. Long branches form at the base of the tassel, and short branches called spikelet pairs cover the long branches and the main spike (Fig. 1A). Each spikelet consists of a pair of leaflike glumes that enclose a pair of florets. The spikelet is the fundamental unit of all grass inflorescences (Clifford, 1987
). Paired spikelets, however, are a defining character of the Andropogoneae, a group of more than 1000 grasses that includes maize, sorghum, and sugarcane (Kellogg, 2000
; G. P. W. G., 2001
). Other grasses outside the Andropogoneae, such as rice and barley, have single spikelets (Shimamoto and Kyozuka, 2002
; Bommert et al., 2005
). Therefore, regulation of the determinacy of axillary meristems that give rise to branches and spikelets has played an important role in the evolution of inflorescence architecture in the grasses (McSteen, 2006
; Kellogg, 2007
). However, the production of multiple orders of axillary meristem before the production of floral meristems is not restricted to the grasses and also occurs in eudicots with panicle or compound umbel inflorescence architecture (Borthwick et al., 1931
; Manning, 1938
; Tucker, 1989
; Prusinkiewicz et al., 2007
).
|
; Irish, 1997
; McSteen et al., 2000
). In hierarchical order, they are branch meristem (BM), spikelet pair meristem (SPM), spikelet meristem (SM), and floral meristem (FM) (Fig. 1D). First, the inflorescence meristem produces a few primary axillary meristems, BMs, which become the long branches at the base of the tassel (Fig. 1B). Then, the inflorescence meristem (and the branches) produce many additional primary axillary meristems called SPMs (Fig. 1B). Each of the SPMs gives rise to two secondary axillary meristems, the SMs (Fig. 1C). Each SM produces two leaflike glumes enclosing two tertiary axillary meristems, the FMs (Fig. 1D). Finally, each FM initiates the floral organs. The early stages of tassel and ear development are very similar except that ears do not produce BMs. The mechanism by which the SPMs produce the SMs and the SMs produce the FMs is unknown (McSteen and Leyser, 2005
). Two alternative models for inflorescence branching have been proposed (Irish, 1997
; Chuck et al., 1998
; Irish, 1998
). In the lateral branching model, the SPM is proposed to give rise to the two SMs by lateral branching, leaving a residual SPM between the two SMs (Chuck et al., 1998
). In the conversion model, the SPM is proposed to initiate the first SM by lateral branching and to produce the second SM by a meristem conversion event (Irish, 1997
, 1998
). Despite the recent cloning of several meristem identity genes, this controversy remains unresolved (Chuck et al., 2002
; Vollbrecht et al., 2005
; Bortiri et al., 2006
; Chuck et al., 2007
).
A large class of mutants has been identified that fail to produce axillary meristems in maize (McSteen et al., 2000
). Two of these mutants, barren inflorescence2 (bif2) and barren stalk1 (ba1), have been cloned and have been shown to be required for the initiation of axillary meristems in the inflorescence (Gallavotti et al., 2004
; McSteen et al., 2007
). Both mutants fail to produce ear shoots, branches, spikelets, florets, and floral organs, all of which arise from axillary meristems (McSteen and Hake, 2001
; Ritter et al., 2002
). The gene bif2 encodes a co-ortholog of PID that regulates auxin transport in A. thaliana (McSteen et al., 2007
). There is evidence that bif2 may regulate auxin transport in maize (Carraro et al., 2006
; McSteen et al., 2007
) and that the rice ortholog, OsPID, may regulate auxin transport in rice (Morita and Kyozuka, 2007
). The gene ba1 encodes an atypical bHLH transcription factor that is not present in A. thaliana (Gallavotti et al., 2004
). Overexpression of the rice ortholog of ba1, lax panicle1, causes defects indicative of a role for this gene in auxin action (Komatsu et al., 2001
, 2003
). Therefore, auxin transport may also play a role in axillary meristem initiation in the inflorescence of maize and rice, though this has not yet been shown directly.
To determine whether auxin transport is required for axillary meristem initiation during maize inflorescence development, we treated normal plants with different concentrations of NPA at multiple stages of inflorescence development. Scanning electron microscopy (SEM) analysis and RNA in situ hybridization using kn1 as a marker for meristems showed that auxin transport is required for axillary meristem initiation, irrespective of meristem identity. We then utilized these plants treated with auxin transport inhibitors to dissect the auxin transport pathway by testing the expression of bif2 and ba1 by RNA in situ hybridization. Although the phenotypes of bif2 mutants and NPA-treated plants are very similar, bif2 was expressed in inflorescences of NPA-treated plants. This suggests that bif2 is expressed upstream of auxin transport. However, ba1 was not expressed after NPA treatment, indicating that ba1 expression depends on polar auxin transport. This analysis provides insight into the genetic and hormonal regulation of axillary meristem initiation in maize. Furthermore, the effects of NPA on spikelet initiation resolves the controversy over the mechanism of inflorescence branching in maize and provides insight into the mechanism of inflorescence branching in all plants.
MATERIALS AND METHODS
Plant growth conditions
Maize (Zea mays) B73 seeds were planted in 10-cm pots containing Scotts Metromix 360 soil (Griffin Greenhouse, Morgantown, Pennsylvania, USA). Plants were grown in the greenhouse with supplemental lighting during winter/spring. Inflorescence meristems were dissected and observed with the stereomicroscope to determine the developmental stage. We used three developmental stages: (1) When the apical meristem was transitioning to inflorescence development (Fig. 2A), which occurred after about 4 wk of growth (inflorescences were 
1–2 mm tall); (2) when branch meristems were initiating (Fig. 2F), which occurred after about 5 wk of growth (inflorescences were 3–5 mm tall); and (3) when spikelet pair meristems were initiating, which occurred after about 6 wk of growth (inflorescences were 5–7 mm tall) (Fig. 2K).
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Cautionary note: A trace amount of ß-NPA contaminates NPA during commercial production (A. Murphy, Purdue University, personal communication). Within the plant, ß-NPA is potentially hydrolyzed by aminopeptidases to ß-naphthylamine, which is a carcinogen (Reznikoff et al., 1993
; Murphy and Taiz, 1999
). Therefore, plant tissues treated with NPA must be treated as potentially hazardous.
Scanning electron microscopy (SEM)
Meristems from each treatment were critical point dried with liquid CO2 (BAL-TEC CPD 030, Techno Trade, Manchester, New Hampshire, USA), sputter coated with Au-Pd (BAL-TEC SCD 050, Techno), and viewed at 20 kV accelerating voltage by SEM (JSM5400, JEOL, Peabody, Massachusetts, USA).
RNA in situ hybridization
After fixing and dehydration, inflorescence meristems were embedded in paraffin wax (Paraplast, Oxford Labware, St. Louis, Missouri, USA). Sections 8 µm thick were cut with a Finesse paraffin microtome (Thermo Fisher, Waltham, Massachusetts, USA) and mounted on slides (Probe-on plus, Thermo Fisher, Waltham, Massachusetts, USA). The kn1 and bif2 probes were previously described (Jackson et al., 1994
; McSteen et al., 2007
). The ba1 probe was generated by PCR from genomic DNA (primer 1: 5'-GAT GCA GCA CGA GGA AGG ATG CCA T-3', primer 2: 5'-ATA CGG TGT ATC ATC TGC GAT CAG CGG A-3'). This 524-bp sequence from the 3' end of ba1, excluding the bHLH domain, was subcloned into the pGEM-TEasy vector (Promega, Madison, Wisconsin, USA). The plasmid was digested with NdeI to generate an antisense probe with T7 RNA polymerase. RNA in situ hybridization was performed according to Jackson et al. (1994)
.
Histology
For histological analysis, 8-µm sections were treated with Histoclear (National Diagnostics, Atlanta, Georgia, USA) to remove wax and then hydrated through an ethanol series. Sections were stained in 0.05% toluidine blue O (TBO) for 30 s, dehydrated through an ethanol series, mounted using Histosolve and Histomount (Thermo Shandon, Waltham, Massachusetts, USA), and observed in bright field with a light microscope. The vascular bundles were counted, and the data from meristems treated with 40, 80, 100, and 120 µM NPA were pooled because there was no statistical difference in vascular bundle number among samples treated with different concentrations of NPA. T-test analysis was performed using Minitab version 15 software (Minitab, State College, Pennsylvania, USA).
RESULTS
NPA inhibits the initiation of axillary meristems in the inflorescence
To determine whether auxin transport was required for axillary meristem initiation in maize, we treated normal plants with NPA via daily watering for 2 wk. Control plants were mock treated with water containing a small amount of the solvent used to dissolve NPA (Fig. 2B, G, L). Three days after treatment, the roots became agravitropic, indicating that the NPA was effective and had similar effects on root development as seen in other species (Rashotte et al., 2000
) (Fig. S1, see Supplemental Data with online version of this article). Two weeks of treatment was required for sufficient growth and development to have occurred to see the effect of NPA on the inflorescence. After 2 wk, inflorescences were dissected, fixed, and viewed by SEM. Because development proceeds in a defined spatial and temporal order, the effect of NPA on different axillary meristems types could be investigated by starting the treatment at different times and hence different stages of development (Fig. 2A, F, K). As development proceeds, younger meristems are located closer to the tip of the inflorescence and older meristems toward the base.
Upon the transition to reproductive development, the shoot apical meristem stops initiating leaves and elongates to form the transition stage inflorescence meristem (Fig. 2A) (Cheng et al., 1983
; McSteen et al., 2000
). This transition occurred after 4 wk of growth in our greenhouse. A few days later, branch meristems (BMs) became visible at the base of the inflorescence (Fig. 2F). As the main rachis and branches elongated, bract primordia became visible (Fig. 2K). After more than 5 wk of growth, spikelet pair meristems (SPMs) arose in the axils of bract primordia, which were suppressed (Fig. 2B, G, K).
To determine whether auxin transport was required for primary axillary meristem initiation (BM and SPM), we treated plants at the transition stage of inflorescence development (Fig. 2A, 4 wk old) with NPA for 2 wk. In contrast to untreated controls (Fig. 2B), BMs and SPMs did not develop on the flanks of the inflorescence after NPA treatment (Fig. 2C–E). However, the apical inflorescence meristem appeared to be unaffected by the treatment and continued to elongate. SPM initiation was also inhibited when plants that had begun to initiate BMs were treated (Fig. 2F, 5 wk of age). In this case, inhibition of SPM initiation was seen in the apical region of the inflorescence, where the youngest meristems would have initiated (Fig. 2H–J). If BMs had already initiated before the treatment took effect, they continued to elongate but did not produce SPMs on their flanks (arrows in Fig. 2I, N, O). Occasionally, secondary branches formed at the base of the primary branches, and similarly, they elongated without producing SPMs (Fig. 3C). Additionally, the surface of the inflorescence rachis was not completely smooth because ridges were visible (Fig. 2H–J). These ridges formed rings surrounding the inflorescence stem (close-up shown in Fig. 3C, D). In conclusion, NPA inhibited the production of the first axillary meristems (BM or SPM) by the inflorescence but did not affect the elongation of the apical inflorescence meristem.
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To determine whether NPA blocked the initiation of BMs and SPMs or whether these meristems initiated and then aborted, we performed RNA in situ hybridization using kn1 as a marker for meristems. In normal plants, kn1 is expressed in the inflorescence meristem and all axillary meristems (Fig. 4A) (Jackson et al., 1994
). The first indication of bract leaf initiation is the absence of kn1 expression on the flanks of the inflorescence meristem (Fig. 4A, arrow). The first indication of BM and SPM initiation occurs in a few cells in the axil of the bract primordia that express kn1 before a protrusion is visible (Fig. 4A). The expression of kn1 then expands as the axillary meristem grows out (Fig. 4A). In the inflorescences of NPA-treated plants, kn1 was expressed in the apical inflorescence meristem and in the stem and vasculature as normal (Fig. 4B). However, kn1 was not expressed on the flanks of the inflorescence (Fig. 4B, arrow). These results indicate that NPA treatment does not inhibit the initiation of bract primordia and that the ridges visible after NPA treatment likely correspond to bract primordia. Furthermore, because no expression of kn1 was visible on the flanks of the inflorescence meristem, we infer that BMs and SPMs do not initiate after NPA treatment and that auxin transport is required for the initiation of BMs and SPMs. These results are similar to those obtained in studies of the expression of kn1 in bif2 and ba1 mutants in maize (McSteen and Hake, 2001
; Ritter et al., 2002
).
|
). However, the effects of NPA on secondary axillary meristems (SMs) depended on which of the models for inflorescence branching in maize is correct, and in fact afforded an opportunity to distinguish between these models. If the lateral branching model for inflorescence branching is correct, NPA treatment should inhibit SPMs from initiating both SMs, leading to the production of arrested SPMs. However, if the conversion model is correct, NPA treatment should inhibit the initiation of the first SM but might not inhibit the conversion from SPM to SM identity, leading to the production of single spikelets. The meristem can be identified as a SM rather than a SPM by its ability to produce glumes (Fig. 3A). To distinguish between these models for inflorescence branching and to determine the effects of NPA on spikelet meristem (SM) initiation, we applied NPA to plants in which SPMs had already begun to initiate (Fig. 2F, K). In control plants, the SPM produced two SMs, which give rise to the larger pedicellate spikelet and the smaller sessile spikelet (Fig. 3A). In support of the conversion model for inflorescence branching, NPA-treated plants produced inflorescences with single instead of paired spikelets (Figs. 2H–J, 2M–O, 3B). If plants were treated at 5 wk of age, single spikelets were found at the base of the inflorescence (Fig. 2H–J). If plants were treated at 6 wk of age, single spikelets were produced toward the apex while the older spikelets at the base of the inflorescence, which had presumably initiated before the treatment took effect, were still paired (Fig. 2M–O). Similarly, single spikelets were also observed near the tips of branches (Fig. 2M, arrow). A dose response was apparent with increasing concentrations of NPA causing more SPMs to produce single SMs. Only the SMs near the tip of the apex were inhibited by 40 µm NPA (Fig. 2M). With 80 µm NPA, usually a third of the apical part of the inflorescence was affected, producing single spikelets (Fig. 2N). The most severe phenotype was seen when plants were treated with 120 µM NPA; in some cases, the entire inflorescence produced single spikelets (Fig. 2O). The production of single spikelets shows that, although NPA inhibits the ability of the SPM to initiate an SM, it does not inhibit the ability of the SPM to convert to SM identity as evidenced by the production of glumes.
In NPA-treated inflorescences, spikelets that formed were borne on elongated pedicels similar to pedicellate spikelets (Fig. 3B). However, the SMs that formed were more elongated than normal (Fig. 3D). This elongation was clearly evident in RNA in situ hybridization experiments using kn1. In control inflorescences, kn1 was strongly expressed in SPMs and SMs (Fig. 4C). However, in the inflorescences of NPA-treated plants, kn1 expression was seen in SMs that were more elongated relative to normal (Fig. 4D). Therefore, although NPA inhibits the initiation of axillary meristems, it does not inhibit the growth of axillary meristems that have already formed.
NPA causes defects in lateral organ development in the inflorescence
In normal inflorescences, the first lateral organs to be produced by the SMs are the glumes (Cheng et al., 1983
). First, an outer glume becomes visible on the pedicellate spikelet followed by the appearance of an outer glume on the sessile spikelet (Figs. 1C, 3A). Then, an inner glume becomes visible on both the pedicellate and sessile spikelet (Fig. 3A). When spikelets formed in inflorescences treated with NPA (at 5 or 6 wk old), they had several defects in glume development depending on when NPA took effect. Occasionally, glumes did not appear to initiate when viewed by SEM (Fig. 3E, F, arrows). Glumes were absent on both single spikelets (Fig. 3E) and on the sessile SM of paired spikelets (Fig. 3F). However, because downregulation of kn1 is evident on the flanks of elongated SMs (Fig. 4D), glumes may actually initiate but fail to grow out. This inhibition of glume outgrowth was more apparent at increasing NPA concentrations (compare glume size on spikelets at the base of Fig. 2H–J). Glumes that had formed usually had defects in organ separation. Frequently, the glumes formed a ring surrounding the SM as if there were defects in separation of the inner and outer glumes (Fig. 3D,H). Occasionally, the glumes of neighboring spikelets fused together (Fig. 3G). In older inflorescences, NPA treatment usually had no effect on floral organ development (Fig. 3I). In a few cases, however, there were defects in floral organ development, such as inhibition and fusion of organ primordia, similar to the effects of NPA on glume development (Fig. 3J). These results imply that continued auxin transport is required for the normal development of lateral organs in the inflorescence, similar to the role of auxin transport in lateral organs during leaf development (Scanlon, 2003
).
NPA reduces vascular development in the inflorescence stem
Auxin plays a fundamental role in vascular development (Aloni, 2004
). In A. thaliana, NPA treatment causes an increase in vasculature in the inflorescence stem (Galweiler et al., 1998
). However, in bif2 mutants of maize which have reduced auxin transport, vasculature is reduced (McSteen et al., 2007
). To determine if this difference in effect is due to differences between monocots and eudicots, we determined the effects of NPA on vascular development in the maize inflorescence. Plants at the transition stage of development were treated with NPA for 2 wk. Transverse sections through inflorescences were stained with toluidine blue O (TBO). Vascular bundles were visible as oval shaped, darkly staining groups of cells (Fig. 5A). The vascular bundles near the base of the inflorescence stem were counted in NPA-treated plants and in untreated controls (Fig. 5A, B). The number of vascular bundles was divided by the cross sectional area of the stem to adjust for the slight difference in width between NPA-treated and control inflorescences. This analysis showed that NPA-treated inflorescences had significantly fewer vascular bundles than the controls (t = 2.71, df = 10, P = 0.01, Fig. 5C). Therefore, NPA treatment reduced the number of vascular bundles in the inflorescence stem of maize, which is the opposite effect to what was observed in A. thaliana (Galweiler et al., 1998
).
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). Because NPA treatment of normal maize plants mimics the bif2 mutant phenotype, we tested whether bif2 was expressed in the inflorescences of NPA-treated plants. In normal plants, bif2 is expressed on the flanks of the inflorescence before SPMs arise and is subsequently expressed in all axillary meristems (McSteen et al., 2007
). In TIBA-treated plants, which look normal, bif2 was expressed as normal in SPMs as they arose (Fig. 7A). Surprisingly, bif2 was expressed in NPA-treated plants (Fig. 7B). The bif2 gene was expressed in a ring surrounding the inflorescence meristem (Fig. 7B, arrow). However, bif2 was not expressed at the base of the inflorescence, indicating that bif2 expression was not maintained (Fig. 7B). Similar results were seen in inflorescences of HFCA-treated plants (not shown). Therefore, early expression of bif2 does not depend on active auxin transport.
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; Gallavotti et al., 2004
). To test whether the early expression of ba1 depends on polar auxin transport, we performed RNA in situ hybridization of ba1 in the inflorescences of NPA-treated plants. In normal plants, ba1 is expressed on the adaxial side of initiating BMs and SPMs (Gallavotti et al., 2004
). In TIBA-treated plants, ba1 was expressed as normal (Fig. 7C). However, no ba1 transcript was detected in the inflorescences of NPA- (not shown) or HFCA-treated plants (Fig. 7D). These results suggest that, unlike bif2, the initial expression of ba1 requires polar auxin transport during BM and SPM initiation. Therefore, NPA treatment allows us to dissect the pathway for axillary meristem initiation in maize. DISCUSSION
We showed that treatment with auxin transport inhibitors prevents axillary meristem initiation in the maize inflorescence. In A. thaliana, treatment with auxin transport inhibitors prevents the initiation of floral meristems (FMs), which are the primary axillary meristems produced by the inflorescence (Okada et al., 1991
; Long and Barton, 2000
). In maize, the BM and SPM are the primary axillary meristems produced by the inflorescence meristem (McSteen et al., 2000
). Therefore, auxin transport inhibitors prevent the initiation of axillary meristems, irrespective of meristem identity. Furthermore, auxin transport inhibitors also inhibit the initiation of secondary axillary meristems, resulting in the production of single instead of paired spikelets in maize. These results provide insight into the mechanism of inflorescence branching in grasses and have significance for the regulation of inflorescence architecture in all plants with branched inflorescences. In addition, analysis of the expression of bif2 and ba1 in the inflorescence of plants treated with auxin transport inhibitors enabled us to dissect the pathway for axillary meristem initiation in maize.
Implications for models of inflorescence branching
A striking effect of NPA treatment of maize is the production of single instead of paired spikelets in the inflorescence. These results strongly support the conversion model for spikelet initiation. We propose that in NPA-treated inflorescences, inhibition of auxin transport prevents the SPM from initiating an SM but does not affect the ability of the SPM to convert to SM identity as evidenced by its ability to produce glumes. If the lateral branching model for spikelet initiation is correct, auxin transport inhibition would have resulted in the production of aborted spikelet pair meristems, which was rarely seen. These results imply that, although auxin transport is required for the initiation of axillary meristems, auxin transport is not required for the meristem to convert to a new meristem identity.
Paired spikelets are a defining character of the Andropogoneae, while grasses outside the Andropogoneae, such as rice and barley have single spikelets (Clifford, 1987
; Kellogg, 2000
; G. P. W. G., 2001
). The presence of single spikelets is also characteristic of bif2 and ba1 mutants in maize (McSteen and Hake, 2001
; Gallavotti et al., 2004
). This phenotype suggests that auxin transport or response is required for the formation of paired spikelets in maize. Therefore, the regulation of auxin transport may have played a role in the evolution of inflorescence architecture in the grasses. Furthermore, because branched inflorescence architecture in all plants can be explained by a similar mechanism (Prusinkiewicz et al., 2007
), our results support the idea that meristem conversion events also occur in other plants in which the inflorescence produces multiple orders of axillary meristem.
NPA does not inhibit the elongation of apical or axillary meristems
A notable feature of the NPA experiments is that the apical inflorescence meristem is unaffected by NPA treatment. The inflorescence meristem continues to elongate even though it cannot initiate axillary meristems or lateral primordia. This effect on the apical inflorescence meristem is also seen in other plants treated with NPA (Okada et al., 1991
; Reinhardt et al., 2000
) as well as in auxin-transport mutants in A. thaliana and maize (Christensen et al., 2000
; Vernoux et al., 2000
; McSteen and Hake, 2001
). We suggest that NPA fails to affect the inflorescence meristem because the inflorescence meristem, or more correctly, the central zone at the tip of the inflorescence meristem, is auxin insensitive as proposed by de Reuille et al. (2006)
. This difference in the effects of NPA on apical vs. axillary meristems could be due to the identity of the meristem as apical rather than axillary. However, our results show that axillary meristems also continue to elongate if they had initiated before NPA treatment. In maize inflorescences treated with NPA, BMs that had initiated continue to elongate without initiating lateral organs. BMs normally elongate, so the more significant result is that SPMs or SMs, which normally do not elongate, also continue to grow without producing lateral primordia. Hence, once a meristem has initiated, whether it is apical or axillary, its ability to elongate is not inhibited and in fact may be promoted by NPA treatment. Perhaps active auxin transport is required in the peripheral zone of the meristem but not in the central (and rib) zones.
Polar auxin-transport inhibitors differ in their effectiveness
Both NPA and HFCA inhibited axillary meristem initiation. However, unlike results in eudicots, an additional auxin transport inhibitor, TIBA, did not inhibit axillary meristem initiation (Okada et al., 1991
; Mattsson et al., 1999
; Sieburth, 1999
) though it did affect root gravitropism. We suggest that this is due to the difference in the mechanism of action of TIBA vs. NPA and HFCA (Thomson et al., 1973
; Petrasek et al., 2003
). TIBA is less effective than NPA at inhibiting auxin transport (Thomson et al., 1973
; Katekar and Geissler, 1977
). Moreover, the chemical structure of TIBA differs from NPA, but has similarities with that of the most common auxin, indole-3-acetic acid (Katekar and Geissler, 1977
, 1980
). Therefore, TIBA may act as an auxin antagonist (Katekar and Geissler, 1980
), while NPA may act as an inhibitor of the auxin efflux carrier (Lomax et al., 1995
; Muday, 2000
; Muday and Murphy, 2002
). HFCA has been proposed to have a mechanism of action similar to that of NPA (Thomson and Leopold, 1974
; Katekar and Geissler, 1980
).
By contrast, TIBA had the same affects as NPA on development in A. thaliana (Okada et al., 1991
; Mattsson et al., 1999
; Sieburth, 1999
). We propose that there may be differences in how monocots and eudicots respond to TIBA. For example, although NPA and TIBA have similar effects on leaf development in eudicots (Mattsson et al., 1999
; Sieburth, 1999
), NPA and TIBA differ in their effects on leaf development in maize (Tsiantis et al., 1999
; Scanlon, 2003
).
Model for axillary meristem initiation in maize
Because bif2 mutants have the same phenotype as NPA-treated plants, we propose that bif2 and polar auxin transport are both required for axillary meristem initiation (Fig. 8). The similarity of the phenotypes of NPA-treated plants and bif2 mutants includes the failure to initiate axillary meristems, the production of single spikelets, the reduction in vasculature, and the lack of an effect on the apical inflorescence meristem (McSteen and Hake, 2001
; McSteen et al., 2007
). Similar to bif2 mutants, NPA-treated plants produce circular ridges that do not express kn1 (McSteen and Hake, 2001
). We propose that these are bract primordia that do not separate, showing that phyllotaxy is abolished in bif2 mutants and in NPA-treated inflorescence meristems. There is evidence that bif2 and its co-orthologs in other species regulate auxin transport (Benjamins et al., 2001
; Friml et al., 2004
; Lee and Cho, 2006
; McSteen et al., 2007
; Morita and Kyozuka, 2007
). The expression of bif2 in NPA-treated meristems shows that bif2 is expressed independently of polar auxin transport.
|
). The regular pattern of bract primordia initiation suggests that auxin transport is normal in ba1 mutants and that ba1 acts downstream of polar auxin transport. In support of this, RNA in situ hybridization shows that ba1 is not expressed in NPA-treated meristems early in development. This result suggests the polar auxin transport is required for the initial expression of ba1 (Fig. 8). Hence, we propose a model in which bif2 acts upstream and ba1 acts downstream of polar auxin transport in the regulation of axillary meristem initiation in the maize inflorescence. Future work on the genetic and biochemical interaction between bif2 and ba1 will further resolve this model.
FOOTNOTES
1 The authors thank S. Li for help with initial experiments, T. Omeis for plant care in the Biology Department greenhouse, M. Hazen and R. Haldeman of the Huck Institutes Electron Microscopy Facility for advice on SEM analysis and sectioning, T. Yang and S. Roths of the Statistical Consulting Center for advice on statistical analysis, S. Hake for advice at the beginning of the project, and H. Ma and members of the McSteen and Braun laboratories for critical reading of the manuscript. The research was funded by the National Science Foundation (IBN-0416616). 
2 Author for correspondence (e-mail: pcm11{at}psu.edu
)
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