| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online January 9, 2009; doi:10.3732/ajb.0800268 American Journal of Botany 96: 420-430 (2009) © 2009 Botanical Society of America, Inc. |
What's this? |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Developmental Biology and Developmental Genetics |
2 Department of Biology, The Pennsylvania State University, 208 Mueller Laboratory, University Park, Pennsylvania 16802 USA 3 Department of Biology, Swarthmore College, 106 Martin Hall, Swarthmore, Pennsylvania 19081 USA
Received for publication 5 August 2008. Accepted for publication 28 October 2008.
ABSTRACT
Axillary meristems, which give rise to branches and flowers, play a critical role in plant architecture and reproduction. To understand how axillary meristems initiate, we have screened for mutants with defects in axillary meristem initiation to uncover the genes controlling this process. These mutants, called the barren class of mutants in maize (Zea mays), have defects in axillary meristem initiation during both vegetative and reproductive development. Here, we identify and characterize a new member of the barren class of mutants named Developmental disaster1 (Dvd1), due to the pleiotropic effects of the mutation. Similar to the barren mutants, Dvd1 mutants have fewer branches, spikelets, florets, and floral organs in the inflorescence due to defects in the initiation of axillary meristems. Furthermore, double mutant analysis with teosinte branched1 shows that dvd1 also functions in axillary meristems during vegetative development. However, unlike the barren mutants, Dvd1 mutants are semidwarf due to the production of shorter internodes, and they produce leaves in the inflorescence due to the outgrowth of bract leaf primordia. The suite of defects seen in Dvd1 mutants, together with the genetic interaction of Dvd1 with barren inflorescence2, suggests that dvd1 is a novel regulator of axillary meristem and internode development.
Key Words: barren • bract • Developmental disaster1 • inflorescence • internode • maize • meristem • mutation • Poaceae • Zea mays
In all plants, shoot growth is modular and is based on repeating units called phytomers (Steeves and Sussex, 1989
; McSteen and Leyser, 2005). Phytomers consist of a leaf; a node, which is the attachment site for the leaf; an axillary meristem, which is produced in the axil of the leaf; and an internode, which forms the stem between the nodes. During vegetative development, the internodes are short, the leaves are large, and the axillary meristems are often suppressed. In maize, the outgrowth of vegetative axillary meristems is suppressed by the teosinte branched1 (tb1) gene (Doebley et al., 1997; Hubbard et al., 2002). In tb1 mutants, all basal branches grow out to produce vegetative branches called tillers. In many plants, the transition to flowering triggers a rapid elongation of internodes, suppression of leaves to form bract leaves, and the outgrowth of axillary meristems to produce flowers or flowering branches called inflorescences. Therefore, the regulation of the relative growth and activity of the components of the phytomer controls plant morphology.
Maize produces highly modified phytomers in the inflorescence (Irish, 1997; McSteen and Leyser, 2005). There are two types of inflorescence in maize; the male inflorescence, called the tassel, is produced after the conversion of the shoot apical meristem to an inflorescence meristem, and the female inflorescence, called the ear, is produced from an axillary meristem in the axil of a leaf on the main stalk (Kiesselbach, 1949). During inflorescence development, four types of axillary meristem are produced (Cheng et al., 1983; Irish, 1997; McSteen et al., 2000). Branch meristems (BMs) give rise to the long branches at the base of the tassel. Spikelet pair meristems (SPMs) produce short branches bearing two spikelets. Spikelet meristems (SMs) produce the spikelets, which consist of two florets enclosed by two leaf-like glumes. Lastly, floral meristems (FMs) produce the floral organs. In the inflorescence, the phytomers have very short internodes, and the subtending leaves are suppressed. For example, BMs and SPMs form in the axils of bract leaf primordia, which do not grow out (McSteen and Leyser, 2005).
Genes required for the initiation of axillary meristems in the inflorescence have been identified by the characterization of the barren class of mutants in maize. The Barren inflorescence1 (Bif1), barren inflorescence2 (bif2), barren stalk1 (ba1), and sparse inflorescence1 (spi1) mutants produce fewer branches, spikelets, florets, and floral organs in the tassel, fewer kernels in the ear, and fewer ears overall (McSteen and Hake, 2001; Ritter et al., 2002; Barazesh and McSteen, 2008; Gallavotti et al., 2008a). The analogous mutants in Arabidopsis have pinformed-like inflorescences (Okada et al., 1991; Bennett et al., 1995; Przemeck et al., 1996; Cheng et al., 2006). Both the barren and pinformed-like mutants are caused by defects in auxin biosynthesis, transport, or response (Galweiler et al., 1998; Hardtke and Berleth, 1998; Christensen et al., 2000; Benjamins et al., 2001; Zhao et al., 2001; Gallavotti et al., 2004; McSteen et al., 2007; Barazesh and McSteen, 2008; Gallavotti et al., 2008a). The bif2 gene encodes a serine/threonine protein kinase co-orthologous to PINOID, which regulates auxin transport in Arabidopsis (Christensen et al., 2000; Benjamins et al., 2001; Friml et al., 2004; Lee and Cho, 2006; McSteen et al., 2007; Michniewicz et al., 2007), while Bif1 has a very similar phenotype to bif2 and is proposed to regulate auxin transport (Barazesh and McSteen, 2008; Gallavotti et al., 2008b). The spi1 gene encodes a YUCCA-like flavin monooxygenase involved in auxin biosynthesis (Gallavotti et al., 2008a), and ba1 encodes a bHLH transcription factor that functions in axillary meristem initiation (Gallavotti et al., 2004). Although the relationship of ba1 with auxin transport is debated (Wu and McSteen, 2007; Gallavotti et al., 2008b), biochemical, cellular, and genetic evidence suggests that BA1 is a target of BIF2 (Skirpan et al., 2008).
In addition, the barren mutants have defects in vegetative development. For example, bif2, ba1, and spi1 produce fewer tillers in double mutant combination with tb1, indicating that they also function in vegetative axillary meristems (Ritter et al., 2002; McSteen et al., 2007; Gallavotti et al., 2008a). In addition, Bif1, bif2, and spi1 mutants are slightly shorter than normal due to the production of fewer leaves (McSteen et al., 2007; Barazesh and McSteen, 2008; Gallavotti et al., 2008a). Double mutant combinations of either Bif1 or spi1 with bif2 have synergistic effects that result in dwarfed plants, illustrating the redundant roles of Bif1, bif2, and spi1 in vegetative development (Barazesh and McSteen, 2008; Gallavotti et al., 2008a).
Our understanding of how axillary meristems develop has been greatly enhanced by characterizing the barren class of mutants. Here, we introduce a new member of this class of mutants, Developmental disaster 1 (Dvd1), so named because of the pleiotropic defects in plant development caused by the mutation. Dvd1 mutants have defects in axillary meristem formation during vegetative and reproductive development similar to the barren mutants, with the exception that bract leaves grow out in the inflorescence. In addition, unlike the barren mutants, the semidwarf stature of Dvd1 mutants is due to the production of shorter internodes rather than fewer leaves. The suite of defects in Dvd1 mutants together with the interaction of Dvd1 with bif2 suggest that we have identified a novel regulator of axillary meristem, internode, and bract leaf development. Moreover, the Dvd1 phenotype suggests that these three aspects of phytomer development are coordinately regulated in the control of plant morphology.
MATERIALS AND METHODS
Dvd1 origin and mapping
The Dvd1 mutant of maize (Zea mays) was previously identified as a semidominant reversed germ orientation (rgo) mutant in a screen of the Mutator Maize-Targeted Mutagenesis (MTM) population (Kaplinsky, 2002; May et al., 2003). The mutation was provisionally mapped to the short arm of chromosome 5 by the Maize Mapping Project (http://www.maizemap.org) with simple sequence repeat (SSR) primers (Kaplinsky, 2002). To more accurately map Dvd1, we constructed new mapping populations and identified other SSR markers from the Maize Genetics and Genomics Database (MaizeGDB; http://www.maizegdb.org) (Lawrence et al., 2005) and from BAC contigs in the region (http://www.maizesequence.org and http://www.genome.arizona.edu/fpc) (Coe et al., 2002). Additional insertion deletion polymorphism (IDP) markers in the region were identified from the MAGI database (http://magi.plantgenomics.iastate.edu/) (Emrich et al., 2004). For the first mapping population, Dvd1 was backcrossed into the inbred line Mo17 six times, crossed to a divergent inbred line, B73, and then backcrossed to B73. Plants were grown in the field in Rocksprings, PA during the summer, leaf tissue was collected for mapping when the plants were a few weeks old, and the plants were scored for phenotype at maturity. Mapping with this population revealed that Dvd1 resides between markers umc1870 (3/791 recombinants) and umc1591 (41/1335 recombinants). However, the region around Dvd1 was not polymorphic, indicating that Dvd1 possibly arose in the B73 background. Hence, Dvd1 introgressed into Mo17 (eight times) was used as a second mapping population that allowed us to map Dvd1 to between idp3995 (9/1039 recombinants) and bnlg1902 (22/963 recombinants) on chromosome 5 bin 3.
Mature phenotype data
Dvd1 was backcrossed seven times into both the B73 and Mo17 inbred lines before phenotypic analysis. Data were collected from field-grown plants at maturity (10–12 wk). Tassel branches were counted in two families from both B73 (N = 76) and Mo17 (N = 110) genetic backgrounds. Five individuals from each genetic class (normal, heterozygous mutant, and homozygous mutant) were also used to quantify inflorescence phenotypes before anthesis. For each individual, all spikelets from the branches and main spike were counted and scored as single, paired, or triple. The spikelets were then dissected to count the florets and stamens. Visible ears were counted on all individuals from two B73 families (N = 97) and two Mo17 families (N = 58). Kernels were counted on mature open-pollinated ears from two Mo17 families (N = 16).
Plant height, from the ground to the tip of the tassel, was measured at maturity for two B73 families (N = 97). Leaves were counted every few weeks beginning soon after germination so that senesced leaves would be included in the total leaf count (N = 85). For internode length quantification, 10 individuals from each genetic class were collected, leaves were removed, and the lengths of the internodes from the base of one node to the base of the next node were measured and recorded by position.
SEM and histology
Developing tassels from 5- to 6-wk-old greenhouse-grown plants were dissected from segregating families and immediately fixed in FAA (3.7% formalin, 50% ethanol, 5% glacial acetic acid). Samples were kept in fixative at 4°C overnight then dehydrated through an ethanol series. Basal internodes below the tassel were also obtained from these plants and cut into approximately 0.5–1.0 cm pieces before being fixed and dehydrated in the same manner.
For SEM, samples stored in 100% ethanol were critical point dried, sputter coated, and mounted as described previously (Wu and McSteen, 2007). Samples were viewed and photographed using a JSM 5400 scanning electron microscope (JEOL, Peabody, Massachusetts, USA) at an accelerating voltage of 10–20 keV.
For histology, samples in 100% ethanol were embedded into wax, sectioned, mounted on slides, de-waxed, and stained with toluidine blue O as previously described (Barazesh and McSteen, 2008). Images were obtained on a Nikon Eclipse 80i upright microscope under bright field conditions with a DXM1200F digital camera (Nikon, Melville, New York, USA). Internode cell size was determined by measuring the length and width of 10 cells per section of five internode sections of each genetic class.
Double mutant analyses
Double mutant families were grown to maturity (10–12 wk) in two summer field seasons. Data presented here are representative of one field season.
Dvd1; tb1
Double mutant families were generated using the tb1-ref allele (Doebley et al., 1997) in the B73 genetic background. Segregating F2 families were planted in two separate field locations and grown to maturity (N = 209 and 172). Genotyping for tb1 was performed as previously described (Hubbard et al., 2002). Visible primary and secondary tillers were counted at maturity (N = 49).
Dvd1; bif2
Double mutant families were generated using the bif2-77 allele in the B73 genetic background (McSteen et al., 2007). Four segregating F2 families were planted (N = 205). Individuals were genotyped for bif2 as previously described (Skirpan et al., 2008). Plant height (N = 200) was measured and leaves (N = 199) were counted as described for Dvd1 single mutants. Tassels were collected to count spikelets and bract leaves (N = 54).
Statistical analysis
Students t tests were performed using the program Minitab v.15 (State College, PA, USA) and 95% confidence intervals. In all graphs, bars represent the mean of each data set and error bars represent the standard error of the mean.
RESULTS
Dvd1 maps to chromosome 5
Dvd1 was previously mapped to the short arm of chromosome 5 with simple sequence repeat (SSR) markers (Kaplinsky, 2002). We fine mapped Dvd1 using additional public molecular genetic markers and narrowed the region to two BAC contigs between umc1870 (0.38 cM) and bnlg1902 (2.28 cM) in bin 5.03. No mutants with similar phenotypes have been mapped in this region, indicating that Dvd1 is a novel mutant. To analyze the effects of the mutation, we backcrossed Dvd1 into two different inbred lines: B73 and Mo17. Analysis of the segregation ratio showed that Dvd1 is a semidominant mutation with homozygous Dvd1/Dvd1 individuals having a more severe phenotype than heterozygous Dvd1/+ mutants (Fig. 1).
|
Statistical analysis confirmed a significant reduction in tassel branch number in Dvd1 heterozygous and homozygous mutants in both B73 (Fig. 2A, t = 17.40, df = 21, P < 0.001 and t = 17.40, df = 21, P < 0.001) and Mo17 backgrounds (Fig. 2B, t = 5.49, df = 84, P < 0.001 and t = 10.14, df = 42, P < 0.001). Because the tassel phenotype of Dvd1 mutants was more severe in B73, subsequent analysis was carried out using this genetic background. Total spikelet number was significantly reduced in both Dvd1/+ and Dvd1/Dvd1 mutant tassels (Fig. 2E, t = 14.84, df = 4, P < 0.001 and t = 18.16, df = 4, P < 0.001). In normal plants, spikelets form in pairs. However, in Dvd1/+ mutants some spikelets formed singly and in triplets, while all the spikelets produced on Dvd1/Dvd1 mutant tassels formed singly (Fig. 2F). Defects were also seen within spikelets: fewer florets per spikelet were produced in Dvd1/Dvd1 mutants although the reduction was not statistically significantly different from normal (Fig. 2G, t = 2.47, df = 4, P = 0.069). Furthermore, Dvd1/Dvd1 mutants had a significant reduction in the number of stamens per floret compared to normal (Fig. 2H, t = 5.14, df = 4, P = 0.007). In addition, bract leaves that would otherwise be suppressed from growing out in normal individuals developed in Dvd1 mutant tassels (Fig. 1C). There was a significant increase in the number of visible bract leaves in Dvd1/+ and Dvd1/Dvd1 mutants compared to zero in normal plants (Fig. 2I, t = –2.36, df = 4, P = 0.078 and t = –10.49, df = 4, P < 0.001). The failure of Dvd1 mutants to produce the normal number of tassel branches, spikelets, florets, and floral organs suggests that either the initiation or maintenance of all types of axillary meristems is defective in Dvd1 inflorescences.
|
; Cheng et al., 1983). In Mo17, however, Dvd1 heterozygous ears had irregular kernel rows (Fig. 1E), reduced size (70% of normal length) and reversed germ orientation (rgo) (Fig. 1F, asterisk). The number of kernels in Dvd1/+ mutants was also significantly reduced compared to normal (Fig. 2J, t = 4.50, df = 12, P = 0.001). In homozygous Dvd1 mutants, ears that were occasionally produced were small and had very few kernels (Figs. 1E, 2J). Bract leaf outgrowth was also visible in the ear (arrow in Fig. 1F). Hence, Dvd1 tassels and ears had similar defects in bract leaf and axillary meristem development although the inflorescence defects were more severe in B73 than in Mo17.
Dvd1 mutants have defects in axillary meristem formation during inflorescence development
To investigate whether the reduced number of branches and spikelets in Dvd1 mutants was due to defective BM and SPM formation, we performed scanning electron microscopy (SEM) on tassel inflorescences from the B73 background at various stages of development. Early in development, normal inflorescences developed lateral branches at the base of the main spike and the flanks of both the branches and main spike were covered by SPMs (visible as bumps) in regular rows (Fig. 3A). Both Dvd1/+ and Dvd1/Dvd1 inflorescences at similar developmental stages had no evidence of BM initiation (Fig. 3B, C). SPM formation was observed in Dvd1/+ mutants, although there were patches without SPMs (Fig. 3B). In Dvd1/Dvd1 mutants, SPMs often failed to initiate (Fig. 3C). Bract primordia were visible in regular phyllotaxy in Dvd1/Dvd1 mutants (Fig. 3C) but were hidden by SPMs in normal inflorescences. Furthermore, unlike normal, bract leaves continued to grow out in Dvd1/Dvd1 mutants (Fig. 3C).
|
In summary, similar to the barren mutants, Dvd1 mutants have defects in the initiation and outgrowth of all axillary meristems produced during inflorescence development. However, distinct from the barren mutants, Dvd1 mutants have abnormal outgrowth of bract leaves.
Dvd1 mutants have defects in vegetative development
In addition to defects in inflorescence development, Dvd1 mutants also had defects during vegetative development; they were semidwarf. In both B73 and Mo17 inbred lines, Dvd1 mutant plants were markedly shorter than their normal siblings, with homozygous mutants having an even more severe height reduction than heterozygotes (B73 shown in Fig. 4A). Quantification of mature plant height in B73 showed that Dvd1/Dvd1 mutants were less than half the height of normal siblings and confirmed that there was a statistically significant reduction in plant height in both Dvd1/+ and Dvd1/Dvd1 compared to normal (Fig. 4B, t = 5.24, df = 35, P < 0.001 and t = 9.46, df = 25, P < 0.001).
|
Because leaf number was not affected in Dvd1 mutants, the defect in plant height was further investigated by analyzing the internodes. Leaves were removed from individuals at maturity, which revealed that the internodes from heterozygous and homozygous mutants were shorter and much more variable in length compared to those of normal individuals (Fig. 5A). Mature internode length differed significantly at all (except one) internodes measured for Dvd1/Dvd1 individuals compared to normal (Fig. 5B; Appendix S1, see Supplemental Data with online version of this article) and was significantly reduced in half of the internodes in Dvd1/+ individuals compared to normal (Fig. 5B; online Appendix S1). Thus, the reduction of plant height in Dvd1 mutants is due to defects in internode length.
|
|
; Hubbard et al., 2002). In Dvd1; tb1 double mutants, we observed a severe reduction in overall tiller number (Fig. 7A). Genotyping individuals for tb1 revealed that Dvd1 often completely suppressed the tb1 phenotype. Quantification of tiller number confirmed a statistically significant reduction of tiller number in both Dvd1/+; tb1/tb1 and Dvd1/Dvd1; tb1/tb1 double mutants compared to tb1/tb1 single mutants (Fig. 7B, Primary tiller number t = 7.39, df = 9, P < 0.001 and t = 7.0, df = 2, P = 0.02). Thus, Dvd1 also plays a role in axillary meristem formation during vegetative development.
|
; Gallavotti et al., 2008a). For example, Bif1 and spi1, which regulate auxin transport and biosynthesis respectively, produce extremely dwarf plants in combination with bif2 (Barazesh and McSteen, 2008; Gallavotti et al., 2008a). To test whether Dvd1 had a similar interaction with bif2, we constructed double mutants. Tassels of the bif2 mutant have similarities to those of Dvd1 including fewer branches, spikelets, florets, and floral organs (Fig. 8A). Dvd1/Dvd1; bif2/bif2 double mutants produced barren tassels with no lateral branches, no spikelets, and a few bract leaves (Fig. 8A). Quantification revealed that the number of spikelets was not significantly different from Dvd1/Dvd1 (Fig. 8B, t = –0.18, df = 4, P = 0.868), but the number of bract leaves was significantly reduced compared to Dvd1/Dvd1 (Fig. 8C, t = 11.98, df = 11, P < 0.001). Furthermore, there was no enhancement of vegetative phenotypes as Dvd1/Dvd1; bif2/bif2 double mutants were the same height as Dvd1/Dvd1 (Fig. 8D, t = –0.76, df = 3, P = 0.5) and had the same number of leaves as bif2 (Fig. 8E, t = –0.26, df = 3, P = 0.813). We interpret these data as an additive interaction, suggesting that dvd1 functions independently of bif2.
|
We have identified and characterized a novel maize mutant with defects in both vegetative and reproductive development. We show using SEM analysis that Dvd1 mutants produce fewer branches, spikelets, florets, and floral organs due to defects in the production of axillary meristems in the inflorescence. SEM analysis also shows that the leaves visible in the inflorescence of Dvd1 mutants are due to the outgrowth of bract leaf primordia, which are normally suppressed. In addition, Dvd1 mutants have defects during vegetative development due to shortened internodes. Genetic interaction studies with tb1 further illustrate the function of Dvd1 in axillary meristems during vegetative development. Therefore, we have identified a novel player in the regulation of axillary meristem, bract leaf, and internode development, illustrating that all three aspects of phytomer development are under common genetic control.
Role of dvd1 in axillary meristem development
Dvd1 mutants are most similar to the barren class of mutants in maize, which have defects in axillary meristem initiation during vegetative and inflorescence development. The inflorescence defects of Dvd1 mutants such as fewer branches, spikelets, florets, and floral organs are also seen in Bif1, bif2, ba1, and spi1 mutants (McSteen and Hake, 2001; Ritter et al., 2002; Barazesh and McSteen, 2008; Gallavotti et al., 2008a). Single spikelets and fewer organs at the center of the floret, which are observed in Dvd1 mutants, are also characteristic of the barren class of mutants. During ear development, fewer ear shoots and fewer kernels in the ear are seen in the barren mutants; in particular, ba1 mutants never produce an ear shoot (Ritter et al., 2002), which is similar to the effects of the Dvd1 mutation in B73. Dvd1 mutants produce aborted SPMs as also seen when normal plants are treated with auxin transport inhibitors (Wu and McSteen, 2007). Furthermore, in double mutant combinations between Dvd1 and tb1, Dvd1 mutants have defects in vegetative axillary meristems. The magnitude of the effect of Dvd1 on tiller outgrowth is very similar to the effect of bif2 and ba1 double mutant combinations with tb1 (Ritter et al., 2002; McSteen et al., 2007). Thus, Dvd1 is a new member of the barren class of mutants.
The barren mutants all have defects in auxin biosynthesis, transport, or response (McSteen et al., 2007; Wu and McSteen, 2007; Barazesh and McSteen, 2008; Gallavotti et al., 2008a, b; Skirpan et al., 2008). A key difference between the mutants that are defective in auxin transport or biosynthesis (Bif1, bif2, and spi1) and the ba1 mutant, is the phenotype of the inflorescence rachis. In ba1 mutants, bract primordia are produced with normal phyllotaxy resulting in the production of regular bumps along the surface of the inflorescence rachis (Ritter et al., 2002). On the other hand, in Bif1, bif2, and spi1 mutants, bract primordia are not visible in a regular pattern on the surface of the inflorescence rachis, resulting in a smooth or ridged inflorescence rachis (McSteen and Hake, 2001; Barazesh and McSteen, 2008; Gallavotti et al., 2008a; Skirpan et al., 2008). The interpretation of the Bif1, bif2, and spi1 inflorescence rachis phenotype is that phyllotaxy is abolished due to the defects in auxin biosynthesis and transport, resulting in auxin being unavailable to specify the position of the bract primordia (Reinhardt et al., 2003; Skirpan et al., 2008). The interpretation of the ba1 bract phenotype is that auxin transport is normal during the initiation of bract primordia, and subsequently, there are defects in the initiation of SPMs in the axils of bract primordia (Gallavotti et al., 2008b; Skirpan et al., 2008). In this paper, SEM analysis shows that homozygous Dvd1 mutants differ from Bif1, bif2, and spi1 mutants and instead, are more similar to ba1 mutants because bract primordia are produced with regular phyllotaxy along the surface of the inflorescence.
Another mechanism to distinguish the barren mutants from each other is through their genetic interaction with bif2. The Bif1 and spi1 mutants have a synergistic interaction with bif2, while ba1; bif2 double mutants resemble bif2 (Barazesh and McSteen, 2008; Gallavotti et al., 2008a; Skirpan et al., 2008). Double mutants between Dvd1 and bif2 do not have synergistic defects and instead appear to be somewhat additive. The genetic interaction of Dvd1 with bif2 suggests that the Dvd1 mutant does not have general defects in auxin transport or biosynthesis as do Bif1 and spi1 mutants.
Role of dvd1 in bract leaf outgrowth
The compensatory relationship between the axillary meristem and the subtending leaf has long been recognized (Steeves and Sussex, 1989). For example, during vegetative development, the leaf is large and the axillary meristem is suppressed, while during floral development, the axillary meristem is large and the subtending bract leaf is suppressed (Steeves and Sussex, 1989; Long and Barton, 2000). The compensatory relationship between the axillary meristem and the subtending bract leaf was experimentally demonstrated in Arabidopsis by expressing diphtheria toxin under the control of the leafy (lfy) promoter, which is expressed in floral meristems (Nilsson et al., 1998). Ablation of floral meristems in these plants resulted in the outgrowth of bract leaves.
The compensatory relationship between the axillary meristem and the subtending bract leaf is also seen in the maize inflorescence as demonstrated by the ba1 mutant which has defects in axillary meristem initiation and larger than normal bract primordia (Ritter et al., 2002). However, Dvd1 mutants are distinct from ba1 mutants because bract primordia do not grow out to produce bract leaves in ba1 mutants. The suppression of bract leaves in maize is controlled by the tasselsheath1 (tsh1) gene (McSteen and Hake, 2001). The tsh1 mutants have elongated bract leaves subtending the branches and the spikelet pairs at the base of the tassel (McSteen and Hake, 2001). The ba1; tsh1 double mutants produce a ba1 tassel with bract leaves (P. McSteen, unpublished results), indicating that tsh1 suppresses bract leaf outgrowth in ba1 mutants. We propose that the differences between the Dvd1 and ba1 mutant phenotypes can be explained by the expression of tsh1 in ba1 but not in Dvd1, which could be tested after tsh1 is cloned. A further indication that the Dvd1 mutant is different from ba1 is that ba1 has an epistatic interaction with bif2, while Dvd1 has an additive interaction with bif2. Therefore, Dvd1 represents a distinct type of barren mutant.
Role of dvd1 in germ orientation
The Dvd1 mutant was originally isolated based on the rgo phenotype in the ear. An rgo phenotype can develop in one of three ways, which can be explained by defects in the development of florets. Normally, the spikelet produces two florets, an upper and a lower floret. Due to the alternate phyllotaxis of floret initiation, these florets are mirror images of each other. In the ear, the lower floret aborts, leaving only the upper floret (Cheng et al., 1983). Analysis of rgo1 mutants in maize showed that an rgo phenotype can occur due to the production of three florets (Kaplinsky and Freeling, 2003). In this case, the lower two florets abort, leaving the third floret in an inverse orientation compared to normal so that when the ovary is pollinated, the embryo (germ) forms on the opposite face of the endosperm (Kaplinsky and Freeling, 2003). Another way of obtaining an rgo phenotype is through the production of single florets. If only the lower floret forms and it does not abort, then the germ would be in an inverse orientation compared to normal. This phenotype is seen in Bif1 and bif2 mutants (P. McSteen, unpublished results). A third mechanism of obtaining an rgo phenotype is through changes in floral symmetry. If the floret is twisted compared to normal or if the axis of adaxial–abaxial symmetry is not set up correctly, as in the wandering carpel mutant of maize, then an rgo phenotype could form (Irish et al., 2003). Because Dvd1 mutants have fewer florets than normal, the rgo phenotype in Dvd1 is probably caused by the production of single florets.
Role of dvd1 in internode development
Dvd1 plants are semidwarf due to the production of shorter internodes. Because the cells in mutant internodes are significantly larger than normal, we infer that the defect in Dvd1 is caused by reduced cell proliferation and that the cells expand to compensate for the reduction. Many examples of reduced cell proliferation have been shown to result in compensatory increases in cell expansion (Haber and Foard, 1964; Hemerly et al., 1995; Doonan, 2000; Shpak et al., 2004).
Mutants with defects in various hormone pathways cause plants to be shorter than normal due to a reduction in the size of the internodes. Dwarf mutants in rice and wheat are caused by defects in gibberellic acid (GA) or brassinosteroid pathways (Hedden, 2003; Morinaka et al., 2006). However, short internodes in these mutants are caused by reduced cell elongation in contrast to Dvd1 mutants, which have larger cells. Furthermore, Dvd1 does not have other characteristics of GA- or brassinosteroid-insensitive mutants, indicating that dvd1 is unlikely to be involved in GA or brassinosteroid hormone pathways.
Multiple mutants have been identified that are dwarf due to reduced auxin transport. The brachytic2 (br2) mutants in maize and dwarf3 mutants in sorghum are semidwarf due to the reduced length of internodes (Multani et al., 2003). The br2 gene encodes an ABC transporter protein that functions in regulating auxin transport. The roughsheath2 (rs2) and semaphore (sem) mutants in maize also have short internodes and reduced polar auxin transport (Schneeberger et al., 1998; Tsiantis et al., 1999; Scanlon et al., 2002). Furthermore, treatment of maize plants with auxin transport inhibitors causes dwarfism (Tsiantis et al., 1999). Mutants with short internodes and defects in auxin transport have also been seen in Arabidopsis (Gil et al., 2001; Geisler et al., 2003). Because auxin is known to control cell expansion (Jones et al., 1998; Christian et al., 2006), some of these cases have been shown to be caused by reduced cell elongation (Multani et al., 2003). However, auxin also plays a role in regulating cell division (del Pozo et al., 2005; Li et al., 2005; Vanneste et al., 2005; Hartig and Beck, 2006; David et al., 2007). We speculate that many of the defects in Dvd1 mutants could be explained by the dvd1 gene functioning in auxin-mediated cell proliferation.
Conclusions
Dvd1 mutants have pleiotropic defects in phytomers produced during both vegetative and reproductive development. There are differences in the severity of the defects in two different genetic backgrounds, B73 and Mo17, implying that there are other genetic factors influencing the phenotype, which would be interesting to pursue in the future. The defects in axillary meristem initiation and outgrowth indicate that Dvd1 plays an important role in axillary meristems during both vegetative and reproductive development. The defect in bract leaf outgrowth is likely an indirect effect of the lack of axillary meristem initiation. Furthermore, Dvd1 mutants have defects in internode development. The Dvd1 mutant illustrates that axillary meristem and internode development are under common genetic control.
Interestingly, selection on both axillary meristem activity and internode length has been instrumental in the domestication of crop plants. For example, vegetative axillary meristems were suppressed during the domestication of maize leading to a single axis of growth compared to its wild relative teosinte, which is bushy (Doebley et al., 1997). Furthermore, selection of semidwarf varieties of wheat, sorghum, and rice has been critical to reduce lodging (falling over) and increase yield, which led to the "green revolution" in agriculture (Hedden, 2003; Multani et al., 2003; Morinaka et al., 2006). These examples illustrate the importance of understanding the regulation of axillary meristem and internode development for agriculture and for plant morphology in general.
Because Dvd1 is a dominant mutant, it could be either a loss or a gain of function mutation, so the dvd1 gene may be either a positive or a negative regulator of axillary meristem and internode development. We have mapped Dvd1 to two BAC contigs on chromosome 5. Positional cloning of the locus will clarify the mechanism by which the dvd1 gene plays such an important role in vegetative and reproductive development.
FOOTNOTES
1 The authors thank W. S. Harkcom and A. Omeis for plant care in the greenhouse and in the field, X. Wu for providing Fig. 3D, M. Hazen for training on the SEM, and C. Cook for help in the field. The authors thank members of the Braun and McSteen laboratories for discussion and comments on the manuscript. This research was funded by startup funds from Penn State University to P.M. K.A.P. was funded in part by a National Science Foundation Research Experience for Undergraduates Fellowship (NSF DBI-0416616 to P.M.). 
4 Author for correspondence (e-mail: pcm11{at}psu.edu)
LITERATURE CITED
Barazesh, S., AND P. McSteen. 2008. barren inflorescence1 functions in organogenesis during vegetative and inflorescence development in maize. Genetics 179: 389–401.
Benjamins, R., A. Quint, D. Weijers, P. Hooykaas, AND R. Offringa. 2001. The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport. Development 128: 4057–4067.
Bennett, S. R. M., J. Alvarez, G. Bossinger, AND D. R. Smyth. 1995. Morphogenesis in pinoid mutants of Arabidopsis thaliana. Plant Journal 8: 505–520.[CrossRef][Web of Science]
Cheng, P. C., R. I. Greyson, AND D. B. Walden. 1983. Organ initiation and the development of unisexual flowers in the tassel and ear of Zea mays. American Journal of Botany 70: 450–462.[CrossRef][Web of Science]
Cheng, Y. F., X. H. Dai, AND Y. D. Zhao. 2006. Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis. Genes & Development 20: 1790–1799.
Christensen, S. K., N. Dagenais, J. Chory, AND D. Weigel. 2000. Regulation of auxin response by the protein kinase PINOID. Cell 100: 469–478.[CrossRef][Web of Science][Medline]
Christian, M., B. Steffens, D. Schenck, S. Burmester, M. Bottger, AND H. Luthen. 2006. How does auxin enhance cell elongation? Roles of auxin-binding proteins and potassium channels in growth control. Plant Biology 8: 346–352.[CrossRef][Medline]
Coe, E., K. Cone, M. McMullen, S. S. Chen, G. Davis, J. Gardiner, E. Liscum et al.. 2002. Access to the maize genome: An integrated physical and genetic map. Plant Physiology 128: 9–12.
David, K. M., D. Couch, N. Braun, S. Brown, J. Grosclaude, AND C. Perrot-Rechenmann. 2007. The AUXIN-BINDING PROTEIN 1 is essential for the control of cell cycle. Plant Journal 50: 197–206.[CrossRef][Web of Science][Medline]
del Pozo, J. C., M. A. Lopez-Matas, E. Ramirez-Parra, AND C. Gutierrez. 2005. Hormonal control of the plant cell cycle. Physiologia Plantarum 123: 173–183.[CrossRef]
Doebley, J., A. Stec, AND L. Hubbard. 1997. The evolution of apical dominance in maize. Nature 386: 485–488.[CrossRef][Medline]
Doonan, J. 2000. Social controls on cell proliferation in plants. Current Opinion in Plant Biology 3: 482–487.[CrossRef][Web of Science][Medline]
Emrich, S. J., S. Aluru, Y. Fu, T. J. Wen, M. Narayanan, L. Guo, D. A. Ashlock, AND P. S. Schnable. 2004. A strategy for assembling the maize (Zea mays L.) genome. Bioinformatics (Oxford, England) 20: 140–147.[CrossRef]
Friml, J., X. Yang, M. Michniewicz, D. Weijers, A. Quint, O. Tietz, R. Benjamins et al.. 2004. A PINOID-dependent binary switch in apical-basal PIN polar targeting directs auxin efflux. Science 306: 862–865.
Gallavotti, A., S. Barazesh, S. Malcomber, D. Hall, D. Jackson, R. J. Schmidt, AND P. McSteen. 2008a. sparse inflorescence1 encodes a monocot-specific YUCCA-like gene required for vegetative and reproductive development in maize. Proceedings of the National Academy of Sciences, USA 105: 15196–15201.
Gallavotti, A., Y. Yang, R. J. Schmidt, AND D. Jackson. 2008b. The relationship between auxin transport and maize branching. Plant Physiology 147: 1913–1923.
Gallavotti, A., Q. Zhao, J. Kyozuka, R. B. Meeley, M. Ritter, J. F. Doebley, M. E. Pe, AND R. J. Schmidt. 2004. The role of barren stalk1 in the architecture of maize. Nature 432: 630–635.[CrossRef][Medline]
Gälweiler, L., C. H. Guan, A. Müller, E. Wisman, K. Mendgen, A. Yephremov, AND K. Palme. 1998. Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue. Science 282: 2226–2230.
Geisler, M., H. U. Kolukisaoglu, R. Bouchard, K. Billion, J. Berger, B. Saal, N. Frangne et al.. 2003. TWISTED DWARF1, a unique plasma membrane-anchored immunophilin-like protein, interacts with Arabidopsis multidrug resistance-like transporters AtPGP1 and AtPGP19. Molecular Biology of the Cell 14: 4238–4249.
Gil, P., E. Dewey, J. Friml, Y. Zhao, K. C. Snowden, J. Putterill, K. Palme et al.. 2001. BIG: A calossin-like protein required for polar auxin transport in Arabidopsis. Genes & Development 15: 1985–1997.
Haber, A. H., AND D. E. Foard. 1964. Further studies of gamma-irradiated wheat and their relevance to use of mitotic inhibition for developmental studies. American Journal of Botany 51: 151–159.[CrossRef][Web of Science]
Hardtke, C. S., AND T. Berleth. 1998. The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development. EMBO Journal 17: 1405–1411.[CrossRef][Web of Science][Medline]
Hartig, K., AND E. Beck. 2006. Crosstalk between auxin, cytokinins, and sugars in the plant cell cycle. Plant Biology 8: 389–396.[CrossRef][Medline]
Hedden, P. 2003. The genes of the green revolution. Trends in Genetics 19: 5–9.[CrossRef][Web of Science][Medline]
Hemerly, A., J. D. Engler, C. Bergounioux, M. Vanmontagu, G. Engler, D. Inze, AND P. Ferreira. 1995. Dominant-negative mutants of the CDC2 kinase uncouple cell-division from iterative plant development. EMBO Journal 14: 3925–3936.[Web of Science][Medline]
Hubbard, L., P. McSteen, J. Doebley, AND S. Hake. 2002. Expression patterns and mutant phenotype of teosinte branched1 correlate with growth suppression in maize and teosinte. Genetics 162: 1927–1935.
Irish, E. E. 1997. Class II tassel seed mutations provide evidence for multiple types of inflorescence meristems in maize (Poaceae). American Journal of Botany 84: 1502–1515.[Abstract]
Irish, E. E., E. J. Szymkowiak, AND K. Garrels. 2003. The wandering carpel mutation of Zea mays (Gramineae) causes misorientation and loss of zygomorphy in flowers and two-seeded kernels. American Journal of Botany 90: 551–560.
Jones, A. M., K. H. Im, M. A. Savka, M. J. Wu, N. G. DeWitt, R. Shillito, AND A. N. Binns. 1998. Auxin-dependent cell expansion mediated by overexpressed auxin-binding protein 1. Science 282: 1114–1117.
Kaplinsky, N. J. 2002. Combinatorial control of maize meristem identity in maize inflorescences. Ph.D. dissertation, University of California, Berkeley, California, USA.
Kaplinsky, N. J., AND M. Freeling. 2003. Combinatorial control of meristem identity in maize inflorescences. Development 130: 1149–1158.
Kiesselbach, T. A. 1949. The structure and reproduction of corn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA.
Lawrence, C. J., T. E. Seigfried, AND V. Brendel. 2005. The Maize Genetics and Genomics Database. The community resource for access to diverse maize data. Plant Physiology 138: 55–58.
Lee, S. H., AND H. T. Cho. 2006. PINOID positively regulates auxin efflux in Arabidopsis root hair cells and tobacco cells. Plant Cell 18: 1604–1616.
Li, J. S., H. B. Yang, W. A. Peer, G. Richter, J. Blakeslee, A. Bandyopadhyay, B. Titapiwantakun et al.. 2005. Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ development. Science 310: 121–125.
Long, J., AND M. K. Barton. 2000. Initiation of axillary and floral meristems in Arabidopsis. Developmental Biology 218: 341–353.[CrossRef][Web of Science][Medline]
May, B. P., H. Liu, E. Vollbrecht, L. Senior, P. D. Rabinowicz, D. Roh, X. Pan et al.. 2003. Maize-targeted mutagenesis: A knockout resource for maize. Proceedings of the National Academy of Sciences, USA 100: 11541–11546.
McSteen, P., AND S. Hake. 2001. barren inflorescence2 regulates axillary meristem development in the maize inflorescence. Development 128: 2881–2891.
McSteen, P., D. Laudencia-Chingcuanco, AND J. Colasanti. 2000. A floret by any other name: Control of meristem identity in maize. Trends in Plant Science 5: 61–66.[CrossRef][Web of Science][Medline]
McSteen, P., AND O. Leyser. 2005. Shoot branching. Annual Review of Plant Biology 56: 353–374.[CrossRef][Medline]
McSteen, P., S. Malcomber, A. Skirpan, C. Lunde, X. Wu, E. Kellogg, AND S. Hake. 2007. barren inflorescence2 encodes a co-ortholog of the PINOID serine/threonine kinase and is required for organogenesis during inflorescence and vegetative development in maize. Plant Physiology 144: 1000–1011.
Michniewicz, M., M. K. Zago, L. Abas, D. Weijers, A. Schweighofer, I. Meskiene, M. G. Heisler et al.. 2007. Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux. Cell 130: 1044–1056.[CrossRef][Web of Science][Medline]
Morinaka, Y., T. Sakamoto, Y. Inukai, M. Agetsuma, H. Kitano, M. Ashikari, AND M. Matsuoka. 2006. Morphological alteration caused by brassinosteroid insensitivity increases the biomass and grain production of rice. Plant Physiology 141: 924–931.
Multani, D. S., S. P. Briggs, M. A. Chamberlin, J. J. Blakeslee, A. S. Murphy, AND G. S. Johal. 2003. Loss of an MDR transporter in compact stalks of maize br2 and sorghum dw3 mutants. Science 302: 81–84.
Nilsson, O., E. Wu, D. S. Wolfe, AND D. Weigel. 1998. Genetic ablation of flowers in transgenic Arabidopsis. Plant Journal 15: 799–804.[CrossRef][Web of Science][Medline]
Okada, K., J. Ueda, M. K. Komaki, C. J. Bell, AND Y. Shimura. 1991. Requirement of the auxin polar transport system in early stages of Arabidopsis floral bud formation. Plant Cell 3: 677–684.
Przemeck, G. K. H., J. Mattsson, C. S. Hardtke, Z. R. Sung, AND T. Berleth. 1996. Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization. Planta 200: 229–237.[Web of Science][Medline]
Reinhardt, D., E. R. Pesce, P. Stieger, T. Mandel, K. Baltensperger, M. Bennett, J. Traas et al.. 2003. Regulation of phyllotaxis by polar auxin transport. Nature 426: 255–260.[CrossRef][Medline]
Ritter, M. K., C. M. Padilla, AND R. J. Schmidt. 2002. The maize mutant barren stalk1 is defective in axillary meristem development. American Journal of Botany 89: 203–210.
Scanlon, M. J., D. C. Henderson, AND B. Bernstein. 2002. SEMAPHORE1 functions during the regulation of ancestrally duplicated knox genes and polar auxin transport in maize. Development 129: 2663–2673.
Schneeberger, R., M. Tsiantis, M. Freeling, AND J. A. Langdale. 1998. The rough sheath2 gene negatively regulates homeobox gene expression during maize leaf development. Development 125: 2857–2865.[Abstract]
Shpak, E. D., C. T. Berthiaume, E. J. Hill, AND K. U. Torii. 2004. Synergistic interaction of three ERECTA-family receptor-like kinases controls Arabidopsis organ growth and flower development by promoting cell proliferation. Development 131: 1491–1501.
Skirpan, A., X. Wu, AND P. McSteen. 2008. Genetic and physical interaction suggest that BARREN STALK 1 is a target of BARREN INFLORESCENCE2 in maize inflorescence development. Plant Journal 55: 787–797.[CrossRef][Web of Science][Medline]
Steeves, T., AND I. Sussex. 1989. Patterns in plant development. Cambridge University Press, Cambridge, UK.
Tsiantis, M., M. I. N. Brown, G. Skibinski, AND J. A. Langdale. 1999. Disruption of auxin transport is associated with aberrant leaf development in maize. Plant Physiology 121: 1163–1168.
Vanneste, S., L. Maes, I. De Smet, K. Himanen, M. Naudts, D. Inzé, AND T. Beeckman. 2005. Auxin regulation of cell cycle and its role during lateral root initiation. Physiologia Plantarum 123: 139–146.[CrossRef]
Wu, X., AND P. McSteen. 2007. The role of auxin transport during inflorescence development in maize, Zea mays (Poaceae). American Journal of Botany 94: 1745–1755.
Zhao, Y. D., S. K. Christensen, C. Fankhauser, J. R. Cashman, J. D. Cohen, D. Weigel, AND J. Chory. 2001. A role for flavin monooxygenase-like enzymes in auxin biosynthesis. Science 291: 306–309.
CiteULike Complore Connotea Del.icio.us Digg Facebook Reddit Technorati Twitter What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
