Abstract
The leaf surfaces of almost all plant species possess specialized epidermal cell types that form hairs or trichomes. Maize leaves produce three distinct types of hairs, the most prominent being the macrohairs that serve as a marker for adult leaf identity and may contribute to insect resistance. This report describes the maize macrohairless1 (mhl1) locus, which promotes macrohair initiation specifically in the leaf blade. Each of seven recessive mhl1 mutant alleles significantly reduces or eliminates macrohairs in the leaf blade. The mhl1 mutations block macrohair initiation rather than interfering with macrohair morphogenesis. Genetic mapping placed mhl1 within bin 4 on chromosome 9. A second independently segregating locus was found to partially suppress the mhl1 mutant phenotype in certain genetic backgrounds. Macrohair density was observed to increase during early adult vegetative development and then progressively decline, suggesting macrohair initiation frequency is affected by factors that act throughout shoot development. Genetic analyses demonstrated that mhl1 acts in the same pathway but downstream of factors that either promote or repress adult leaf identity. Thus, mhl1 plays a key role in integrating developmental programs that regulate leaf identity during shoot development with those that specify macrohair initiation within the leaf blade.
HAIRS or trichomes are present on the leaf surfaces of nearly all plant species. The density, morphology, and chemical composition of leaf hairs vary widely and these factors contribute to their diverse physiological functions. Leaf hairs may act as physical and chemical deterrents to insect feeding, may provide hydrorepellency and reflective properties to the leaf, and in some xeromorphic species may limit water loss due to transpiration (Esau 1977). Leaf hairs form through the specialized differentiation of epidermal cells and may be unicellular or be composed of many cells. In many plant species, multiple different types of leaf hairs whose production varies during shoot development are observed, with more than one type often present within the same leaf.
Three different types of hairs are found on maize leaves. Macrohairs, prickle hairs, and bicellular microhairs are produced in patterned files of cells within the adaxial leaf epidermis, beginning with the fifth or sixth leaf (Figure 1). The prominent macrohairs (MHs) thus serve as a readily visible marker for adult leaf identity in vegetative development (Poethig 1990) as well as for dorsoventral polarity within the leaf (Nelsonet al. 2002). Although the function of maize MHs remains unclear, they have been reported to influence oviposition by the insect pests Heliothis zea (Widstromet al. 1979) and Chilo partellus (Durbey and Sarup 1982).
Glabrous mutants in which leaf hairs or trichomes are greatly reduced or absent have been identified in many plant species. Molecular analyses of such mutants in Arabidopsis have revealed much about the genes that regulate trichome patterning, initiation, and morphogenesis (reviewed in Szymanskiet al. 2000). However, insights from studies of the molecular mechanisms that regulate trichome development in Arabidopsis have not yet been extended to other plant species. Glabrous mutant varieties have been identified in many cereal crop species, including rice (Foster and Rutger 1978), wheat (Leisle 1974), barley (Sato and Takeda 1992), oats (Sarkarung and Collins 1977), pearl millet (Kumar and Andrews 1993), sugarcane (Jagathesan 1977), and sorghum (Gibson and Maiti 1983). In each of these species, leaf hairs have been implicated as important factors for insect resistance. Surprisingly, similar mutations have not previously been described in maize, the best developed system among the cereals for the molecular genetic analysis of leaf development.
We report here the identification and characterization of mutations that define the maize macrohairless1 (mhl1) locus, which specifically promotes MH initiation in the leaf blade. Mutant alleles of mhl1 greatly reduce or eliminate MH initiation in the leaf blade without affecting other aspects of leaf or plant development. We mapped mhl1 to the long arm of maize chromosome 9 and show that the mhl1 phenotype can be partially suppressed by an independently segregating modifier locus in certain genetic backgrounds. Mhl1 is the first mutation that specifically affects a marker for adult leaf identity in maize and we demonstrate that it functions in the same pathway and downstream of factors that either promote an adult leaf identity (such as gibberellic acids) or repress adult leaf identity (glossy15). The mhl1 mutation thus defines a gene that integrates the regulation of leaf identity during shoot development with the initiation of MH differentiation within the leaf blade.
MATERIALS AND METHODS
Genetic stocks: The mhl1-R allele was discovered as a spontaneous mutation in the inbred line K55 obtained from Paul Sisco, which was confirmed in a second K55 accession (Ames 22754) obtained from the North Central Regional Plant Introduction Station (Ames, Iowa). The A632, 4Co63, NC89, and W64A inbred lines were also obtained from Paul Sisco and used in crosses with K55 to generate the F2 and BC1 populations from which the phenotypic segregation ratios that are reported in Tables 1 and 2 were obtained. The wx1 gl15-L,mhl1-R or wx1,gl15-m1,mhl1-R triple-mutant stocks were generated by crossing K55 to either a wx1,gl15-L stock initially obtained from the Maize Genetics Cooperation Stock Center or a wx1,gl15-m1 stock (Moose and Sisco 1994). Mutant wx1 kernels were selected from F2 ears, and seedlings that produced glossy leaves without MHs beginning at leaf 3 were self-pollinated.
Four additional mhl1 alleles (mhl1-411, mhl1-330, mhl1-561, and mhl1-249) were recovered from a targeted Mutator transposon mutagenesis experiment where Mutator-active plants were crossed as males onto wx1,gl15-L,mhl1-R females. A total of 47,000 F1 progeny were screened for the macrohairless phenotype, 22,000 in a 1998 summer nursery and an additional 25,000 in a 2002 summer nursery. Putative Mutator-induced mhl1 alleles resulting in a macrohairless phenotype in combination with the mhl1-R allele were propagated by selfing and outcrossing as males to both the W64A inbred and the wx1,gl15-L,mhl1-R parental lines. The heritability of these alleles and their allelism with mhl1-R was confirmed in the progeny of these crosses.
Six mutant lines with reduced leaf macrohair density were recovered from a screen for leaf epidermal differentiation defects in an M2 population of EMS-mutagenized plants and were generously provided to us by Laurie Smith (University of California, San Diego). Hence these alleles are designated as mhl*-LS(number). These mutations were tested for allelism with mhl1 by crossing to both the K55 and the wx1,gl15-L,mhl1-R stocks.
The mhl1-R, mhl1-330, and mhl1-411 alleles were each backcrossed at least three times into the maize inbred line W64A. Resultant backcross plants were selfed to generate segregating populations that were phenotyped to generate the data in Table 3. For mhl1-R, phenotypic selection for the intermediate MH density and macrohairless phenotypes was conducted by selfing heterozygous plants and visually scoring MH phenotypes segregating in the resulting progeny.
Stocks for the dwarf1 mutation and those harboring the TB-9Sb, TB-9Sd, or TB-9Lc B-A translocations were obtained from the Maize Genetics Cooperation Stock Center. A dwarfl; gl15-m1 double-mutant stock was generated by crossing dwarf1 to a gl15-m1 stock (Moose and Sisco 1994) and selfing double-mutant F2 progeny with short stature and adult leaf epidermal traits beginning at leaf 3.
Phenotypic analyses: Glue slide leaf impressions were made as suggested by L. Smith (University of California, San Diego) and described in Nelson et al. (2002) from odd-numbered leaf blades beginning with leaf three until formation of the tassel. Since proximodistal gradients for MH density occur within leaves, data are reported from glue slides prepared from the same allometric position on all leaves. Impressions were made equidistant from the margin and midrib 2 in. distal to the ligule on the adaxial blade. MH density was counted from at least two different 1.13-cm2 fields of each glue impression using a Zeiss Stemi-2000C dissecting microscope at ×40 magnification. The number of bulliform cell files across the width of the field of view was also counted to normalize for potential differences in MH initiation due to the relative density of these cell files. However, the number of bulliform cell files per unit leaf width was not observed to vary significantly among leaves of the genotypes examined in this study. Means and standard errors of the mean were calculated for MH density values from each leaf and phenotypic class.
mhl1 genetic mapping: TB-A translocation tests: K55 and wx,gl15-L,mhl1-R stocks were crossed as females by plants carrying TB-9Lc (breakpoint between centromere and gl15), TB-9Sd (breakpoint between the centromere and wx1), and TB-9Sb (breakpoint distal to wx1). All translocation-bearing plants used in crosses were verified to contain the translocation both by the morphological effects associated with hypoploidy (narrow leaves, semisterility in pollen) and by crossing plants to tester stocks containing markers distal to the translocation breakpoints (TB-9Lc, virescent1 or gl15; TB-9Sb, colorless1; and TB-9Sd, wx1). F1 progeny from crosses of mhl1-R by translocation stocks were scored for the macrohairless phenotype, which was observed only in the crosses with TB-9Lc.
Three-point linkage test: A wx1,gl15-L,mhl1-R plant was crossed to the inbred Gaspé Flint, and F1 plants were backcrossed to the wx1,gl15-L,mhl1-R parental line. A total of 181 progeny were scored visually for waxy1 kernel, gl15 seedling (loss of juvenile wax), and macrohairless phenotypes.
Molecular marker mapping: Two populations, one segregating for mhl1-411 in a W64A background and F2 progeny from the NC89 × K55 cross, were examined for linkage between a macrohairless leaf phenotype and molecular markers for gl15, umc1120, and umc95 on the long arm of chromosome 9.
Double-mutant analyses: Families simultaneously segregating for gl15 and mhl1 were generated by crossing K55, which carries the mhl1-R allele, to the wx1,gl15-L stock introgressed into a W64A inbred background (Moose and Sisco 1994). The gl15 phenotype was scored as the visual loss of juvenile epicuticular wax at leaf 3. The mhl1 phenotype was scored visually as the absence or significant reduction in MH density on the abaxial leaf surface of fully adult leaves. Glue slide leaf impressions, MH counts, and data analysis were conducted as described above.
Families segregating for dwarf1 and mhl1-R in a gl15 mutant background were produced by first crossing the d1; gl15-m1 stock to the wx1,gl15-m1,mhl1-R stock. F1 plants were then backcrossed to the wx1,gl15-m1,mhl1 stock. Eight backcross progeny were self-pollinated and one family that segregated for d1 and mhl1 in a gl15-m1 background was used for phenotypic analyses. Macrohair frequencies were estimated from glue slide impressions as described above.
RESULTS
Identification and characterization of macrohairless1: A macrohairless leaf phenotype was observed in the maize inbred line K55, grown in the greenhouse during the fall of 1994 in Raleigh, North Carolina. The macrohairless phenotype is environmentally stable, as K55 plants grown in summer nurseries at four locations with different growing conditions (Clayton, North Carolina; San Diego; Mystic, Connecticut; and Urbana, Illinois) have shown the same phenotype. The K55 inbred line was initially released in 1942 and is derived from the open-pollinated Kansas variety “Pride of Saline” (Gerdeset al. 1993). It is unlikely that the macrohairless phenotype arose recently in our K55 stock, as leaves from plants of a second K55 seed source obtained from the North Central Regional Plant Introduction Station were also macrohairless.
—The macrohairless1 mutant phenotype. Micrographs from normal Mhl1 (A and C) and mhl1-R mutant (B and D) plants are shown. (A and B) Micrographs of glue slide impressions prepared from adaxial surface of mature leaf 9. (C and D) Micrographs of glue slide impressions prepared from the basal 0.5–1.0 cm of leaf 9 early in its development. The region where macrohairs initiate is shown. mh, macrohair; ph, prickle hair; bm, bicellular microhair; bc, bulliform cell files. Bars, 100 μm.
Macrohairs are produced only on adult leaves (Poethig 1990), raising the possibility that the macrohairless phenotype in K55 was due to a prolonged expression of juvenile leaf identity. However, like most other maize inbred lines, K55 produced juvenile leaf waxes only through leaf 8, demonstrating that the transition from juvenile to adult leaf identity occurred normally in K55, but that adult leaves lacked MHs. Consistent with this conclusion, all K55 leaves beyond leaf 7 possessed each of the other cellular characteristics associated with normal adult leaf identity, including presence of bulliform cells, prickle hairs, and bicellular microhairs, as well as intercostal cells that possessed highly invaginated cell walls (Figure 1, A and B) and stained aqua with toluidine blue (data not shown).
The macrohairless1 mutation affects MHs only on leaf blades. The macrohairs on the margins and collar of the leaf sheath and the variably sized macrohair-like hairs present on the auricles and at the very edges of the leaf blade, both of which are present in most maize inbred lines, were not affected in K55 macrohairless leaves (data not shown). Further, the macrohairless1 mutation has no discernible effects on root hairs, which are affected by mutations in some plant hair initiation genes from Arabidopsis (data not shown; Walkeret al. 1999; Schellmannet al. 2002).
K55 did occasionally produce a few MHs, most often near the leaf tip or margins. The normal morphology of these MHs suggested that the macrohairless phenotype was more likely to be a defect in MH initiation rather than in morphogenesis. This idea is supported by the observation that the group of differentiated cells that normally proliferate at the base of MHs, the pedestal cells (Payne 1978), is also absent in K55 leaves, indicating a defect in the entire program of MH differentiation. Confirmation that the macrohairless phenotype is due to a block in MH initiation was obtained by examining the developing leaf blade epidermis at the stage where MH initiation occurs (the basal 0.5–1 cm). Macrohair initiation is the first observable specialized cell type to differentiate in the epidermis of the adult leaf blade and begins as an outgrowth of a single cell perpendicular to the sheet of otherwise undifferentiated epidermal cells (Figure 1C). These cellular outgrowths fail to occur in macrohairless leaves, demonstrating that the macrohairless phenotype is due to a block prior to or at the earliest observable sign of MH initiation (Figure 1D).
Inheritance of the mhl1 phenotype
The inheritance of the macrohairless phenotype was examined by crossing K55 plants to each of three inbred lines with normal MHs. The phenotypes of the F1 progeny from each cross indicated recessive inheritance, because all plants exhibited a normal frequency and distribution of MHs beginning with leaves 5 or 6. F1 plants were self-pollinated and also backcrossed as males to the K55 parent. The resulting progeny segregated into two distinct classes of plants, those with a normal MH density and macrohairless plants (Table 1). The proportions of the normal and macrohairless classes in both the F2 and the backcross progenies closely followed those expected if the macrohairless phenotype was conditioned by a single recessive locus, which we have named macrohairless1 (mhl1). Because the mhl1 allele present in K55 is the first to be described at this locus, we have named this allele mhl1-reference, abbreviated as mhl1-R.
The NC89 inbred was chosen for crosses to mhl1-R because it possesses a relatively high density of leaf MHs, particularly on the medial area of the leaf sheath just below the ligule. This “hairy sheath” phenotype is rare among maize inbred lines and is not present in K55, although it is often observed in the wild relative of maize, teosinte (Lauter 2001). A larger number of F2 progeny were scored from the NC89 × K55 cross to assess the role of mhl1-R in promoting medial leaf sheath MHs. We observed that mhl1-R was fully penetrant for the leaf blade MH phenotype (Table 1). The hairy sheath phenotype showed a quantitative mode of inheritance, although a few F2 individuals that lacked sheath MHs were observed. No clear association was observed between the macrohairless leaf blade phenotype and sheath MH density. Some plants with macrohairless leaf blades still expressed the hairy sheath phenotype, while others exhibited very few MHs on the medial portion of their sheaths.
A dominant suppressor of mhl1: Crosses of K55 (homozygous for mhl1-R) to the inbred line W64A produced three phenotypic classes in the F2 progeny: plants with normal macrohair density, completely macrohairless plants, and plants with intermediate MH density (Figure 2; Table 2). Similarly, backcrosses of W64A × K55 F1 plants to K55 also produced plants with a normal density of MHs, no MHs, or an intermediate density of MHs. Combining the intermediate and no MH phenotypic classes represented ∼25% of the F2 progeny and 50% of the backcross progeny, suggesting that mhl1 continued to be inherited as a simple recessive factor in these crosses but was partially suppressed by a modifier locus inherited from the W64A inbred parent. Comparisons of the proportions of macrohairless to intermediate MH density plants in both F2 and backcross progenies suggested that W64A is homozygous for a dominant factor, which we designate in this study as Suppressor of macrohairless1 (Smh1), that segregates independently from mhl1 and partially suppresses the macrohairless phenotype. In this scenario, F2 progeny would be expected to segregate into three phenotypic (genotypic) classes with the following proportions: 12/16 normal (Mhl1/–; Smh1/–), 3/16 intermediate MH density (mhl1/mhl1; Smh1/–), and 1/16 macrohairless (mhl1/mhl1; smh1/smh1). Similarly, progeny of F1 plants (presumed Mhl1/mhl1; Smh1/smh1 genotype) backcrossed to K55 (mhl1/mhl1; smh1/smh1) would be expected to segregate 1/2 normal (Mhl1/–; Smh1/–), 1/4 intermediate MH density (mhl1/mhl1; Smh1/–), and 1/4 macrohairless (mhl1/mhl1; smh1/smh1). The observed proportions of these phenotypic classes closely fit those expected for the hypothesis that W64A is homozygous for Smh1, which is unlinked to mhl1 and partially suppresses the mhl1 phenotype. Chi-square tests for goodness of fit could not reject this hypothesis at the 0.05 significance level for either F2 or backcross data (Table 2).
Segregation of the suppressor of macrohairless locus identified in W64A
—Variation in the mhl1-R phenotype. Macrohair phenotypes on blades of leaf 11 in (A) W64A, (B) a plant with the intermediate macrohair density phenotype associated with mhl1-R and Smh1 alleles, and (C) a plant with the completely macrohairless phenotype associated with mhl1-R and smh1 alleles are shown. Bars, 1.0 mm.
Further support for the above hypothesis was obtained by examining the segregation of MH phenotypes among progeny from crosses involving plants that exhibited either an intermediate MH density or a macrohairless phenotype, which were initially derived from the backcross of W64A × K55 F1 plants to K55 (Table 2). When plants with an intermediate MH density were selfed, they segregated ∼75% intermediate and 25% macrohairless plants. Backcrossing these intermediate plants again to K55 resulted in equal segregation for intermediate and macrohairless phenotypes. When macrohairless plants were similarly selfed and backcrossed, they produced all completely macrohairless progeny. These ratios are consistent with the intermediate parent plants having an mhl1/mhl1; Smh1/smh1 genotype and the macrohairless parent plants having an mhl1/mhl1; smh1/smh1 genotype. Chi-square tests for goodness of fit could not reject this hypothesis at the 0.05 significance level (Table 2).
Genetic mapping of mhl1: Among 979 F2 individuals from crosses between K55 and a stock homozygous for mutant alleles at the linked gl15 and waxy1 (wx1) loci, only 1 plant was observed to exhibit glossy leaves beginning with leaf 3 and no MHs on any leaf blades, the phenotype expected for gl15, mhl1 double mutants. Repulsion phase linkage, rather than epistasis, appeared to be the most likely explanation because ∼25% (239/979) of the F2 progeny exhibited a macrohairless phenotype and many more macrohairless plants were observed from nonwaxy (203) compared to wx1 mutant seeds (36). The mhl1 locus appeared to be more closely associated with gl15 compared to wx1 in these crosses, indicating that mhl1 is nearer to gl15 on the long arm of chromosome 9 than to wx1 on the short arm. To further test this hypothesis, K55 was crossed to different B-A translocation stocks with breakpoints on chromosome arms 9S and 9L. The mhl1 phenotype was observed only among hemizygous plants from crosses of K55 to TB-9Lc, whose breakpoint is located between the centromere and gl15. Attempts to map the smh1 locus using B-A translocation stocks were unsuccessful, perhaps due to the presence of other modifiers of the macrohairless phenotype in the genetic backgrounds harboring the B-A translocations.
The single wx1,gl15-L,mhl1-R plant recovered from the F2 progeny of crosses between wx1,gl15-L and mhl1-R was selfed and the resulting triple-mutant stock was used to perform a three-point linkage test. The segregation ratios of the different phenotypic classes among 181 testcross progeny indicated that mhl1 mapped ∼16 cM distal to wx1 (29 recombinants) and 5 cM distal to gl15 (10 recombinants). No double crossovers involving gl15 were observed between wx1 and mhl1. Nineteen recombinants were observed between wx1 and gl15, which is consistent with the 10-cM distance previously reported for these loci (Howellet al. 1991). A more precise map location and markers distal to mhl1 were determined by molecular marker mapping in 28 plants with a macrohairless phenotype from the NC89 × K55 F2 population. These efforts produced the following map order with genetic distances in Haldane centimorgans: glossy15 [restriction fragment length polymorphism (RFLP)]—6.7 cM—mhl1—14.7 cM—umc1120 (simple sequence repeat)—6.3 cM—umc95 (RFLP).
Macrohair density varies during both normal and mhl1 mutant shoot development: One feature of the intermediate MH density phenotype was that it was often difficult to macroscopically distinguish from normal plants in early adult leaves (leaves 7–9), but became readily apparent in later adult leaves. Closer examination of MH densities in W64A and mhl1 mutant plants provided an explanation. Macrohairs were first observed at leaf 7 and increased in density until leaf 9, after which MH density progressively declined until leaf 13 (Table 3). Macrohair densities in the mhl1 mutant plants exhibiting either the macrohairless or the intermediate MH density phenotype showed the same pattern of variation during shoot development as W64A plants, although the density of MHs was reduced in all leaves of the mutants relative to W64A (Table 3). As suggested by macroscopic observations, the differences in MH densities between plants with the intermediate MH density phenotype and wild-type plants were greater after leaf 9 (Figure 2).
Leaf blade macrohair density varies during shoot development
The data in Table 3 demonstrate node-to-node variation in macrohair density during shoot development. This same trend is evident in tip-to-base comparisons within single leaf blades (data not shown). For example, MH density within leaf 8 is lower at the tip than at the base. Since the base of a leaf develops later than its tip, the early adult trend toward greater pubescence is manifested within leaf 8. Likewise, MH density is higher at the tip of leaf 12 than at its base, illustrating the late adult tendency toward decreasing hair density. These examples demonstrate the importance of allometric sampling when comparing MH densities. This is particularly important in transition and early adult maize leaves because they have the longest blades, such that the tip of leaf 10 often forms earlier in a plant's life than the base of leaf 8 does (Lauter 2001).
Additional macrohairless mutants were generated by transposon and EMS mutagenesis: The presence of a few MHs on leaf blades of K55 plants suggested that perhaps the mhl1-R allele was weakly functional. The spontaneous nature of mhl1-R also did not immediately suggest a strategy to molecularly clone the mhl1 gene. Thus, we generated four additional mhl1 alleles in a Mutator transposon tagging experiment. To date, only two of these putative Mutator-induced mhl1 alleles (mhl1-330 and mhl1-411) have been sufficiently introgressed into the W64A background to permit comparisons with mhl1-R. The mhl1-330 and mhl1-411 mutant phenotypes are very similar to those previously described for mhl1-R, but all leaves are essentially bald throughout shoot development (Table 3). The more severe macrohairless phenotype observed in the mhl1-330 and mhl1-411 mutants compared to mhl1-R suggests that mhl1-330 and mhl1-411 could be null alleles, whereas mhl1-R may be a weak allele. This idea is supported by the observation that after introgression of mhl1-330 or mhl1-411 into the W64A inbred background, which is homozygous for Smh1, intermediate MH density phenotypes are not observed. Thus, Smh1 appears capable of suppressing the mhl1-R allele, but not mhl1-330 or mhl1-411.
Six lines with macrohairless or reduced leaf macrohair density phenotypes were recovered from screens of an EMS-mutagenized population and were kindly provided to us by Laurie Smith. The mhl*-LS4 and mhl*-LS14 lines failed to complement mhl1-R in allelism tests and their macrohairless phenotypes were linked to gl15 (data not shown), indicating that they represent additional mhl1 mutant alleles hereafter designated mhl1-LS4 and mhl1-LS8. Among F2's derived from crosses of the mhl-LS4 and mhl-LS14 lines to W64A, plants that were homozygous for either the mhl1-LS4 or the mhl1-LS14 alleles exhibited totally macrohairless leaf phenotype, suggesting that they may both be null mhl1 alleles. Each of the other four EMS mutant lines (mhl*-LS8, mhl*-LS9, mhl*-LS10, and mhl*-LS11) showed weak, partially penetrant phenotypes on their own, characterized by near normal macrohair densities in early adult leaves followed by intermediate MH density phenotypes in later leaves. Tests for allelism between these mutants and mhl1 suggest that these mutants are not allelic to mhl1 (data not shown). The phenotypic variability of the mhl*-LS8, mhl*-LS9, mhl*-LS10, and mhl*-LS11 mutants may be due in part to genetic background effects; thus these mutations are being introgressed into multiple inbred lines to clarify their phenotypic effects and modes of inheritance.
mhl1 is epistatic to glossy15: The gl15 gene encodes a putative transcription factor that both promotes the expression of juvenile leaf epidermal traits (e.g., waxes) and represses adult leaf epidermal cell differentiation, including MHs (Moose and Sisco 1996). Macrohairs are produced precociously within gl15 mutant shoots, beginning at leaf 2 or 3 instead of leaf 5 or 6 (Evanset al. 1994; Moose and Sisco 1994). To investigate whether mhl1 is required for macrohair production in leaves 3–6 of gl15 mutant seedlings, MH production was observed in families segregating for gl15 and mhl1 (Table 4).
MH density in nonmutant plants shows a pattern similar to that previously observed, where MH production begins at leaf 7, peaks at leaf 9, and declines until leaf 13. Macrohair production begins at leaf 3 in gl15 single mutants, progressively increases until peaking at leaf 9, and then declines in later adult leaves (Table 4). As expected, MH production was increased only in leaves 3–7 of gl15 mutants relative to nonmutant plants. The gl15-L parental stock used to generate this segregating family had been introgressed into W64A and thus had an Smh1/Smh1 genotype, explaining why the mhl1 single mutants in this family exhibited MH frequencies throughout shoot development that are typical for intermediate MH density plants (Tables 2 and 4). Plants homozygous for both gl15-L and mhl1-R did not produce MHs in leaves 3 or 4 as in gl15 single-mutant siblings. MHs were observed beginning with leaf 5 and continuing through leaf 13, but their frequency was significantly reduced in gl15; mhl1 double mutants relative to gl15 single mutants and was instead similar to mhl1 single-mutant siblings (Table 4).
MH density among a family segregating for mhl1-R and gl15
Results similar to those reported here were also observed either when mhl1-R was combined with the gl15-m1:dSpm allele or when mhl1-411 was combined with gl15-L, except that the mhl1-411; gl15-L double mutants did not produce any MHs (data not shown). The gl15; mhl1 double-mutant phenotype demonstrates that mhl1 is required for the early onset of MH production in gl15 mutants, which indicates that mhl1 acts in the same pathway as gl15 and that juvenile leaf identity is determined in part through the repression of mhl1 by gl15.
The gibberellin class of plant growth regulators promote macrohair initiation and act through mhl1: The gibberellin class of plant growth regulators (GAs) promote adult leaf identity and, therefore, MH production, in maize (Evans and Poethig 1995). Trichome production in Arabidopsis leaves has also been shown to be sensitive to GAs (Chien and Sussex 1996; Telferet al. 1997). Although GAs clearly influence the production of MH within the context of vegetative phase change, their role in promoting MH initiation within adult leaves has not been directly investigated. To determine if GAs act as positive regulators of MH initiation in maize, we measured MH frequencies in dwarf1 (d1) mutant plants, which synthesize greatly reduced amounts of bioactive GAs (Sprayet al. 1996). We also investigated the potential requirement for mhl1 in the response of MH initiation to GA reduction by examining d1; mhl1 double mutants. Both of the above investigations were carried out in a family segregating for mhl1 and d1 in a gl15 mutant background (Table 5), to separate potential effects of GA on MH initiation from the known role of GAs in promoting vegetative phase change.
Throughout shoot development, MH frequencies in gl15 single mutants and gl15, mhl1 double mutants were similar to those previously observed (Table 4), with MH production beginning as expected at leaf 3 but being significantly reduced when mhl1 is also present. The d1; gl15 double mutants showed the typical short stature, delayed flowering, and reproductive phenotypes expected for d1, as well as glossy leaves beginning with leaf 3. However, MHs were not observed in leaves 3 or 4, and their frequency was significantly reduced in leaves 5–9 relative to gl15 single mutants (Table 5). The differences in MH frequency between gl15 and d1; gl15 plants became progressively less with successive leaves. By leaf 11 and continuing through leaf 15 (the last leaf where comparisons could be made) MH frequencies for d1; gl15 plants were similar to gl15 single-mutant siblings. These observations suggest that GAs do have a positive effect on MH initiation, with this effect being mainly limited to early adult leaves. Even in early adult leaves, GAs are not required for MH initiation because MHs are produced in both d1 and d1; gl15 plants. As observed previously, MH production was essentially abolished in gl15, mhl1 double mutants (Table 4). MH production was also nearly eliminated in d1; gl15, mhl1 triple mutants, indicating that the formation of MHs in d1; gl15 double mutants would also require a functional allele of mhl1.
DISCUSSION
We have described a locus in maize, macrohairless1, which functions specifically in MH initiation within the leaf blade. Genetic analyses indicate mhl1 is a major regulator of macrohair initiation, but is not the only genetic factor associated with this specific cellular differentiation pathway. Because MHs are a marker for adult leaf identity, analysis of MH initiation in the context of shoot development and the effects of the mhl1 mutation on this process provide additional insights into the regulation of leaf identity in maize.
MH density among a family segregating for mhl1-R and dwarf1 in a gl15 mutant background
Leaf MH density varies during maize shoot development: One consistent observation in the studies reported here is that MH initiation frequency in maize progressively increases from the onset of adult leaf identity through leaf 9. In contrast, adult leaves produced later in shoot development show a progressive decline in MH density until leaf 13, when MH initiation frequency appears to reach a minimum stable level. In Arabidopsis, trichome density and distribution in leaves also vary quantitatively during shoot development (Martínez-Zapateret al. 1995) and are coordinated with the reproductive development of the shoot (Chien and Sussex 1996). Genetic analyses indicate that reduced adaxial trichome production in bracts (leaves produced during inflorescence development) is associated with the onset of inflorescence development (Telferet al. 1997). MH initiation frequency in maize may be similarly reduced by the onset of inflorescence development, as the transition from increasing to decreasing MH initiation frequency appears to be correlated with the vegetative node that produces the uppermost branch terminating in a female inflorescence, which occurs at nodes 9–11 in the genotypes examined here. The trends in MH initiation frequency are also consistent with changes in leaf size during shoot development, which increases until the uppermost ear node and then decreases (Greysonet al. 1982). However, the fact that leaf 5 of gl15 mutants produces the same or greater frequencies of MHs as leaf 13 from the same plants (Table 4), despite leaf 5 being considerably smaller than leaf 13 (data not shown), argues that MH initiation frequency is more closely correlated with inflorescence development than with leaf size per se.
Because the same patterns of variation in MH initiation frequency during shoot development were observed in mhl1-R plants with an intermediate MH density phenotype (Table 3), the factors that regulate leaf MH initiation frequency during shoot development must act upstream of mhl1. Indeed, both GAs and gl15 act upstream of mhl1 in their antagonistic regulation of the onset of MH production in transition and early adult leaves (Tables 4 and 5). However, while both of these factors affect the macrohair response to the juvenile-to-adult vegetative phase change, neither of them seems to affect MH initiation during late adult vegetative development (Tables 4 and 5).
These observations suggest that MH initiation in maize is stimulated by at least two pathways, one dependent on GAs and acting in early adult leaves and the other becoming increasingly active during shoot development and operating independently from GAs. One candidate for a gene acting in a second MH-stimulating pathway is viviparous8 (vp8), which like GAs functions to promote the transition from juvenile to adult vegetative development (Evans and Poethig 1997). As observed for d1; gl15 double mutants (Table 5), the early adult leaves of vp8; gl15 double mutants also express all other adult traits except MHs (Evans and Poethig 1997). The peak MH frequencies observed near leaf 9 suggest that both of these pathways may converge to maximize MH initiation during early adult vegetative development.
mhl1 and the specification of adult leaf identity in maize: The mhl1 mutation represents the first characterized defect in a cellular differentiation pathway specific to adult leaf identity in maize. The mhl1 mutant phenotype demonstrates that MH initiation can be uncoupled from the differentiation of other adult leaf epidermal cell types such as bulliform cells and prickle hairs (Figure 1, A and B). Detailed analyses of MH initiation throughout adult vegetative development in normal and mhl1 mutant plants also shows that even this single cellular differentiation pathway is regulated by multiple factors during shoot development.
Double-mutant analyses (Tables 4 and 5) demonstrate that both GAs and gl15 regulate adult leaf identity in opposite ways by acting through mhl1, which defines the primary pathway through which these factors influence MH initiation. We propose here a model (Figure 3) where GAs cooperate with other factors (possibly defined by the vp8 and smh1 loci) to stimulate mhl1 activity and/or MH initiation in adult leaves, whereas in normal juvenile leaves gl15 instead acts as a repressor of MHL1 function. The gl15 gene acts downstream of GAs and vp8 (Evans and Poethig 1995, 1997) but upstream of mhl1 (Table 4). Therefore, gl15 could suppress MH initiation either by repressing mhl1 activity or by interfering with the response of mhl1 to upstream factors. In this model, juvenile leaves are competent to express mhl1 and produce MHs in response to GAs, but fail to do so because gl15 represses mhl1 or some relatively late component of the GA signal transduction pathway leading to adult leaf identity. When gl15 activity is reduced or lost, either by mutation or by its downregulation in adult leaves (Moose and Sisco 1996), the mhl1 gene is capable of responding to activation by GAs. The antagonistic interactions between GAs and gl15 appear to regulate MH initiation only in transition leaves that express both juvenile and adult traits (e.g., leaf 7) and the earliest adult leaves, but have little effect on MH initiation in later adult leaves, which are instead promoted by other factors whose identities are currently unknown.
—A model for the regulation of MH initiation in maize. Juvenile leaves (dark gray) lack macrohairs because gl15 activity represses mhl1, which is required to initiate MH differentiation. GAs promote the transition to adult vegetative development and also stimulate mhl1 activity in early adult leaves (light gray). Other factors, possibly defined by the viviparous8 (vp8) or suppressor of macrohairless1 (smh1) loci, also stimulate mhl1 activity and MH initiation in adult leaves. While vp8 is known to act at the whole-shoot level, it is likely that smh1 acts further downstream and possibly at the level of mhl1. The activating arrow for GAs is thicker than the repressing T-bars for gl15 to indicate the greater sensitivity of mhl1 to GAs compared to gl15.
The proposed model predicts that GAs would have their greatest effect on promoting MH initiation in gl15 mutant leaves, where mhl1 would be stimulated by GAs in the absence of gl15. The drastic reduction in MHs of d1; gl15 double mutants compared to gl15 single mutants supports this view (Table 5). The onset of MH production in transition leaves of normal plants prior to the loss of juvenile traits also suggests that mhl1 activity is more sensitive to GAs than to gl15. Consistent with this idea, treatment of dwarf3; Teopod1 double mutants with exogenously applied GA3 affected MH production more than the expression of juvenile epicuticular waxes (Evans and Poethig 1995).
Possible functions for mhl1 and smh1: The mhl1 mutations exhibit mutant phenotypes only in the leaf blade, suggesting that either MHL1 function is limited to the leaf blade or there is redundancy for MHL1 function in the initiation of MHs elsewhere in the plant (for example, on leaf sheaths). The mhl1 locus is likely to encode a regulatory gene that specifically programs MH initiation in the leaf blade epidermis. This conclusion is based on the observations that mhl1 conditions a very early block in MH initiation, mhl1 does not affect MH morphogenesis, and other aspects of cellular differentiation are normal in the mhl1 mutant leaf epidermis, except for the failure to proliferate the multicellular pedestal associated with the base of each MH (Figure 1, A and B).
In Arabidopsis, a complex of interacting proteins from the MYB domain (GLABROUS1), basic helix-loop-helix (GLABRA3), and WD-40 repeat (TRANSPARENT TESTA GLABRA1) families has been shown to program trichome initiation (reviewed in Scheres 2002). Thus, it is possible that mhl1 encodes one of the members of an MYB-bHLH-WD-40 protein complex that may program macrohair initiation in the maize leaf blade. A number of similarities between maize and Arabidopsis epidermal hair formation support this hypothesis. In both taxa, hair initiation is promoted by GAs, varies both qualitatively and quantitatively during shoot development, and is influenced by the transition to inflorescence development (Chien and Sussex 1996; Telferet al. 1997; Tables 3,4,5). In addition, the placement of both types of hairs within the epidermis is affected by a minimum distance spacing mechanism (Larkinet al. 1996; N. Lauter and S. Moose, unpublished observations). Finally, several cases of apparent coregulation of anthocyanins and macrohairs have been documented in a close wild relative of maize, Zea mays ssp. mexicana (Lauter 2001; N. Lauter, C. Gustus, A. Westerbergh and J. Doebley, unpublished observations). Since anthocyanins are regulated by MYB, bHLH, and WD-40 repeat family members in both maize and Arabidopsis (Chandleret al. 1989; Coneet al. 1993; Selinger and Chandler 1999; Walkeret al. 1999; Borevitzet al. 2000; Nesi et al. 2000, 2001), the coregulation observations further support the hypothesis that a similar protein complex may regulate maize MH initiation.
If such a macrohair initiation complex does exist in maize, we believe mhl1 would be most likely to represent a defect in the MYB domain-containing partner orthologous to Arabidopsis GLABROUS1 (GL1). The phenotypes of GL1 (Koorneef 1982; Oppenheimeret al. 1991) and mhl1 mutants (Figure 1) are similar in that both are specifically defective in hair initiation on the medial blade without affecting the hairs at the margins of the blade. In addition, both GL1 (Perazzaet al. 1998) and mhl1 (Table 5) activities are required for the positive effects of GA on MH initiation. Finally, the reductions in both trichome size and density conditioned by GLA-BRA3 mutations in Arabidopsis are more similar to the weaker phenotypes seen in our macrohairless mutants that are nonallelic to mhl1 (Payneet al. 2000). It should be noted that although many MYB-, bHLH-, and WD-40-encoding DNA sequences are known for Z. mays, no clear orthologs of GL1, GL3, or TTG1 have emerged in Blast searches using either the expressed sequence tag or the genomic survey sequence database.
The Smh1 allele present in the W64A, but not the A632, 4Co63, or NC89 inbred backgrounds, acted as a dominant suppressor of mhl1 that conditioned an intermediate density of leaf macrohairs (Tables 1 and 2). Notably, plants with the intermediate MH density phenotype show the same changes in MH frequencies during shoot development as normal plants do, suggesting that the smh1 locus functions at the level of MH initiation rather than at the level of shoot developmental programs. Since Smh1 plants homozygous for the putative null alleles of mhl1 are glabrous while Smh1 plants homozygous for mhl1-Ref display an intermediate MH density phenotype (Table 3), the Smh1 allele likely requires some MHL1 protein to cause macrohair initiation. Together, these observations suggest that the Smh1 allele characterized here could represent a dominant hypermorphic allele of a macrohair initiation regulator that is capable of stimulating MH initiation despite reduced levels of MHL1 protein.
If the smh1 locus were to encode an ortholog of an Arabidopsis gene affecting trichome initiation, we think it would be a gene like GL3, which is directly involved in the initiation process and, when overexpressed, can condition hairs even when other members of the initiation-promoting protein complex are present at reduced levels (Payneet al. 2000). Alternatively, smh1 could encode an ortholog of the Reduced Trichome Number (RTN) locus, which is known as a quantitative trait locus difference between the Columbia (Col) and Landsberg erecta (Ler) ecotypes of Arabidopsis (Larkinet al. 1996). Although the exact action of RTN remains unclear, it could be argued that the Col allele of RTN is hypermorphic, acting semidominantly to increase trichome density, which is similar to our interpretation of the action of the Smh1 allele from maize inbred W64A. However, GL1 and TTG1 mutations are not suppressed by the Col allele of RTN.
In addition to the mhl1 and smh1 loci, the mhl*-LS8, mhl*-LS9, mhl*-LS10, and mhl*-LS11 mutations, which are not allelic to mhl1, also appear to regulate MH initiation in maize. Each of these mutations exhibits a weak, variably penetrant macrohairless phenotype. Another mutation with a weak macrohairless leaf phenotype, which we have named macrohairless2 (mhl2), was identified in a Mutator transposon-active population and mapped to the long arm of chromosome 4 by B-A translocation tests (J. Nelson and M. Freeling, personal communication). Interestingly, mhl2 mutants show a reduction in both MH density and size, which is reminiscent of the GLABRA3 mutant phenotype in Arabidopsis (N. Lauter, J. Nelson, M. Freeling and S. Moose, unpublished observations). Given this similarity and the fact that smh1 has not yet been mapped, it remains possible that smh1 and mhl2 are the same locus. Allelism testing of these macrohairless mutations, genetic mapping, and tests for their interactions with mhl1 are ongoing.
Prospects: The availability of putative transposon-tagged mhl1 alleles and candidate genes from Arabidopsis that may be orthologous to mhl1 in maize should facilitate the eventual molecular cloning of the mhl1 gene. Once cloned, it should be possible to elucidate the molecular mechanisms by which mhl1 promotes leaf blade MH initiation and how mhl1 responds to factors that act upstream of mhl1 to regulate MH production during shoot development.
In many other cereal crops, studies of glabrous mutant varieties that lack MHs have led to the suggestion that MHs contribute to insect resistance (e.g., Ringlund and Everson 1968; Sosa 1990). In maize, a similar function for leaf MHs has been proposed on the basis of associations between quantitative variation in leaf macrohair density and insect behavior or feeding (Widstromet al. 1979; Durbey and Sarup 1982). Our study has identified mhl1 mutant alleles that appear to completely eliminate leaf blade MHs, which should permit direct tests of their role in insect resistance and in other physiological functions in maize.
Acknowledgments
We thank Paul Sisco for the K55, 4Co63, NC89, and W64A inbred stocks; the North Central Regional Plant Introduction Station (Ames, Iowa) for providing an additional source of the K55 inbred; the Maize Genetics Cooperation Stock Center for the glossy15-L stock and lines harboring B-A translocations; and Laurie Smith (University of California, San Diego), Jennifer Nelson, and Michael Freeling (University of California, Berkeley) for generously providing additional macrohairless mutants. We thank Andy Gaspar, Stephanie Halbig, Paulina Jazwierska, and Magen Starr for their assistance with phenotyping macrohair densities. We are grateful to DEKALB Genetics Corporation for providing nursery space to perform the initial screen for Mutator -tagged macrohairless1 alleles in 1999. Finally, we thank Marty Sachs and two anonymous reviewers for helpful comments on the manuscript. This research was supported by a grant from the Illinois Agricultural Experiment Station (ILLU-15-0359).
Footnotes
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Communicating editor: V. Sundaresan
- Received February 20, 2003.
- Accepted December 2, 2003.
- Copyright © 2004 by the Genetics Society of America
