Leaves 1 through 7 in most maize inbred lines exhibit juvenile traits. The juvenile phase is most easily identified by the expression of leaf epidermal traits such as bluish, highly hydrophobic waxes and the absence of leaf hairs. Using these traits as well as toluidine blue-O staining and cell wall morphology as markers, I have observed that all juvenile leaves have similar phenotypes and therefore appear developmentally equivalent with respect to the juvenile-to-adult phase transition. The phenotypes of glossy15 (gl15) mutations, however, do not support this hypothesis. In gl15 mutant seedlings, adult traits are observed beginning with leaf 3 but do not affect the juvenile character of leaves 1 and 2. This phenotype could reflect the possibility that the available gl15 mutant alleles are hypomorphic rather than null mutations, where Gl15 gene function is reduced but not absent; or it may represent the effects of an additional, independently regulated juvenile epidermal program which only operates in leaves 1 and 2 (Evans et al., Development 120: 1971-1981, 1994; Moose and Sisco, Plant Cell 6: 1343-1355, 1994).

In this report, I present three new findings which support the hypothesis that the juvenile epidermal phenotypes in leaves 1 through 7 represent the composite effects of two similar, but independently regulated developmental programs. These programs appear to act in distinct spatial/temporal domains which overlap at leaf 3. The early juvenile program operates in leaves 1 - 3, while the late juvenile program acts in leaves 3 through 7 and is regulated by Gl15.

Phenotypic null alleles of gl15 : I have examined the phenotypes of seven different gl15 mutant alleles, none of which affect the juvenile traits in leaves 1 and 2. The origin of four of these gl15 alleles is known. The unstable gl15-m1 allele is the result of a 2.1-kb dSpm insertion and therefore probably conditions a null phenotype. This hypothesis is supported by the observation that gl15-m1 did not exhibit dosage effects expected of a hypomorphic allele. Hypoploid gl15-m1 plants generated by crossing gl15-m1 to a TB-9Lc stock had leaf epidermal phenotypes which were qualitatively similar to their gl15-m1 parents. I have also identified three independent stable derivative alleles from gl15-m1. The molecular features of these alleles were examined using a cloned portion of the gl15-m1 gene as a probe. One derivative allele from gl15-m1 appears to have sustained a 600-bp deletion within the dSpm element, which has led to a stabilized insertion. Even if this dSpm is inserted in a non-coding region of the Gl15 gene, it is unlikely that this allele would encode a functional Gl15 protein. Therefore this gl15-stable allele is probably a phenotypic null.

Cg1 extends the effects of the early juvenile program: Figure 1 shows a typical leaf 3 from gl15-m1 plants. Revertant juvenile wax sectors can be partially obscured by the diffuse pattern of transition waxes often found at the tip of leaf 3, which is similar to that observed in leaf 6 of normal plants. This result suggests that the deposition of these apparently similar juvenile waxes in leaf 3 is regulated independently. The diffuse waxes appear to be produced by the action of the early juvenile program, while the underlying sector depends on Gl15 function. In Cg1/+, gl15-m1 plants, revertant sectors in leaves 3 - 8 are also obscured by a diffuse layer of juvenile waxes (Fig. 1). These waxes are not present in subsequent leaves which exhibit defined juvenile and adult sectors. This observation indicates that the effects of the early juvenile program may be extended through leaf 8 in the presence of Cg1.

Adult trait expression in revertant juvenile sectors from gl15-m1: Microscopic examination of gl15-m1 plants revealed two types of revertant sectors, those which express only juvenile traits and others which express both juvenile and adult traits. The phenotypes of the two types of sectors suggested that the reactivation of Gl15 and juvenile traits during leaf development was not always capable of suppressing adult cellular differentiation, and that the timing of the reversion event was important. A possible explanation for these sector phenotypes is that the early reactivation of Gl15 during leaf development can suppress adult traits, but later transposition events cannot suppress adult differentiation which has been previously initiated. Upon further examination of many more sectors, contradictions to this hypothesis were observed. I have identified large revertant sectors from leaves 4 and 5 which were coincident in both the abaxial and adaxial epidermis and expressed juvenile waxes, but also possessed epidermal hairs. These sectors represent early transposition events since they affect a large number of cells within the leaf, yet both juvenile and adult traits are present. Closer examination of these sectors revealed that the two different sector phenotypes were distributed with a defined polarity. These sectors showed only juvenile traits near the tip of the leaf, but both juvenile and adult traits were present near the base of the leaf. These observations argued that the presence of adult traits was influenced by the relative position of the sector from the base of the shoot.

The effects of leaf position on the presence of adult traits within gl15-m1 revertant juvenile sectors were further examined by determining where epidermal hairs appeared in adjacent mutant and revertant sectors which extended the entire length of the leaf blade. Such sectors were expected to be derived from early transposition events, and all cells within these sectors should share a common lineage. At least 10 sectors were selected from each of leaves 3 through 5 of gl15-m1 plants backcrossed three times into the W64A inbred background. A schematic representation of these observations appears in Figure 2. No epidermal hairs were observed in leaf 2 or revertant sectors from leaf 3. These tissues showed a complete juvenile wax phenotype. gl15 mutant sectors from leaf 3 expressed epidermal hairs throughout their length except at the leaf tip, where they were absent. As described earlier, the tip of leaf 3 still shows some juvenile wax expression, and the absence of hairs was correlated with the presence of juvenile wax. In leaves 4 and 5, gl15 mutant sectors expressed epidermal hairs throughout their entire length. However, adjacent revertant sectors from these same leaves were found to express both juvenile and adult traits. The epidermal hairs within these revertant sectors were distributed with a defined polarity. Hairs were present at the base of the sector, and their frequency gradually declined towards the leaf tip. Leaf 4 sectors expressed epidermal hairs for approximately the basal quarter of the sector's length, while in leaf 5 hairs were present in the basal half of a sector. By leaf 6, sectors expressing juvenile waxes had hairs along their entire length like gl15 mutant sectors (data not shown). These results demonstrate that the appearance of epidermal hairs within a gl15-m1 revertant sector is dependent upon leaf position. Adult differentiation appears to be suppressed in revertant sectors along the entire length of leaf 3 and progressively less in leaves 4 and 5. 

The proposed early juvenile program clearly conditions juvenile epidermal phenotypes in leaves 1 and 2. Therefore, it might be expected to influence the differentiation of other leaf cells which were present at the same time during development. In mature W64A embryos, cells which will become the upper portions of leaves 4 and 5 and the majority of leaves 1, 2, and 3 are all present and may therefore be considered developmentally equivalent. Adult traits in these leaf tissues are completely absent and their suppression is independent of Gl15 activity. These observations provide support for the existence of an early juvenile program. The distribution of epidermal hairs within revertant sectors from leaves 4 and 5 of gl15-m1 plants suggests that the action of the early juvenile program is restricted to a defined spatial/temporal domain at the base of the developing shoot, and that somatic reactivation of Gl15 only suppresses adult traits when the early program is also active. The remainder of juvenile leaf epidermal cell differentiation is independent from the early juvenile program and is controlled by Gl15. It is still not clear why adult traits are completely suppressed in leaves 4 - 6 of normal plants but do appear within Gl15-active somatic sectors. Such phenotypes suggest that Gl15 directly activates juvenile traits, but only plays an indirect role in the suppression of adult epidermal cell differentiation.

Figure 1: Cg1 Extends the Effects of the Early Juvenile Program. Schematic drawings of representative leaf phenotypes are shown. A single large Gl15-revertant sector expressing juvenile waxes is within the left half of each gl15-m1 leaf. Epidermal tissues expressing juvenile waxes appear grey, adult waxes white. Stipples indicate the presence of epidermal hairs.

Figure 2: Wax and Epidermal Hair Phenotypes of gl15-m1 Leaf Sectors. Schematic drawings of representative leaf phenotypes are shown. A single large Gl15-revertant sector expressing juvenile waxes is within the left half of each gl15-m1 leaf. Epidermal tissues expressing juvenile waxes appear grey, adult waxes white. Stipples indicate the presence of epidermal hairs. Note how epidermal hairs are progressively distributed towards the leaf tip in revertant sectors from successive leaves.

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