Developmental morphology of leaves using UV light-induced autofluorescence of cell wall components

--Anne W. Sylvester, Zac Cande and Mike Freeling

The pattern of cell wall deposition is both developmentally and spatially regulated in the maize leaf. We are using the fluorescence emission differences among wall components as developmental markers in several leaf mutants. The purpose of this analysis is to characterize the time when cells in the maize leaf are programmed to follow one developmental pathway or another. It is clear that differentiation of leaf parts in maize begins early during growth of the primordium. Cells of the blade, sheath, and ligular region differentiate from one another around plastochron 2 - 3, before the ligule itself is visible on the adaxial epidermal surface (Sylvester et al., Development, 1990, Nov. issue). The availability of developmentally specific markers in addition to epidermal cell markers (i.e. hairs, waxes, wall shapes, etc.) will allow for more precise analysis of stages when specific genes are acting during development.

Components of the phenylpropanoid pathway, particularly the monomers ferulic acid and p-coumaric acid, and the polymers, lignin and suberin, are known to emit excess energy in the form of autofluorescence when excited by UV light. Ferulic acid and p-coumaric acid absorb at around 310 nm and emit at 415 - 445 nm (Goulas et al., Photo. Res. 25:299-307, 1990), whereas lignin shows a maximum emission at 358 nm (Lundquist et al., Holzforschung 32:27-32, 1978). This shift in spectrum is useful for recognizing the time when lignin deposition begins. In the Gramineae, ferulic acid is bound to all cell walls (Harris and Hartley,. Nature 259:508-510, 1976) and can be recognized early in development by its characteristic fluorescence spectrum. Lignin on the other hand is deposited specifically in vascular tissue and in adjacent sclerenchyma cells later in development when cell growth has slowed. Using a standard Zeiss filter combination (exciter filter at 365 nm and a longwave pass filter at 418 nm), ferulic acid and associated monomeric phenylpropanoids appear white-blue whereas walls containing lignin appear dark purple-black. Histochemical tests using the standard metachromatic stain toluidine-blue O (TBO) at low pH confirmed that the observed fluorescence shift to purple-black corresponds to the presence of lignin. TBO staining, however, does not exhibit the subtle color gradations associated with the esterification of ferulic acid that is permitted by the use of UV excitation.

The lignin deposition pattern, based on shifts in UV emission, was characterized in seedlings and mature leaves of normal and heterozygous mutant siblings of Lg3 plants. Seedling plants bearing a total of 8 -10 leaves were dissected, hand-sectioned, excited by UV light and photographed. Representative sections were fixed in buffered formaldehyde for histochemical staining. The blue fluorescence of walls in each of several leaf parts including the sheath, the ligular region, and the blade was recorded and compared. Preliminary observations show that the white-blue fluorescence characteristic of ferulic acid is visible as early as plastochrons 2-3 in normal plants. The blue intensity increases in sclerenchyma cells on either side of the vascular bundles (possibly due to esterification of pre-lignin monomers). By plastochron 6 the characteristic dark-purple fluorescence of lignin is first visible in local regions of the leaf. Sclerenchyma near the tip of the blade lignify first, in accordance with the age gradient of the leaf. Cells that are subjacent to vascular bundles in the adaxial sclerenchyma of the midrib lignify initially. As the leaf grows, lignification spreads until the adaxial midrib has a continuous band of lignified sclerenchyma. The adaxial and abaxial surfaces lignify at different rates with the abaxial sclerenchyma lignifying before the adaxial. There is also a lateral gradient of age: the shift from white-blue to bright-blue to purple-black occurs first at the midrib and then proceeds gradually toward the blade margin.

This method should prove useful for the analysis of developmental mutants that show alterations in the identity of entire regions of cells. For example, plants heterozygous for Lg3 show alterations in the position of the ligule over the midrib region. By UV light analysis, Lg3 plants show distinct changes in the lignification pattern. Deposition is delayed in the adaxial sclerenchyma but only over the midrib portion of the blade. The abaxial surface of the midrib appears to lignify normally as do both surfaces of the blade proper. There is an extra accumulation of lignified sclerenchyma cells at the junction between midrib and blade on the adaxial surface. This delay in lignification of the adaxial midrib continues past the time of ligule outgrowth, such that the ligule never joins at the midrib. Instead, the ligule stops at the site of extra lignin accumulation at the midrib/blade border. The continuous band of lignin at the adaxial midrib is absent except near the tip of the blade. This method of using wall autofluorescence should prove useful for further characterization of Lg3 plants and other mutants with similar phenotypes. More detailed analysis of the fluorescence spectra for developmental study is underway.

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