A morphological analysis of the liguleless2-2757 mutant, a
new allele at the lg2 locus
-- Katrina Snyder and R. Bertrand-Garcia
In normal maize leaves, the ligule is a landmark structure delineating the blade from the sheath. The ligule is initiated as a result of periclinal divisions (it projects out from the plane of the leaf) and traverses the entire width.
The recessive liguleless2 mutation was first described by Brink (J. Hered. 24:325, 1933). This spontaneous mutation removes ligule from the lower leaves of the plant and produces only partial ligules in the upper leaves.
We have characterized the phenotype of a new lg2 allele recovered in a Mutator transposon background. This new lg2 allele (liguleless2-2757) was recovered from a field of heterozygotes (Lg2/lg2) in a Mutator background. The new allele was confirmed by Southern analysis and allelism was determined by test crossing to the spontaneous lg2 mutant (M. Freeling, M lab unpublished data). The mutant allele removes the ligule from the first two leaves and retards its development in subsequent leaves. When the ligule first appears it is fragmented and only present on the edges of the leaf. In later leaves the ligule progressively becomes more complete but never fully develops. Often the ligule is present in discontinuous segments progressing diagonally down the leaf. Even in the uppermost leaves, where the ligule is almost complete, the ligule is never joined across the mid vein and is often displaced along the leaf.
We have examined the ligules of both wildtype and lg2-2757 siblings in a B73 background. Leaves 1, 2, 5, 7 and 12 were removed during various stages of leaf development and examined using SEM. Our results show that ligule development is arrested just after the onset of periclinal division in leaves 1 and 2. In addition it appears that blade cells in these leaves are also arrested before fully forming (Figure 1). These blade-like cells are elongated with only slight crenulations and appear to be in an intermediate state of development (between blade and sheath).
Leaves 5 and 7 show a progressive increase in the cellular organization of the blade-sheath boundary, however, these leaves never show a distinct blade and sheath region. Further, these leaves have projections of sheath invading the normal blade tissue (Figure 2).
In the ligular region of leaf 12 the cells are not clearly separated into distinct blade and sheath regions as they are in the wildtype. There are distinct hair and blade-like cells that develop below the ligule. In addition the ligule on these leaves is not entirely normal and has hairs developing on the ligule surface (Figure 3).
These results suggest that the lg2-2757 mutant causes the leaf to lose the ability to establish a normal blade-sheath boundary. The transition from blade to sheath is disrupted both temporally and spatially. These results suggest that the wildtype Lg2 gene product may play a key role not only in the formation of the ligule but also in setting up the proper boundaries between blade and sheath.
The degree of disorganization may result in fewer cells being able to respond to the ligule signal or a disruption in the signal itself. Ligule development is initiated, even in the lower most leaves, suggesting that a few cells in the first leaves are competent to receive the signal for ligule formation, however, most are not. These smaller leaves mature faster and may have a smaller window in which to see the ligule signal. Later leaves, that take longer to mature may have cells that remain competent longer and therefore (1) have more time to set up a boundary and set up cellular order, and (2) have more cells that remain competent to receive the ligule signal. This would explain why the later leaves show more ligule formation.
We are unsure whether this cellular disorder is caused by temporal factors, spatial factors or both. We are exploring these possibilities by placing the lg2-2757 allele in various time-to-flowering backgrounds. Preliminary results suggest that there is a difference in both the amount of ligule formed, and the leaves in which it is formed, depending on the plant's time-to-flowering.
Figure 1. Shows the ligular region of a mature second leaf from a lg2-2757 homozygote. The arrow shows cells that have been arrested during the onset of periclinal division. Cells surrounding this region are sheath-like in appearance. (190X)
Figure 2. Shows the ligular region of a mature seventh leaf from a lg2-2757 homozygote. The arrow indicates a transition zone between blade cells (B) and sheath cells (S). This sheath tissue is invading the blade, in a region that would normally give rise to ligule. (260X)
3. A partially formed ligule (L) on a mature twelfth leaf of a lg2-2757
homozygote. The arrows show hairs developing both beneath the ligule and
on the ligule itself. Those cells producing hair at the base of the ligule
appear blade-like (showing crenulations along the margin and a cuboidal
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