New mutants with aberrant ligule/auricle formation
--Kloeckener-Gruissem, B, Freeling, M

The maize leaf develops three morphologically distinct domains: i. the sheath which wraps around the stem, ii. the blade which angles off the stem and serves as the major photosynthetically active region and iii. the region that separates those two domains, occupied by the triangular shaped auricles, which act as a hinge for the blade and the ligule, a fringe-like epidermal structure that grows out of the leaf’s plane. So far, two genes have been identified by recessive mutants that play an essential role in the proper development of the ligule/auricle region, namely liguleless1 (lg1) and liguleless2 (lg2). For both genes, many mutant alleles have been isolated. Certainly, other genes must be involved in the development of the ligule/auricle region. Past screens for liguleless mutants focused on the altered, upright stature of the plants, due to the lack of the auricles, and hence it was easy to miss any mutants in which only the ligule, but not the auricle is affected. Therefore we decided to investigate the ligule structure on every plant while screening mutagenized families. Although screening of transposon Mutator (Mu) families facilitates the isolation of the mutated gene, the frequency with which new Mu- induced mutations occur might be about ten fold lower than that of chemically induced mutations. Therefore, we chose to screen ethylmethanesulfonate (EMS) mutagenized material. Using the method of EMS treatment of pollen, Jay Hollick and Vicki Chandler, University of Oregon, Eugene, generated highly mutagenic seed stocks. They generously shared 840 M2 families with us. We grew 30 kernels from each of these families in the summer of 1998 in San Jose, California, and screened for families segregating plants with deficient ligules or auricles.

Here we report the isolation of two novel mutants that seem to affect exclusively the ligule/auricle region. We found one family that segregated (2/15) plants with a phenotype similar to that of lg1 mutant plants, but also with several important differences. Like lg1, mutant plants showed the upright leaf stature, due to the lack of auricles. Closer examination of the ligule/auricle region revealed a novel phenotype. On each leaf, a varying number of small patches of sectors with normal appearing ligules were found. The size of the sector can range from less than one to five millimeters. If a larger sector is located at or near the leaf margin, the development of an auricle can be recognized. Whether the other ligule sectors were also developing auricles requires Scanning Electron Microscopy (SEM) analysis. The exact pattern of the sectors did not appear on sequential leaves, suggesting that they are not of meristematic nature. Another novel aspect of this phenotype is in the location of sector development. Sectors can be found irregularly spaced over a distance of up to 2 cm in the proximo-distal dimension of mature, juvenile leaves. The existence of such broad "line" suggests that the normal function responsible for defining the fine line at which the ligule normally develops is deficient. This is in contrast to revertant sectors of the lg1-m1 allele (Ac-induced) (Moreno et al. 1997, Genes & Development 11, 616.), where all sectors, also irregularly spaced, lie on a sharply drawn line from margin to midrib. The novel liguleless phenotype described here suggests that the mutant might not be another allele of lg1 or lg2. We performed complementation tests by crossing pollen from the new liguleless mutant plants to homozygous lg1 as well as lg2 plants. Progeny was scored for a liguleless phenotype at the seedling stage. The lg2 mutant complemented the new mutant, suggesting that they are not allelic. The progeny of the lg1 cross did not yield a clear result: 50% of the seedlings were normal while the other half showed the phenotype of the new mutant. These results suggest two points: lg1 can complement the new mutant but interactions exist between the new mutant and lg1. We also examined the progeny of the new mutant crossed to inbred lines and all were normal, suggesting the original mutant plants were not heterozygous for a dominant allele. We are in the process of obtaining progeny from self-pollinations.

We identified another family segregating a ligule phenotype. In 2 out of 15 plants, a ligule formed on the abaxial surface of the leaf. A normal ligule/auricle developed at its normal place on the adaxial side of the leaf. The abnormal, abaxial ligule does not span the entire width of the leaf but is rather restricted to the region at the tip of the triangular-shaped auricle on both sides of the leaf, near the midrib. Just proximal to the ligule fringe, reaching a few millimeters into the sheath, one can recognize a small region of cells of shiny appearance, which is clearly distinct from the neighboring sheath cells. Similarly, distal to the fringe there extends a sector of distinct appearance into the blade. The length of such sectors varies over 5 to 10 cm. Whether such sectors share characteristic features of normal auricle, blade, or sheath will be determined by SEM analysis. Possibly this mutant is deficient in the identification of polarity with respect to the ab- and ad-axial side of the leaf as well as the proximo-distal dimension. The formation of this abaxial ligule is sensitive to the development of the plant: only leaves 5 through 9 are affected.

We are continuing the analysis of both mutants by introgression into various genetic backgrounds as well as the construction of double mutants with other mutants that affect the ligule/auricle region.

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