The original Knotted mutation (Bryan and Sass, 1941, J. Hered. 32:343), Kn1-0, specifies a constellation of dominant phenotypes depending on the genetic background: mild dwarfism, outpocketings of tissue at foci along lateral veins of the blade (knots), twisting of the midrib and stem, and a condition that has been called "ligule displacement" (Gelinas, Postlethwait and Nelson, 1969, Amer. J. Bot. 56:671), where the normal ligule is disrupted and bits of epidermal "fringe" grow from thickened tracts above lateral veins. Other Kn mutants specify different phenotypes.
The ligule is a dispensable organ. In suitable genetic backgrounds, a Kn1-0 mutant gene specifies a paired fringe structure that we have diagrammed below.
The fringe appears to be like the inside structure of the ligule. To test for this homology directly, we constructed double mutants of liguleless-2 and Knotted. Kn1-0/+, lg2/lg2 plants showed no such fringes whereas siblings with ligules showed both ligule disruption and fringes spread up the blade. Knots were unaffected. We conclude that the fringe is homologous to ligule and that fringes and knots above the same vein are separable. Among about 100 Kn1-0 heterozygotes, we found two leaves that showed a particularly informative morphology: the ligule was perfect as was the lower leaf blade, but an area of distorted tissue occurred about one-third up from the ligule. Distal to this area were copious bits of fringe material. Clearly, these ligule bits originated de novo rather than as displaced cells of the original ligule.
In order to test whether de novo ligule formation is a cell autonomous effect of Kn1-0, we have used x-rays to disconnect the long arm of chromosome 1 carrying Kn, leaving a segmental monosomic carrying kn marked by a white stripe (lw, 1.5 mu from Kn1). De novo ligule formation and epidermal thickening above veins is cell
autonomous. Our six clones were white in all layers and extended to the periphery of the leaf. Methods--500-1000 rads 225 Kv x-ray through 0.35 mm Cu at 150 rads/min acute onto 4 day seedlings--were adapted from S. Poethig (1982,
MNL 56:53); sectors 1/8-1/32 of a leaf width occurred. We do not yet have definitive fate maps for knots, or other components of Knotted phenotype.
Our most informative experiments involved constructing sibling plants that differed in the dose of kn (kn = + = normal) gene in a background containing one dose of Kn1-0. We used the TB-1La translocation to add or remove + as a part of the entire arm; we used a pair of reciprocal 1-3 translocations to add an extra + on an 18 mu segment. Stocks were marked with Adh1 allozymes (Adh1 is less than 0.1 mu from Kn) by J. Birchler. All details will be reported elsewhere. Our results were a double surprise.
Surprise 1. Kn/+/+ had less and developmentally later de novo ligule formation than Kn/+ sibs. However, total degree of knotting and twisting did not differ significantly between these groups. This result was repeated using the TB-1La translocation and once more using the segmental addition method.
Surprise 2. Kn1-0/deletion heterozygotes had no Knotted phenotype at all. Surprise 1 seems to-exclude an "overproducer" hypothesis. We are left with a possible case of transvection.
With respect to ligule disruptions, Kn1-0 apparently specifies a wrong product, but a product similar to normal kn product, that leads to de novo epidermal thickenings and ligule formation. Other Kn mutations seem to behave differently from Kn1-O in most of these tests. When we obtain a Kn clone, we should be able to derive meaning from DNA sequence and sequence arrangements.
M. G. Neuffer and M. Zuber have given us new Kn mutants that are proving extremely valuable. If you have any mutant that encodes knots, rough sheath, wiggly veins, liguleless, or the like, we would very much appreciate hearing about it. Thank you.
Michael Freeling and Sarah Hake
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