--John Fowler and Michael Freeling

The ligular region of maize provides an excellent model system for the study of development in plants. This region occurs on the maize leaf at the junction of its two major organ-components: the blade, which bends outward from the stem, and the sheath, which wraps tightly around the stalk. The boundary between these two morphologically distinct tissues is sharply defined by the ligule, a small fringe of tissue angled upward on the inner surface of the leaf, and by the auricle, a yellowish, thickened area which lies just above the ligule. By using mutations which specifically alter the morphology of this region, and therefore define genes which act to control the decisions made during its development, it will be possible to gain insight into the mechanisms by which plants control those decisions. Here we describe a new mutation, Liguleless 4, which appears to affect a gene involved in this process.

Lg4 (originally designated Lg*-403) was originally isolated by Dr. Scott Poethig in a cross from a Robertson's Mutator male parent to a W23 standard inbred line. The mutation was crossed into the standard inbreds W23, Mo17Ht, and B73 to observe background effects on expression of the mutant phenotype, and mutant progeny were backcrossed to continue introgression into these lines. Lg4 has now been backcrossed into these inbred lines four times, and the morphological characteristics described here are seen in all lines in which the mutation shows penetrance. The phenotype of Lg4 is similar to that of Lg3, showing sporadic fingerlike intrusions of transformed tissue from the sheath into the blade, and elimination of the ligule and auricle in the region where these transformations occur. Vestigial fringes of ligule and accompanying auricle can sometimes be seen displaced into the blade along the transformed tissue, and the transformations appear to be associated with the lateral veins in the blade (Figure 1). The ìfingersî can appear anywhere along the width of the leaf, although they occur most commonly near the leaf margins and very rarely at the midrib, in direct contrast with Lg3, which affects the blade region around the midrib of the leaf. Preliminary evidence from sector analysis of the mutant suggests that Lg4 acts cell-autonomously.

Hand sections of transformed regions of the blade revealed characteristics which are generally associated with the sheath. These characteristics include few intermediate veins, closely spaced lateral veins, thickened tissue, and fewer chloroplasts. When examined using Scanning Electron Microscopy, the morphology of the epidermal cells of the transformed tissue was found to be similar to that of sheath epidermal cells, and distinct from blade epidermal cell morphology. These results indicate that Lg4 causes a transformation of blade cells into cells which adopt a sheath cell fate, altering the normal blade-sheath boundary in the maize leaf.

Another interesting characteristic of Lg4 is that its expressivity is highly affected by genetic background. By the second generation of backcrossing into the B73 inbred line Lg4 is totally suppressed, but continued outcrossing of a B73-suppressed Lg4 plant to lines which allow penetrance will restore expression of the phenotype. In the Mo17Ht and W23 inbreds Lg4 shows high penetrance, but the timing of its expression is modified by both lines. In W23, Lg4 is expressed most severely on the lowest leaves of the plant and is often absent from the topmost leaves. However, in the Mo17Ht background the pattern is reversed and Lg4 is more severe at the top of the plant (Table 1).

Table 1. Expressivity of Lg4 heterozygotes in W23 and Mo17Ht inbred lines. Each leaf on plants expressing the phenotype was assayed for appearance of transformed blade tissue. Severity from 0-5 was assigned based on an estimate of % blade tissue transformed to sheath, 0 being none and 5 being ~25%. Families assayed had been backcrossed into the respective lines four times, and segregated 21 Lg:26 + in the W23 family and 18 Lg:15 + in the Mo17Ht family.

Leaf #	W23		Mo17Ht
	# Leaves Affected	Avg. Severity	# Leaves Affected	Avg. Severity
2	5/6	nd	0/13	-
3	11/11	nd	0/17	-
4	18/18	nd	0/18	-
5	18/18	nd	1/18	.06
6	18/18	3.0	7/18	.39
7	18/18	2.7	10/18	.61
8	18/18	2.8	14/18	.94
9	18/18	2.5	16/18	1.2
10	17/18	2.3	18/18	1.6
11	18/18	2.1	18/18	2.1
12	17/18	1.6	18/18	2.2
13	15/18	1.3	18/18	2.8
14	14/18	1.1	18/18	2.9
15	10/18	.83	18/18	3.4
16	9/18	.67	18/18	3.6
17	4/10	.70	16/16	3.5
18	1/3	.33	4/4	3.5

Table 2. Mapping Lg4 to 8L with wx translocations. Numbers indicate the total number of progeny of the indicated genotype. Plants 378-3, 379-4, and 379-5 were heterozygous for Lg and the translocation indicated; family 358 was a homozygous wx tester line.

Cross	Translocation	Wx Lg	Wx +	wx Lg	wx +
378-3x358-3	8L.09; 9L.16	53	28	6i	32
379-4x358-L3	8L.35; 9S.31	31	18	nd	nd
379-5x358-L1	8L.35; 9S.31	79	17	9	45

Table 3. Confirming linkage to 8L with RFLPs. Linkage of Lg4 to 8L was confirmed using the RFLP loci UMC89 and UMC120. 27 Lg4 individuals from a family segregating for Lg4 and RFLPs from the W23 and Mo17Ht inbreds were scored for linkage using Southern analysis. Calculated map distances are given in the text.

Progeny Type	#
UMC120-W23 - Lg4 - UMC89-W23	20 (parental)
UMC120-Mo17 - Lg4 - UMC89-W23	6 (single recombinant)
UMC120-W23 - Lg4 - UMC89-Mo17	1 (single recombinant)
UMC120-Mo17 - Lg4 - UMC89-Mo17	0 (double recombinant)

Finally, Lg4 has been mapped to chromosome 8L. The mutation was placed on 8L by linkage to the waxy translocations T8-9d and T8-9(6673) and confirmed by RFLP analysis. Available data place Lg4 between UMC120 and UMC89, 22 + 2.2 map units from UMC120 and 3.7 + 0.98 map units from UMC89 (Tables 2 and 3). Mapping to conventional genetic markers on 8L is in progress.

Figure 1. Leaf expressing Lg4 in a W23 background. Note dispacement of ligule fringes into blade at the boundaries of the transformed tissue.

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