Yale University
Cold Spring Harbor Laboratory

Leafbladeless1 is allelic to raggedseedling1 and is required for dorsal cell identity
--Timmermans, M; Schultes, N; Martienssen, R; Nelson, T

The leafbladeless1 (lbl1) mutation was isolated by Donald Miles from selfed Mu-active stocks. A preliminary characterization of the lbl1 mutant phenotype (Miles, MNL 63:66-67, 1989) revealed that homozygous lbl1 plants showed a complete or partial loss of leaf lamina development. In the most extreme phenotype, leaves formed no lamina tissue but developed as radial, thread-like organs. In the weaker phenotype, leaf lamina development varied and leaves often bifurcated along the midrib. In order to investigate further the lbl1 defect in leaf initiation and development, lbl1 and wild type sibling plants were examined using SEM and histological techniques. The adaxial/dorsal epidermis of wild type leaves can be distinguished by the presence of bulliform cells and macrohairs. SEM of severely affected, radial lbl1 leaves showed that the epidermis of such leaves lacks bulliform cells and macrohairs and appeared abaxial/ventral in nature. Transverse sections through wild type leaf blade illustrate the dorsoventral asymmetry of maize leaves by the adaxial positioning of lamina relative to the midrib and of vascular xylem relative to phloem, as well as by the presence of the relatively larger bulliform cells in the dorsal epidermis. Transverse sections through the distal blade regions of radial lbl1 leaves showed a complete loss of asymmetry and a lack of bulliform cells. Such lbl1 leaves appeared to consist of a central, irregular vascular cylinder surrounded by concentric rings of bundle sheath, mesophyll, and ventral epidermis.

These observations suggest that the loss of lamina development in lbl1 plants could result from a defect in the establishment of dorsal cell identity. Recently, a similar mutation has been described in Antirrhinum (Waites, and Hudson, Development 121:2143-2154, 1995). Because lbl1 is a recessive mutation, the ventral nature of radial lbl1 leaves indicates that dorsal cell identity is imposed on the primordia. In the absence of LBL1 activity, the epidermis adopts a ventral identity.

Consistent with the idea that Lbl1 is required for dorsal cell identity, adaxial ectopic margins were found on weakly phenotypic lbl1 leaves. Loss of Lbl1 function later in leaf development would result in patches of ventral cells adjacent to dorsal cells. Because marginal outgrowth occurs at the boundary between dorsal and ventral cell types, patches of ventral cells on the dorsal side of the leaf result in the formation of extra margins.
Dorsoventrality of the maize leaf is in part established during the recruitment of founder cells into the primordia. SEM and transverse sections of apices of lbl1 plants showed a strong reduction in primordial leaf width as compared to normal sibs, consistent with a role for Lbl1 in founder cell recruitment.

As noted above, lbl1 leaves often bifurcate. SEM of lbl1 shoot apices showed the bifurcation of leaf primordia as early as plastochron stage 3. Such bifurcated primordia appear to establish multiple proximodistal axes, each determining its own sheath/blade boundary. In addition, a single leaf was found that had developed a midrib in each half leaf, presumably the result of bifurcation prior to midrib differentiation. If the establishment of dorsoventrality is required for leaf initiation and outgrowth, patchy Lbl1 expression may result in the formation of multiple proximodistal axes per plastochron.

In many respects the lbl1 phenotype resembles the phenotype of ragged seedling1 (rgd1) (Kramer, MNL 31:120-121, 1957). Preliminary observation indicated linkage to white endosperm (y1) (6L), and further RFLP linkage analysis placed lbl1 on chromosome arm 6S near rgd1 (Fig. 1). Allelism tests have confirmed that lbl1 is an allele of rgd1.

Figure 1. Chromosomal location of lbl1 and rgd1. The genetic map was based upon information presented in the MNL of 1993 and 1996; numbers indicate map positions. The RFLP linkage map was obtained using lbl1/lbl1 individuals of selfed progeny from outcrosses of lbl1/+ siblings to B73. Numbers in this case represent map distances in cM 

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