Early event in maize leaf epidermis formation as revealed by cell lineage studies

Sergio Cerioli 1 Adriano Marocco 1 Massimo Maddaloni 0 Mario Motto 0 Francesco Salamini 0 0 Istituto Sperimentale per la Cerealicoltura , I-24100 Bergamo , Italy 1 Istituto di Botanica e Genetica Vegetale, Universita Cattolica del S. Cuore , I-29100 Piacenza , Italy SUMMARY The epidermal cells of the juvenile leaves of maize are covered by a wax layer. glossy mutants are known which reduce drastically wax deposition. We have used the somatically unstable glossy-1 mutable 8 allele to study the distribution on the epidermis of spontaneous revertant sectors of wild-type tissues. Sectors tend to start and end at positions that correlate with the location on the epidermis of the long costal cells of ribs. It is concluded that in the protoderm only a few cells have a role and position in the generation of each of the developmental modules located between leaf midrib and margin. The module consists of an The epidermis of the grass leaf is a single cell layer organized as longitudinally oriented parallel rows of cells that are elongated in the direction base to leaf tip. Starting from the primordium, the cells of the epidermal layer divide according to planes that allow the epidermis to grow in the transverse direction, via longitudinal anticlinal divisions, or in the longitudinal direction, via transverse anticlinal divisions. Both anatomical studies (Esau, 1977) and the type of leaf sectoring reported in grass species (Tilney-Basset, 1986; Klekowski, 1988) support the view that longitudinal anticlinal divisions are mainly restricted to early stages of leaf development. The organization of cells in longitudinal rows is typical also for the internal layers of the leaf. In maize, for example, the leaf is divided into longitudinal units by parallel veins located in the mesophyll layer (Sharman, 1942; Esau, 1943; Russell and Evert, 1985; reviewed by Langdale et al., 1989 and Freeling, 1992). Veins have been classified as mid, lateral, intermediate and small (Sharman, 1942). They derive from the central layer of the leaf primordium (Langdale et al., 1989). The adaxial and the abaxial epidermis and the middle mesophyll layer show coordinate development (Freeling, 1992; Freeling and Lane, 1993): cells with a particular shape are present on the epidermis in positions corresponding to the location of veins. In short, when maize leaves are viewed from above, vein positions are marked on the epidermis by costal epidermal strip of cells bordered by two lateral ribs. The module originates from at least 4 cells, with one cell being the progenitor of the other three. Data are provided describing the mode of longitudinal anticlinal epidermal cell divisions within the module that are responsible for the increase in leaf width. The results suggest the existence of a clonal type of development during early leaf epidermis formation. long cells (Freeling and Lane, 1993), which constitute the structures known as ribs. The epidermis of the young maize shoot is covered by the juvenile wax layer (Salamini, 1963). Mutations mapping to at least 8 different genetic loci modify the shape and drastically reduce the wax layer (summarized by Bianchi et al., 1985). Wax extrusion is cell autonomous, as is evident from the variegation pattern of leaves of somatically unstable mutants (Maddaloni et al., 1990). Mutable alleles of the glossy-1 locus revert both somatically and germinally to the wild-type phenotype (Maddaloni et al., 1990; Bossinger et al., 1992). We have used the mutant glossy-1 mutable 8 (gl1-m8) to study the width and distribution of revertant sectors. The leaf epidermis is quite suited to such analyses because it originates from a single meristematic layer (Sharman, 1942), and because the start and end of revertant sectors can be defined with respect to landmark signals represented by vein-rib boundaries. When using transposon-induced sectors in cell lineage analysis, a requisite is that the excision of the transposon is not developmentally regulated. To avoid this, only sectors that originate in the apical meristem before leaf promordia are formed should be studied. The inception of leaf primordia, in fact, induces differentiation between cells. Steffensen (1968) has shown that sectors with a size from 5 to 34% of the midrib to margin space fully comparable with those studied by us always appear in more than one leaf, and are assumed to have been generated by cells present on the shoulder of the meristem before leaf primordia inception. Additional proof that in our system excisions are random in position is given by Maddaloni et al. (1990), who noted that single cell sectors were spread randomly over the epidermal surface, while sectors as large as those studied in this paper appeared in more than one leaf, as was the case for the sectors described by Steffensen (1968). Three developmental problems are addressed. The first concerns the existence of leaf epidermal compartments. The concept of compartment (Garcia-Bellido and Merriam, 1971; Garcia-Bellido et al., 1973; 1976) refers to the observation that cellular clones do not cross a line that defines the border between morphologically distinct domains. It is accepted that epidermal segments of Drosophila are subdivided into compartments, developmental units expressing a specific set of homeotic genes (Brower, 1985; Lawrence and Morata, 1993). In our system candidates for compartment boundaries are the epidermal ribs. The second question addresses the possibility of recognizing the number of cells that have a founder role during the early development of the leaf epidermis. This role can be clarified because of the particular position these cells occupy with respect to the ribs in the primordia or in adult leaves; as founders they generate groups of cells that are clonally related. Thirdly, we have attempted to gain information on the polarity of longitudinal anticlinal cell divisions needed to add, in the transverse direction of the leaf, cells to leaf width. Starting with one cell the problem is to establish which out of several models of cell division (see Fig. 1), fits best the distribution and width of revertant sectors observed. MATERIALS AND METHODS The maize mutant gl1-m8 was isolated in an attempt to tag the Glossy1 locus by transposon mutagenesis (Maddaloni et al., 1990). The stock waxy mutable 7 (wx-m7), where an active copy of the activator (Ac) transposon (McClintock, 1951) is inserted into the Wx gene, was the transposon donor. The allele gl1-m8, however, was not generated by the insertion of Ac but, nevertheless, it behaved autonomously, i.e. another self-excising element was present at the locus (Maddaloni et al., 1990). The gl1-m8 mutant is characterized by its somatic instability: reversions to the wild-type phenotype (longitudinal sectors) are present on both the leaf sheath and blade (Bossinger et al., 1992). Four hundred seedlings of the gl1-m8 strain were grown at 25C under natural light conditions supplement (...truncated)