All of the above ground parts of plants develop from an indeterminate group of cells, the shoot apical meristem (SAM). The SAM is formed during embryogenesis, and after seed germination it continuously generates various organs and tissues, such as leaves, stems and flowers, throughout the life of the plant. The SAM is not only the ultimate source of new tissues and organs of the above ground part of plants, but also maintains and replenishes itself (Steeves & Sussex 1989).
Previous morphological studies indicated that various angiosperms share some structural features in the internal organization of the SAM (Steeves & Sussex 1989). The SAM can be divided into three histologically distinct zones, the central zone, the peripheral zone and the rib zone, based on the cell size and the arrangement of the cell divisions (Esau 1977; Lyndon 1990; Steeves & Sussex 1989). The central zone, occupying the centre of the SAM, consists of relatively large cells that result from slower cell divisions in this region, and is thought to act as a source of new undifferentiated cells to replace those that are gradually lost from other zones of the SAM. The peripheral zone is located in the flanking regions of the SAM and consists of smaller, more rapidly dividing cells. Lateral organs such as leaf primordia and axillary buds are generated within this zone. Finally, the rib zone, which is located at the base of the other two zones, gives rise to the pith of the stem.
SAM is also organized into a few fundamental cell layers (Schmidt 1924). The tunica consists of one to three cell layers and covers the underlying corpus. This tunica/corpus structure is a consequence of differences in the orientation of cell division in different parts of the SAM. Cell divisions within the tunica are ordinarily restricted to the anticlinal plane, thus maintaining a layered arrangement of the cells. Within the corpus, the plane of cell division is more variable. Despite extensive knowledge of the complex structural organization of the SAM, very little is known about the molecular mechanisms underlying meristem function in such processes as lateral organ formation.
Many homeobox genes have now been cloned from various plant species. Comparative analysis of the homeodomains and other conserved motifs of the plant homeobox genes has revealed that the genes fall into several classes (Kerstetter et al. 1994; Lincoln et al. 1994; Vollbrecht et al. 1991). These classes include the kn1-type (HD-kn1), the homeodomain leucine zipper (HD-ZIP: Ruberti et al. 1991; Schena & Davis 1992), the plant homeodomain finger (PHD-finger: Bellmann & Werr 1992; Korfhage et al. 1994; Schindler et al. 1993), the GLABRA2-like homeodomain (HD-GL2: Lu et al. 1996; Rerie et al. 1994), and the BELL1-like homeodomain proteins (HD-BEL1: Quaedvlieg et al. 1995; Reiser et al. 1995). The most extensively characterized gene family is the kn1-type, all members of which share homeodomain sequences similar to that of the maize knotted1 gene (kn1), the first homeobox gene to be identified in plants (Vollbrecht et al. 1991). Based on sequence comparisons, the members of this family fall into two subclasses, class 1 and 2 (Kerstetter et al. 1994). The class 1 genes show higher similarity to kn1 in the homeodomain and are primarily expressed in the SAM and developing stem, whereas the class 2 genes have lower similarity to kn1 in the homeodomain and are expressed in most organs (Kerstetter et al. 1994). Class 1 genes, such as maize kn1 (Sinha et al. 1993; Vollbrecht et al. 1991), tobacco NTH15 (Tamaoki et al. 1997a), and Arabidopsis KNAT1 (Lincoln et al. 1994), show strong expression within the SAM. Ectopic expression of class 1 genes in spontaneous mutants, or in transgenic plants carrying copies of the genes expressed from the cauliflower mosaic virus 35S promoter, causes dramatic alterations in leaf and flower morphology. Furthermore, mutant plants with recessive alleles of the Arabidopsis SHOOT MERISTEMLESS (STM) gene, another member of the class 1 gene family, fail to develop a SAM during embryogenesis (Long et al. 1996). These findings strongly suggest that the class 1 kn1-type genes are responsible for meristem maintenance and/or lateral organ formation from the SAM (Hake et al. 1995).
During organ differentiation in animal embryos, families of homeobox genes with similar structures are often involved in a series of related developmental processes (Gehring 1987; Ingham 1988; McGinnis & Krumlauf 1992). In plants, by analogy, a battery of class 1 kn1-type homeobox genes may cooperate in meristem maintenance and/or lateral organ formation from the SAM. Indeed, it has been reported that the maize class 1 kn1-type homeobox genes comprise a small multi-gene family and that these genes are highly expressed in the shoot apex (Jackson et al. 1994; Kerstetter et al. 1994). With this in mind, we attempted to isolate as many different class 1 homeobox genes from tobacco as possible, and then used in situ hybridization and GUS histochemical staining of transgenic tobacco to compare their expression patterns within the SAM.