Periclinal chimeras (L1 gives rise to the epidermis, L2 the mesophyll and L3 the midvein and vasculature)
Clonal analysis has also been used to examine the growth dynamics of leaves by inducing albino sectors at different stages of leaf development. The cell lineages revealed provide information about the timing, location and extent of cell division within the leaf. Larger sectors indicate more extensive growth following sector induction. This analysis has been conducted for tobacco and maize. Both show similar patterns of leaf growth. The leaf can be traced to files of cells in the apex and each file gives rise to characteristic portions of the leaf. Files in the center of the leaf extend to the tip of the leaf while files on the sides are sequentially displaced to the margins. As the leaf grows, the tip is the first part to mature (cease dividing and differentiate) while active growth continues in the base of the leaf. Sectors also show that there are no marginal meristems but that cell divisions occur throughout the developing leaves.
In the process of organ identity determination, the source of the determining signal appears to be the meristem. If fern leaf primordia are separated from the meristem by the insertion of mica chips or removed and grown in culture, the primordia develop as shoots. As the primordia grow and mature, they become determined as leaves such that by P10, when the primordium is about 1mm long, they all formed leaves in culture. (Steeves, 1966).
One key to acquiring leaf identity appears to be turning off meristem genes (eg. KN1) and turning on differentiation genes (whatever they may be). The rough sheath2 gene of maize is important for this process. RS2 is expressed in the leaf primordia at the same time that KN1 is turned off and in rs2 mutants there is ectopic expression of KN1 in leaves, leading to severe growth deformations.
Leaves are bilaterally symmetrical with 3 axes: proximal-distal, lateral, and abaxial-adaxial (dorsoventral). Initially radial outgrowths become flattened in a plane tangential to the meristem at the point of attachment. Surgical incisions that separate incipient primordia from the meristem often result in determinate leaves with radial symmetry suggesting the apex is important for determining the dorsiventrality of leaves. Dorsiventrality consists of establishing the two leaf surfaces. The adaxial surface is the side adjacent to the leaf axil, where the axillary meristem is located. The abaxial surface is the back surface away from the plant axis (usually the lower leaf surface).
There are inconsistencies among experiments that study determination of organ identity and dorsiventrality. The fern leaf primordia explants grown in culture developed normal dorsiventrality with no contact with the meristem. In higher plants organ identity is determined earlier, by the time of leaf buttress formation, yet dorsiventrality is not yet established. There are yet other examples of developmental oddities such as a begonia that forms small leaves from the upper surface of its leaves. These secondary leaves have leaf identity and normal dorsiventrality and yet were never in contact with the meristem. The general conclusions are that determination of organ identity and bilateral symmetry occur separately, with offset timing in different plant groups, and that while the apical meristem has a clear influence on these processes, there are likely to be other factors also at work.
Several genes have been identified that are important for dorsiventrality. A number of mutants, such as phantastica in snapdragon, give a radially symmetrical leaf covered with just abaxial epidermal cell types. The vascular elements also show radial symmetry. Yabby genes such as FIL (Filamentous flower) are expressed in abaxial side of leaf. Ectopic expression causes abaxial cell types to form on adaxial leaf surface (Siefried, 1999).
The determination of regional identity within leaf primordia was studied by surgically removing portions of pea leaf primordia at various developmental stages. When portions were removed at the earliest visible stage of primordium protrusion, the primordium was able to regenerate the missing portions and form a normal leaf. When primordia were about 30mm long, there was incomplete regeneration of missing portions indicating that regions of the primordia were beginning to be determined to form certain regions of the leaf. When leaves were 1 plastochron old (about 70 mm long) there was no regeneration of missing portions indicating that the fate of different regions of the primordium to form certain parts of the leaf was fully determined by this time. Thus the regional identities were established in the primordium as it matured.
Examination of the narrow sheath mutant suggests that maize establishes domains of the leaf as founder cells are recruited in the SAM. In this mutant, the leaves are narrow because part of the lamina (blade) toward the margin is missing. Normal leaf primordia grow to encircle the SAM and there is a downregulation of KNOX genes (KNOTTED homeobox genes) in cells that participate in primordia formation (founder cells). In ns mutants the primordia only grow part way around the SAM and downregulation of KNOX genes does not completely encircle the SAM. Thus the marginal blade region of the leaf is established in the meristem before the primordium even grows out (Scanlon, 1996).
Simple vs. compound.
Some leaves just produce a single blade (simple leaves) while other leaves produce a central rachis with secondary leaflets (compound leaves) and higher orders of compounding are also possible. One key difference between simple and compound leaves appears to be that unlike simple leaves, compound leaves express KNOX genes in the leaf primordia. The highest levels of expression are seen in the leaflet primordia. Overexpression of KN1 and other KNOX genes causes extreme compounding of tomato leaves (Janssen et al, 1998).
Janssen BJ, Lund L, Sinha N (1998) Overexpression of a homeobox gene, LeT6, reveals indeterminate features in the tomato compound leaf. Plant Physiol 117: 771-786
Scanlon MJ, Schneeberger RG, Freeling M (1996) The maize mutant narrow sheath fails to establish leaf margin identity in a meristematic domain. Development 122: 1683-1691
Siefried et al (1999) Members of the YABBY gene family specify abaxial cell fate in Arabidopsis. Development 126:4117-28.