Leaves of plants are three-dimensionally designed for efficiently performing their functions. In grasses, sheath and blade are differentiated along proximal-distal axis, and ligule and auricle are formed at the boundary. In dicot species, petiole and blade differentiate along this axis. In the transverse direction, central, lateral and marginal regions are discriminated, and a large midrib occupies the central region in many grasses. These two leaf axes have been genetically investigated using available mutants in several species. On the other hand, as for the regulation of adaxial-abaxial pattern formation, almost nothing is known, although a few mutants have been reported (McConnell 1998, Timmermans 1998). Here, we describe two mutants affecting the adaxial-abaxial pattern of rice leaves.

We have identified two recessive mutants from M2 population of rice cv. Taichung 65 mutagenized with MNU. In the mutants, adaxial characteristics of leaf blade were also detected in the abaxial side. Since allelism test revealed that these mutations were nonallelic, they were designated adaxialized leaf 1 (adl1) and adaxialized leaf 2 (adl2), respectively.

Both adl1 and adl2 plants were dwarf, and their leaves were rolled in reverse direction (abaxial side became inside) and frequently twisted. In many traits, adl2 showed

more modified phenotypes than adl1. The adl1 plants set almost normal flowers, while adl2 plants died before the reproductive stage. In rice, adaxial epidermis of leaf blade is characterized by the presence of bulliform cells. In adl1 and adl2, bulliform cells were also observed in the abaxial epidermis, as well as in the adaxial side (Fig. 1). Thus, the epidermis of these mutants seems to be adaxialized. Abnormalities were also detected in the inner tissues. In normal leaf blade, mesophyll cells in the abaxial side are interrupted in the central-marginal direction by sclerenchymatous cells connecting vascular bundle and epidermis. In both adl1 and adl2, mesophyll cells occupied the space between small

vascular bundle and abaxial epidermis. However, it is not clear if the phenotype of mesophyll cells reflects adaxialization. Thus, a mutant in which abnormal phenotype is confined to either adaxial or abaxial mesophyll cells is needed. By screening of M2 population, we identified a mutant (TCM2380) in which adaxial side of leaf blade appeared white whereas abaxial side was normal green. Sectioning of fresh leaf blade showed that most of the adaxial mesophyll cells lacked chloroplasts, while all abaxial mosophyll cells were green. This indicates that chloroplast development is differently regulated between adaxial and abaxial mesophyll cells. Thus, the albino cells of this mutant can be used as adaxial marker of mesophyll. Then, we made TCM2380 adl2 double mutant for confirming the adaxialization of mesophyll cells in adl2 mutant. The double mutant showed albino cells distributed in both adaxial and abaxial sides of mesophyll, indicating that in adl2, mesophyll cells are adaxialized. Accordingly, adl is a mutation that causes the loss of abaxial identity and the gain of adaxial identity in abaxial leaf cells. Since the arrangement of xylem and phloem in each vascular bundle was normal, it appears that the polarity of vascular bundle is determined by the mechanism unrelated to adaxial-abaxial pattern of leaf.

Since leaf development is closely related to the organization of shoot apical meristem (SAM), we examined the SAM and leaf primordia in adl1 and adl2. In mature embryos, the SAM of adl mutants was flat. In the vegetative phase, a conspicuous abnormality was observed in the shoot apices of adl1 and adl2. In the adl mutants, the leaf primordia initiated at extremely distal position of the SAM compared to that of the wild type (Fig. 2). Then we made in situ hybridization experiment using a rice homeobox gene, OSH1, as a probe, which is expressed in indeterminate cells of the SAM but down-regulated in leaf founder cells. The expression pattern of OSH1 in adl1 was normal except the site of down-regulation. These results suggest that the adaxial-abaxial patterning of leaves may be closely associated with the position of leaf primordial initiation.

In conclusion, ADL genes are required for the specification of abaxial cell identity in leaf, and the loss of function mutation causes the adaxialization of leaf tissues.

References

McConnell J. R. and M. K. Barton, 1998. Leaf polarity and meristem formation in Arabidopsis. Development 125: 2935-2942.

Timmermans-Marja, C.P., N.P. Schultes, J.P. Jankovsky and T. Nelson, 1998. Leafbladeless1 is required for dorsoventrality of lateral organs in maize. Development 125: 2813-2823.