The fdl (fused leaves) mutation affects shoot apex organization and seedling growth

--Adamo, A, Dall’Aglio, C, Dolfini, S, Gavazzi, G, Consonni, G

Maize embryogenesis results in the formation of a massive cotiledonary structure, the scutellum, and a well differentiated embryonic axis, comprising the root and the shoot apex. The coleoptilar ring starts from a ridge on the face of the scutellum encircling the shoot apex, and consists of a sheathing structure surrounding the stem tip and the embryonic leaves, except for a small pore. At germination, the plumule and the coleoptile elongate. Initially the coleoptile grows faster than the plumule, but when it reaches the surface of the soil and is thus exposed to light, it stops growing and the plumule is extruded from the tip.

The fused leaves (fdl) mutant was isolated from an active Spm line by Dr. Jane Langdale (Oxford University), and behaves as a monogenic recessive mutant. The main effects of the mutation are detectable starting from the plumule emergence to the four leaves stage. Homozygous fdl seedlings are retarded in their germination and growth when compared to wild-type siblings. In addition, the first two mutant leaves appear curly, as a result of their growth while still enclosed within the coleoptile. Opening of the mutant coleoptile occurs with an irregular lateral fracture in contrast to the clear-cut hole formed in the wild-type. Regions of fusion between the coleoptile and the first leaf and, occasionally, between the first and the second leaf are also observed. At the emergence of the fourth leaf, the plant acquires a normal phenotype with expanded leaves, thus appearing indistinguishable from wild-type plants. Homozygous mutant plants can be grown to maturity and selfed.

A histological analysis was performed to find out if embryo development is also affected. We examined transversal sections of mutant and wild-type shoot apex at 14 dap (days after pollination). Interestingly in the mutant embryo, a file of coleoptilar cells occupied an ectopic position, invading the central region of the shoot apex, which is normally occupied by the leaf primordia. Accordingly, in embryo rescue tests, excised 12 dap embryos transferred to an MS medium, yielded the expected fdl phenotype.

We also investigated the effects of endogenous auxin application (IAA) on development of immature wild-type and mutant embryos (12 dap). Embryos were excised from segregating ears and transferred to MS medium or to MS medium containing IAA. IAA did not show a significant effect on wild-type shoot and root growth, whereas in the fdl mutants an increase in both seedling and primary root elongation was observed, particularly evident in the primary root. Wild-type and mutant embryos were cultivated on media containing the synthetic auxin 2,4-D. The fdl mutant phenotype can be mimicked in wild-type embryos grown in the presence of 2,4-D, but no phenotypic effects were detected in mutant embryos.

Taken together these observations suggest that the mutation has a pleiotropic effect on both embryo and seedling growth. fdl has been located, by SSR analysis, on the long arm of chromosome 7 between umc1342 and umc1125. Molecular genetic analysis indicates that the mutation is caused by insertion of a Spm element in the fdl locus. Efforts aimed at cloning the gene are underway.