| Aerenchyma formation in maize roots has been characterized morphologically, physiologically and chemically, but not genetically. The first step is gene identification. To that end, RNA was extracted from the primary root tips (1 cm) of Zea mays L. cv TX 5855 seedlings that were mechanically impeded for 1 h at 100 kPa, from root tips of seedlings that were untreated, and from the root tips of seedlings that were hypoxically treated (4% O2) for 2 d. RNA pools extracted from mechanically impeded and untreated roots were compared by differential display. Seven differential display fragments were induced by hypoxia and one by mechanical impedance. Sequence homology of these fragments identified two classes of retroelements, two unknowns, tRNA-leucyl synthetase, a transmembrane phosphatase, and a 60 S ribosomal protein (L23A). Degenerate-primer RT-PCR was used to clone an ACC synthase-like cDNA and a cellulase cDNA. A cDNA library was constructed from RNA from hypoxically-treated primary root tips. The library was probed with differential display fragments, RT-PCR cDNAs and heterologous probes. Genes previously associated with apoptosis were discovered. They include homologs to a serine protease inhibitor induced by treatment with tunicamycin, a hypoxically-induced tonoplast integral protein (TIP), defender against apoptotic cell death ( Dad1), seven in absentia homolog (Siah) and a cysteine protease. A unique gene, Granddad, was induced by tunicamycin treatment and submergence. The Granddad transcript has 2 reading frames. The first frame encodes a probable G protein-coupled receptor. The second frame may be an unspliced Dad, with 3 introns and a small sequence repeat (SSR). Granddad was re-cloned by RT-PCR, and a partial genomic clone confirmed the sequence. Other cDNAs were also sequenced and tested including an ACC oxidase encoding fragment and a beta-glucosidase cDNA. The expression patterns and sequence homology of these genes are consistent with cell death programs in plants and with apoptosis in animals. Based on these findings and prior evidence it was concluded that aerenchyma, formation in maize roots is a form of programmed cell death. These findings suggest that programmed cell death in plants is analogous to apoptosis in animals. |
