A tobacco homologue of the Ri-plasmid orf13 gene causes cell proliferation in carrot root discs.

The tobacco genome contains genes, called cellular rol (c-rol) genes, that are very similar in sequence to genes present in the T-DNA of the Agrobacterium rhizogenes Ri-plasmid. We have cloned two homologues (torf13-1 and torf13-2) of the Ri-plasmid orf13 gene from Nicotiana tabacum L. cv. Havana 425. The clone torf13-1 has a 594-bp open reading frame (ORF) which is similar in sequence (77-82% for DNA and 67-77% for the deduced amino acid sequence) to orf13 genes of the agropine, mikimopine, and mannopine Ri-plasmids and the N. glauca homologue Ngorf13. Southern analyses showed that there are at least two torf13 genes derived from the N. tomentosiformis ancestor of tobacco, strongly suggesting that torf13 resulted from an ancient transfer between ancestors of modern A. rhizogenes and tobacco. Steady-state expression of torf13 mRNA is high in sepals, petals, shoot tips and in younger leaves, but considerably lower in stem tissues, lower leaves and roots. Treatment of cultured leaf discs for 5-20 days on medium containing auxin (10.7 microM alpha-naphthaleneacetic acid) and cytokinin (1.4 microM kinetin) resulted in a marked down-regulation of torf13 mRNA accumulation. Therefore, torf13 is transcriptionally active in normal tobacco tissues and the steady-state mRNA level is regulated. Inoculation of carrot-root discs with A. tumefaciens strains carrying the mannopine Ri-plasmid orf13 and torf13-1 regulated by the strong cauliflower mosaic virus 35S RNA promoter induced the formation of dense green callus on the disc surface. These findings indicate that at least one function of the orf13 ORF is conserved in the tobacco homologue, and provide direct evidence that a c-rol gene can influence cell proliferation.

Class I [beta]-1,3-Glucanases in the Endosperm of Tobacco during Germination.

Rupture of the seed coat and rupture of the endosperm are separate events in the germination of Nicotiana tabacum L. cv Havana 425 seeds. Treatment with 10-5 M abscisic acid (ABA) did not appreciably affect seed-coat rupture but greatly delayed subsequent endosperm rupture by more than 100 h and resulted in the formation of a novel structure consisting of the enlarging radicle with a sheath of greatly elongated endosperm tissue. Therefore, ABA appears to act primarily by delaying endosperm rupture and radicle emergence. Measurements of [beta]-1,3-glucanase activity, antigen content, and mRNA accumulation together with reporter gene experiments showed that induction of class I [beta]-1,3-glucanase genes begins just prior to the onset of endosperm rupture but after the completion of seed-coat rupture. This induction was localized exclusively in the micropylar region of the endosperm, where the radicle will penetrate. ABA treatment markedly inhibited the rate of [beta]-1,3-glucanase accumulation but did not delay the onset of induction. Independent of the ABA concentration used, onset of endosperm rupture was correlated with the same [beta]-1,3-glucanase content/seed. These results suggest that ABA-sensitive class I [beta]-1,3-glucanases promote radicle penetration of the endosperm, which is a key limiting step in tobacco seed germination.

Developmental, hormonal, and pathogenesis-related regulation of the tobacco class I beta-1,3-glucanase B promoter.

The class I beta-1,3-glucanases are antifungal vacuolar proteins implicated in plant defense that show developmental, hormonal, and pathogenesis-related regulation. The tobacco enzymes are encoded by a small gene family with members derived from ancestors related to the present-day species Nicotiana sylvestris and N. tomentosiformis. We studied the expression in transgenic tobacco plants of a chimeric beta-glucuronidase (GUS) reporter gene fused to 1.6 kb of upstream sequence of the tobacco class I beta-1,3-glucanase B (GLB) gene, which is of N. tomentosiformis origin. Expression of the GUS reporter gene and the accumulation of class I beta-1,3-glucanase and its mRNA showed very similar patterns of regulation. In young seedlings the reporter gene was expressed in the roots. In mature tobacco plants it was preferentially expressed in lower leaves and roots and was induced in leaves by ethylene treatment and by infection with tobacco mosaic virus (TMV). Furthermore, it was down-regulated in cultured leaf discs by combinations of the hormones auxin and cytokinin. Histological studies of GUS activity showed that the GLB promoter shows highly localized expression in roots of seedlings. It is also expressed in a ring of cells around necrotic lesions induced by TMV infection, but not in cells immediately adjacent to the lesions or in the lesions themselves. The results of deletion analyses suggest that multiple positive and negative elements in the GLB promoter regulate its activity. The region from -1452 to -1193 containing two copies of the heptanucleotide AGCCGCC, which is highly conserved in plant-stress and defense-related genes, is necessary for high level expression in leaves. Additional regions important for organ-specific and regulated expression were: -568 to -402 for ethylene induction of leaves; -402 to -211 for expression in lower leaves and cultured leaf discs and for TMV induction of leaves; and -211 to -60 for expression in roots.