Decreased GA1 content caused by the overexpression of OSH1 is accompanied by suppression of GA 20-oxidase gene expression

Abstract

We previously reported that overexpression of the rice homeobox gene OSH1 led to altered morphology and hormone levels in transgenic tobacco (Nicotiana tabacum L.) plants. Among the hormones whose levels were changed, GA1 was dramatically reduced. Here we report the results of our analysis on the regulatory mechanism(s) of OSH1 on GA metabolism. GA53 and GA20, precursors of GA1, were applied separately to transgenic tobacco plants exhibiting severely changed morphology due to overexpression of OSH1. Only treatment with the end product of GA 20-oxidase, GA20, resulted in a striking promotion of stem elongation in transgenic tobacco plants. The internal GA1 and GA20 contents in OSH1-transformed tobacco were dramatically reduced compared with those of wild-type plants, whereas the level of GA19, a mid-product of GA 20-oxidase, was 25% of the wild-type level. We have isolated a cDNA encoding a putative tobacco GA 20-oxidase, which is mainly expressed in vegetative stem tissue. RNA-blot analysis revealed that GA 20-oxidase gene expression was suppressed in stem tissue of OSH1-transformed tobacco plants. Based on these results, we conclude that overexpression of OSH1 causes a reduction of the level of GA1 by suppressing GA 20-oxidase expression.

Figure 1

Severe-phenotype transgenic tobacco plants expressing OSH1.

Figure 2

Stem elongation of severe-phenotype transgenic tobacco plants treated with GA₅₃ or GA₂₀. Ten microliters of a 10 or 100 μm solution of GA₅₃ or GA₂₀ in 5% acetone was applied to the shoot apex of severe-phenotype transformants once a week.

Figure 3

Levels of GA₁₉, GA₂₀, and GA₁ in leaves of wild-type and severe-phenotype OSH1-transformed tobacco plants. fw, Fresh weight.

Figure 4

Nucleotide and deduced amino acid sequences of tobacco GA 20-oxidase cDNA. Regions corresponding to the degenerate primers used in PCR amplification are underlined.

Figure 5

RNA-blot analysis of GA 20-oxidase gene expression. ³²P-Labeled GA 20-oxidase cDNA or XbaI/SacI fragment of OSH1 cDNA was hybridized with 10 μg of total RNA extracted from mature leaves, vegetative stem, developed flowers, and developing siliques (a), and stem tissue treated without (−) or with (+) 100 μm GA₃ for 8 h (b), and stem tissue of wild-type (left) and severe-phenotype OSH1-transformed (right) tobacco plants (c).