Production of dwarf lettuce by overexpressing a pumpkin gibberellin 20-oxidase gene

Abstract

We investigated the effect of overexpressing a pumpkin gibberellin (GA) 20-oxidase gene encoding an enzyme that forms predominantly biologically inactive products on GA biosynthesis and plant morphology in transgenic lettuce (Lactuca sativa cv Vanguard) plants. Lettuce was transformed with the pumpkin GA 20-oxidase gene downstream of a strong constitutive promoter cassette (El2-35S-Omega). The transgenic plants in which the pumpkin gene was detected by polymerase chain reaction were dwarfed in the T(2) generation, whereas transformants with a normal growth phenotype did not contain the transgene. The result of Southern-blot analysis showed that the transgene was integrated as a single copy; the plants segregated three dwarfs to one normal in the T(2) generation, indicating that the transgene was stable and dominant. The endogenous levels of GA(1) and GA(4) were reduced in the dwarfs, whereas large amounts of GA(17) and GA(25), which are inactive products of the pumpkin GA 20-oxidase, accumulated in these lines. These results indicate that a functional pumpkin GA 20-oxidase is expressed in the transgenic lettuce, resulting in a diversion of the normal pathway of GA biosynthesis to inactive products. Furthermore, this technique may be useful for controlling plant stature in other agricultural and horticultural species.

Figure 1

Structure of T-DNA region of pSGΩ. Pnos, 5′-upstream region of nopaline synthase gene. NPT-II, Coding region of nopaline synthase gene. Tnos, Polyadenylation region of nopaline synthase gene. El2, 5′-upstream region of CaMV 35S promoter (−419 to −90) × 2. P35S, 5′-upstream region of CaMV promoter (−90 to −1). Ω, 5′-Untranslated region of tobacco mosaic virus.

Figure 2

Southern-blot analysis of transgene in SGΩ-4 plants. Genomic DNA extracted from wild-type or SGΩ-4 plants was digested with EcoRI, HindIII, or XbaI, respectively, and 10 μg of digested DNA was loaded per lane. An EcoRI-digested PCR fragment of the pumpkin GA 20-oxidase gene, including a 3′-specific region (368 bp), was used as a probe.

Figure 3

Northern-blot analysis of transgene expression. Total RNA was extracted from mature leaves of wild-type and SGΩ-4 plants, and 15 μg of total RNA per lane was blotted onto a membrane. Probe for hybridization was as described in Figure 2.

Figure 4

Morphological changes in SGΩ-4 plants. A, Seedlings (3 weeks after sowing) of wild-type (right) and SGΩ-4 plants (normal, center and dwarf type, left). B, Mature plants (wild type, right. SGΩ-4 plants: normal, center and dwarf type, left).

Figure 5

Inhibitory effect of UCZ treatment on plant growth of wild-type lettuce plants. Seedlings of lettuce plants (dwarf type of SGΩ-4, left and wild type, center-left, center-right, and right) were treated with 0.001% (w/v, center-right) and 0.01% (w/v, right) or without (left and center-left) UCZ. Each concentration of 10 mL of UCZ solution was added to the soil. The plants were photographed at 1 month after the treatment.

Figure 6

GA biosynthesis pathway showing reactions catalyzed by GA 20-oxidase (GA₅₃ to GA₂₀ and GA₁₂ to GA₉) and 3β-hydroxylase (GA₂₀ to GA₁ and GA₉ to GA₄). Conversion of GA₁₇ from GA₁₉ and GA₂₅ from GA₂₄, catalyzed by the pumpkin GA 20-oxidase, are shown with heavy arrows.