Activation tagging of a dominant gibberellin catabolism gene (GA 2-oxidase) from poplar that regulates tree stature

Abstract

We identified a dwarf transgenic hybrid poplar (Populus tremula x Populus alba) after screening of 627 independent activation-tagged transgenic lines in tissue culture, greenhouse, and field environments. The cause of the phenotype was a hyperactivated gene encoding GA 2-oxidase (GA2ox), the major gibberellin (GA) catabolic enzyme in plants. The mutation resulted from insertion of a strong transcriptional enhancer near the transcription start site. Overexpression of the poplar GA2ox gene (PtaGA2ox1) caused hyperaccumulation of mRNA transcripts, quantitative shifts in the spectrum of GAs, and similarity in phenotype to transgenic poplars that overexpress a bean (Phaseolus coccineus) GA2ox gene. The poplar PtaGA2ox1 sequence was most closely related to PsGA2ox2 from pea (Pisum sativum) and two poorly known GA2oxs from Arabidopsis (AtGA2ox4 and AtGA2ox5). The dwarf phenotype was reversible through gibberellic acid application to the shoot apex. Transgenic approaches to producing semidwarf trees for use in arboriculture, horticulture, and forestry could have significant economic and environmental benefits, including altered fiber and fruit production, greater ease of management, and reduced risk of spread in wild populations.

Figure 1.

Wild-type (WT) and mutant (stumpy) plants at the same age and grown under the same conditions. Stumpy is on the left-hand side of each frame; WT is on the right. A, Tree size after 14 months of growth in a greenhouse. B, Short internodes of stumpy. C, Dark foliage of stumpy.

Figure 2.

Map of the EcoRI-rescued activation tagging plasmid from stumpy (A) and PtaGA2ox1 accumulation in stumpy and WT poplars (B).

Figure 3.

Alignment (A) and phylogenetic tree (B) of putative poplar PtaGA2ox1 with GA2oxs from Arabidopsis, rice, pea, bean, wild cucumber, and lettuce. Sequences were aligned using ClustalW (http://www.ebi.ac.uk/clustalw/), and the output was produced using BoxShade (http://www.ch.embnet.org/software/BOX_form.html). Black shading indicates identity, and gray shading indicates similarity, of amino acid residues. Phylogenetic analysis was performed using MEGA2 software (http://www.megasoftware.net/). Sites containing alignment gaps were excluded from further analysis, and the distance between sequences represent the proportion of amino acid sites at which the two sequences compared are different. The unrooted tree was constructed using the neighbor-joining method. The bootstrap percentage indicated at each joint point was created from 1,000 data samples. Nodes with less than 50% bootstrap confidence were collapsed.

Figure 4.

Reversion of mutant phenotype in greenhouse-grown plants by application of GA₃. The photograph represents a typical response observed in five experimental plants in each of the GA and control treatments.

Figure 5.

Overexpression of bean GA 2-oxidase (PcGA2ox1, accession no. AJ132438) causes stumpy-like phenotype. Photographs represent plants grown for 1 month in tissue culture under the same conditions. Lower and upper tiers represent the same plants photographed from above and sideways.