Green revolution trees: semidwarfism transgenes modify gibberellins, promote root growth, enhance morphological diversity, and reduce competitiveness in hybrid poplar.

Semidwarfism has been used extensively in row crops and horticulture to promote yield, reduce lodging, and improve harvest index, and it might have similar benefits for trees for short-rotation forestry or energy plantations, reclamation, phytoremediation, or other applications. We studied the effects of the dominant semidwarfism transgenes GA Insensitive (GAI) and Repressor of GAI-Like, which affect gibberellin (GA) action, and the GA catabolic gene, GA 2-oxidase, in nursery beds and in 2-year-old high-density stands of hybrid poplar (Populus tremula × Populus alba). Twenty-nine traits were analyzed, including measures of growth, morphology, and physiology. Endogenous GA levels were modified in most transgenic events; GA(20) and GA(8), in particular, had strong inverse associations with tree height. Nearly all measured traits varied significantly among genotypes, and several traits interacted with planting density, including aboveground biomass, root-shoot ratio, root fraction, branch angle, and crown depth. Semidwarfism promoted biomass allocation to roots over shoots and substantially increased rooting efficiency with most genes tested. The increased root proportion and increased leaf chlorophyll levels were associated with changes in leaf carbon isotope discrimination, indicating altered water use efficiency. Semidwarf trees had dramatically reduced growth when in direct competition with wild-type trees, supporting the hypothesis that semidwarfism genes could be effective tools to mitigate the spread of exotic, hybrid, and transgenic plants in wild and feral populations.

Populus CEN/TFL1 regulates first onset of flowering, axillary meristem identity and dormancy release in Populus.

Members of the CENTRORADIALIS (CEN)/TERMINAL FLOWER 1 (TFL1) subfamily control shoot meristem identity, and loss-of-function mutations in both monopodial and sympodial herbaceous plants result in dramatic changes in plant architecture. We studied the degree of conservation between herbaceous and woody perennial plants in shoot system regulation by overexpression and RNA interference (RNAi)-mediated suppression of poplar orthologs of CEN, and the related gene MOTHER OF FT AND TFL 1 (MFT). Field study of transgenic poplars (Populus spp.) for over 6 years showed that downregulation of PopCEN1 and its close paralog, PopCEN2, accelerated the onset of mature tree characteristics, including age of first flowering, number of inflorescences and proportion of short shoots. Surprisingly, terminal vegetative meristems remained indeterminate in PopCEN1-RNAi trees, suggesting the possibility that florigen signals are transported to axillary mersitems rather than the shoot apex. However, the axillary inflorescences (catkins) of PopCEN1-RNAi trees contained fewer flowers than did wild-type catkins, suggesting a possible role in maintaining the indeterminacy of the inflorescence apex. Expression of PopCEN1 was significantly correlated with delayed spring bud flush in multiple years, and in controlled environment experiments, 35S::PopCEN1 and RNAi transgenics required different chilling times to release dormancy. Considered together, these results indicate that PopCEN1/PopCEN2 help to integrate shoot developmental transitions that recur during each seasonal cycle with the age-related changes that occur over years of growth.

Precocious flowering in trees: the FLOWERING LOCUS T gene as a research and breeding tool in Populus.

Expression of FLOWERING LOCUS T (FT) and its homologues has been shown to accelerate the onset of flowering in a number of plant species, including poplar (Populus spp.). The application of FT should be of particular use in forest trees, as it could greatly accelerate and enable new kinds of breeding and research. Recent evidence showing the extent to which FT is effective in promoting flowering in trees is discussed, and its effectiveness in poplar is reported. Results using one FT gene from Arabidopsis and two from poplar, all driven by a heat-inducible promoter, transformed into two poplar genotypes are also described. Substantial variation in flowering response was observed depending on the FT gene and genetic background. Heat-induced plants shorter than 30 cm failed to flower as well as taller plants. Plants exposed to daily heat treatments lasting 3 weeks tended to produce fewer abnormal flowers than those in heat treatments of shorter durations; increasing the inductive temperature from 37 degrees C to 40 degrees C produced similar benefits. Using optimal induction conditions, approximately 90% of transgenic plants could be induced to flower. When induced FT rootstocks were grafted with scions that lacked FT, flowering was only observed in rootstocks. The results suggest that a considerable amount of species- or genotype-specific adaptation will be required to develop FT into a reliable means for shortening the generation cycle for breeding in poplar.

RNAi of AGAMOUS genes in sweetgum alters reproductive organ identity and decreases fruit persistence.

Sweetgums (Liquidambar), members of the family Altingiaceae (Altingiales), have inflorescences and floral organs that are distinctive in structure compared with other angiosperms in which the roles of floral homeotic genes have been studied. To begin to understand the role of AGAMOUS (AG)-a floral homeotic gene that has a major role in stamen and carpel development-in development of the monosexual flowers of sweetgum, we used RNAi to reduce the expression of two members of the AG subfamily. Because AG suppression should induce floral sterility, RNAi might also provide a tool to mitigate the risks of invasiveness-and to reduce the production of its nuisance fruits or allergenic pollen-when sweetgum is used as an exotic shade or forest tree. We tested 33 independent transgenic events and non-transgenic controls during 10 years in the field. The RNAi-AG sweetgum trees maintained normal growth, phenology, and vivid fall coloration during the 10 years of study, but 8 insertion events had highly modified inflorescence and floral morphology. The modified flowers had anthers and carpels that were converted to flat leaf-like structures lacking pollen grains and ovules, respectively. The female inflorescences developed into dry papery structures that failed to produce seeds. These infructescences were smaller than control infructescences, and lost a greater percentage of biomass in a controlled decay assay. RNAi against AG genes was highly effective at impairing fertility and modifying reproductive development without significant vegetative effects in sweetgum and gave phenotypes distinct from, but similar to, that of AG loss of function in other angiosperms.

Efficiency of gene silencing in Arabidopsis: direct inverted repeats vs. transitive RNAi vectors.

We investigated the efficiency of RNA interference (RNAi) in Arabidopsis using transitive and homologous inverted repeat (hIR) vectors. hIR constructs carry self-complementary intron-spliced fragments of the target gene whereas transitive vectors have the target sequence fragment adjacent to an intron-spliced, inverted repeat of heterologous origin. Both transitive and hIR constructs facilitated specific and heritable silencing in the three genes studied (AP1, ETTIN and TTG1). Both types of vectors produced a phenotypic series that phenocopied reduction of function mutants for the respective target gene. The hIR yielded up to fourfold higher proportions of events with strongly manifested reduction of function phenotypes compared to transitive RNAi. We further investigated the efficiency and potential off-target effects of AP1 silencing by both types of vectors using genome-scale microarrays and quantitative RT-PCR. The depletion of AP1 transcripts coincided with reduction of function phenotypic changes among both hIR and transitive lines and also showed similar expression patterns among differentially regulated genes. We did not detect significant silencing directed against homologous potential off-target genes when constructs were designed with minimal sequence similarity. Both hIR and transitive methods are useful tools in plant biotechnology and genomics. The choice of vector will depend on specific objectives such as cloning throughput, number of events and degree of suppression required.