Figure 4. Ectopic expression of CO1 and CO2 individually (Pro_35S:CO1 or Pro_35S:CO2) or together (Pro_35S:CO1/CO2) in poplar (P. tremula × P alba).

(A) When compared with controls at age 5, Pro_35S:CO1 or Pro_35S:CO2 trees did not differ in reproductive onset, spring reproductive and vegetative bud break, and fall bud set. Pro_35S:FT2 trees showed year-round active growth. Red arrows denote the emerging inflorescence in the spring, whereas black arrows point the dormant terminal vegetative bud in the fall. Unlike wild-type and vector controls, Pro_35S:CO1 or Pro_35S:CO2 trees (1, 2, and 3) significantly overproduced CO1 or CO2 transcripts when analyzed via qRT-PCR in leaves sampled in April. While the expression of FT1 was undetectable, that of FT2 fluctuated with no clear trend in controls and CO1- or CO2-overexpressing trees. Letters above the bars showing the abundance of CO1 or CO2 transcripts indicate statistically significant (P≤0.001) differences. Error bars indicate SD about the mean. (B) When Pro_35S:CO1 and Pro_35S:CO2 were co-expressed in the same trees, no difference between the transformants and controls was observed in spring bud break and fall bud set in two years. However, Pro_35S:FT2 trees showed a non-dormant phenotype. Black arrows indicate the terminal vegetative bud, whereas purple arrows point to the axillary vegetative bud. The axillary vegetative buds were opening and preformed leaves were emerging from the control and co-expressing transgenic trees on March 23. Unlike wild-type and vector-control plants, co-expressing transgenic trees (1, 2, 3, and 4) significantly overproduced CO1 and CO2 transcripts in leaves sampled in April. While the expression of FT1 was undetectable, that of FT2 fluctuated with no clear trend in controls and CO1/CO2 overexpressing trees. Letters above the bars showing the abundance of CO1 or CO2 transcripts indicate statistically significant (P≤0.001) differences. Error bars indicate SD about the mean.