Functional analysis of Poplar genes regulating flowering and vegetative growth

Poplar (Populus spp. and hybrids) are used for pulp, paper and solid wood products. Furthermore, poplar is being developed as a dedicated biomass crop for biofuels and biomaterials. Thus, methods to accelerate genetic improvement to improve woody biomass yield, quality and optimal growth on marginal lands are of considerable interest. One approach is to identify genes that could be manipulated through breeding or biotechnology to achieve these goals. I studied two sets of candidate genes for improving biomass, growth and manipulating flowering time. First, I studied the functions of PopNAC154 and PopNAC156, co-orthologs of Arabidopsis SECONDARY CELL WALL NAC DOMAIN2 (SND2), which are putative regulators of wood cell wall synthesis, the source of lignocellulosic biomass. Second, I studied PopCEN1, PopCEN2, and PopBFT, members of the TERMINAL FLOWER1 (TFL1)/CENTRORADIALIS (CEN) gene family that act as flowering repressors in Arabidopsis and many plants.

I studied INRA 717-1B (P. tremula x P. alba) transgenics with an artificial microRNA (AmiRNA) downregulating PopNAC154 and PopNAC156 (AmiSND2 trees). In a field trial, AmiSND2 trees showed higher mean height and diameter than wild-type (WT). We also observed that AmiSND2 transgenics showed delayed leaf senescence and leaf drop. After conducting controlled environment studies with AmiSND2 trees, I was able to confirm that downregulation of PopNAC154 and PopNAC156 genes does not alter the short daylength-induced bud set and growth cessation but it delays the low temperature induced leaf senescence and leaf drop. Further I was able to show that down regulation of the PopNAC154 and PopNAC156 genes resulted in significantly higher mean plant heights and delayed bud set compared to the WT plants under low soil nutrient conditions. Wood chemistry data analysis of field grown AmiSND2 trees showed that they have a significantly higher cellulose content a lower lignin content compared to that of the WT. Thus, these results show that downregulating the PopNAC154 and PopNAC156 genes has the potential to increase biomass yield and quality.

In a previous study, simultaneous downregulation of PopCEN1 and PopCEN2 genes using RNA interference (RNAi) method caused poplar trees to flower only after two years of growth in the field. I used CRISPR/CAS9 method to knock-out each paralog individually as well as the related gene, PopBFT. The popcen1 mutant trees developed flowers even under in vitro conditions, but popcen2 mutants did not show an obvious phenotype. popbft mutant trees also did not show an obvious phenotype under standard growing conditions. However, when soil nutrient availability was allowed to deplete, the popbft mutants showed lower mean plant height compared to the WT and also showed lower root length and root volume under low Nitrogen conditions in an in vitro assay compared to the WT. These results prove that PopCEN1 gene is directly involved in repressing flowering in poplar and allele-specific mutation should be tested as an approach to accelerate breeding. PopCEN2 and PopBFT might not have a role in regulation of flowering time, and though additional studies are needed, PopBFT appears to have a role in regulating growth in response to nutrient availability.