Branching control mechanisms in the model tree Populus: analyzing the role of strigolactones and BRANCHED1

Plants exhibit a large degree of phenotypic plasticity. Modifications of their genetically pre-defined body plan allow them to flexibly react to a wide range of environmental conditions. This includes changes in plant architecture, which are facilitated by the modular composition of the shoot. In the leaf axils of the primary stem, axillary buds are formed. Each of these buds has the potential to grow into a secondary stem, i.e. a branch. However, bud outgrowth is restricted and most buds are kept in a dormant state. To make the decision whether a bud is released from dormancy and grows into a branch, many endo- and exogenous factors are integrated in a complex network of hormones and transcription factors. This includes strigolactones (SLs), a novel class of phytohormones, which generally suppress bud outgrowth. The inhibitory effect of SLs is discussed to be mediated by flux modulation of the phytohormone auxin and/or regulation of other downstream targets directly within the bud. The most prominent example for a bud-specific SL-regulated gene is BRANCHED1 (BRC1), whose transcript levels are positively influenced by SLs. It encodes a transcription factor which represses bud outgrowth, most likely by regulating cell cycling. SLs and BRC1 were extensively studied in model species such as Arabidopsis (Arabidopsis thaliana), pea (Pisum sativum), petunia (Petunia hybrida) and rice (Oryza sativa). In contrast, our knowledge of the genes and pathways in woody perennial species, such as the model tree poplar (Populus sp.), is limited. In this project, poplar orthologs of genes involved in SL biosynthesis (MAX4) and SL signaling (MAX2) were identified to investigate an anticipated role for SLs in controlling tree architecture. There are two orthologs each in poplar. To study their function, expression analysis was performed and transgenic lines were generated for amiRNA-mediated knockdowns of the individual orthologs, as well as simultaneous silencing of both. MAX2 knockdowns were only partially successful and no phenotype could be observed, most likely due to a redundant function of the non-silenced ortholog. In contrast, MAX4 double knockdowns were successful and typical SL-deficiency phenotypes were observed in the corresponding amiMAX4-1+2 lines. This includes highly increased shoot branching, reduced plant height, reduced internode length and increased adventitious rooting. Direct quantification of SLs generally is difficult due to their low abundance, high instability and large diversity. Furthermore, standards and references for poplar SLs are not available, making measurements not feasible. Indirect evidence for SL-deficiency in amiMAX4-1+2 plants was gathered instead, including successful complementation of the shoot phenotypes by grafting. Tree-specific aspects of bud dormancy, especially winter dormancy, were also addressed. However, an influence of SLs could not be shown, indicating that SLs only appear to suppress bud outgrowth during the vegetative period. As a downstream target of SLs and, therefore, another important component of branching control, a poplar BRC1 ortholog was identified. This gene exhibited the typical expression patterns reported for other species and a significant down-regulation in the putatively SL-deficient amiMAX4-1+2 lines. In addition, a poplar BRC2 ortholog was found based on sequence and expression analysis. Both genes may control branching in poplar, integrating different environmental factors. Taken together, the data generated in this study supports a role for SLs and BRC1 as important regulators of bud outgrowth in poplar. The findings underline the high degree of conservation of fundamental processes involved in the control of plant architecture among a range of species, including trees. Beside of being a useful tool for discovering the role of SLs and BRC1 in poplar, the highly branching lines generated in this project may be economically valuable for the use on short rotation coppices, where they may exhibit improved re-sprouting and canopy closure after coppicing.