Comparative and reverse genetic analysis of the cytokinin response regulator gene family in Populus

Cytokinins are important plant hormones that influence a diverse array of physiological and developmental processes such as root and shoot morphogenesis. However, little is known with respect to whether or how cytokinin action is connected to biomass distribution in any forest tree. A better understanding of the molecular mechanisms by which cytokinin signaling is connected to specific traits can provide the genetic tools to improve agronomic characteristics (e.g., stem wood production per unit root biomass). Cytokinin signaling resembles two-component systems from bacteria and yeast in which an external signal is sensed by a histidine kinase (HK) and then transferred to a response regulator (RR). Because of their ability to activate transcription and regulate protein activity, RRs have been proposed to coordinate most of physiological processes regulated by cytokinin.;I identified, annotated and characterized at the transcript level 11 type-As, 11 type-Bs and 11 pseudo cytokinin response regulators (RRs) in Populus balsamifera ssp. trichocarpa (Torr. and Gray) genotype Nisqually-1. Developmental and cytokinin-responsive expression of the Populus RRs indicate that while the type-As and type-Bs are preferentially expressed in nodes, pseudo-RRs are preferentially expressed in mature leaves.;Next I investigated the in vivo role of a particular Populus RR using a reverse genetic approach. Transgenic lines with ectopic expression of a constitutively active form of PtRR13 (Delta DDKPtRR13) were found to exhibit a delay in rootability during propagation. Microarray analysis in non transgenic (NT) plants evidenced a massive transcriptome remodeling during the 24 h following excision with approximately 30% of the nuclear genes differentially regulated. During this time gene networks involved in wound and stress responses showed significant regulation while genes with potential roles in root morphogenesis were significantly regulated later during the 24 to 48 hour interval. Misregulated genes in Delta DDKPtRR13 included COV1, a negative regulator of vascularization; PDR9, an auxin transporter; two genes with sequence similarity to TINY1; and BELL1, encoding a homeodomain protein. I also observed a time point-specific influence of Delta DDKPtRR13 expression on the transcriptome at 24 h where 273 genes were differentially regulated.;Results obtained show organ-preferred expression patterns of Populus RRs, suggesting possible roles for the type-As and type-Bs in development and pseudo-RRs in integration of environmental signals with plant function. I confirmed the negative role of cytokinin action in root developmental processes previously hypothesized in other plant systems, and obtained direct evidence that links a specific type RR (PtRR13) with inhibition of adventitious root formation. I propose that the inhibitory effects of PtRR13 on adventitious rooting are manifest physiologically, as reflected by transcriptome shifts, 24 h after shoot excision. This defines a discrete time frame during which cytokinin may act in adventitious root formation in vivo. Finally, putative direct and indirect targets of the constitutively active PtRR13 transcription factor imply that cross-talk between cytokinin, auxin and ethylene are important during adventitious rooting in cuttings.