Cloning and characterization of the genes involved in cambial growth in response to elevated [CO2]

Aspen (Populus tremuloides) trees growing under elevated [CO2] at a free-air CO2 enrichment (FACE) site have produced significantly more biomass compared to control trees. The molecular mechanisms underlying the observed increase in biomass productivity was investigated by producing transcriptomic profiles of the vascular cambium zone (VCZ) and leaves, followed by a comparative study to identify genes and pathways that showed significant changes following long-term exposure to elevated [CO2]. This study is mainly to verify if genetic modification of a few selected candidate genes including CAP1, CKX6, and ASML2 that are expressed in vascular cambium in response to elevated [CO2] can cause the changes in plant growth and development. To this end, these three genes were cloned into both sense and antisense constructs. Then antisense and sense transgenic lines of above-mentioned genes were developed. 15 events were generated for 5 constructs, which were confirmed with regular PCR and RT-PCR. Confirmed plants were planted in greenhouse for growth and phenotypic characterization. The expression of CAP1, CKX6 and ASML2 in antisense plants was measured by real-time RT-PCR, and the changes caused by gene interference in cambial growth were studies by analyzing the microscopic sections made from the antisense transgenic plants. It has been found that 1) CAP1 is mainly expressed in xylem and root. 2) RNAi suppression of CAP1 significantly affected height and diameter. 3) CAP1, ASML2 and CKX6 affected xylem and phloem cell proliferation and elongation. Due to the delay in regenerating sense transgenic plants, the characterization of sense transgenic plants is limited to growth only.

Regulation of woody plant development by auxin and polar auxin transport

Auxin is a key regulator in plant growth and development. This dissertation examines the role of auxin and polar auxin transport in woody growth and development. Strategies of promoter reporter system, microarray expression analysis, transgenic modification, physiological assays, anatomical analysis, and histochemical/biochemical assays were employed to improve our understanding of auxin study in Populus. The results demonstrate various aspects of auxin regulation on shoot growth, root development, wood formation, and gravitropism in woody tissues. We describe the behavior of the DR5 reporter system for measuring auxin concentrations and response in stably transformed Populus trees. Our study shows that DR5 reporter system can be efficiently used in Populus to study auxin biology at a cellular resolution. We investigated the global gene expression in responding to auxin in Populus root. The results revealed groups of IBA up- and down- regulated genes involved in various biological processes including cell wall modification, root growth and lateral root formation, transporter activity and hormone crosstalk. We also verify two of the identified genes' function by transgenic modification in Populus, which encode auxin efflux carrier PtPIN9 and transcription factor PtERF72. We investigated the role of PtPIN9 in woody growth and development, especially in wood formation and gravitropic response in woody stem. We found that overexpressing PtPIN9 enhanced several growth parameters while suppression of PtPIN9 has inhibited tension wood formation. Our results show that PIN9 and other members from PIN family could be possible useful tools for increasing biomass productivity, wood quality, or in modifying plant form.