Transgenic Poplar Plants for the Investigation of ABA-Dependent Salt and Drought Stress Adaptation in Trees

Important functions of the plant hormone abscisic acid (ABA) in stress reactions, growth and photosynthetic processes are extensively studied in the model plant Arabidopsis thaliana. This paper investigates the importance of Moco-sulphurase ABA3 and aldehyde oxidase (AO) on ABA-biosynthesis in Populus × canescens. ABA3 is essential for activation of the molybdenum enzymes AO and xanthine dehydrogenase (XDH). AO itself catalyzes the last step in ABA-biosynthesis. Generation of transgenic poplar plants altered in ABA3 and AO-activity using RNAi knock down and overexpression was performed. Whereas RNAi-AO plants show a specific loss of AO activity, the RNAi-ABA3 plants has a strongly reduced activity of both molybdenum enzymes: AO and XDH. Constructs of AO and ABA3-promoters fused to β-glucuronidase provide the basis to investigate transcriptional regulation of ABA-biosynthetic processes under stress conditions. Application of high salt concentrations and different drought stress intensities does change the endogenous AO or XDH neither on the side of transcription nor on protein activity. On phytohormone level however, water loss leads to increased ABA-amounts regardless of whether transgenic or wildtype plants are studied. Salt application resulted in higher ABA-levels in all analyzed plant lines. The down regulation of AO in the two different RNAi-plant lines strongly prevented a wildtype-like increase of ABA-levels. Whereas the WT plants accumulated up to 6000 ng ABA g-1 FW-1 after 16 h of salt stress exposure, plants of the RNAi lines revealed a markedly lower increase of only up to 2000 ng ABA g-1 FW-1. Opposing to these observations, ABA-levels increased during drought without any influence by the RNAi-effect. These results revealed that although stresses did not result in a visible increased AO-activity, ABA-production was influenced by AO and ABA3 at least under salinity.

Temporal and spatial changes in Bt toxin expression in Bt-transgenic poplar and insect resistance in field tests

Extensive planting of Bacillus thuringiensis （Bt）-transgenic plants economically benefits society; how-ever, the potential risk they pose is receiving increasing attention. This study used enzyme-linked immunosorbent assay and fluorescence quantitative PCR （RT-PCR） to monitor the temporal and spatial dynamics of the expression of Bt toxic protein in a forest of 6- to 8-year-old trees of transgenic insect-resistant poplar 741 for three consec- utive years. The enrichment, distribution, and degradation of Bt toxic protein and the influence of transgenic poplars on the targeted insect population, Hyphantria cunea, were investigated. The content of CrylAc toxic protein dynamically changed in transgenic poplar. During the annual growth cycle, the content initially increased, then decreased in the long and the short branches of the crown and in the root system, peaking in August. During the study, the protein did not accumulate overtime. The mRNA transcription of gene CrylAc was almost consistent with the level of the protein, but transcription peaked in July. In the transgenic and control forestland, microscale levels of the CrylAc toxic protein were detected from the soil, but increased accumulation was not observed with the planting year of transgenic poplar. Meanwhile, Bt was isolated and detected molecularly from the soil in the experimental forestland. A systematic investigation of the density of H. cunea in the experimental transgenic poplar forest indi- cated that transgenic Pb29 poplar could resist insects to a certain degree. At peak occurrence of the targeted insects, the density of H. cunea in the experimental forest was significantly lower than in the nontransgenic poplar forest.

Assessment of Rhizospheric Microorganisms of Transgenic Populus tomentosa with Cowpea Trypsin Inhibitor (CpTI) Gene

To have a preliminary insight into biosafety of genetically transformed hybrid triploid poplars （Populus tomentosa × P bolleana）× P. tomentosa with the cowpea trypsin inhibitor （CpTD gene, two layers of rhizospheric soil （from 0 to 20cm deep and from 20 to 40cm deep, respectively） were collected for microorganism culture, counting assay and PCR analysis to assess the potential impact of transgenic poplars on non-target microorganism population and transgene dispersal. When the same soil layer of suspension stock solution was diluted at both 1：1000 and 1：10000 rates, there were no significant differences in bacterium colony numbers between the inoculation plates of both transgenic and non-transgenic poplars. The uniform results were revealed for both soil layer suspension solutions of identical poplars at both dilution rates except for non-transgenic poplars at 1：10000 dilution rates from the same type of soil. No significant variation in morphology of both Gram-positive and Gram-negative bacteria was observed under the microscope. The potential transgene dispersal from root exudates or fallen leaves to non-target microbes was repudiated by PCR analysis, in which no CpTI gene specific DNA band was amplified for 15 sites of transgenic rhizospheric soil samples. It can be concluded that transgenic poplar with the CpTI gene has no severe impact on rhizospheric microorganisms and is tentatively safe to surrounding soil micro-ecosystem.

Loosening Xyloglucan Accelerates the Enzymatic Degradation of Cellulose in Wood

In order to create trees in which cellulose, the most abundant component in biomass, can be enzymatically hydrolyzed highly for the production of bioethanol, we examined the saccharification of xylem from several transgenic poplars, each overexpressing either xyloglucanase, cellulase, xylanase, or galactanase. The level of cellulose degradation achieved by a cellulase preparation was markedly greater in the xylem overexpressing xyloglucanase and much greater in the xylems overexpressing xylanase and cellulase than in the xylem of the wild-type plant. Although a high degree of degradation occurred in all xylems at all loci, the crystalline region of the cellulose microfibrUs was highly degraded in the xylem overexpressing xyloglucanase. Since the complex between microfibrils and xyloglucans could be one region that is particularly resistant to cellulose degradation, loosening xyloglucan could facilitate the enzymatic hydrolysis of cellulose in wood.