Culture-independent assessment of Rhizobiales-related alphaproteobacteria and the diversity of Methylobacterium in the rhizosphere and rhizoplane of transgenic eucalyptus.

The rhizosphere is an ecosystem exploited by a variety of organisms involved in plant health and environmental sustainability. Abiotic factors influence microorganism-plant interactions, but the microbial community is also affected by expression of heterologous genes from host plants. In the present work, we assessed the community shifts of Alphaproteobacteria phylogenetically related to the Rhizobiales order (Rhizobiales-like community) in rhizoplane and rhizosphere soils of wild-type and transgenic eucalyptus. A greenhouse experiment was performed and the bacterial communities associated with two wild-type (WT17 and WT18) and four transgenic (TR-9, TR-15, TR-22, and TR-23) eucalyptus plant lines were evaluated. The culture-independent approach consisted of the quantification, by real-time polymerase chain reaction (PCR), of a targeted subset of Alphaproteobacteria and the assessment of its diversity using PCR-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Real-time quantification revealed a lesser density of the targeted community in TR-9 and TR-15 plants and diversity analysis by principal components analysis, based on PCR-DGGE, revealed differences between bacterial communities, not only between transgenic and nontransgenic plants, but also among wild-type plants. The comparison between clone libraries obtained from the transgenic plant TR-15 and wild-type WT17 revealed distinct bacterial communities associated with these plants. In addition, a culturable approach was used to quantify the Methylobacterium spp. in the samples where the identification of isolates, based on 16S rRNA gene sequences, showed similarities to the species Methylobacterium nodulans, Methylobacterium isbiliense, Methylobacterium variable, Methylobacterium fujisawaense, and Methylobacterium radiotolerans. Colonies classified into this genus were not isolated from the rhizosphere but brought in culture from rhizoplane samples, except for one line of the transgenic plants (TR-15). In general, the data suggested that, in most cases, shifts in bacterial communities due to cultivation of transgenic plants are similar to those observed when different wild-type cultivars are compared, although shifts directly correlated to transgenic plant cultivation may be found.

SAGE transcript profiling of the juvenile cambial region of Eucalyptus grandis.

Despite the importance of Eucalyptus spp. in the pulp and paper industry, functional genomic approaches have only recently been applied to understand wood formation in this genus. We attempted to establish a global view of gene expression in the juvenile cambial region of Eucalyptus grandis Hill ex Maiden. The expression profile was obtained from serial analysis of gene expression (SAGE) library data produced from 3- and 6-year-old trees. Fourteen-base expressed sequence tags (ESTs) were searched against public Eucalyptus ESTs and annotated with GenBank. Altogether 43,304 tags were generated producing 3066 unigenes with three or more copies each, 445 with a putative identity, 215 with unknown function and 2406 without an EST match. The expression profile of the juvenile cambial region revealed the presence of highly frequent transcripts related to general metabolism and energy metabolism, cellular processes, transport, structural components and information pathways. We made a quantitative analysis of a large number of genes involved in the biosynthesis of cellulose, pectin, hemicellulose and lignin. Our findings provide insight into the expression of functionally related genes involved in juvenile wood formation in young fast-growing E. grandis trees.

Proteomic analysis of the cambial region in juvenile Eucalyptus grandis at three ages.

Recent advances in genomics and proteomics have provided an excellent opportunity to understand complex biological processes such as wood formation at the gene and protein levels. The aim of this work was to describe the proteins participating in the processes involved in juvenile wood formation by isolating proteins from the cambial region of Eucalyptus grandis, at three ages of growth (6-month-old seedlings, 3- and 6-year-old trees), and also to identify proteins differentially expressed. Using a 2-D-LC-MS/MS strategy we identified a total of 240 proteins, with 54 corresponding spots being present in at least two ages. Overall, nine proteins classified into the functional categories of metabolism, cellular processes, and macromolecular metabolism showed significant changes in expression. Proteins were classified into seven main functional categories, with metabolism representing 35.2% of the total proteins identified. The comparison of the reference maps showed not only differences in the expression pattern of individual proteins at each age, but also among isoforms. The results described in this paper provide a dynamic view of the proteins involved in the formation of juvenile wood in E. grandis.

Production of transgenic Eucalyptus grandis x E. urophylla using the sonication-assisted Agrobacterium transformation (SAAT) system.

A method for genetic transformation of germinating seeds and seedlings of Eucalyptus grandis × E. urophyllais described using the sonication-assisted Agrobacterium-mediated transformation (SAAT) system. Seeds germinated for 2 d, and 15-d-old seedlings, sonicated for 30 s, had the highest percentage of ß-glucuronidase (GUS) transient expression (21.7 and 37.4%, respectively). Pre-sonication greatly enhanced the efficiency of transformation. The differential transformation of tissues was also investigated, with seeds imbibed for 2 d having over 90% of the blue sectors localised in cotyledons and in the intersection of the hypocotyls and roots, whereas in 5-d-old seedlings, 70% of GUS activity was detected in cotyledons. However, 15-17-d-old seedlings had around 60% of transformed sectors localised in the first pair of leaves. The efficiency of the method was also assessed using a chimeric construct containing the Lhcb1*2 gene of the 28 kDa chlorophyll a/b binding pea protein from the LHCII antenna. Four stable transformants were confirmed by genomic blotting.