MYB transcription factors are differentially expressed and regulated during secondary vascular tissue development in hybrid aspen.

More than 120,000 poplar ESTs have been sequenced from 20 different cDNA libraries by the Swedish Centre for Tree Functional Genomics. We screened this EST collection for MYB transcription factors involved in secondary vascular tissue formation, and genes assigned as PttMYB3Ra, PttMYB4a and PttMYB21a were selected for further characterisation. Three MYB genes showed different expression patterns in various organs, tissues and stem sub-sections representing different developmental stages of vascular tissue formation. Furthermore, the analysis showed that PttMYB21a expression was much higher in secondary cell wall formation zone of xylem and phloem fibers than in other developmental zones. Transgenic hybrid aspen plants, expressing the 3'-part of the PttMYB21a gene in antisense orientation were generated to assess the function of PttMYB21a gene in vascular tissue formation and lignification. All transgenic lines showed reduced growth and had fewer internodes compared to the wild-type. The analysis of selected lines showed that acid soluble lignin present in the bark was higher in transgenic lines as compared to wild-type plants. Moreover a higher transcript level of caffeoyl-CoA 3-O-methyltransferase [CCoAOMT]; EC 2.1.1.104) was found in the phloem of the transgenic plants, suggesting that PttMYB21a might function as a transcriptional repressor.

Characterization of a PttRPS18 promoter active in the vascular cambium region of hybrid aspen.

By screening 273 hybrid aspen plants transformed with a luciferase-based promoter trap T-DNA vector, one plant was found in which the reporter gene (luxF2) was activated only in cells of the cambial region, i.e., vascular cambium, phloem and differentiating xylem. Southern blot analysis revealed that this plant denoted #24 had a single T-DNA insert. The chromosomal regions flanking the T-DNA were cloned by plasmid rescue. A 757 bp DNA fragment, originating from the rescued plasmid and covering the genomic region immediately upstream from the right-border sequence of the T-DNA, was used as a probe to isolate the corresponding chromosomal region from a wild-type hybrid aspen genomic library. A hybrid aspen small ribosomal protein gene, PttRPS18, was then isolated. By screening a wt cambial region-specific cDNA library, two cDNA clones encoding a putative 152 amino acid PttRPS18 protein were isolated. Comparison of the DNA sequence immediately flanking the T-DNA insert in #24 with the corresponding wild-type sequence showed that only a minor deletion occurred during the T-DNA integration. Northern analysis revealed that the PttRPS18 gene was expressed mainly in the cambial region. By RT-PCR and DNA sequencing analysis, the exact structures of the PttRPS18 and luxF2 transcripts were determined. Finally, the hybrid aspen PttRPS18 promoter was fused to the uidA reporter gene and transformed into hybrid aspen plants. Histochemical analysis of GUS activity showed that the PttRPS18 promoter was expressed in the cambial region in the same manner as the luciferase reporter gene in the initial screening.

Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen.

Indole acetic acid (IAA/auxin) profoundly affects wood formation but the molecular mechanism of auxin action in this process remains poorly understood. We have cloned cDNAs for eight members of the Aux/IAA gene family from hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) that encode potential mediators of the auxin signal transduction pathway. These genes designated as PttIAA1-PttIAA8 are auxin inducible but differ in their requirement of de novo protein synthesis for auxin induction. The auxin induction of the PttIAA genes is also developmentally controlled as evidenced by the loss of their auxin inducibility during leaf maturation. The PttIAA genes are differentially expressed in the cell types of a developmental gradient comprising the wood-forming tissues. Interestingly, the expression of the PttIAA genes is downregulated during transition of the active cambium into dormancy, a process in which meristematic cells of the cambium lose their sensitivity to auxin. Auxin-regulated developmental reprogramming of wood formation during the induction of tension wood is accompanied by changes in the expression of PttIAA genes. The distinct tissue-specific expression patterns of the auxin inducible PttIAA genes in the cambial region together with the change in expression during dormancy transition and tension wood formation suggest a role for these genes in mediating cambial responses to auxin and xylem development.