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.

Several isoforms of superoxide dismutase (SOD) with a high isoelectric point (pI) have been identified by isoelectric focusing chromatography in protein extracts from Scots pine (Pinus sylvestris) needles. One of these isoforms, a CuZn-SOD with a pI of about 10 and thus denoted hipI-SOD, has been isolated and purified to apparent homogeneity. A cDNA encoding the hipI-SOD protein was cloned and sequenced. Northern hybridization of mRNA isolated from different organs and tissues showed that hipI-SOD has a markedly different pattern of expression compared with chloroplastic and cytosolic SOD. Furthermore, the transcript levels of hipI-SOD and cytosolic SOD were found to respond differently to mechanical wounding, treatment with oxidized glutathione, paraquat, and ozone. Immunogold electron microscopy localized the hipI-SOD in the plasma membrane of sieve cells and the Golgi apparatus of albuminous cells. Moreover, high protein density was also detected in extracellular spaces such as secondary cell wall thickenings of the xylem and sclerenchyma and in intercellular spaces of parenchyma cells.

A special form of a CuZn-superoxide dismutase with a high isoelectric point (hipI-SOD; EC 1.15.1.1) and hydrogen peroxide (H2O2) production were studied during the secondary cell wall formation of the inducible tracheary element cell-culture system of Zinnia elegans L. Confocal microscopy after labelling with 2',7'-dichlorofluorescin diacetate showed H2O2 to be located largely in the secondary cell walls in developing tracheary elements. Fluorescence-activated cell sorting analysis showed there were lower levels of H2O2 in the population containing tracheary elements when H2O2 scavengers such as ascorbate, catalase, and reduced glutathione were applied to the cell culture. Inhibitors of NADPH oxidase and SOD also reduced the amount of H2O2 in the tracheary elements. Furthermore, addition of these compounds to cell cultures at the time of tracheary element initiation reduced the amount of lignin and the development of the secondary cell walls. Analysis of UV excitation under a confocal laser scanning microscope confirmed these results. The expression of hipI-SOD increased as the number of tracheary elements in the cell culture increased and developed. Additionally, immunolocalization of a hipI-SOD isoform during the tracheary element differentiation showed a developmental build-up of the protein in the Golgi apparatus and the secondary cell wall. These findings suggest a novel hipI-SOD could be involved in the regulation of H2O2 required for the development of the secondary cell walls of tracheary elements.

SummaryHigh-resolution, magic angle spinning, proton nuclear magnetic resonance (