Phenolic compounds and expression of 4CL genes in silver birch clones and Pt4CL1a lines.

A small multigene family encodes 4-coumarate:CoA ligases (4CLs) catalyzing the CoA ligation of hydroxycinnamic acids, a branch point step directing metabolites to a flavonoid or monolignol pathway. In the present study, we examined the effect of antisense Populus tremuloides 4CL (Pt4CL1) to the lignin and soluble phenolic compound composition of silver birch (Betula pendula) Pt4CL1a lines in comparison with non-transgenic silver birch clones. The endogenous expression of silver birch 4CL genes was recorded in the stems and leaves and also in leaves that were mechanically injured. In one of the transgenic Pt4CL1a lines, the ratio of syringyl (S) and guaiacyl (G) lignin units was increased. Moreover, the transcript levels of putative silver birch 4CL gene (Bp4CL1) were reduced and contents of cinnamic acid derivatives altered. In the other two Pt4CL1a lines changes were detected in the level of individual phenolic compounds. However, considerable variation was found in the transcript levels of silver birch 4CLs as well as in the concentration of phenolic compounds among the transgenic lines and non-transgenic clones. Wounding induced the expression of Bp4CL1 and Bp4CL2 in leaves in all clones and transgenic lines, whereas the transcript levels of Bp4CL3 and Bp4CL4 remained unchanged. Moreover, minor changes were detected in the concentrations of phenolic compounds caused by wounding. As an overall trend the wounding decreased the flavonoid content in silver birches and increased the content of soluble condensed tannins. The results indicate that by reducing the Bp4CL1 transcript levels lignin composition could be modified. However, the alterations found among the Pt4CL1a lines and the non-transgenic clones were within the natural variation of silver birches, as shown in the present study by the clonal differences in the transcripts levels of 4CL genes, soluble phenolic compounds and condensed tannins.

Phenolic compounds in ectomycorrhizal interaction of lignin modified silver birch.

BACKGROUND: The monolignol biosynthetic pathway interconnects with the biosynthesis of other secondary phenolic metabolites, such as cinnamic acid derivatives, flavonoids and condensed tannins. The objective of this study is to evaluate whether genetic modification of the monolignol pathway in silver birch (Betula pendula Roth.) would alter the metabolism of these phenolic compounds and how such alterations, if exist, would affect the ectomycorrhizal symbiosis. RESULTS: Silver birch lines expressing quaking aspen (Populus tremuloides L.) caffeate/5-hydroxyferulate O-methyltransferase (PtCOMT) under the 35S cauliflower mosaic virus (CaMV) promoter showed a reduction in the relative expression of a putative silver birch COMT (BpCOMT) gene and, consequently, a decrease in the lignin syringyl/guaiacyl composition ratio. Alterations were also detected in concentrations of certain phenolic compounds. All PtCOMT silver birch lines produced normal ectomycorrhizas with the ectomycorrhizal fungus Paxillus involutus (Batsch: Fr.), and the formation of symbiosis enhanced the growth of the transgenic plants. CONCLUSION: The down-regulation of BpCOMT in the 35S-PtCOMT lines caused a reduction in the syringyl/guaiacyl ratio of lignin, but no significant effect was seen in the composition or quantity of phenolic compounds that would have been caused by the expression of PtCOMT under the 35S or UbB1 promoter. Moreover, the detected alterations in the composition of lignin and secondary phenolic compounds had no effect on the interaction between silver birch and P. involutus.

The seasonal activity and the effect of mechanical bending and wounding on the PtCOMT promoter in Betula pendula Roth.

In this study, 900-bp (signed as p including nucleotides -1 to -886) and partly deleted (signed as dp including nucleotides -1 to -414) COMT (caffeate/5-hydroxyferulate O-methyltransferase) promoters from Populus tremuloides Michx. were fused to the GUS reporter gene, and the tissue-specific expression patterns of the promoters were determined in Betula pendula Roth along the growing season, and as a response to mechanical bending and wounding. The main activity of the PtCOMTp- and PtCOMTdp-promoters, determined by the histochemical GUS assay, was found in the developing xylem of stems during the 8th-13th week and in the developing xylem of roots in the 13th week of the growing season. The GUS expression patterns did not differ among the xylem cell types. The PtCOMT promoter-induced GUS expression observed in phloem fibres suggests a need for PtCOMT expression and thus syringyl (S) lignin synthesis in fibre lignification. However, the PtCOMTdp-promoter induced GUS expression in stem trichomes, which may contribute to the biosynthesis of phenylpropanoid pathway-derived compounds other than lignin. Finally, a strong GUS expression was induced by the PtCOMT promoters in response to mechanical stem bending but not to wounding. The lack of major differences between the PtCOMTp- and PtCOMTdp-promoters suggests that the deleted promoter sequence (including nucleotides -415 to -886) did not contain a significant regulatory element contributing to the GUS expression in young B. pendula trees.

Does lignin modification affect feeding preference or growth performance of insect herbivores in transgenic silver birch (Betula pendula Roth)?

Transgenic silver birch (Betula pendula Roth) lines were produced in order to modify lignin biosynthesis. These lines carry COMT (caffeate/5-hydroxyferulate O-methyltransferase) gene from Populus tremuloides driven by constitutive promoter 35S CaMV (cauliflower mosaic virus) or UbB1 (ubiquitin promoter from sunflower). The decreased syringyl/guaiacyl (S/G) ratio was found in stem and leaf lignin of 35S CaMV-PtCOMT transgenic silver birch lines when compared to non-transformed control or UbB1-PtCOMT lines. In controlled feeding experiments the leaves of transgenic birch lines as well as controls were fed to insect herbivores common in boreal environment, i.e., larvae of Aethalura punctulata, Cleora cinctaria and Trichopteryx carpinata (Lepidoptera: Geometridae) as well as the adults of birch leaf-feeding beetles Agelastica alni (Coleoptera: Chrysomelidae) and Phyllobius spp. (Coleoptera: Curculionidae). The feeding preferences of these herbivores differed in some cases among the tested birch lines, but these differences could not be directly associated to lignin modification. They could as well be explained by other characteristics of leaves, either natural or caused by transgene site effects. Growth performance of lepidopteran larvae fed on transgenic or control leaves did not differ significantly.

Cutaneous lymphocyte antigen expression on human effector B cells depends on the site and on the nature of antigen encounter.

In contrast to T cells, information on skin-homing B cells expressing the cutaneous lymphocyte antigen (CLA) is sparse. CLA expression on human B cells was investigated among circulating immunoglobulin-secreting cells (ISC) and among antigen-specific antibody-secreting cells (ASC) elicited by parenteral, oral or rectal primary immunization, or by parenteral or oral secondary immunization with Salmonella typhi Ty21a. CLA expression was examined by combining cell sorting with an enzyme-linked immunospot assay. Among all ISC, the proportion of CLA(+) cells was 13-21%. Parenteral immunization induced antigen-specific ASC of which 13% were CLA(+), while oral and rectal immunizations were followed by only 1% of CLA(+) ASC (p<0.001). Oral re-immunization was followed by an up-regulation of CLA (34-48%) regardless of the route of priming. Parenteral re-immunization elicited ASC of which 9-14% were CLA(+). In conclusion, the expression of CLA on human effector B cells depends on the site of antigen encounter: intestinal stimulation elicits cells with no CLA, while parenteral encounter elicits significant numbers of CLA(+) cells. Even though primary antigen encounter in the intestine failed to stimulate CLA expression, up-regulation of CLA was found upon intestinal antigen re-encounter. These findings may be of relevance in the pathogenesis of some cutaneous disorders.

Expression of Vitreoscilla haemoglobin in hybrid aspen (Populus tremula x tremuloides).

We describe the first ever expression of Vitreoscilla haemoglobin (VHb) in an economically important boreal woody plant hybrid aspen (Populus tremula x tremuloides). VHb has mainly been expressed in biotechnologically important unicellular organisms of both prokaryotic and eukaryotic origin. VHb expression, in this study, was analysed under different greenhouse cultivation conditions and under elevated UV-B illumination. Microscope analyses of leaves grown under optimized conditions revealed significant differences both in cell structure and size when the transgenic VHb lines were compared with the control lines. VHb lines displayed a higher relative volume of mitochondria and a significantly enhanced accumulation of starch in chloroplasts, all of which pointed towards changes in cellular energy production. Under elevated UV-B illumination, the differences between VHb lines became evident. Some specific VHb lines had elevated levels of total flavonoids, individual quercetin, kaempferol- and myricetin-derivatives relative to controls and other transgenic lines. This observation may reflect the availability of extra energy resources for secondary metabolite production and possibly an enhanced protection ability of these transgenic lines against UV-B illumination. Thus, all these findings point to changes in the energy metabolism of VHb lines. In the cultivation conditions tested this observation did not, however, result in a general improvement of elongation growth.