An approach to the biochemical mechanisms of heartwood formation using cell cultures

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(1). Biochimistry of lignificationLignification process has been investigated in this project since it occurs prior to the heartwood formation in forest tree cells. A peroxidase isoenzyme was found to catalyze substrate specifically the dehydrogenetive polymerization of sinapyl alcohol. This enzyme was isolated and characterized from cell cultures of Populus alba.(2). Establishment of cell cultures that produce heartwood constituentsCallus cultures of Cupressus lusitanica (Cupressanceae) and Pinus spp. produced their heartwood constituents, beta-thujaplicin and pinosylvins, respectively, although no heartwood constituents were detected in calluses of Cryptomeria japonica and Chamaecyparis obtusa. Thus, the culture of C.lusitanica cells is regarded as a good experimental material for studying phenomena of heartwood formation.(3). Elicitor of beta-thujaplicin accumulation in callus culturesThe addition of yeast extract to callus cultures of C.lusitanica leads to a large increase in the p … More roduction of beta-thujaplicin that act as a phyoalexin of the treated cultures. The extract was fractionated by means of ethanol precipitation, ribonucleasetreatment, gel permiation choromatography and Cu-complexation. The most effective fraction was a polysaccharide composed of maily mannose and glucose.(4). Biosythetic pathway of beta-thujaplicinCallus cultures of C.lusitanica was fed with ^<14>C-mevaronate, ^<14>C-glucose or ^<14>C-malonate by the shot-gun method. Elicitor was added to the callus at the same time. The results proved that beta-thujaplicins synthsized via the mevaronate pathway.(5). An attempt to isolation of the key enzyme(s) for beta-thujaplicin biosynthesisWe tried to isolate any enzyme(s) that catalyze biosynthesis of beta-thujaplicin from isopentenyl pyrophosphate. However, such enzyme(s) have not been detected in cell-free extract of callus cultures of C.lusitanica. Isoltion of the key enzyme(s), its characterization and proof of its presence in transition zone of C.lustanica trees should be subjects to be solved in the near future. Less