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

(1)。木质化的生物化学本项目研究了木质化过程，因为木质化过程发生在林木细胞的心材形成之前。发现过氧化物酶同工酶能催化底物芥子醇的脱氢聚合。该酶是从白杨细胞培养物中分离得到的。(2)建立产生心材成分的细胞培养物吕氏柏愈伤组织培养和松树愈伤组织培养。在日本柳杉和钝顶青霉的愈伤组织中未检测到心材成分，但分别产生了其心材成分--β-胡萝卜苷和松脂素。因此，鹿茸细胞的培养被认为是研究心材形成现象的良好实验材料。愈伤组织中β-胡芦巴蛋白积累的诱导子添加酵母膏能显著提高胡芦巴愈伤组织的p-…更多的贝塔-胡黄素的产生，作为处理的培养物的植保素。提取液采用乙醇沉淀、核糖核酸处理、凝胶渗透层析和铜络合等方法进行分离。最有效的部位是主要由甘露糖和葡萄糖组成的多糖组分。用弹枪法对白蜡梅愈伤组织培养的生物合成途径进行了补加。同时在愈伤组织中加入激发子。结果证明，β-胡黄连蛋白是通过甲伐他酸途径合成的。β-胡黄素生物合成关键酶(S)的分离我们尝试从焦磷酸异戊烯中分离出催化合成β-胡黄素的任何酶(S)。然而，在红豆杉愈伤组织培养的无细胞提取液中没有检测到这种酶(S)。关键酶(S)的分离、鉴定以及证明其存在于黄连木过渡带是近期亟待解决的课题。较少

(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