Simultaneous Down-Regulation of Caffeic/5-Hydroxy Ferulic Acid-O-Methyltransferase I and Cinnamoyl-Coenzyme A Reductase in the Progeny from a Cross between Tobacco Lines Homozygous for Each Transgene. Consequences for Plant Development and Lignin Synthesis

Inhibition of specific lignin biosynthetic steps by antisense strategy has previously been shown to alter lignin content and/or structure. In this work, homozygous tobacco (Nicotiana tabacum) lines transformed with cinnamoyl-coenzyme A reductase (CCR) or caffeic acid/5-hydroxy ferulic acid-O-methyltransferase I (COMT I) antisense sequences have been crossed and enzyme activities, lignin synthesis, and cell wall structure of the progeny have been analyzed. In single transformed parents, CCR inhibition did not affect COMT I expression, whereas marked increases in CCR activity were observed in COMT I antisense plants, suggesting potential cross talk between some genes of the pathway. In the progeny, both CCR and COMT I activities were shown to be markedly decreased due to the simultaneous repression of the two genes. In these double transformants, the lignin profiles were dependent on the relative extent of down-regulation of each individual enzyme. For the siblings issued from a strongly repressed antisense CCR parent, the lignin patterns mimicked the patterns obtained in single transformants with a reduced CCR activity. In contrast, the specific lignin profile of COMT I repression could not be detected in double transformed siblings. By transmission electron microscopy some cell wall loosening was detected in the antisense CCR parent but not in the antisense COMT I parent. In double transformants, immunolabeling of non-condensed guaiacyl-syringyl units was weaker and revealed changes in epitope distribution that specifically affected vessels. Our results more widely highlight the impact of culture conditions on phenotypes and gene expression of transformed plants.

此前已有研究证实，通过反义策略（antisense strategy）抑制木质素生物合成的特定步骤，可改变木质素的含量及/或结构。本研究中，研究人员将携带肉桂酰辅酶A还原酶（cinnamoyl-coenzyme A reductase, CCR）或咖啡酸/5-羟基阿魏酸-O-甲基转移酶I（caffeic acid/5-hydroxy ferulic acid-O-methyltransferase I, COMT I）反义序列的纯合烟草（Nicotiana tabacum）株系进行杂交，并对子代的酶活性、木质素合成及细胞壁结构展开了分析。在单基因转化的亲本株系中，CCR的抑制并不会影响COMT I的表达；而在COMT I反义植株中，CCR活性出现显著升高，这提示该生物合成通路的部分基因间存在潜在的串扰效应。在子代株系中，由于两个基因同时被抑制，CCR与COMT I的活性均出现显著下降。在这些双转化株系中，木质素谱特征取决于每种酶下调的相对幅度。对于源自反义CCR强抑制亲本的子代株系，其木质素谱与CCR活性降低的单基因转化株系的木质素谱相一致；与之相反，在双转化子代株系中无法检测到COMT I抑制所特有的木质素谱特征。通过透射电子显微镜（transmission electron microscopy）观察，反义CCR亲本株系中可检测到一定程度的细胞壁松弛现象，而反义COMT I亲本株系中则未出现该现象。在双转化株系中，非缩合愈创木基-紫丁香基单元（non-condensed guaiacyl-syringyl units）的免疫标记信号较弱，且其表位分布出现了特异性影响导管组织的变化。本研究结果进一步凸显了培养条件对转化植株表型及基因表达的影响。