We investigated feasibility of the Full-length complementary DNA OvereXpression (FOX) system as a mutagenesis approach in poplar, using developing xylem tissue. The main goal was to assess the overall mutation rate and if the system will increase instances of mutants affected in traits linked to the xylem tissue. Indeed, we found a high mutation rate of 17.7%, whereas 80% of all mutants were significantly affected in cellulose, lignin and/or hemicellulose. Cell wall biosynthesis is a major process occurring during xylem development. Enrichment of mutants affected in cell wall composition suggests that the tissue source for the FOX library influenced the occurrence of mutants affected in a trait linked to this tissue. Additionally, we found that FLcDNAs from mutants affected in cell wall composition were homologous to genes known to be involved in cell wall biosynthesis and most recovered FLcDNAs corresponded to genes whose native expression was highest in xylem. We characterized in detail a mutant line with increased diameter. The phenotype was caused by a poplar homolog of LONELY GUY 1 (LOG1), which encodes an enzyme in cytokinin biosynthesis and significantly increased xylem proliferation. The causative role of LOG1 in the observed phenotype was further reaffirmed by elevated cytokinin concentration in the mutant and recapitulation overexpression experiment wherein multiple independent lines phenocopied the original FOX mutant. Our experiments show that the FOX approach can be efficiently used for gene discovery and molecular interrogation of traits specific to woody perennial growth and development.

中文翻译：

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转基因杨树中正在发育的木质部 cDNA 文库的过度表达会产生木材形成特有的高突变率。

我们利用发育中的木质部组织，研究了全长互补 DNA 过度表达 (FOX) 系统作为杨树诱变方法的可行性。主要目标是评估总体突变率，以及系统是否会增加受木质部组织相关性状影响的突变体实例。事实上，我们发现 17.7% 的高突变率，而所有突变体中 80% 的纤维素、木质素和/或半纤维素受到显着影响。细胞壁生物合成是木质部发育过程中发生的主要过程。细胞壁组成受影响的突变体的富集表明，FOX 文库的组织来源影响了与该组织相关的性状受影响的突变体的出现。此外，我们发现来自受细胞壁组成影响的突变体的FLcDNA与已知参与细胞壁生物合成的基因同源，并且大多数回收的FLcDNA对应于木质部中天然表达最高的基因。我们详细描述了直径增加的突变系。该表型是由 LONELY GUY 1 (LOG1) 的杨树同源物引起的，该基因编码细胞分裂素生物合成中的一种酶，并显着增加木质部增殖。突变体和重演过表达实验中细胞分裂素浓度升高进一步证实了LOG1在观察到的表型中的致病作用，其中多个独立品系复制了原始FOX突变体的表型。我们的实验表明，FOX 方法可以有效地用于基因发现和对木本多年生生长和发育特有性状的分子询问。