Wagner Doris, Wellmer Frank, Dilks Kieran, William Dilusha, Smith Michael R, Kumar Prakash P, Riechmann José Luis, Greenland Andrew J, Meyerowitz Elliot M
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Plant J. 2004 Jul;39(2):273-82. doi: 10.1111/j.1365-313X.2004.02127.x.
We have developed a versatile floral induction system that is based on ectopic overexpression of the transcription factor LEAFY (LFY) in callus. During shoot regeneration, flowers or floral organs are formed directly from root explants without prior formation of rosette leaves. Morphological and reporter gene analyses show that leaf-like structures are converted to floral organs in response to LFY activity. Thus, increased levels of LFY activity are sufficient to bypass normal vegetative development and to direct formation of flowers in tissue culture. We found that about half of the cultured cells respond to inducible LFY activity with a rapid upregulation of the known direct target gene of LFY, APETALA1 (AP1). This dramatic increase in the number of LFY-responsive cells compared to whole plants suggested that the tissue culture system could greatly facilitate the analysis of LFY-dependent gene regulation by genomic approaches. To test this, we monitored the gene expression changes that occur in tissue culture after activation of LFY using a flower-specific cDNA microarray. Induction of known LFY target genes was readily detected in these experiments. In addition, several other genes were identified that had not been implicated in signaling downstream of LFY before. Thus, the floral induction system is suitable for the detection of low abundance transcripts whose expression is controlled in an LFY-dependent manner.
我们开发了一种通用的花诱导系统，该系统基于转录因子LEAFY（LFY）在愈伤组织中的异位过表达。在芽再生过程中，花或花器官直接从根外植体形成，而无需先形成莲座叶。形态学和报告基因分析表明，叶状结构会响应LFY活性而转化为花器官。因此，增加的LFY活性水平足以绕过正常的营养发育，并在组织培养中直接引导花的形成。我们发现，大约一半的培养细胞会对可诱导的LFY活性做出反应，使LFY的已知直接靶基因APETALA1（AP1）迅速上调。与完整植株相比，LFY反应性细胞数量的这种显著增加表明，该组织培养系统可以极大地促进通过基因组方法对LFY依赖性基因调控的分析。为了验证这一点，我们使用花特异性cDNA微阵列监测了LFY激活后组织培养中发生的基因表达变化。在这些实验中很容易检测到已知LFY靶基因的诱导。此外，还鉴定出了其他几个以前未涉及LFY下游信号传导的基因。因此，花诱导系统适用于检测其表达以LFY依赖性方式受到控制的低丰度转录本。
