Functional characterization of auxin-mediated dark stimulation of adventitious root formation in Petunia

茎尖插穗不定根的形成是园艺作物无性繁殖的重要基础。这也适用于矮牵牛，矮牵牛在世界范围内排名第一，并越来越多地被开发为园艺作物研究的模式植物。我们以前对矮牵牛‘米切尔’的研究表明，在种植前对插条进行黑暗孵育强烈加速了AR的形成，从而可以显着减少随后在加热温室中培养的时间。暗刺激的不定根形成在茎基部与生长素吲哚-3-乙酸的局部增强的积累和编码生长素外排转运蛋白PIN 1和生长素信号转导链的几个组分的基因的上调相关。茎基部生长素积累的增强取决于上部茎段，而生长素信号传导也受到扦插的氮含量的影响。拟议的项目旨在阐明关键的分子因素和过程，在整个插条的水平，决定黑暗增强生长素输送到茎基和刺激生长素信号。黑暗的潜在影响生长素源上切割部分，竞争生长素汇，生长素运输效率和生长素与代谢产物和其他激素的相互关系将进行调查。为此，将比较两种基因组测序的亲本矮牵牛属物种对黑暗的生根反应。一个专门设计的微阵列，包括所有已知的生长素，茉莉酸和独脚金内酯相关基因的矮牵牛和定量RT-PCR将结合激素分析，生长素运输研究和内部和细胞内PIN定位，以确定暗介导的植物激素稳态和信号传导的分子和生化过程在不同的切割器官的水平。药理学工具将用于操纵植物激素和代谢物稳态或信号。利用农杆菌介导的转化和突变技术，利用RNA引导的Cas内切酶，研究了暗促进不定根形成的候选基因的功能及其组织特异性表达。该项目的目的是扩大了解黑暗/光介导的内源性控制生长素稳态和信号在整个切割水平，并开辟了新的观点，这可以对齐的目标值“根再生”，使输入的热能和合成生长素可以减少无性繁殖。

Adventitious root formation in shoot tip cuttings is the most important basis for vegetative propagation of horticultural crops. This applies also to petunia, which ranks among the top bedding plants worldwide and has increasingly been developed as model plant for research on horticultural crops. Our previous work with Petunia hybrida ‘Mitchell’ has shown, that dark incubation of cuttings before planting strongly accelerates AR formation so that the time of subsequent cultivation in heated greenhouses can be significantly reduced. The dark-stimulated adventitious root formation in the stem base is associated with locally enhanced accumulation of the auxin indole-3-acetic acid and upregulation of genes that encode the auxin efflux transporter PIN1 and several components of the auxin signal transduction chain. The enhanced auxin accumulation in the stem base depends on the upper shoot section, while the auxin signalling is also subject to the nitrogen content of the cutting. The proposed project aims to elucidate the critical molecular factors and processes at the level of whole cuttings that determine the dark-enhanced auxin delivery towards the stem base and the stimulation of auxin signalling. Potential effects of darkness on the auxin source in the upper cutting section, on competing auxin sinks, on auxin transport efficiency and the auxin interrelationships with metabolites and other hormones will be investigated. To this end, two genome-sequenced parental petunia species that contrast in the rooting response to darkness will be compared. A specially designed microarray, that covers all known auxin-, jasmonic acid- and strigolactone-related genes in petunia and quantitative RT-PCR will be combined with hormone analysis, auxin transport studies and inter- and intracellular PIN localization to identify the molecular and biochemical processes underlying the dark-mediated plant hormone homeostasis and signalling at the level of distinct cutting organs. Pharmacological tools will be used to manipulate plant hormone and metabolite homeostasis or signalling. The function of selected candidate genes for dark-promoted adventitious root formation and their tissue specific expression are investigated with the help of Agrobacterium-mediated transformation and mutagenesis using RNA-guided Cas endonucleases. The project is intended to expand the understanding of the dark-/light-mediated endogenous control of auxin homeostasis and signalling at whole cutting level and open up new perspectives on how this can be aligned to the target value “root regeneration” so that inputs of heating energy and of synthetic auxins can be reduced in vegetative propagation.