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

茎尖插条不定根的形成是园艺作物无性繁殖的最重要基础。这也适用于矮牵牛，它是世界顶级卧铺植物之一，越来越多地被发展为园艺作物研究的模式植物。我们之前对矮牵牛‘Mitchell’的研究表明，插穗在种植前的黑暗孵化强烈地加速了AR的形成，因此可以显著减少随后在加热温室中栽培的时间。暗刺激下不定根在茎基部的形成与生长素吲哚-3-乙酸的局部增强积累和生长素外流转运体Pin1和生长素信号转导链的几个组成部分的基因上调有关。生长素在茎基中的增强积累取决于上部茎段，而生长素信号也受制于插穗的含氮量。该项目旨在阐明整个插条水平上的关键分子因素和过程，这些因素和过程决定了黑暗增强的生长素向茎基运输和生长素信号的刺激。研究黑暗对上切段生长素来源、竞争生长素汇、生长素运输效率的潜在影响，以及生长素与代谢产物和其他激素的相互关系。为此，将比较两种对黑暗的生根反应不同的基因组测序的亲本矮牵牛。一个特别设计的微阵列，涵盖了矮牵牛所有已知的生长素、茉莉酸和斯特里内酯相关基因和定量RT-PCR，将与激素分析、生长素运输研究和细胞内和细胞内PIN定位相结合，以确定在不同切割器官水平上暗调节植物激素稳态和信号传递的分子和生化过程。药理工具将被用来操纵植物激素和代谢产物的动态平衡或信号传递。通过农杆菌介导的转化和RNA引导的Cas内切酶诱变，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。该项目旨在扩大对暗/光介导的内源控制生长素动态平衡和信号在整个插条水平上的理解，并为如何将其与目标值“根再生”相结合开辟新的视角，以便在无性繁殖中减少热能和合成生长素的输入。

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.