The function of cytokinin in regulating flowering time

细胞分裂素对开花的促进作用在近50年前这种植物激素被发现后不久就被描述出来了。然而，尽管在了解激素的新陈代谢和信号传递方面取得了很大进展，但这种活性背后的分子机制仍然难以捉摸。本研究的目的是研究细胞分裂素调节开花时间的程度和分子途径。这项工作的大部分将在拟南芥中完成，但也将研究转移到油菜(Brassica napus L.)的可行性，因为开花时间是一个重要的育种目标。在第一部分中，将对大多数~60个细胞分裂素代谢和信号基因丧失功能对开花时间的影响进行详细的研究，以确定功能相关的基因。我们的初步工作已经确定了细胞分裂素受体AHK2和AHK3以及转录因子ARR10和ARR12在介导细胞分裂素作用中发挥核心作用。这一分析将得到一项关于不同组织细胞分裂素状态相关性的研究，该研究使用具有组织特异性改变的细胞分裂素状态的转基因植物，重点是地上部顶端分生组织、叶片、韧皮部和根。这项研究包括比较已知的开花途径基因在不同日长下的转录水平以及在短时间的日间轮班后的转录水平。将测试不同环境信号(光、营养)对细胞分裂素在调节开花时间中作用的影响，以了解在哪些情况下它的作用特别相关。对转录数据的挖掘已经产生了关于细胞分裂素在已知开花途径中的调节作用的几个假说，包括对花抑制基因ATC、miR156、miR172的调控以及与赤霉素和海藻糖-6-磷酸途径的相互作用，这些将被分子和遗传学方法进一步探索。这些分析和ARR10和ARR12下游靶标的确定旨在将细胞分裂素的作用与开花途径的已知成分联系起来。此外，还选择了一种遗传方法来获得调节细胞分裂素活动的新成分。强烈的细胞分裂素缺乏导致在短日照下不开花，这可以被从抑制子筛选中分离出来的显性功能获得突变体逆转。应该确定新的影响开花途径的突变体，并用基于图谱的方法克隆相应的基因。最后，将利用在不同组织中具有细胞分裂素状态的转基因品系来探索细胞分裂素在调节甘蓝型油菜开花中的作用。这项研究应该揭示调控开花时间的机制是否在这种重要的农作物中被保守，并可能被用作调控其开花行为的育种目标。

The promotive activity of cytokinin on flowering was already described soon after the discovery of this plant hormone almost fifty years ago. However, the molecular mechanisms underlying this activity remain elusive despite great progress in understanding the metabolism and signalling of the hormone. The objective of this research proposal is to study the extent and the molecular pathways by which cytokinin regulates flowering time. Most of this work will be done in Arabidopsis thaliana but the transferability to oilseed rape (Brassica napus L.), where flowering time is an important breeding target, will be studied as well. In a first part, a detailed study of the consequences of loss-of-function of most of the ~60 cytokinin metabolism and signalling genes on flowering time will be performed to identify the functionally relevant genes. Our preliminary work has identified the cytokinin receptors AHK2 and AHK3 as well as the transcription factors ARR10 and ARR12 to play central roles in mediating cytokinin action. This analysis will be complemented by a study on the relevance of the cytokinin status of different tissues using transgenic plants with a tissue-specifically altered cytokinin status, focussing on the shoot apical meristem, the leaves, the phloem and the root. The investigation includes a comparison of the transcript levels of known flowering pathway genes under different day lengths and after a short day-to-long day shift. The influence of different environmental cues (light, nutrition) on cytokinin action in regulating flowering time will be tested to understand under which circumstances its role is particularly relevant. Mining of transcriptomic data has yielded several hypotheses about a modulatory role of cytokinin in known flowering pathways, including regulation of the flower repressor gene ATC, miR156, miR172 and interactions with the gibberellin and trehalose-6-phosphate pathways, which will be explored further by molecular and genetic approaches. These analyses and the identification of downstream targets of ARR10 and ARR12 aim to link the action of cytokinin to known components of flowering pathways. Furthermore, a genetic approach is chosen to obtain access to novel components regulating the action of cytokinin. Strong cytokinin deficiency causes lack of flowering under short days, which can be reversed by dominant gain-of-function mutants isolated from a suppressor screen. Novel mutants specifically affecting the flowering pathway should be identified and the respective genes cloned by a map-based approach. Finally, the role of cytokinin in regulating flowering in Brassica napus will be explored using transgenic lines with a genetically engineered cytokinin status in different tissues. This study should reveal whether mechanisms regulating flowering time have been conserved in this important crop plant and may be used as a breeding target to modulate its flowering behaviour.