Establishing an integrated network model for secondary wall thickening in anther development.

次生植物细胞壁增厚对于植物生长的许多方面都至关重要，特别是对于产生用于水运输和支持的机械组织，但当需要其他方面的机械力时，例如花药开裂，也至关重要。次生细胞壁由纤维素、半纤维素和木质素组成。在了解这些壁组分的生物合成方面已经取得了进展，但对控制这些途径的因素知之甚少。这种调控网络倾向于启动多种细胞壁组分的生物合成，解剖这些调控网络可能允许这些途径的分离，未来的目标是控制细胞壁基质的特定组分与其他组分分离。了解这些过程对于操纵二次增厚以改变木材质量、生物燃料至关重要，而且对于操纵需要这种机械力的过程也至关重要，例如花药开裂和控制雄性生育力以实现杂交发育和控制基因流动。次生增厚发生在花药内膜，对花药打开所需的物理力量至关重要，正如我们的拟南芥雄性不育突变体ms35所证明的那样，它产生正常的花粉，但由于花粉不释放，因此是雄性不育的。我们已经证明这种缺陷是由MYB26的突变引起的，MYB26是一种与MYB转录调节因子家族成员同源的基因。我们已经证明MYB26的过表达可以在通常不产生继发性增厚的组织中开启继发性增厚（异位继发性增厚）。在这些过表达细胞系中，我们看到许多基因的表达增加，包括转录因子，以及次生增厚途径的生物合成成分。我们已经构建了这一途径的初步模型，并假设两个NAC结构域基因可能是MYB26的直接靶点。我们还发现了大量尚未进入这一途径的基因。我们建议测试我们目前的模型并确定MYB26的主要调控靶点，同时扩展我们的模型以包括在我们的突变体中改变表达的其他基因。我们将在MYB26启动后对这些基因进行时间过程分析。这将确定那些在MYB26表达后很快开启的MYB26的直接靶标基因，以及那些在该途径中稍后开启的基因，可能是由MYB26以外的因素开启的。我们将利用这些信息与数学家/系统生物学家合作，为这一途径开发一个综合模型。然后，我们将通过选择被预测为该途径关键调控点的基因来测试该模型，并研究敲除（突变）或过度表达它们的影响。我们将通过分析这些背景下下游和上游基因的表达来检查我们网络的顺序，并确定随后在这些背景下表达MYB26对基因表达的影响。我们还将通过研究敲除或过表达细胞系中细胞壁结构的变化来确定这些基因的功能影响。我们还发现了一种似乎与MYB26结合的蛋白质，可能有助于激活它，使其发挥正常功能。我们将测试这种蛋白在哪里表达，以及它是否激活MYB26蛋白。这将为确定MYB26活性的MYB26通路的第一步提供有价值的信息。

Secondary plant cell wall thickening is vital for many aspects of plant growth, typically for the production of mechanical tissues for water transport and support, but is also critical when other aspects of mechanical force are required, for example anther dehiscence. Secondary cell walls are composed of cellulose, hemicellulose and lignin. Advances have been made towards understanding the biosynthesis of these wall components, however little is known about the factors that control these pathways. Such regulatory networks tend to initiate the biosynthesis of multiple wall components, dissecting these regulatory networks may permit the separation of these pathways with the future goal of controlling specific components of the cell wall matrix in isolation from other components. Understanding such processes is vital for the manipulation of secondary thickening for modification of wood quality, biofuels, but also for the manipulation of processes requiring such mechanical forces, for example anther dehiscence and the control of male fertility for hybrid development and control of gene flow. Secondary thickening occurs in the anther endothecium and is vital for the physical forces needed for anther opening, as demonstrated by our Arabidopsis male sterile mutant ms35 which produces normal pollen but is male sterile because the pollen is not released. We have shown that this defect is caused by a mutation in MYB26, a gene that has homology to members of the MYB family of transcriptional regulators. We have shown that over-expression of MYB26 can switch on secondary thickening in tissues that do not normally produce secondary thickening (ectopic secondary thickening). In these over-expressing lines we see increases in the expression of a number of genes, including transcription factors, but also biosynthetic components of the secondary thickening pathway. We have constructed a preliminary model for this pathway and hypothesise that two NAC domain genes may be direct targets for MYB26. We have also identified a large number of genes that have not yet been placed into this pathway. We propose to test our current model and determine the primary regulatory targets for MYB26, but also to extend our model to include the other genes that have altered expression in our mutant. We will do this by carrying out a time-course analysis of these genes after MYB26 is switched on. This will identify those genes that are direct targets of MYB26 which are switched on very soon after MYB26 expression, but also those genes that are switched on later in the pathway, possibly by factors other than MYB26. We will use this information in collaboration with mathematicians/systems biologists to develop an integrated model for this pathway. We will then test this model by selecting genes that are predicted to be key regulatory points in the pathway and investigate the effects of knocking-out (mutating) or over-expressing them. We will check the order of our network by analysing the expression of down and up-stream genes in these backgrounds and determine the effect on gene expression of subsequently expressing MYB26 in these backgrounds. We will also establish the functional effects of these genes by investigating changes to the structure of the cell wall in the knockout, or overexpression lines. We have also found a protein that appears to binds to MYB26 and may serve to activate it to enable it to carry out its normal function. We will test where this protein is expressed and whether it activates the MYB26 protein. This will provide valuable information on the first step in the MYB26 pathway in determining the activity of MYB26.