Forestan Cristian, Varotto Serena
Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, Padova, Italy.
Methods Mol Biol. 2013;959:223-33. doi: 10.1007/978-1-62703-221-6_15.
The major naturally occurring auxin, indol-3 acetic acid (IAA), coordinates many growth and differentiation processes by modulating gene expression during plant development. The sites of IAA biosynthesis and its polar transport (PAT) routes determine auxin accumulation and distribution during growth. From many studies on the model plant Arabidopsis thaliana over the last years, it has become evident that the expression and sub-cellular localization of multiple transport proteins are required to initiate and maintain directional auxin flows within plant organs and tissues, creating the auxin concentration gradients that regulate plant development. For this reason, the understanding of auxin dependent pattern formation also relies on the possibility to directly visualize auxin concentration and distribution in the tissues. The production and isolation of antibodies highly specific for IAA provide the means to detect and localize free IAA in different plant species during tissues differentiation and organ development. The immunolocalization protocol presented here uses a monoclonal anti-IAA specific antibody that can be used to visualize auxin accumulation in different organs and tissues during plant development. We successfully used this protocol to determine IAA maxima during kernel and inflorescence development in maize, highlighting also alterations in auxin accumulation patterns in a mutant with reduced auxin accumulation capacity and in plants treated with an inhibitor of auxin transport.
主要的天然生长素吲哚 - 3 - 乙酸（IAA）通过在植物发育过程中调节基因表达来协调许多生长和分化过程。IAA生物合成的位点及其极性运输（PAT）途径决定了生长过程中生长素的积累和分布。过去几年对模式植物拟南芥的许多研究表明，多种转运蛋白的表达和亚细胞定位对于启动和维持植物器官和组织内的定向生长素流动是必需的，从而形成调节植物发育的生长素浓度梯度。因此，对生长素依赖性模式形成的理解也依赖于直接可视化组织中生长素浓度和分布的可能性。针对IAA产生和分离的高度特异性抗体为在不同植物物种的组织分化和器官发育过程中检测和定位游离IAA提供了手段。这里介绍的免疫定位方案使用一种单克隆抗IAA特异性抗体，可用于可视化植物发育过程中不同器官和组织中的生长素积累。我们成功地使用该方案确定了玉米籽粒和花序发育过程中的IAA最大值，同时也突出了生长素积累能力降低的突变体和用生长素运输抑制剂处理的植物中生长素积累模式的变化。
