Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Porto, Portugal.
Biosystems and Integrative Sciences Institute - BioISI, Porto, Portugal.
Ann Bot. 2023 May 15;131(5):827-838. doi: 10.1093/aob/mcad046.
Morphogenesis occurs through accurate interaction between essential players to generate highly specialized plant organs. Fruit structure and function are triggered by a neat transcriptional control involving distinct regulator genes encoding transcription factors (TFs) or signalling proteins, such as the C2H2/C2HC zinc-finger NO TRANSMITTING TRACT (NTT) or the MADS-box protein SEEDSTICK (STK), which are important in setting plant reproductive competence, feasibly by affecting cell wall polysaccharide and lipid distribution. Arabinogalactan proteins (AGPs) are major components of the cell wall and are thought to be involved in the reproductive process as important players in specific stages of development. The detection of AGPs epitopes in reproductive tissues of NTT and other fruit development-related TFs, such as MADS-box proteins including SHATTERPROOF1 (SHP1), SHP2 and STK, was the focus of this study.
We used fluorescence microscopy to perform immunolocalization analyses on stk and ntt single mutants, on the ntt stk double mutant and on the stk shp1 shp2 triple mutant using specific anti-AGP monoclonal antibodies. In these mutants, the expression levels of selected AGP genes were also measured by quantitative real-time PCR and compared with the respective expression in wild-type (WT) plants.
The present immunolocalization study collects information on the distribution patterns of specific AGPs in Arabidopsis female reproductive tissues, complemented by the quantification of AGP expression levels, comparing WT, stk and ntt single mutants, the ntt stk double mutant and the stk shp1 shp2 triple mutant.
These findings reveal distinct AGP distribution patterns in different developmental mutants related to the female reproductive unit in Arabidopsis. The value of the immunofluorescence labelling technique is highlighted in this study as an invaluable tool to dissect the remodelling nature of the cell wall in developmental processes.
形态发生是通过关键参与者之间的精确相互作用发生的,以生成高度特化的植物器官。果实的结构和功能是由涉及不同调节基因的转录控制引发的,这些调节基因编码转录因子(TFs)或信号蛋白,例如 C2H2/C2HC 锌指 NO TRANSMITTING TRACT(NTT)或 MADS-box 蛋白 SEEDSTICK(STK),它们在设定植物生殖能力方面很重要,可能通过影响细胞壁多糖和脂质的分布来实现。阿拉伯半乳糖蛋白(AGPs)是细胞壁的主要成分,被认为是生殖过程中的重要参与者,在发育的特定阶段发挥重要作用。本研究的重点是检测 NTT 和其他与果实发育相关的 TF(如 MADS-box 蛋白包括 SHATTERPROOF1(SHP1)、SHP2 和 STK)的生殖组织中的 AGP 表位。
我们使用荧光显微镜对 stk 和 ntt 单突变体、ntt stk 双突变体和 stk shp1 shp2 三突变体进行免疫定位分析,使用特定的抗 AGP 单克隆抗体。在这些突变体中,还通过定量实时 PCR 测量了选定的 AGP 基因的表达水平,并与相应的 WT 植物的表达水平进行了比较。
本免疫定位研究收集了拟南芥雌性生殖组织中特定 AGP 分布模式的信息,并用 WT、stk 和 ntt 单突变体、ntt stk 双突变体和 stk shp1 shp2 三突变体的 AGP 表达水平的定量来补充。
这些发现揭示了与拟南芥雌性生殖单位相关的不同发育突变体中不同的 AGP 分布模式。本研究强调了免疫荧光标记技术的价值,它是研究发育过程中细胞壁重塑性质的一种非常有价值的工具。