Plant Cytogenetics and Molecular Biology Group, Institute of Biology, Biotechnology and Environmental Protection, Faculty of Natural Sciences, University of Silesia in Katowice, 40-032 Katowice, Poland.
Animal Histology and Embryology Group, Institute of Biology, Biotechnology and Environmental Protection, Faculty of Natural Sciences, University of Silesia in Katowice, 40-032 Katowice, Poland.
Int J Mol Sci. 2021 Jul 14;22(14):7548. doi: 10.3390/ijms22147548.
As cell wall proteins, the hydroxyproline-rich glycoproteins (HRGPs) take part in plant growth and various developmental processes. To fulfil their functions, HRGPs, extensins (EXTs) in particular, undergo the hydroxylation of proline by the prolyl-4-hydroxylases. The activity of these enzymes can be inhibited with 3,4-dehydro-L-proline (3,4-DHP), which enables its application to reveal the functions of the HRGPs. Thus, to study the involvement of HRGPs in the development of root hairs and roots, we treated seedlings of with 250 µM, 500 µM, and 750 µM of 3,4-DHP. The histological observations showed that the root epidermis cells and the cortex cells beneath them ruptured. The immunostaining experiments using the JIM20 antibody, which recognizes the EXT epitopes, demonstrated the higher abundance of this epitope in the control compared to the treated samples. The transmission electron microscopy analyses revealed morphological and ultrastructural features that are typical for the vacuolar-type of cell death. Using the TUNEL test (terminal deoxynucleotidyl transferase dUTP nick end labelling), we showed an increase in the number of nuclei with damaged DNA in the roots that had been treated with 3,4-DHP compared to the control. Finally, an analysis of two metacaspases' gene activity revealed an increase in their expression in the treated roots. Altogether, our results show that inhibiting the prolyl-4-hydroxylases with 3,4-DHP results in a vacuolar-type of cell death in roots, thereby highlighting the important role of HRGPs in root hair development and root growth.
作为细胞壁蛋白，富含羟脯氨酸的糖蛋白（HRGPs）参与植物的生长和各种发育过程。为了发挥其功能，HRGPs，特别是伸展蛋白（EXTs），通过脯氨酰-4-羟化酶发生脯氨酸的羟化。这些酶的活性可以被 3,4-脱氢-L-脯氨酸（3,4-DHP）抑制，这使其能够应用于揭示 HRGPs 的功能。因此，为了研究 HRGPs 在根毛和根发育中的作用，我们用 250µM、500µM 和 750µM 的 3,4-DHP 处理幼苗。组织学观察表明，根表皮细胞及其下方的皮层细胞破裂。用 JIM20 抗体（识别 EXT 表位）进行的免疫染色实验表明，与处理过的样品相比，对照样品中这种表位的丰度更高。透射电子显微镜分析显示了形态和超微结构特征，这些特征是液泡型细胞死亡的典型特征。使用 TUNEL 试验（末端脱氧核苷酸转移酶 dUTP 缺口末端标记），我们显示与对照相比，用 3,4-DHP 处理的根中受损 DNA 的细胞核数量增加。最后，对两种类半胱氨酸蛋白酶基因活性的分析表明，处理过的根中它们的表达增加。总之，我们的结果表明，用 3,4-DHP 抑制脯氨酰-4-羟化酶会导致根中的液泡型细胞死亡，从而突出了 HRGPs 在根毛发育和根生长中的重要作用。
