Characterization and functional studies of a plant class II glycine-rich protein LsGRP1 in plant defense
Date Issued
2014
Date
2014
Author(s)
Lin, Chia-Hua
Abstract
Salicylic acid- and Botrytis elliptica-inducible defense-related LsGRP1 gene in lily presumably encodes a plant class II glycine-rich protein (GRP). In this study, the results of western blot detection and tandem mass spectrometer analysis revealed that three LsGRP1 variants of 14, 16 and 23 kDa expressed in the leaves of lily specifically, and the expression of 14 and 16 kDa LsGRP1 remained a similar level at different growth stages while the amount of 23 kDa LsGRP1 decreased at the senescence stage. As investigated by immunohistochemistry, LsGRP1 was found to accumulate in the epidermal and the phloem tissues of leaves. The subcellular localization assayed by EGFP imaging and protein extraction analysis revealed that 14 kDa LsGRP1 was located in the plasma membrane whereas 16 and 23 kDa LsGRP1 were weakly bound to the cell wall. Additionally, the accumulation of 14 kDa LsGRP1 and ubiquitin antibody-recognizable 23 kDa LsGRP1 was triggered by salicylic acid and B. elliptica, suggesting that 23 kDa LsGRP1 comes from mono-ubiquitinated 14 kDa LsGRP1 and is related to the occurrence of induced resistance in lily. This is a novel trait never reported for other plant class II GRPs. On the other hand, the failure in LsGRP1 expression using Escherichia coli system suggested the presence of antimicrobial activity in certain region of LsGRP1, and LsGRP1C corresponding to the cysteine-rich C-terminal region was considered an antimicrobial peptide according to its broad-spectrum and effective antimicrobial activity as assayed using chemically synthesized LsGRP1-derived peptides. Furthermore, the inhibition effect of LsGRP1C on fungal growth is possibly via alteration of the integrity of cell membrane and induction of programmed cell death-like phenomenon as revealed by SYTOX Green, H2DCFDA and DAPI staining assays. Moreover, immunofluorescence of LsGRP1C present at the outer layer of fungal cells was indicated and implied that plant cell surface-localized LsGRP1 might retard pathogen via the antimicrobial activity conferred by its C-terminal region. Thus, defense-related LsGRP1 playing an important role in the induced resistance of lily against B. elliptica was assumed; in addition, LsGRP1C derived from LsGRP1 is an antimicrobial peptide with a potential for practical use.
Subjects
防禦相關蛋白LsGRP1
雙重次細胞定位
泛素化
植物第二型富含甘胺酸蛋白
富含半胱胺酸抗菌胜肽
膜通透性
類細胞程序性死亡現象
Type
thesis
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