Overexpression of ginseng patatin-related phospholipase pPLAIIIβ alters the polarity of cell growth and decreases lignin content in Arabidopsis

Abstract

Background: The patatin-related phospholipase AIII family (pPLAIIIs) genes alter cell elongation and cell wall composition in Arabidopsis and rice plant, suggesting diverse commercial purposes of the economically important medicinal ginseng plant. Herein, we show the functional characterization of a ginseng pPLAIII gene for the first time and discuss its potential applications.

Methods: pPLAIIIs were identified from ginseng expressed sequence tag clones and further confirmed by search against ginseng database and polymerase chain reaction. A clone showing the highest homology with pPLAIIIβ was shown to be overexpressed in Arabidopsis using Agrobacterium. Quantitative polymerase chain reaction was performed to analyze ginseng pPLAIIIβ expression. Phenotypes were observed using a low-vacuum scanning electron microscope. Lignin was stained using phloroglucinol and quantified using acetyl bromide.

Results: The PgpPLAIIIβ transcripts were observed in all organs of 2-year-old ginseng. Overexpression of ginseng pPLAIIIβ (PgpPLAIIIβ-OE) in Arabidopsis resulted in small and stunted plants. It shortened the trichomes and decreased trichome number, indicating defects in cell polarity. Furthermore, OE lines exhibited enlarged seeds with less number per silique. The YUCCA9 gene was downregulated in the OE lines, which is reported to be associated with lignification. Accordingly, lignin was stained less in the OE lines, and the expression of two transcription factors related to lignin biosynthesis was also decreased significantly.

Conclusion: Overexpression of pPLAIIIβ retarded cell elongation in all the tested organs except seeds, which were longer and thicker than those of the controls. Shorter root length is related to auxin-responsive genes, and its stunted phenotype showed decreased lignin content.

Keywords: Auxin; Cell elongation; Lignin; Panax ginseng; Patatin-related phospholipase.

Fig. 1

Ginseng-derived PgpPLAIII proteins are closely related to other pPLAIII proteins. (A) Phylogenetic tree of PgpPLAIII proteins with the closest homologous proteins from Arabidopsis and rice. The phylogenetic tree was constructed using the ClustalX program (neighbor-joining method). At, Arabidopsis thaliana; Os, Oryza sativa; Pg, Panax ginseng. The GenBank accession numbers are AtpPLAIIIs: pPLAIIIα, (At2g39220), pPLAIIIβ (At3g54950), At4g29800 (pPLAIIIγ), and pPLAIIIδ (At3g63200) and OspPLAIIIs: OspPLAIIIα (LOC_Os03g14950), OspPLAIIIβ (LOC_Os03g43880), OspPLAIIIγ (LOC_Os03g57080), OspPLAIIIδ (LOC_Os06g46350), OspPLAIIIε (LOC_Os07g05110), and OspPLAIIIζ (LOC_Os12g41720). The bar represents 0.1 substitution per amino acid position. (B) Alignment of PgpPLAIIIβ protein with its closest homologs. The red-dotted box motifs represent the anion-binding box, esterase box, and catalytic dyad–containing motif.

Fig. 1

Ginseng-derived PgpPLAIII proteins are closely related to other pPLAIII proteins. (A) Phylogenetic tree of PgpPLAIII proteins with the closest homologous proteins from Arabidopsis and rice. The phylogenetic tree was constructed using the ClustalX program (neighbor-joining method). At, Arabidopsis thaliana; Os, Oryza sativa; Pg, Panax ginseng. The GenBank accession numbers are AtpPLAIIIs: pPLAIIIα, (At2g39220), pPLAIIIβ (At3g54950), At4g29800 (pPLAIIIγ), and pPLAIIIδ (At3g63200) and OspPLAIIIs: OspPLAIIIα (LOC_Os03g14950), OspPLAIIIβ (LOC_Os03g43880), OspPLAIIIγ (LOC_Os03g57080), OspPLAIIIδ (LOC_Os06g46350), OspPLAIIIε (LOC_Os07g05110), and OspPLAIIIζ (LOC_Os12g41720). The bar represents 0.1 substitution per amino acid position. (B) Alignment of PgpPLAIIIβ protein with its closest homologs. The red-dotted box motifs represent the anion-binding box, esterase box, and catalytic dyad–containing motif.

Fig. 2

Organ-specific expression pattern of PgpPLAIIIβ. (A) Differential expression pattern of PgpPLAIIIβ in 2-year-old ginseng plants by the qRT-PCR. Exact organ nomenclature for 2-year-old ginseng plants is indicated on the right inset. (B) Differential expression pattern of PgpPLAIIIβ in 4-year-old ginseng roots. The data are presented as mean ± SD of three independent replicates. qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; SD, standard deviation.

Fig. 3

Heterologous overexpression of PgpPLAIIIβ caused dwarf plant with altered cell polarity in the trichome. (A) Transcript level of PgpPLAIIIβ from 4-week-old leaves of three overexpressing lines. The data are presented as mean ± SE of three independent replicates at P < 0.05 (*) and P < 0.01 (**). (B) Transgenic plants were smaller than Col-0 plants. (C) Overexpressing lines exhibited stunted and dwarf phenotype with smaller and less number of leaves. Scale bar = 1 cm. Construction map of overexpression under the 35S promoter is depicted in inset. (D) Cell growth patterns are altered in the OE lines. Scale bar = 100 μm. All surface images were taken using a low-vacuum scanning electron microscope (JSM-IT300, JEOL Korea) at 10.8-mm working distance and 20.0 kV. SE, standard error.

Fig. 4

Quantification of gibberellin oxidases by the overexpression of PgpPLAIIIβ. Relative gene expression patterns of four gibberellin oxidases (GA2ox1, GA2ox2, GA3ox1, and GA20ox1) in 6-week grown plant stems. The data are presented as mean ± SE of three independent replicates at P < 0.05 (*) and P < 0.01 (**).

Fig. 5

Germination rate is regulated by the function of PgpPLAIIIβ with the increase in seed size. (A) The number of individual mature seeds increased but the number of siliques reduced in the OE lines. Scale bar = 1 mm. (B) Seed length and width were measured from mature seeds n = 35–51. Scale bar = 1 mm. (C) Seed number per each silique was reduced in OE lines. n = 19–27. (D) Germination was faster in the OE lines after 20 h and 24 h of germination under light condition. n = 36. The data are presented as mean ± standard error (SE) of three independent replicates at P < 0.05 (*) and P < 0.01 (**).

Fig. 6

Reduced primary root length of PgpPLAIIIβ-OE line is regulated by auxin-responsive genes. (A) Eight-day-old roots were shorter in OE lines than those in Col-0. Scale bar = 1 cm. (B) Root length and (C) the number of lateral roots in the OE lines. n = 11. (D) Transcript level of auxin-responsive genes was quantified by qRT-PCR using 8-day-old seedlings. (E) Transcript level of YUCCA8 and YUCCA9 was quantified in 7-week-old stems by qRT-PCR. The data are presented as mean ± SE of three independent replicates at P < 0.05 (*) and P < 0.01 (**). qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; SE, standard error.

Fig. 7

Lignin content in the stems of PgpPLAIIIβ-OE lines decreased. (A and B) Histochemical staining of cross sections from second internode of 7-week-old stems from Col-0 and PgpPLAIIIβ-OE lines using phloroglucinol-HCl. Scale bars = 100 μm. (C) Mäule staining for S lignin staining. Scale bars = 100 μm. (D) Lignin content in 7-week-old stems determined using acetyl bromide. (E) Relative gene expression of transcription factors, MYB58 and MYB63, involved in lignin biosynthesis decreased. The data are presented as mean ± SE of three independent replicates at P < 0.05 (*) and P < 0.01 (**). SE, standard error.