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Characterization of Secretory Phospholipase A2 with Phospholipase A1 Activity in Tobacco, Nicotiana tabacum (L.)

  • Original Article
  • Published:
Lipids

Abstract

A cDNA encoding protein with homology to plant secretory phospholipase A2 (sPLA2), denoted as Nt1 PLA2, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature form of Nt1 PLA2 has 12 cysteines, Ca2+ binding loop and catalytic site domain that are commonly conserved in plant sPLA2s. The recombinant Nt1 PLA2 was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease to obtain the mature form. Interestingly, Nt1 PLA2 could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for the sn-1 and -2 positions of glycerophospholipids required Ca2+ essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA2 mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.

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Abbreviations

AGPC:

Acid guanidinium thiocyanate-phenol–chloroform

PLA:

Phospholipase A

POPC:

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

POPE:

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine

POPG:

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol

RACE:

Rapid amplification of cDNA ends

RT:

Reverse-transcription

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Authors and Affiliations

  1. Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-8528, Japan

    Yukichi Fujikawa, Ritsuko Fujikawa, Noriaki Iijima & Muneharu Esaka

Authors
  1. Yukichi Fujikawa
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  2. Ritsuko Fujikawa
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  3. Noriaki Iijima
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  4. Muneharu Esaka
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Corresponding author

Correspondence to Muneharu Esaka.

Additional information

Y. Fujikawa and R. Fujikawa contributed equally to this work.

Electronic Supplementary Material

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Supplementary material 1 (DOC 40 kb)

11745_2011_3632_MOESM2_ESM.pdf

Supplemental Figure 1. A phylogenetic tree of various plants and animals sPLA2s. For abbreviations and accession numbers see Fig. 1. Nt1 PLA2 was boxed (PDF 29 kb)

11745_2011_3632_MOESM3_ESM.pdf

Supplemental Figure 2. Elution profile of recombinant Nt1 PLA2 on ion exchange HPLC (a) and SDS-PAGE of recombinant Nt1 PLA2 (b) and a The mixture digested with factor Xa was applied to a POROS HS/M column (4.6 × 100 mm, Perseptive Biosystems, Framingham, MA, USA) which was pre-equilibrated with 20 mM MES–NaOH (pH 6.0). The elution of protein was followed by monitoring the absorbance at 280 nm (solid line). The dashed line indicates the linear gradient of NaCl. The flow rate was 2 mL/min. The pooled fraction is indicated by an inverted triangle. b Molecular mass of marker proteins (right panel) from top to bottom: 250, 75, 50, 37, 25, 20, 15 and 10 kDa. The protein was stained with a coomassie brilliant blue. Lane 1; E. coli cells expressing thioredoxin, lane 2; E. coli cells expressing thioredoxin fusion Nt1 PLA2, lane 3; the crude extract prepared from E. coli expressing thioredoxin fusion Nt1 PLA2, lane 4; the crude extract after treating with Factor Xa proteinase, lane 5; the purified Nt1 PLA2. The recombinant protein is indicated by the triangle. Trx, thioredoxin; Trx:Nt1PLA2, thioredoxin fusion Nt1 PLA2 (PDF 395 kb)

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Fujikawa, Y., Fujikawa, R., Iijima, N. et al. Characterization of Secretory Phospholipase A2 with Phospholipase A1 Activity in Tobacco, Nicotiana tabacum (L.). Lipids 47, 303–312 (2012). https://doi.org/10.1007/s11745-011-3632-3

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  • DOI: https://doi.org/10.1007/s11745-011-3632-3

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