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Planta

, Volume 240, Issue 6, pp 1253–1267 | Cite as

Identification of three proteins involved in fertilization and parthenogenetic development of a brown alga, Scytosiphon lomentaria

  • Jong Won Han
  • Tatyana A. Klochkova
  • Junbo Shim
  • Chikako Nagasato
  • Taizo Motomura
  • Gwang Hoon KimEmail author
Original Article

Abstract

Metabolic pathways of cell organelles may influence the expression of nuclear genes involved in fertilization and subsequent zygote development through a retrograde regulation. In Scytosiphon lomentaria, inheritance of chloroplast is biparental but mitochondria are maternally inherited. Male and female gametes underwent different parthenogenetic outcomes. Most (>99 %) male gametes did not differentiate rhizoid cells or survived beyond four-cell stage, while over 95 % of female gametes grew into mature asexual plants. Proteomic analysis showed that the protein contents of male and female gametes differed by approximately 1.7 %, 12 sex-specific proteins out of 700 detected proteins. Three sex-specific proteins were isolated and identified using CAF-MALDI mass spectrometry and RACE-PCR. Among them, a male gamete-specific homoaconitate hydratase (HACN) and a female gamete-specific succinate semialdehyde dehydrogenase (SSADH) were predicted to be the genes involved in mitochondrial metabolic pathways. The expression level of both mitochondrial genes was dramatically changed at the fertilization event. During parthenogenetic development the male-specific HACN and GTP-binding protein were gradually down-regulated but SSADH stayed up-regulated up to 48 h. To observe the effect of chemicals on the expression of these genes, male and female gametes were treated with γ-aminobutyric acid (GABA), hydrogen peroxide and l-ascorbic acid. Among them GABA treatment significantly reduced SSADH gene expression in female gamete but the same treatment induced high upregulation of the gene in male gamete. GABA treatment affected the behavior of gametes and their parthenogenetic development. Both gametes showed prolonged motile stage, retarded settlement and subsequent parthenogenetic development. Our results suggest that male and female gametes regulate mitochondrial metabolic pathways differentially during fertilization, which may be the reason for their physiological and behavioral differences.

Keywords

Brown algae Fertilization GABA Maternal inheritance Parthenogenetic development Retrograde signaling 

Abbreviations

CAF-MALDI

Chemically assisted fragmentation–matrix assisted desorption/ionization

GABA

γ-Aminobutyric acid

HACN

Homoaconitate hydratase

RACE-PCR

Rapid amplification of cDNA ends polymerase chain reaction

SSADH

Succinate semialdehyde dehydrogenase

2-DE

Two-dimensional electrophoresis

Notes

Acknowledgments

We appreciate Prof. Joe Zuccarello for his careful revision and many helpful comments. This research was supported by Golden Seed Project, Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA) and Korea Forest Service (KFS).

Supplementary material

425_2014_2148_MOESM1_ESM.tif (3.7 mb)
Supplementary material 1 (TIFF 3741 kb) Fig. S1 Sequence alignment of S. lomentaria TypA/BipA GTP binding protein with its homologs. The alignment was performed using ClustalW program. Triangles: putative GEF interaction site [polypeptide binding], Lines: GTP/Mg2+ binding site [chemical binding], Arrows: Domains
425_2014_2148_MOESM2_ESM.tif (3.8 mb)
Supplementary material 2 (TIFF 3853 kb) Fig. S2 Sequence alignment of S. lomentaria SSADH with its homologs. The alignment was performed using ClustalW program. Triangles indicate SSA binding sites. Circles indicate residues involved in enzyme catalysis
425_2014_2148_MOESM3_ESM.tif (2.8 mb)
Supplementary material 3 (TIFF 2861 kb) Fig. S3 Sequence alignment of S. lomentaria HACN with its homologs. The alignment was performed using ClustalW program. Triangles indicate substrate binding sites.[chemical binding] Circles indicate ligand binding site [chemical binding]
425_2014_2148_MOESM4_ESM.tif (396 kb)
Supplementary material 4 (TIFF 396 kb) Fig. S4 Prediction of subcellular localization of Arabidiosis thaliana mitochondiral proteins. (a) TargetP prediction, (b) SubLoc prediction, (c) MitoP prediction. All analysis results were collected from the website, Arabidopsis Mitochondrial Protein Database (http://www.plantenergy.uwa.edu.au/ampdb/)
425_2014_2148_MOESM5_ESM.xls (2.6 mb)
Supplementary material 5 (XLS 2683 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Jong Won Han
    • 1
  • Tatyana A. Klochkova
    • 1
  • Junbo Shim
    • 1
  • Chikako Nagasato
    • 2
  • Taizo Motomura
    • 2
  • Gwang Hoon Kim
    • 1
    Email author
  1. 1.Department of BiologyKongju National UniversityKongjuKorea
  2. 2.Muroran Marine Station, Field Science Centre for Northern BiosphereHokkaido UniversityMuroranJapan

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