Peptomics, identification of novel cationic Arabidopsis peptides with conserved sequence motifs

Few plant peptides involved in intercellular communication have been experimentally isolated. Sequence analysis of the Arabidopsis thaliana genome has revealed numerous transmembrane receptors predicted to bind proteinacious ligands, emphasizing the importance of identifying peptides with signaling function. Annotation of the Arabidopsis genome sequence has made it possible to identify peptide-encoding genes. However, such annotational identification is impeded because small genes are poorly predicted by gene-prediction algorithms, thus prompting the alternative approaches described here. We initially performed a systematic analysis of short polypeptides encoded by annotated genes on two Arabidopsis chromosomes using SignalP to identify potentially secreted peptides. Subsequent homology searches with selected, putatively secreted peptides, led to the identification of a potential, large Arabidopsis family of 34 genes. The predicted peptides are characterized by a conserved C-terminal sequence motif and additional primary structure conservation in a core region. The majority of these genes had not previously been annotated. A subset of the predicted peptides show high overall sequence similarity to Rapid Alkalinization Factor (RALF), a peptide isolated from tobacco. We therefore refer to this peptide family as RALFL for RALF-Like. RT-PCR analysis confirmed that several of the Arabidopsis genes are expressed and that their expression patterns vary. The identification of a large gene family in the genome of the model organism Arabidopsis thaliana demonstrates that a combination of systematic analysis and homology searching can contribute to peptide discovery.

[1]  D. Marshall,et al.  Analysis of Arabidopsis genome sequence reveals a large new gene family in plants , 1999, Plant Molecular Biology.

[2]  C. Wilkerson,et al.  Identification and analysis of Arabidopsis expressed sequence tags characteristic of non-coding RNAs. , 2001 .

[3]  K. Nakamura,et al.  Diversity of Arabidopsis genes encoding precursors for phytosulfokine, a peptide growth factor. , 2001, Plant physiology.

[4]  B. Rost.  PHD: predicting one-dimensional protein structure by profile-based neural networks. , 1996, Methods in enzymology.

[5]  C. Curie,et al.  The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1α: Molecular cloning, characterization and expression , 1989, Molecular and General Genetics MGG.

[6]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[7]  James W. Fickett,et al.  ORFs and Genes: How Strong a Connection? , 1995, J. Comput. Biol..

[8]  I. Bancroft.  Duplicate and diverge: the evolution of plant genome microstructure. , 2001, Trends in genetics : TIG.

[9]  R. Hancock,et al.  The role of cationic antimicrobial peptides in innate host defences. , 2000, Trends in microbiology.

[10]  L. Kaminsky,et al.  Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR. , 1996, BioTechniques.

[11]  G. Pearce,et al.  RALF, a 5-kDa ubiquitous polypeptide in plants, arrests root growth and development , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Andreas Schaller,et al.  Modulation of Plasma Membrane H+-ATPase Activity Differentially Activates Wound and Pathogen Defense Responses in Tomato Plants , 1999, Plant Cell.

[13]  P. Macheroux,et al.  LeSBT1, a Subtilase from Tomato Plants , 2000, The Journal of Biological Chemistry.

[14]  Anders Gorm Pedersen,et al.  Neural Network Prediction of Translation Initiation Sites in Eukaryotes: Perspectives for EST and Genome Analysis , 1997, ISMB.

[15]  M. Kanehisa,et al.  A knowledge base for predicting protein localization sites in eukaryotic cells , 1992, Genomics.

[16]  S. Brunak,et al.  SHORT COMMUNICATION Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites , 1997 .

[17]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[18]  Y. Matsubayashi,et al.  Peptide signals and their receptors in higher plants. , 2001, Trends in plant science.

[19]  G. Pearce,et al.  Production of multiple plant hormones from a single polyprotein precursor , 2001, Nature.

[20]  Christophe Geourjon,et al.  SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments , 1995, Comput. Appl. Biosci..

[21]  S. Brunak,et al.  Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. , 2000, Journal of molecular biology.

[22]  Joseph R. Ecker,et al.  CTR1, a negative regulator of the ethylene response pathway in arabidopsis, encodes a member of the Raf family of protein kinases , 1993, Cell.

[23]  J. Thornton,et al.  Influence of proline residues on protein conformation. , 1991, Journal of molecular biology.

[24]  Erik Andreasson,et al.  Arabidopsis MAP Kinase 4 Negatively Regulates Systemic Acquired Resistance , 2000, Cell.

[25]  K. Nakamura,et al.  Oryza sativa PSK gene encodes a precursor of phytosulfokine-alpha, a sulfated peptide growth factor found in plants. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[27]  J. Cock,et al.  A large family of genes that share homology with CLAVATA3. , 2001, Plant physiology.

[28]  C. Dumas,et al.  Two large Arabidopsis thaliana gene families are homologous to the Brassica gene superfamily that encodes pollen coat proteins and the male component of the self-incompatibility response , 2001, Plant Molecular Biology.