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Planta

, Volume 226, Issue 1, pp 159–167 | Cite as

Independently silencing two JAR family members impairs levels of trypsin proteinase inhibitors but not nicotine

  • Lei Wang
  • Rayko Halitschke
  • Jin-Ho Kang
  • Albrecht Berg
  • Falk Harnisch
  • Ian T. Baldwin
Original Article

Abstract

Jasmonic acid (JA)–amino acid conjugates are important JA metabolites that activate JA responses. However, our understanding of their involvement in herbivore defenses is limited. We identified a new Arabidopsis jasmonate resistant 1 (JAR1) homologue in Nicotiana attenuata (N. attenuata) and named it jasmonate resistant 6 (JAR6). JAR6 clustered closely with Arabidopsis JAR1 and the recently reported jasmonate resistant 4 (JAR4), another JAR1 homologue in N. attenuata, in a phylogenic analysis. The strong elicitation of JAR6 transcripts by wounding and treatment with Manduca sexta (M. sexta) oral secretions (OS), which mimics herbivore attack, suggests it plays a role in herbivore defense. Independently silencing JAR4 or JAR6 by transforming N. attenuata with inverted repeat JAR4 or JAR6 constructs significantly reduced levels of not only JA–Ile plus JA–Leu but also JA–Val in OS-elicited leaves, suggesting JAR4 and JAR6 are functionally redundant and their amino acid substrates are not highly specific to individual amino acids. A new JA conjugate, JA–Gln, whose levels are much higher than those of the other JA conjugates in WT plants, was not affected in JAR4- or JAR6-silenced lines, implying that another JA-conjugating enzyme exists in N. attenuata. Neither JA–ACC, the second most abundant JA conjugate in Arabidopsis seedlings, nor JA–Met or JA–Trp, was detectable in N. attenuata. Levels of trypsin proteinase inhibitors (TPIs) in JAR4- and JAR6-silenced plants were significantly reduced, but nicotine levels were normal. We conclude that both JAR4 and JAR6 conjugate JA to Ile, Val, and Leu, and that both positively regulate TPI activity.

Keywords

Amino acid conjugates of jasmonic acid Trypsin proteinase inhibitors Nicotine Jasmonic acid signaling Nicotiana attenuata 

Notes

Acknowledgments

We thank Jianqiang Wu for providing cDNA for real-time PCR and assistance in constructing a phylogenic tree; Thomas Hahn for sequencing; Klaus Gase for the construction of vectors for JAR4 and JAR6 silencing; Susan Kutschbach and Antje Wissgott for plant transformation; Eva Rothe for assistance in LC–MS analysis; Jinsong Wu for critical reading of the manuscript and assistance in hybridization; and Emily Wheeler for editorial assistance. This work was supported by the Max Planck Society.

Supplementary material

425_2007_477_MOESM1_ESM.pdf (338 kb)
Fig. S1 Alignment of N. attenuata JAR4 (NaJAR4,DQ359729), JAR6 (NaJAR6, DQ359730) and Arabidopsis JAR (AtJAR1, NP_850453) full-length cDNA sequences with ClustalW method (http://www.ebi.ac.uk/clustalw/). The regions for invert-repeat constructions and hybridization probes are shaded gray. Taqman primers (JAR4, J4F and J4R; JAR6, J6F and J6R) and Taqman probes (JAR4, J4P; JAR6, J6p) are labeled with arrows. Start- and stop-codons are bold and in red. Asterisks indicate nucleotide identity, and dashes, missing nucleotide. (PDF 338kb)
425_2007_477_MOESM2_ESM.pdf (173 kb)
Fig. S2Southern blot of JAR6 in WT plants.10 μg genomic DNA were digested with EcoRI, HindIII, or DraI and blotted onto nylon membranes. The blots were hybridized with JAR6 specific probes (PCR fragments shown in Fig. S1). (PDF 172kb)
425_2007_477_MOESM3_ESM.pdf (34 kb)
Fig. S3 Southern blot of JAR4- and JAR6-silenced plants. 10 μg genomic DNA was digested with EcoRI or DraI and blotted onto nylon membranes. The blots were hybridized with hptII probe. (PDF 34.1kb)

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Lei Wang
    • 1
  • Rayko Halitschke
    • 1
    • 2
  • Jin-Ho Kang
    • 1
    • 3
  • Albrecht Berg
    • 1
  • Falk Harnisch
    • 1
  • Ian T. Baldwin
    • 1
  1. 1.Department for Molecular EcologyMax Planck Institute for Chemical EcologyJenaGermany
  2. 2.Department of Ecology and Evolutionary BiologyCornell UniversityIthacaUSA
  3. 3.Department of Energy-Plant Research LaboratoryMichigan State UniversityEast LansingUSA

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