Abstract
Wounding is required to be made in the walls of the green seed pod of Opium poppy prior exudation of latex. To withstand this kind of trauma plants regulate expression of some metabolites through an induced transcript level. 167 unique wound-inducible ESTs were identified by a repetitive round of cDNA subtraction after 5 hours of wounding in Papaver somniferum seedlings. Further repetitive reverse northern analysis of these ESTs revealed 80 transcripts showing more than two fold induction, validated through semi-quantitative RT-PCR & real time expression analysis. One of the major classified categories among identified ESTs belonged to benzylisoquinoline transcripts. Tissue specific metabolite analysis of benzylisoquinoline alkaloids (BIAs) in response to wounding revealed increased accumulation of narcotine and papaverine. Promoter analysis of seven transcripts of BIAs pathway showed the presence of W-box cis-element with the consensus sequence of TGAC, which is the proposed binding site for WRKY type transcription factors. One of the Wound inducible ‘WRKY’ EST isolated from our subtracted library was made full-length and named as ‘PsWRKY’. Bacterially expressed PsWRKY interacted with the W-box element having consensus sequence TTGACT/C present in the promoter region of BIAs biosynthetic pathway genes. PsWRKY further activated the TYDC promoter in yeast and transiently in tobacco BY2 cells. Preferential expression of PsWRKY in straw and capsule and its interaction with consensus W-box element present in BIAs pathway gene transcripts suggest its possible involvement in the wound induced regulation of BIAs pathway.
Highlights
Papaver somniferum is an important medicinal plant
We have reported eighty novel expressed sequence tags (ESTs), obtained after repetitive cDNA subtractions followed by DNA array-hybridization in response to 5 hours of wound in opium poppy seedlings
Repetitive DNA array hybridization was performed for 167 generated sequences, out of which eighty ESTs were showing more than two fold induction after 5 hours of wounding
Summary
Papaver somniferum is an important medicinal plant. Its medicinal properties are due to benzylisoquinoline alkaloids (BIAs) [1]. BIAs represents approximately 25, 00 elucidated natural product structures found mainly in Papaveraceae, Ranunculaceae, Berberidaceae and Menispermaceae [2]. BIA biosynthesis start with the formation of two L-tyrosine derivatives, 4-hydroxyphenylacetaldehyde (4-HPAA) and dopamine (Figure 1). The synthesis of 4HPAA and dopamine from tyrosine is catalyzed by tyrosine aminotransferase (TyrAT) and tyrosine/DOPA decarboxylase (TYDC) respectively [3,4]. Norcoclaurine synthase (NCS) catalyzes the condensation of 4-HPAA and dopamine yielding (S)norcoclaurine [5]. (S)-methylcoclaurine is synthesized after sequential methylations by norcoclaurine 6-O-methyltransferase (6OMT) and coclaurine N-methyltransferase (CNMT) [6]. (S)-N-Methylcoclaurine-3-hydroxylase (NMCH) catalyzes the 3-hydroxylation of (S)-N-methylcoclaurine to (S)-3-hydroxy-N- (S)-methylcoclaurine is synthesized after sequential methylations by norcoclaurine 6-O-methyltransferase (6OMT) and coclaurine N-methyltransferase (CNMT) [6]. (S)-N-Methylcoclaurine-3-hydroxylase (NMCH) catalyzes the 3-hydroxylation of (S)-N-methylcoclaurine to (S)-3-hydroxy-N-
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