Information Technology Reference
In-Depth Information
Figure.
Confocal image of a nodular cell showing AOC in chloroplasts. Plant tissue
was incubated with a polyclonal antibody anti-AOC and with the secondary
antibody anti-rabbit-Alexa Fluor 488, and further stained with DAPI to confirm the
absence of immunofluorescence signal in the nucleus.
lipoxygenase in metabolic functions of these organelles during organogenic nodule
formation and plantlet regeneration.
In order to enable the study of the transcript pattern during organogenic nodule
formation and to isolate genes related to morphogenesis and wounding response, a cDNA
library was constructed using RNA extracted from induced internodes along 24 h and from
organogenic nodules. The amplified library titter was 1.25x10
10
pfu/ ml. A random in vivo
excision was performed and 60 clones sequenced which showed that full-length cDNAs
could be obtained by screening of this library. Amplification by PCR of cDNA inserts from
the plasmid library revealed that their size was ranged between 400 bp and 2000 bp. The
library redundancy was approximately 40 %.
Sequences clones were checked for identities using the NCBI Database BLAST
program. Homologues of peroxidase, cytochrome P450, metalothionein, and pectinesterase
were isolated among the selected clones. The correspondent mRNAs may be differentially
expressed throughout organogenic nodule formation since they play roles on wound
response, cell division and differentiation processes as well as on biosynthesis of growth
regulators.
A crucial step in the biosynthesis of jasmonic acid is the formation of its
stereoisomeric precursor, cis(+) 12-oxophytodienoic acid, which is catalyzed by allene
oxide cyclase. Study of allene oxide cyclase expression during organogenic nodule
formation revealed that this enzyme is involved in the response of internodes to wounding,
nodule formation and plantlet regeneration from these nodules [5]. A Reverse
Transcriptase- Polimerase Chain Reaction based approach using degenerated primers is
being undertaken in order to clone the AOC homologue from hop. Western blotting
analysis using an antibody raised against allene oxide cyclase from tomato showed elevated
levels of allene oxide cyclase in response to wounding peaking at 24 h after internode
inoculation. Jasmonic acid levels increased at this time (62,2 nmol/ g FW) whereas 12-
oxophytodienoic acid levels reached the highest levels 12 h after wounding (1440,3 nmol/ g
FW). Allene oxide cyclase is mostly present in vascular bundles of inoculated internodes
which may be a first indication that the systemin signalling pathway is operating also in
hop. During prenodule and nodule formation, allene oxide cyclase levels were still high.
Jasmonic acid and 12-oxophytodienoic acid levels decreased respectively to 10 and 118
