Il metabolismo delle ossilipine: caratteristiche biochimiche, localizzazione endocellulare ed attivazione in leguminose in risposta a stress abiotici
Author(s)
De Domenico, Stefania
Date Issued
April 11, 2012
Type
Doctoral Thesis
Abstract
Legumes are important crop in sustainable agriculture because they can provide high value
protein, support meat and dairy production and are unique in their ability to improve soil fertility
through their symbiosis with nitrogen-fixing bacteria. However, legumes are sensitive to a
number of abiotic stresses such as water deficit and soil salinity, which are a main cause of crop
loss worldwide.
Plants have evolved complex cell signalling pathways activating metabolic functions and
developmental switches to cope with environmental stresses. Dissection of molecular
mechanisms controlling plant response to environmental stresses is crucial to provide legume
crops with improved stress tolerance.
In a previous work, microarray analysis showed that among hormone and secondary metabolism
category, four key-genes involved in the oxylipins metabolism were up-expressed in salt tolerant
genotype, M. truncatula Jemalong A17, under salt stress condition. They are lipoxygenase (lox),
hydroperoxide-lyase (hpl), allene oxide synthase (aos) and allene oxide cyclase (aoc).
Furthermore, in another study, SuperSAGE technique was applied to the analysis of gene
expression in chickpea roots in response to drought and salt stress. In both abiotic conditions,
two genes, i.e a lipoxygenase and allene oxide synthase, involved in oxylipins pathway were upregulated.
In this context, an important agronomic grain legume, chickpea (Cicer arietinum), and the model
legume species M. truncatula (Jemalong A17) were subjected to drought and salt stress
respectively to understand the possible involvement of this class of heterogeneous compounds in
the response of legumes to abiotic stresses.
At first, gene silencing experiments were carried out on Medicago plants. A RNA interference
(RNAi) approach adopted to down-regulate the expression of lox, hpl, aos and aoc genes,
yielded transgenic roots whose growth was valuated in presence and in absence of salt. Our
results showed that aoc-RNAi and aos-RNAi roots were less sensitive to the presence of salt in
the medium. Preliminary chemical analysis indicated a reduction in OPDA content in these
roots; the possible absence of oxylipins (JA and JA-Ile) produced from OPDA could indicate an
important role of jasmonats in the “bonsai effect”, a typical plant response to abiotic stresses.
As far as the chickpea is concerned, we took advance of the SuperSAGE information available
for this species to design new Taqman probes to study the expression of key genes involved in
oxylipins metabolism. Therefore, qPCR was carried out on root samples from a drought-tolerant
and a drought-sensitive chickpea variety. Our results indicated a sustained and earlier activation
of a specific lipoxygenase (lox1) gene, two hydroperoxide lyases (hpl1 and hpl2), an allene oxide
synthase (aos) and an oxo-phytodienoate reductase (opr) genes in the drought tolerant variety.
These data were confirmed by the quantification of the main oxylipins derived from the AOS
branch of the pathway. Higher levels of jasmonic acid (JA), its precursor 12-oxophytodienoic
acid (OPDA) and the active form JA-isoleucine (JA-Ile) were detected in the root tissues of the
tolerant variety, suggesting a role of jasmonates in the early signalling and in the tolerance
mechanism in response to drought stress.
This data was supported by HPLC analysis on M. truncatula plants subjected to salinity for
different stress point. In these plants we founded high levels of JA and JA-Ile already at 1 hour
from stress onset.
HPLC quantification of other oxylipins on Cicer arietinum roots, i.e. the aldheydes produced by
the HPL branch, indicated a hexanal accumulation only later during drought stress, thus
suggesting a competition for the same substrate between AOS and HPL enzymes at earlier stress
time points.
In this context, we carried out a biochemical characterisation of the main enzymes of the
oxylipin pathway detected in M. truncatula leaves. Western blot analysis suggested a thylacoid
localisation for MtAOS and MtAOC, and a stromal for MtLOX. An unexpected distribution was
founded for MtHPL: this protein, despite a plastidiali transit peptide (TP59) in N-terminal region,
was not detected in the chloroplast fraction. These results were confirmed by confocal
microscopy analyses obtained expressing some chimeric fluorescent proteins related to the M.
truncatula HPL cDNA.
Token together results from this research indicated an important role of jasmonates in the early
signalling of abiotic stresses.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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