Book of Abstracts - Geyseco

03 - S - Selected Abstracts for Oral Presentations
cence, and the determination of endogenous concentrations of
phytohormones is essential to elucidate the role of a particular
hormone in any physiological process. Availability of a sensitive
and rapid method to quantify multiple classes of plant hormones
simultaneously will greatly facilitate the investigation
of hormone-induced signalling networks in controlling specific
developmental pathways and physiological responses. Due to the
presence of plant hormones at very low concentrations in plant
tissues (10-9 M to 10-6 M) and their different chemistries, the development
of a high-throughput and comprehensive method for
the determination of phytohormones is challenging. The present
work reports a rapid, specific and sensitive method using UPLC-
MS/MS for the quantitative and simultaneous analysis of the
major phytohormones found in plant tissues, including auxins,
cytokinins, gibberellins, abscisic acid, 1-amino-cyclopropane-1-
carboxyic acid (the ethylene precursor), jasmonic acid and salicylic
acid. Sample preparation, extraction procedures and UPLC-
MS/MS conditions were optimized for the determination of all
plant hormones in a single run. This new method is applicable to
the analysis of dynamic changes in endogenous concentrations of
phytohormones in different plant tissues to study plant developmental
processes or plant responses to biotic and abiotic stresses.
An example is shown in which simultaneous analyses of phytohormones
is performed in leaves of plants exposed to salt stress,
both in the model plant, Arabidopsis thaliana and in an aromatic
plant, Salvia officinalis.
S03-001: OXYLIPIN-INDUCED TYROSINE PHOS-
PHORYLATION OF PLANT PROTEINS
Yakusheva, O.* - Karimova, F.
Kazan Institute of Biochemistry and Biophysics, RAS
*Corresponding author: vov1985@mail.ru
Oxylipins are products of oxygenated polyunsaturated fatty
acids, biologically active signaling molecules. Nowadays molecular
mechanisms of oxylipin effects are the object of close
attention. In plant cells the signaling pathway of jasmonates is
the most studied. Earlier the researchers of out Institute showed
that one of the main products of legume lipoxygenase metabolism
is (9Z)-12-hydroxy-9-dodecenoic acid (HDA). It was shown
that HDA is a growth stimulator causing an increase in soybean
callus biomass up to 400%. Previously we showed HDA-induced
Ca 2+ - and cAMP-dependent plant protein phosphorylation for 2h
of exposure. Protein tyrosine phosphorylation is known to be
critical for cell proliferation and differentiation. In this context
plant protein tyrosine phosphorylation is of great interest. We investigated
dynamics of HDA effect in vivo on the tyrosine phosphorylation
level (TPL) of leaf soluble proteins in pea plants. Our
results indicate that TPL quickly changes in control and HDAtreated
plants during different time periods. To detect factors critical
for HDA effect on TPL plants grown on the nutrient solution
without Ca 2+ were used. We showed that Ca 2+ -deficiency in the
growth medium caused a decrease in TPL of all polypeptides in
comparison with control plants grown on the optimal solution.
These data suggest that there is Ca 2+ - dependence of protein
phosphorylation/dephosphorylation enzymes activity. The HDA
effect on TPL was also Ca 2+ -dependent. As known in vertebrate
cells, protein tyrosine phosphatase (PTP) activity is 10-100 times
higher than protein tyrosine kinase activity. Using PTP inhibitor
phenylarsinoxide we showed contribution of PTP activity to
Ca 2+ - dependence of HDA-induced TPL in pea plants.
S03-002: ANALYSIS OF THE ARABIDOPSIS GLYCO-
PROTEOME
Van Der Krol, S.¹* - Song, W.¹ - Mentink, R.² - Henquet, M.³ -
Cordewener, J.³ - Bosch, D.³ - America, T.³
¹Lab. of Plant Physiology, Wageningen University
²Hubrecht Inst. Utrecht Netherlands
³Plant Research International, Wageningen, Netherlands
*Corresponding author: sander.vanderkrol@wur.nl
Arabidopsis contains ~4500 secreted proteins with one or more
of the N-glycosylation consensus site N-x-S/T. However, for
only few glycoproteins has the presence of an Nglycan actually
been confirmed and mapped experimentally. Here we present the
characterization of the glycoproteome from Arabidopsis, as extracted
from leaves, seedlings and developing seeds. Extracted
proteins were first digested with trypsin, after which (activated)
glycopeptides were coupled to Hydrazide resin. After extensive
washing, bound peptides were released by the enzyme PNGaseF
and were measured by LCMS DDA and MS E . Because PNGaseF
converts the N to D, it leaves a ‘glycansignature’ in the peptide
sequence to be analyzed. Moreover, PNGaseF cleaves mannose
glycans (on glycopeptides in ER), but not complex glycans (on
glycopeptides in and from Golgi). This allowed distinguishing
between ER- and Golgi-derived glycoproteomes, by comparing
glycopeptide profiles from WT (only ER-derived glycopeptides)
and glycopeptide profiles from mutant plants without complex
glycans (cgl) (full glycoproteome).
Using this method we confirmed glycan occupancy of over 800
consensus sites on more than 300 proteins. We show that some
glycoproteins (e.g. LRR receptors) have heterogeneous glycosylation
(mannose and complex glycans within the same protein).
N-glycan site occupancy mapping was also used to correct
THMM-predicted membrane protein topology of eight membrane
proteins. The results show that our method allows for High
Through Put proteomics of this important subset of the plant proteome.
This is now used to map changes in the glycoproteome
in response to protein secretion stress in seeds and in pathogeneffector/plant
interactions.
S03-003: A SYSTEM BIOLOGY APPROACH TO UN-
DERSTAND FUNCTIONS OF RAPTOR1 IN ARABIDOP-
SIS THALIANA
Li, Y. * - Caldana, C. - Giavalisco, P. - Leisse, A. - Willmitzer, L.
Max-Planck-Institute of Molecular Plant Physiology
*Corresponding author: yli@mpimp-golm.mpg.de
RAPTOR/KOG1 proteins, conserved WD-40 repeat proteins, are
binding partners of the target of rapamycin (TOR) kinase that
plays a central role in metabolism, such as cellular growth in response
to nutrients, mitogens and growth factors in eukaryotes. In
Arabidopsis, RAPTOR1 interacts with TOR and S6K1 in vivo,
and overexpression of RAPTOR1 rendered the S6K1 osmotic
stress insensitive. We developed computational and experimental
methods to identify RAPTOR1 by using artificial microRNA
lines. It is shown that, by quantitative RT-PCR, RAPTOR1 expression
level decreased after estradiol induction. Interestingly,
amiRaptor1 plants were much smaller after transfer to MS medium
containing estradiol. Further, I will focus on the function of
RAPTOR1 in TOR signaling pathway by tanscriptomics, proteomics
and metabolomics data analysis at system-level.
S03-004: STEADY-STATE 13C METABOLIC FLUX
ANALYSIS: FOCUS ON DEVELOPING BARLEY SEEDS
Krach, C. *
Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung
*Corresponding author e-mail:christian.krach@gmx.de
Metabolic Flux Analysis has become a well-established tool
in microbial metabolic engineering. It has been successfully
adopted to rational redirections of carbon metabolism and so increasing
the yield of desired fermentation products. In contrast
affords to manipulate a plant’s metabolism beyond the scope of
secondary metabolites were less successful, resulting in data hard
to interpret. Accordingly a system-wide analysis and a more general
understanding of metabolic processes are necessary.
A cell’s set of metabolic fluxes represents a very comprehensive
phenotype of its metabolic activity and therefore includes extremely
important information for the targeted improvement of crop
metabolism. Intracellular fluxes themselves cannot be measured
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