Aristotelis Azariadis1, Konstantinos Blazakis1, Mohamed Kouhen1, Khansa Mekkaoui1, Georgios Kostelenos2, and Panagiotis Kalaitzis1
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1Dept of Horticultural Genetics Biotechnology, Mediterranean Agronomic Institute of Chania (MAICh (CIHEAM))
2Kostelenos Olive Nurseries
Comparative characterization of olive prolyl 4hydroxylases and cell wall glycoproteins in Greekolive cultivars under climate change conditions
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Fig. 1The Mediterranean countries, their Mediterranean regions and their different limits(Source: Gaussen & De Philippis – FAO, 2003)
0
20000
40000
60000
80000
100000
120000
140000
0
2000
4000
6000
8000
10000
12000
14000
Salinity in the Meditarrenean
Population(in thousands)
Availability(m3/inh.year)
Fig. 2Availability of water in the Mediterranean(Source: N.V. Paranychianakis, K.S. Chartzoulakis, 2005)
3
2005
Fig. 4 Gravimetric assay of arabinogalactan proteins (AGPs) isolated fromgrowth medium of salt-adapted compared with non-adapted controls. This enablesa comparison of AGPs released by non-adapted and salt-adapted cells. Data fromsingle representative experiments. Solid columns, salt-adapted; hatched columns,non-adapted controls.
1993
Fig. 3 A, B. Localization of AGPs in cultured tobacco cells with 13-glucosylYariv reagent staining. Yellowish-red color is due to Yariv-reagent staining. A So cells10 d after subculture. Insert shows a plasmolyzed cell with Yariv-reagent stainingthe protoplast surface. B S25 cells 15 d after subculture.
Alterations in expression and/or suppression of expression of plant P4Hs
Alterations in frequency of occurrence of proline hydroxylation events in substrate proteins
MECHANISM OF ACTION
Direct or indirect alterations in abiotic stress responses such as salinity
STRUCTURAL ALTERATIONS √ - VARIATION IN PROTEIN CONTENT √ - DEGRADATION ?
TFs (?) Hormone Peptides (?) AGP/Ext proteins √
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Fig. 5Leaf K/Na ratios of nine indigenous olive cultivars grown with 0, 50, 100 and 200mM NaCl. 5
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Salinity (120 mM NaCl) and Heat stress
Barrels of 150 Lt and 90 cm height
Koroneiki
Gaidourolia
Lefkolia
Arvanitolia
The facility
Fig. 6The greenhouse facilities of MAICh (CIHEAM)
Fig. 7Layout of the experimentalgreenhouse facility in thepremises of MAICh(CIHEAM)
The facility
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Temperature and Relative Humidity
Tem
per
atu
re
Rel
ativ
e H
um
idit
y
Max Temperature: 44.03 oCMin Temperature: 21.53 oCAv. Temperature: 31,28 oC
Max Rel. Hum.: 85,81 %Min Rel. Hum.: 16,68 %Av. Rel. Hum.: 49,22 %
Fig. 8 Temperature and Relative Humidity
June July August June July August
The facility
Data collected from period: 21.06.18 – 09.08.18
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a. 90D Control
The morphological analysis was performed by oliveId
b. 90D Salinity
0
1
2
3
4
5
6
45DAYS 90DAYS
Leaf
are
a(cm
2)
KORONEIKI
control
salinity
oliveId
Fig. 10In 90 days the average leaf area of plants thatwere submitted to salinity stress, was reducedby almost 29% in relation to the control plants.
Phenomics
Fig. 9Canopy of olive trees: a. control plants;b. salinity treated for 90 days.
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Fig. 12Dendrogram prepared with the deduced AGP amino acidsequences from olive. The dendrogram was constructedusing MATLAB_R2014b.
Arabinogalactans (AGPs) & Prolyl-4-Hydroxylases (P4Hs)in Olive (O. Europea)
Fig. 11Dendrogram prepared by comparison of the deduced P4Hamino acid sequences from olive, arabidopsis and tomato.The dendrogram was constructed using MATLAB_R2014b.
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P4H4 gene expression in olive roots
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0.5
1
1.5
2
2.5
0D 45D 90D
Arvanitolia
control
salinity
0
0.5
1
1.5
2
0D 45D 90D
Gaidourolia
control
salinity
0
1
2
3
4
5
6
0D 45D 90D
Koroneiki
control
salinity
0
0.5
1
1.5
2
2.5
0D 45D 90D
Lefkolia
control
salinity
P4H4Fig. 13P4H4 gene expression in olive roots
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0
0.5
1
1.5
2
2.5
0D 45D 90D
Arvanitolia
control
salinity
0
1
2
3
4
5
0D 45D 90D
Gaidourolia
control
salinity
P4H7 gene expression in olive roots
0
0.5
1
1.5
2
2.5
0D 45D 90D
Koroneiki
control
salinity
0
1
2
3
4
0D 45D 90D
Lefkolia
control
salinity
P4H7Fig. 14P4H7 gene expression in olive roots
Monoclonal Antibody
Plant of Origin
AntigenEpitope Structure for Carbohydrate Antigen
LM2 Rice
Arabinogalactan
beta-linked glucuronic acid
JIM13 Carrot (beta)GlcA1->3(alpha)GalA1->2Rha
MAC207 Pea (beta)GlcA1->3(alpha)GalA1->2Rha
The Antibodies
Fig. 15The antibodies that were used in order to bind, detect and quantify arabinogalactan proteins (AGPs)Source: E.A. Yates, J. F. Valdor, S.M. Haslam, H.R. Morris, A. Dell, W. Mackie, and J.P. Knox. (1996)
13
14
0 45 90 0 45 90
CONTROL SALINITY
ARVANITOLIA
LM2
ACTIN
250
130
95
72
MW
55
36
0 45 90 0 45 90
CONTROL SALINITY
GAIDOUROLIA
0 45 90 0 45 90
CONTROL SALINITY
KORONEIKI
0 45 90 0 45 90
CONTROL SALINITY
LEFKOLIA
JIM13
ACTIN
250
130
95
72
MW
55
36
MAC207
ACTIN
250
130
95
72
MW
55
36
Fig. 16 Western Blots of AGPs’ content.
Conclusions
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• P4Hs activity is affected by abiotic stress
• The characterization of the molecular response to salinity and heat stress might lead to thedevelopment of new breeding strategies.
• AGPs content is affected by abiotic stress
• Direct or indirect alterations of plant’s responses
Thank YouChania Alsyllio Agrokepio, 1 Makedonias str Chania 73100 Crete GREECE, Greece
0030 2821035000Aristotelis Azariadis
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