Study of the role of L-Idonate Dehydrogenase in tartaric acid biosynthesis during table grape...
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Study of the role of L-Idonate Dehydrogenase in tartaric Study of the role of L-Idonate Dehydrogenase in tartaric acid biosynthesis during table grape development acid biosynthesis during table grape development Cinthya Araneda 1 , Mauricio González-Agüero 2 , Daniel Manríquez 2 , Pablo Muñoz-Robredo 2 and Bruno G. Defilippi 2 . 1 Andrés Bello University, República 217 , Santiago, Chile. 2 Institute of Agricultural Research (INIA-La Platina). P.O. Box 439-3, Santiago, Chile. Email: bdefi[email protected] - A differential behavior in organic acid synthesis was observed for Red Globe and Thompson Seedless varieties. - The reduction on tartaric acid content was concomitant with a decrease in the gene expression of L-IdnDH for both varieties. - As shown by DeBolt et al. (2006), L-IdnDH has a major effect in earlier stages of berry development. So, we will establish if the expression pattern of L-IdnDH gene could explain the behavior of tartaric acid for both varieties. - We will study the factors affecting L-IdnDH gene expression and tartaric acid content, including ethylene, growing conditions and genotype. 1.Titratable acidity determination. We observed differences in titratable acidity evolution between Thompson Seedless and Red Globe table grapes, being this difference higher in early stages of development. Flavor is the most important quality attribute determining consumer preference in fruit, including table grapes. This organoleptic property is directly influenced by pre- and postharvest conditions and the genetic background of each cultivar, feature that in grapes is especially associated with sugars and organic acids ratio. At ripening, tartaric acid is the most important organic acid in grapes, being also one of the major contributors to flavor. It has been observed that an important step in the synthesis of tartaric acid in grapes would be related to the L-Idonate dehydrogenase (L-IdnDH) enzyme; however, the molecular changes underlying this process are unknown for table grapes. Therefore, in order to understand the role of L-IdnDH on tartaric acid biosynthesis, we cloned and characterized the expression pattern of a VvL-IdnDH gene in two commercial varieties of table grapes, i.e. Thompson Seedless and Red Globe. The study was located at ‘Los Andes’ and the sampling was performed weekly starting two weeks before véraison until commercial harvest. In general, as berry fruit development progressed, we observed differences in VvL-IdnDH gene expression levels, which would be affecting the changes and differences measured in tartaric acid (TA) content for both varieties. However, as already shown in the literature, this enzyme would have a major role in earlier stages of berry development. Therefore, further studies will be performed in order to better understand this and other key steps on tartaric acid biosynthesis. Abstract Experimental approach Conclusions and Future work Results 2. Total organic acids. As expected for grapes, total organic acids decreased with the advance in berry development for both varieties (A), with a reduction of malic acid content and an increase of tartaric acid (B). 3. Tartaric acid quantification and expression analyses for L-IdnDH gene in Thompson Seedless and Red Globe. The correlation between the tartaric acid content and the expression pattern of VvL.-IdnDH was characterized in fruits for each variety. The qPCR assays were performed in 6 biological samples for each variety and developmental stage, normalized against a housekeeping gene (VvEF1α) and expressed as a percentage of the highest value of relative abundance. Analysis: • Titratable acidity Titration • Total organics acids • Gene expression qPCR A B C A, Schematic representation of phenological stages; B, Analyses performed; and C, Biosynthetic pathway of tartaric acid and key enzymes: L-Idonate dehydrogenase (L-IdnDH). HPLC Flowering -2 -1 Véraiso n +1 +2 Harvest Postharvest Weeks Season 2009-2010 In progress (season 2010- 2011) A scorbicacid Oxalicacid L-threonate 2-keto-L-idonicacid L-idonicacid 5-keto-D-gluconicacid G lycoaldehyde T artaric acid L-IdnD H L-threo-tetruronate A scorbicacid Oxalicacid L-threonate 2-keto-L-idonicacid L-idonicacid 5-keto-D-gluconicacid G lycoaldehyde T artaric acid L-IdnD H L-threo-tetruronate C itric acid Tartaric acid M alic acid Main acids detected in berry: A B 0% 25% 50% 75% 100% P ercent ofacid 0,0 0,9 1,8 2,6 3,5 g acid/100g grape 0,0 0,9 1,8 2,6 3,5 g acid/100g grape V er-2 V er-1 Ver V er+1 V er+2 H arvest 60d@ 0°C V er-2 V er-1 Ver V er+1 V er+2 H arvest 45d@ 0°C Phenological stages (weeks) 0% 25% 50% 75% 100% P ercent ofacid Red Globe Thompson Seedless T L Tartaric acid content Expression pattern of VvL-IdnDH Red Globe Thompson Seedless Ver-2 V er-1 Ver Ver+1 V er+2 H arvest 60d@ 0°C Ver-2 V er-1 Ver V er+1 V er+2 H arvest 45d@ 0°C Phenological stages (weeks) 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0 0,1 0,2 0,3 0,4 0,5 g acid/ 100g grape Relative expression g acid/ 100g grape Relative expression 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0 0,1 0,2 0,3 0,4 0,5 This work is funded by Fondecyt 1100273 Phenologicalstages (w eeks) 0 1 2 3 4 5 6 T It r a t a b l e a c i d i t Red Glob e Thompson Seedless 0 1 2 3 4 5 6 T It ra t ab l e a c i di t Red Globe Thompson Seedless 0 1 2 3 4 5 6 T itratable acidity R ed G lobe T hom pson Seedless 0 1 2 3 4 5 6 T itratable acidity R ed G lobe T hom pson Seedless
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Transcript of Study of the role of L-Idonate Dehydrogenase in tartaric acid biosynthesis during table grape...
Study of the role of L-Idonate Dehydrogenase in tartaric acid Study of the role of L-Idonate Dehydrogenase in tartaric acid biosynthesis during table grape developmentbiosynthesis during table grape development
Cinthya Araneda 1, Mauricio González-Agüero 2, Daniel Manríquez 2, Pablo Muñoz-Robredo 2 and Bruno G. Defilippi 2.1 Andrés Bello University, República 217 , Santiago, Chile.2 Institute of Agricultural Research (INIA-La Platina). P.O. Box 439-3, Santiago, Chile. Email: [email protected]
- A differential behavior in organic acid synthesis was observed for Red Globe and Thompson Seedless varieties.
- The reduction on tartaric acid content was concomitant with a decrease in the gene expression of L-IdnDH for both varieties.
- As shown by DeBolt et al. (2006), L-IdnDH has a major effect in earlier stages of berry development. So, we will establish if the expression pattern of L-IdnDH gene could explain the behavior of tartaric acid for both varieties.
- We will study the factors affecting L-IdnDH gene expression and tartaric acid content, including ethylene, growing conditions and genotype.
1. Titratable acidity determination. We observed differences in titratable acidity evolution between Thompson Seedless and Red Globe table grapes, being this difference higher in early stages of development.
Flavor is the most important quality attribute determining consumer preference in fruit, including table grapes. This organoleptic property is directly influenced by pre- and postharvest conditions and the genetic background of each cultivar, feature that in grapes is especially associated with sugars and organic acids ratio. At ripening, tartaric acid is the most important organic acid in grapes, being also one of the major contributors to flavor. It has been observed that an important step in the synthesis of tartaric acid in grapes would be related to the L-Idonate dehydrogenase (L-IdnDH) enzyme; however, the molecular changes underlying this process are unknown for table grapes. Therefore, in order to understand the role of L-IdnDH on tartaric acid biosynthesis, we cloned and characterized the expression pattern of a VvL-IdnDH gene in two commercial varieties of table grapes, i.e. Thompson Seedless and Red Globe. The study was located at ‘Los Andes’ and the sampling was performed weekly starting two weeks before véraison until commercial harvest. In general, as berry fruit development progressed, we observed differences in VvL-IdnDH gene expression levels, which would be affecting the changes and differences measured in tartaric acid (TA) content for both varieties. However, as already shown in the literature, this enzyme would have a major role in earlier stages of berry development. Therefore, further studies will be performed in order to better understand this and other key steps on tartaric acid biosynthesis.
Abstract Experimental approach
Conclusions and Future work
Results
2. Total organic acids. As expected for grapes, total organic acids decreased with the advance in berry development for both varieties (A), with a reduction of malic acid content and an increase of tartaric acid (B).
3. Tartaric acid quantification and expression analyses for L-IdnDH gene in Thompson Seedless and Red Globe. The correlation between the tartaric acid content and the expression pattern of VvL.-IdnDH was characterized in fruits for each variety. The qPCR assays were performed in 6 biological samples for each variety and developmental stage, normalized against a housekeeping gene (VvEF1α) and expressed as a percentage of the highest value of relative abundance.
Analysis:
• Titratable acidity Titration
• Total organics acids
• Gene expression qPCR
A
B C
A, Schematic representation of phenological stages; B, Analyses performed; and C, Biosynthetic pathway of tartaric acid and key enzymes: L-Idonate dehydrogenase (L-IdnDH).
HPLCHPLC
Flowering
-2 -1 Véraison
+1 +2 Harvest
Postharvest
Weeks
Season 2009-2010
In progress (season 2010-2011)
Ascorbic acid
Oxalic acid L-threonate
2-keto-L-idonic acid L-idonic acid
5-keto-D-gluconic acid
Glycoaldehyde
Tartaric acid
L-IdnDH
L-threo-tetruronate
Ascorbic acid
Oxalic acid L-threonate
2-keto-L-idonic acid L-idonic acid
5-keto-D-gluconic acid
Glycoaldehyde
Tartaric acid
L-IdnDH
L-threo-tetruronate
0%10%20%30%40%50%60%70%80%90%
100%
Citric acid
Tartaric acid
Malic acid
Main acids detected in
berry:
Main acids detected in
berry:
A
B
0%
25%
50%
75%
100%
Per
cent
of a
cid
Total acids Var Red Globe
0,0
0,9
1,8
2,6
3,5
Ver-2
Ver-1 Ve
r
Ver+
1
Ver+
2
Har
vest
60d@
0°C
g ac
id/ 1
00g
grap
e
Total acids Var Thompson Seedless
0,0
0,9
1,8
2,6
3,5
Ver-2
Ver-1 Ve
r
Ver+
1
Ver+
2
Har
vest
45d@
0°C
g ac
id/ 1
00g
grap
e
0,0
0,9
1,8
2,6
3,5
Ver-2
Ver-1 Ve
r
Ver+
1
Ver+
2
Har
vest
60d@
0°C
0,0
0,9
1,8
2,6
3,5
Ver-2
Ver-1 Ve
r
Ver+
1
Ver+
2
Har
vest
45d@
0°C
Phenological stages (weeks)Phenological stages (weeks)
0%
25%
50%
75%
100%
Per
cent
of a
cid
Red GlobeRed Globe Thompson SeedlessThompson Seedless
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0
0,05
0,1
0,150,2
0,25
0,3
0,35
0,4
Tartaric acid content
L-IdnDH transcriptionalprofile
Rel
ativ
e
exp
ress
ion
Tartaric acid content
Tartaric acid content
Expression pattern of VvL-IdnDH
Expression pattern of VvL-IdnDH
Red GlobeRed Globe Thompson SeedlessThompson Seedless
0,0
0,9
1,8
2,6
3,5
Ver-2
Ver-1 Ve
r
Ver+
1
Ver+
2
Har
vest
60d@
0°C
0,0
0,9
1,8
2,6
3,5
Ver-2
Ver-1 Ve
r
Ver+
1
Ver+
2
Har
vest
45d@
0°C
Phenological stages (weeks)Phenological stages (weeks)
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0
0,1
0,2
0,3
0,4
0,5
g a
cid
/ 100g
gra
pe
g a
cid
/ 100g
gra
pe
Rela
tive e
xp
ressio
nR
ela
tive e
xp
ressio
n
g a
cid
/ 100g
gra
pe
g a
cid
/ 100g
gra
pe
Rela
tive e
xp
ressio
nR
ela
tive e
xp
ressio
n
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0
0,1
0,2
0,3
0,4
0,5
This work is funded by Fondecyt 1100273
Phenological stages (weeks)
0
1
2
3
4
5
6
TItra
table
acid
ity
Red GlobeThompson Seedless
0
1
2
3
4
5
6
TItra
table
acid
ity
Red GlobeThompson Seedless
0
1
2
3
4
5
6
Titra
table
acid
ity
Red Globe ThompsonSeedless
0
1
2
3
4
5
6
Titra
table
acid
ity
Red Globe ThompsonSeedless
