casiroz fall meeting antwerp 2003 participant group 2 w. oßwald manuela c. blumenröther forest...
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Primer design for Rubisco, small subunitTRANSCRIPT
CASIROZ Fall MeetingAntwerp 2003
Participant group 2
W. OßwaldManuela C. Blumenröther
Forest Phytopathology, TU Munich
Objectives/Achievements 2003
b) Gene expression studies for Rubisco and PEPC
RNA extraction, primer design for Rubisco, RT-PCR
a) Enzyme activity measurements for Rubisco and PEPC
Method learned at INRA, Nancy (Prof. Dizengremel)and established in the lab
Primer design for Rubisco, small subunit
Rubisco Exon1 1 TATGGCTTCCTCTATGATTTCCTCGGCCACTGTTGCCACCGTTAGCCGAGCCACCCCGGCTCACGCTACCATGGTTGCAC 80 Rubisco Exon2 1 -------------------------------------------------------------------------------- 1 Rubisco Exon3 1 -------------------------------------------------------------------------------- 1 2Rubisco su FW 1 -------------------------------------------------------------------------------- 1 2Rubisco su RV (RC) 1 -------------------------------------------------------------------------------- 1 Rubisco Exon1 81 CATTCACTGGTCTCAAGTCTACTGCAGCTTTCCCAGCTACCCAAAAGAGCAACAATGACATTACCTCTCTTGCAAGCAAT 160 Rubisco Exon2 1 -------------------------------------------------------------------------------- 1 Rubisco Exon3 1 -------------------------------------------------------------------------------- 1 2Rubisco su FW 1 -------------------------------------------------------------------------------- 1 2Rubisco su RV (RC) 1 -------------------------------------------------------------------------------- 1 Rubisco Exon1 161 GGTGGAAGAGTGCAATGCATGAAGGT 186 Rubisco Exon2 1 --------------------------GTGGCCACCACTTGGTTTGCAGAAGTTTGAGACTCTTTCCTACCTTCCACCACT 54 Rubisco Exon3 1 -------------------------------------------------------------------------------- 1 2Rubisco su FW 1 -----------------CATGAAGGTGTGGCCACCACTT 22 2Rubisco su RV (RC) 1 -------------------------------------------------------------------------------- 1 Rubisco Exon1 186 186 Rubisco Exon2 55 TTCTCTCGAGTCATTGGCTAAGCAAATTGAATACCTTATTTTGAAGGGATGGATTCCTTGCTTGGAATTCGAGCTGGAG 133 Rubisco Exon3 1 -------------------------------------------------------------------------------C 1 2Rubisco su FW 22 22 2Rubisco su RV (RC) 1 ----------------------------------------------------------------------GAGCTGGAGC 10 Rubisco Exon1 186 186 Rubisco Exon2 133 133 Rubisco Exon3 2 ACCCCTTTGTGTACCGTGAGAACAACAGGTCACCAGGGTACTATGATGGACGTTACTGGGTGATGTGGAAGCTGCCCATG 81 2Rubisco su FW 22 22 2Rubisco su RV (RC) 11 ACCCCTTTGTG 21 Rubisco Exon1 186 186 Rubisco Exon2 133 133 Rubisco Exon3 82 TTTGGATGCACCGATGCCACACAGGTGTTGGCGGAGCTCCAGGGGGCCAGTAAGACTTATCCCACTTCCCACATCCGAAT 161 2Rubisco su FW 22 22 2Rubisco su RV (RC) 21 21 Rubisco Exon1 186 186 Rubisco Exon2 133 133 Rubisco Exon3 162 CATCGGATTCGACAACAAGCGTCAAGTGCAGTGCATCAGTTTCATTGCTTACA 214 2Rubisco su FW 22 22 2Rubisco su RV (RC) 21 21
Objectives/Achievements 2003
b) Gene expression studies for Rubisco and PEPC
RNA extraction, primer design for Rubisco, RT-PCR
a) Enzyme activity measurements for Rubisco and PEPC
c) Analysis of the carbohydrate content
Method learned at INRA, Nancy (Prof. Dizengremel)and established in the lab
Methods established, first results for May and June samples
su Rubisco
+Std. -
Sampling-Timetable
50 leaves, all cut in 3 pieces
100 leaves per date, all cut in 3 pieces
50 leaves per time point, all cut in 2 pieces
Σ 150 single samples
Σ 1200 single samples
Σ 300 single samples
June/July/Sept./Oct.: 10 trees, sun & shade crown
July: 10 trees, sun crown, 3 additional time points for diurnal assessment
May: 10 trees, only sun crown
Ap ril M a y J une J uly Aug ust Se p t. O c to b e r
16.19. 29.16.
Variation within the sample branchesJune 2003 sun concentration differences within sample branches
sample no. sucrose glucose fructose sucrose glucose fructose[mg/g dw] [mg/g dw] [mg/g dw] [mg/g dw] [mg/g dw] [mg/g dw]
a 74,31 13,49 50,68reference b 73,94 13,21 50,21
c 74,91 13,61 51,00 0,97 0,40 0,80409_6 66,61 6,70 29,67409_7 77,47 7,39 34,63
tree 409 409_8 75,69 6,75 35,25 10,86 1,06 5,58409_9 72,12 6,86 33,38409_10 67,85 6,33 31,77439_6 71,84 7,61 38,50439_7 74,86 6,87 32,10
tree 439 439_8 64,28 8,12 39,42 30,80 3,96 13,25439_9 72,05 10,83 39,02439_10 95,08 7,43 45,35a 75,50 14,64 51,83
reference b 73,91 13,25 49,89c 75,74 14,01 51,72 1,83 1,39 1,94443_6 46,67 9,89 49,01443_7 56,92 9,31 35,67
tree 443 443_8 66,59 9,63 38,44 24,45 2,91 15,20443_9 70,38 7,89 33,81443_10 71,12 6,98 34,81482_6 60,87 7,27 32,71482_7 51,31 8,33 40,97
tree 482 482_8 56,24 6,59 29,71 17,57 4,65 11,26482_9 68,88 9,32 30,30482_10 60,60 4,68 31,64
Extraction routine is reliable, differences between single samples
are tree/branch specific
Sugar and starch content in May and June leaf samples of adult trees
Glucose, mean values +/-SE
0
4
8
12
16
20
1 x O3 May sun
2 x O3 1 x O3June sun
2 x O3 1 x O3June shade
2 x O3
treatment, sample date & position
gluc
ose
conc
entr
atio
n [m
g/g
DW
]
Fructose, mean values +/-SE
0
20
40
60
1 x O3 May sun
2 x O3 1 x O3June sun
2 x O3 1 x O3June shade
2 x O3
treatment, sample date & position
fruc
tose
con
cent
ratio
n [m
g/g
DW
]
Starch, mean values +/-SE
0
3
6
9
12
1 x O3 May sun
2 x O3 1 x O3June sun
2 x O3 1 x O3June shade
2 x O3
treatment, sample date & position
star
ch c
once
ntra
tion
[mg
gluc
ose
equi
vale
nt/g
DW
]
Sucrose, mean values +/-SE
0
20
40
60
80
1 x O3 May sun
2 x O3 1 x O3June sun
2 x O3 1 x O3June shade
2 x O3
treatment, sample date & position
sucr
ose
conc
entr
atio
n [m
g/g
DW
]
Ozone sensitivity in carbohydrate and starch metabolism visible
Carbohydrate contents for sun leaves on the single tree level (June 2003)
Glucose content in sun leaves (June 2003), means +/-SE
0
4
8
12
3991 x O3
408 409 410 412 4372 x O3
439 443 480 482
tree no.
gluc
ose
conc
entr
atio
n [m
g/gD
W]
Fructose content in sun leaves (June 2003), means +/-SE
0
20
40
60
3991 x O3
408 409 410 412 4372 x O3
439 443 480 482
tree no.
fruc
tose
con
cent
ratio
n [m
g/g
DW
]
Starch content in sun leaves (June 2003), means +/-SE
0
6
12
18
3991 x O3
408 409 410 412 4372 x O3
439 443 480 482
tree no.
star
ch c
once
ntra
tion
[mg
gluc
ose
equi
vale
nt/g
DW
]
Sucrose content in sun leaves (June 2003), means +/-SE
0
30
60
90
3991 x O3
408 409 410 412 4372 x O3
439 443 480 482
tree no.
sucr
ose
conc
entr
atio
n [m
g/g
DW
]
2 x O32 x O31 x O3 1 x O3
2 x O32 x O3 1 x O31 x O3
Is ozone affecting sugar allocation?
...high in the sky...- what about the variation within a canopy?
Sampling for canopy variation assessment (July, 16.)• Sun crown of tree no. 399 devided in 4 segments (N, E, S, W),• 3 branches marked per segment, • 5 leaves per branch collected and cut into 2 halves for enzyme & carbohydrate experiments.
N
W
SE
Carbohydrate variation within the canopy of 399
glucose concentration [mg/g DW] in sun leaves,
mean value s per branch
0
2
4
6
81
2
3
4
5
6
7
8
9
10
11
12
fructose concentration [mg/g DW] in sun leaves,
mean values per branch
0
5
10
15
20
251
2
3
4
5
6
7
8
9
10
11
12
sucrose concentration [mg/g DW],in sun leaves,
mean values per branch
0
20
40
60
80
1001
2
3
4
5
6
7
8
9
10
11
12
N
S
starch concentration [mg glucose equivalent/g DW], in sun leaves, mean values per branch
0,00,51,01,52,02,53,0
1
2
3
4
5
6
7
8
9
10
11
12
Indeed there is variation within the canopy!
In addition...
October, 13.: in contrast to Oct., 07. leaf sampling at sunny weather;
August, 11.: leaf sampling in parallel with gas exchange measurements under saturating light conditions;
Ap ril M a y J une J uly August Sep t. O c to b e r
16.19. 16./29.16.11. 13.
Leaf samples from: • young trees• branch cuvette fumigation chambers
10 trees, 50 leaves, (only carbohydrate and enzyme experiments)
two trees 1 x O3, two trees 2 x O3; sun & shade crown positions, 80 leaves (only carbohydrate and enzyme experiments)
Preview for 2004
• No major changes in sampling time schedule
• Primer design for PEPC
• Continuing carbohydrate and enzyme activity measurements
• Including young tree and cuvette leaf samples