Hormones characterisation and identification of new CK-like substances in transgenic tobacco
plants expressing rolC gene
Dana Tarkowská
Palacky University & Institute of Experimental Botany AS CR
Laboratory of Growth Regulators
Introduction Normally, the plants can actively regulate their endogenous
hormone pools and the phenotypical alterations of transgenic plants are results of abnormal change in the pool size of a particular hormone.
The majority of known genes encoding enzymes involved in plant hormone regulation are of bacterial origin:
• iaaH and iaaM genes from Agrobacterium tumefaciens encode enzymes involved in the biosynthesis of IAA from tryptophan iaaH/iaaM-expressing plants (dwarfs with a strong apical dominance and excessive root
formation) show increased pools of free IAA and IAA conjugates
• ipt gene product also originates from A. tumefaciens and mediates the synthesis of iP nucleotides from IPP and AMP increased levels of cytokinins were detected in ipt-expressing plants (dark green with reduced apical dominance and poor root formation)
rol genesÞ Infection of a plant by A. rhizogenes results in root
formation at the site of the infection due to the transfer of genetic information (T-DNA) carried by Ri plasmids from bacteria to the plant cells
Þ T-DNA of the A. rhizogenes contains four loci involved in the hairy roots formation and are called root loci (rol) A, B, C, and D (White et al., 1985)
Þ rolA, rolB and rolC genes are transfered and expressed in the plant cell and play a central role in the production of hairy root
rolC gene – have been used to trasform:• tobacco (Schmülling et al, 1988; Nillson et al,
1993)• potato (Fladung, 1990)• Atropa belladonna (Kurioka et al, 1992)• hybrid aspen (Nilsson et al, 1996)– has been proposed that -glucosidase is
capable to release free CKs from inactive N- and O-glucose conjugates but quantification data did not support this theory Spena et al, EMBO J, 1987
Phenotype of transgenic tobacco expressing rolC gene
Transgenic tobacco plants carring only the rolC locus under control of cauliflower mosaic virus 35S promoter is more branched with shortened internodes, reduced leaf and flower size and pollen production (Schmülling et al, 1988)
Tobacco plants transgenic for the CaMVC chimaeric gene, where the rolC coding region is under the control of the CaMV 35S promoter, have dwarf and bushy phenotype due to decreased internode length and an increased number of shoots and leaves- CaMVC flowers are tiny and the leaves are small; plants are male sterile, female fertility is reduced
rolC WT
WT rolC
WT
rolC
Hormonal characterisation of rolC tobacco – early 1990´s
Transgenic tobacco (Nicotiana tabacum L.) plants expressing the A. rhizogenes rolC gene under the control of the cauliflower 35S promoter (Nilsson et al, 1993) showed:
- no alteration in IAA pool size and its rate of turnover- almost 6-fold increase if GA19 compared to the WT- drastic reduction of isopentenyladenosine (iPR)- markedly lower ABA levels in rolC leaves being 50% of the concentrations in the
control leaves
Plant material: transgenic tobacco (Nicotiana tabacum L.) plants expressing the A. rhizogenes rolC gene under the control of the CaMV 35S promoter
Auxins - significant alterations in IAA and oxIAA levels in rolC compared to WT (SR1) in apical part with flowers and roots; no changes in leaves
Hormonal characterisation of rolC tobacco – our results
apical part (flowers)
the 1st leaves under
flower (young)
the 2nd leaves under
flower (middle)
the 3rd leaves under
flower (large)
the 4th leaves under
flower
the 5th leaves under
flower
the 6th leaves under
flower
the 7th leaves under
flower
the 8th leaves under
flowerstems roots
SR1 4.25 2.55 0.90 1.34 1.09 1.15 1.24 0.75 0.70 2.01 3.88
rolC 6.15 1.83 0.89 0.54 0.39 1.62 1.07
0.00
1.50
3.00
4.50
6.00
7.50
9.00
IAA
(n
g/g
FW)
IAA
SR1
rolC
apical part (flowers)
the 1st leaves under flower
(young)
the 2nd leaves under flower
(middle)
the 3rd leaves under flower (large)
the 4th leaves under flower
the 5th leaves under flower
the 6th leaves under flower
the 7th leaves under flower
the 8th leaves under flower
stems roots
SR1 0.09 0.30 1.49 0.78 1.53 1.26 1.30 0.74 0.38 0.08 0.18
rolC 0.31 0.16 0.16 0.22 0.11 0.05 0.08
0.00
0.50
1.00
1.50
2.00
2.50
3.00
oxIA
A (
ng/
g FW
)
oxIAA
SR1
rolC
apical part (flowers)
the 1st leaves under flower
(young)
the 2nd leaves under flower
(middle)
the 3rd leaves under flower (large)
the 4th leaves under flower
the 5th leaves under flower
the 6th leaves under flower
the 7th leaves under flower
the 8th leaves under flower
stems roots
SR1 57.64 8.25 0.53 1.76 0.71 0.92 0.98 1.01 1.98 24.31 22.27
rolC 20.40 11.46 5.74 3.08 3.49 35.90 15.95
0.00
20.00
40.00
60.00
80.00
100.00
IAA
/oxI
AA
rat
io
IAA/ oxIAA ratio
SR1
rolC
Plant material: transgenic tobacco (Nicotiana tabacum L.) plants expressing the A. rhizogenes rolC gene under the control of the CaMV 35S promoter
Cytokinins – bases: Significantly high levels of cZR and iPR in rolC roots compared to WT (SR1) and interestingly elevated levels of tZR in rolC stems
Hormonal characterisation of rolC tobacco – our results
WT apical
part
WT the 1st
leaves
WT the 2nd
leaves
WT the 3rd
leaves
WT the 4th
leaves
WT the 5th
leaves
WT the 6th
leaves
WT the 7th
leaves
WT the 8th
leaves
WT stems
WT roots
rolC apical
part
rolC the 1st leaves
rolC the 2nd leaves
rolC the 3rd leaves
rolC the 4th & 5th
leaves
rolC stems
rolC roots
tZR 0.1726 0.2614 0.1805 0.1411 0.1229 0.1593 0.1094 0.0637 0.0679 0.1501 1.2721 0.2619 0.3472 0.3054 0.2682 0.1567 2.8781 0.9270
cZR 2.8166 0.8713 0.7955 0.4810 0.3772 0.3441 0.3501 0.3354 0.3593 0.5361 0.9468 1.6518 0.3889 0.3820 0.3542 0.3358 0.4467 2.5123
DHZR 0.1648 0.1065 0.0840 0.0502 0.0463 0.0415 0.0430 0.0414 0.0431 0.0703 0.1221 0.3172 0.0668 0.0546 0.0439 0.0426 0.0742 0.3339
iPR 1.6583 1.2553 0.8531 0.5505 0.4047 0.3913 0.3958 0.3562 0.3565 0.7551 1.0071 1.4343 0.5470 0.4486 0.4011 0.3582 0.6021 2.5577
0.00
1.00
2.00
3.00
4.00
ISCK
rib
osid
esco
nte
nt(p
mol
/gFW
)
ISCK ribosides content in tobacco WT (SR1) and rolC tissues
tZR cZR
DHZR iPR
Plant material: transgenic tobacco (Nicotiana tabacum L.) plants expressing the A. rhizogenes rolC gene under the control of the CaMV 35S promoter
CK-nucleotides: Extremely high levels of cZR nucleotides in WT apical part compared to rolC and elevated levels of tZR nucleotides in rolC stems corresponding to high tZR levels
Hormonal characterisation of rolC tobacco – our results
WT apical part
WT the 1st
leaves
WT the 2nd
leaves
WT the 3rd
leaves
WT the 4th
leaves
WT the 5th
leaves
WT the 6th
leaves
WT the 7th
leaves
WT the 8th
leaves
WT stems
WT roots
rolC apical part
rolC the 1st leaves
rolC the 2nd
leaves
rolC the 3rd leaves
rolC the 4th & 5th
leaves
rolC stems
rolC roots
tZR nucleotides 0.1360 0.1517 0.1420 0.1106 0.0481 0.0488 0.0731 0.0443 0.0410 0.1549 0.8516 0.3631 0.3434 0.2403 0.1233 0.1110 1.5440 0.4736
cZR nucleotides 13.946 2.8696 1.2401 0.6648 0.4044 0.3265 0.3414 0.3340 0.3346 0.3769 0.9197 2.3030 0.3565 0.3807 0.4010 0.3572 0.3848 2.8315
iPR nucleotides 0.7760 1.1965 0.6992 0.4079 0.3277 0.3251 0.3308 0.3156 0.3193 0.5007 0.8802 1.6179 0.4591 0.4291 0.3967 0.3567 0.5122 2.2625
DHZR 1.1106 0.3396 0.1680 0.0862 0.0511 0.0412 0.0417 0.0426 0.0426 0.0462 0.1284 1.1530 0.0878 0.0842 0.0638 0.0606 0.0673 0.3215
0.00
4.00
8.00
12.00
16.00
20.00
ISCK
nuc
leot
ides
con
tent
(p
mol
/g F
W)
ISCK nucleotides content in tobacco WT (SR1) and rolC tissues
tZR nucleotidescZR nucleotidesiPR nucleotidesDHZR
Unknown CK-like compounds in WT and rolC tobacco plants
Calibration 50pmol CK/1.0 pmol 2H-CK/5uL
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
AU
0.0
2.0e-3
4.0e-3
6.0e-3
060718_Calib16 9: Diode Array 268 1.00Da
Range: 5.547e-2
3.55
2.832.593.433.13
4.303.98
3.87
7.246.86
4.60 5.334.97
4.76 5.146.215.695.86 6.50
SR 1_4
SR 1_3
SR 1_2
SR 1_1
2.4
9
2.2
5
2.0
7
1.64
2.4
9
2.2
4
2.0
7
1.64
1.5
3
2.2
5
2.0
7
1.9
61.5
4
1.64
2.4
91.5
4
2.0
7
2.2
5
1.9
5
1.64
Tobacco shoots SR 1_fraction bases_UV chromatograms at 268 nm_July 2006
iPRBAPR
iPBA
P
oT
R
oT
iP9
G
BA
P9
G
KR
mT
R
oT
9G
pT
R+
K
dH
ZR
+cZ
R+
mT
pT
mT
9G
cZ
dH
Z
tZ
dH
Z9
G
Z9
G
CK standards
blank sample
Calibration 50pmol CK/1.0 pmol 2H-CK/5uL
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
AU
0.0
2.0e-3
4.0e-3
6.0e-3
060718_Calib16 9: Diode Array 268 1.00Da
Range: 5.547e-2
3.55
2.832.59
3.433.13
4.30
3.98
3.87
7.24
6.864.60 5.33
4.974.76
5.146.21
5.695.86 6.50
Tobacco shoots (CaMVC-X)_fraction bases_UV chromatograms at 268 nm_July 2006
(CaMVC-X)_3
(CaMVC-X)_2
(CaMVC-X)_1
2.242.49
1.94
1.64
1.16; 1.26
2.491.94
1.64
1.26
2.08
1.65
1.28
iPR
BAPR
iP
BA
P
oT
R
oT
iP9
G
BA
P9
G
KR
mT
R
oT
9G
pT
R+
K
dH
ZR
+cZ
R+
mT
pT
mT
9G
cZ
dH
Z
tZ
dH
Z9
G
Z9
G
CK standards
blank sample
WT (SR1) rolC
Spectral characterisation of new CK-like compounds in WT and rolC tobacco plants
Tobacco Shoots (SR1) 2g Base Inj.15/30
nm210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400
AU
0.0
5.0e-3
1.0e-2
1.5e-2
2.0e-2
2.5e-2
3.0e-2
3.5e-2
4.0e-2
4.5e-2
5.0e-2
5.5e-2
6.0e-2
6.5e-2
7.0e-2
7.5e-2
316.47
290.47
235.47
SR 1_4
SR 1_3
SR 1_2
SR 1_1
Tobacco shoot_SR 1_fraction bases_UV spectra of a peak at 1.64 min_July 2006
292.47
318.47
291.47
318.47
293.47
317.47
Tobacco Shoots (CaMVC-X) 2g Base Inj.15/30
nm210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400
AU
0.0
2.0e-3
4.0e-3
6.0e-3
8.0e-3
1.0e-2
1.2e-2
1.4e-2
1.6e-2
1.8e-2
320.47
234.47221.47
(CaMV-X)_3
(CaMV-X)_2
(CaMV-X)_1
Tobacco shoot_rolC_fraction bases_UV spectra of a peak at 1.64 min_July 2006
293.47
319.47
291.47
317.47
UV spectrum of the first unknown peak from SR1 UV spectrum of the first unknown peak from rolC
Spectral characterisation of new CK-like compounds in WT and rolC tobacco plants
M+H+ = 251
Elemental Composition Report
Single Mass Analysis Tolerance = 20.0 PPM / DBE: min = -1.5, max = 10.0Selected filters: None
Monoisotopic Mass, Even Electron Ions624 formula(e) evaluated with 3 results within limits (up to 50 closest results for each mass)Elements Used: C: 0-50 H: 0-100 N: 0-10 O: 0-10 Minimum: -1.5Maximum: 5.0 20.0 10.0Mass Calc. Mass mDa PPM DBE i-FIT Formula472.2405 472.2407 -0.2 -0.4 6.5 259.6 C20 H34 N5 O8 472.2367 3.8 8.0 2.5 1034.4 C15 H34 N7 O10 472.2479 -7.4 -15.7 2.5 1190.1 C14 H34 N9 O9
6.00000000 tobacco rolC roots, harvest Sept2009, no previously HPLC fractionated
m/z 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000
%
0
100 100216_HPLC_MS-POS_Tob110-113-B 283 (15.530) AM (Cen,4, 80.00, Ar,5000.0,362.93,1.00,LS 10); Sm (SG, 2x1.00); Sb (1,40.00 ); Cm (282:288 -(103:168+460:479)) 1: TOF MS ES+
6.52e4 472.2405 65201
470.2248 696 161.1095
70 58.7184
3 371.1943 10
211.3715 1
294.3364 1
473.2471 19349
474.2503 3691
555.3118 1332 484.2395
424 636.3592 48
868.2130 2
664.8049 1 958.0555
0
Spectral characterisation of new CK-like compounds in WT and rolC tobacco plants
M+H+ = 472
Quantification of new CK-like compounds in WT and rolC tobacco plants
WT apical part
WT the 1st leaves
WT the 2nd
leaves
WT the 3rd
leaves
WT the 4th leaves
WT the 5th leaves
WT the 6th leaves
WT the 7th leaves
WT the 8th leaves
WT stems
WT roots
rolC apical part
rolC the 1st leaves
rolC the 2nd
leaves
rolC the 3rd
leaves
rolC the 4th & 5th
leaves
rolC stems
rolC roots
compound 250 0.1905 0.3361 0.2242 0.1355 0.0706 0.0683 0.0925 0.0708 0.0717 0.0667 0.1019 0.1285 0.0520 0.0000 0.0000 0.0000 0.0184 0.6641
0.00
0.20
0.40
0.60
0.80
1.00
Com
pou
nd
wit
h M
r 25
0 g.
mol
-1co
nte
nt
(pm
ol)
Content of compound with Mr 250 g·mol-1 in tobacco WT (SR1) and rolC tissue - fraction of bases
WT apical
part
WT the 1st
leaves
WT the 2nd
leaves
WT the 3rd
leaves
WT the 4th
leaves
WT the 5th
leaves
WT the 6th
leaves
WT the 7th
leaves
WT the 8th
leaves
WT stems
WT roots
rolC apical
part
rolC the 1st leaves
rolC the 2nd leaves
rolC the 3rd leaves
rolC the 4th & 5th
leaves
rolC stems
rolC roots
compound 250 3.1585 7.4550 1.9028 1.6906 0.2288 0.1754 0.0667 0.0654 0.0384 0.0508 0.1299 0.4097 0.0631 0.0630 0.0393 0.0000 0.0000 0.1837
0.00
2.00
4.00
6.00
8.00
10.00
Con
ten
t of
com
pou
nd
wit
h M
r 2
50
g.m
ol-1
(pm
ol)
Content of compound with Mr 250 g·mol-1 in tobacco WT (SR1) and rolC tissue- fraction of nucleotides
WT apical
part
WT the 1st
leaves
WT the 2nd
leaves
WT the 3rd
leaves
WT the 4th
leaves
WT the 5th
leaves
WT the 6th
leaves
WT the 7th
leaves
WT the 8th
leaves
WT stems
WT roots
rolC apical
part
rolC the 1st leaves
rolC the 2nd
leaves
rolC the 3rd leaves
rolC the 4th & 5th
leaves
rolC stems
rolC roots
compound 471 2.6391 1.1838 0.7250 0.2726 0.0959 0.0695 0.1526 0.1372 0.1002 0.1906 0.0830 0.8248 0.0744 0.0532 0.0339 0.0326 0.0339 0.4035
0.00
0.80
1.60
2.40
3.20
Com
pou
nd
wit
h M
r 47
1 g.
mol
-1
con
ten
t (p
mol
)
Content of compound with Mr 471 g·mol-1 in tobacco WT (SR1) and rolC tissue- fraction of bases
WT apical part
WT the 1st
leaves
WT the 2nd
leaves
WT the 3rd
leaves
WT the 4th
leaves
WT the 5th
leaves
WT the 6th
leaves
WT the 7th
leaves
WT the 8th
leaves
WT stems
WT roots
rolC apical part
rolC the 1st leaves
rolC the 2nd
leaves
rolC the 3rd leaves
rolC the 4th & 5th
leaves
rolC stems
rolC roots
compound 471 0.7096 0.4219 0.1950 0.1183 0.0511 0.0364 0.0335 0.0330 0.0333 0.0348 0.0723 0.2154 0.0354 0.0351 0.0359 0.0336 0.0328 0.2322
0.00
0.20
0.40
0.60
0.80
Con
ten
t of
com
pon
ud
wit
h M
r 4
71
g.m
ol-1
(pm
o
Content of compound with Mr 471 g.mol-1 in tobacco WT (SR1) and rolC tissue- fraction of nucleotides
Conclusions• hormonal characterisation of WT tobacco (Nicotiana tabacum) plants
and transgenic rolC tobacco is in agreement with previously published findings concerning the auxin and isoprenoid cytokinin level in leaf tissues
• Newly was quantified cytokinin and auxin content in apical part of the WT and rolC plants as well as in the plant stems and roots
• Ratio of active auxin IAA and its main inactive degradation product oxIAA is higher in rolC tissue only in case of the plant stem, in all the other studied tissues is this ratio higher for WT plants than for rolC
• Very high levels of cZR and iPR were surprisingly observed in rolC roots compared to the WT. Extremely high levels of tZR were found in rolC stems
• 2 new CK-like substances were characterised using MS so far without elucidation of their structures – compunds with Mr 250 and 471; 471 is closely related to 250
• Using external calibration, content of newly found substances were quantified – the highest levels of compound with Mr 250 are present in nucleotide fraction of the youngest WT tobacco leaves extract, while substance with Mr 471 could be observed at highest levels in fraction of bases of WT tobacco apical part