dana tarkowská

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 genes 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

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)

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


flower


flower


flower


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


the 1st leaves under flower

(young)

the 2nd leaves under flower

(middle)

the 3rd leaves under flower (large)

the 4th 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


the 1st leaves under flower

(young)

the 2nd leaves under flower

(middle)

the 3rd leaves under flower (large)






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


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


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


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


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

ten

t (p

mo

l/g

FW)

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

2.492.25

2.07

1.64

2.492.24

2.07

1.64

1.53

2.25

2.07

1.96

1.54

1.64

2.491.

54

2.07 2.25

1.95

1.64

Tobacco shoots SR 1_fraction bases_UV chromatograms at 268 nm_July 2006

iPRBAPR

iPBA

P

oTR

oT

iP9G

BA

P9G

KR

mTR

oT9G

pTR

+K

dHZR

+cZR

+mT

pT

mT

9G

cZ

dHZ

tZ

dHZ9

G

Z9G

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

2.832.593.433.13

4.30

3.98

3.87

7.246.86

4.60 5.334.97

4.765.14

6.215.695.86 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

iPRBAPR

iPBA

P

oTR

oT

iP9G

BA

P9GKR

mTR

oT9G

pTR

+K

dHZR

+cZR

+mT

pT

mT9

G

cZ

dHZ

tZ

dHZ9

G

Z9G

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

316.47290.47

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


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


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

dana tarkowská

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