1. 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

2. 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)

3. 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 rolABCD  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 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

4. 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 rolCWT rolC WT rolC

5. 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 GA 19 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

6. 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

7. 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

8. 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

9. Unknown CK-like compounds in WT and rolC tobacco plants WT (SR1)rolC

10. Spectral characterisation of new CK-like compounds in WT and rolC tobacco plants UV spectrum of the first unknown peak from SR1 UV spectrum of the first unknown peak from rolC

11. Spectral characterisation of new CK-like compounds in WT and rolC tobacco plants  M+H  + = 251

12. Elemental Composition Report Single Mass Analysis Tolerance = 20.0 PPM / DBE: min = -1.5, max = 10.0 Selected filters: None Monoisotopic Mass, Even Electron Ions 624 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: -1.5 Maximum: 5.0 20.0 10.0 Mass Calc. Mass mDa PPM DBE i-FIT Formula 472.2405 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 Spectral characterisation of new CK-like compounds in WT and rolC tobacco plants  M+H  + = 472

13. Quantification of new CK-like compounds in WT and rolC tobacco plants

14. 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 M r 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 M r 250 are present in nucleotide fraction of the youngest WT tobacco leaves extract, while substance with M r 471 could be observed at highest levels in fraction of bases of WT tobacco apical part