Zeatin Cis-Trans Isomerasein Plants

Tomáš HLUSKADepartment of Molecular Biology

Centre of the Region Haná for Biotechnological and Agricultural Research

Zeatin interconversions

N

N

NH

CH3

CH3

N

NH

N

N

NH

CH3

N

NH

OH

N

N

NH CH3

N

NH

OH

N

N

NH

N

NH

CH3

OH

N

N

NH

N

NH

CH3

CH2

tRNA degradation

de novobiosynthesis

proved in plantagene is known

iP

tZ

cZ

DHZ

???

hypothesized

proved in vitroactivity detected

Zeatin Cis-Trans Isomerase

Zeatin Cis-Trans Isomerase

Existence of the enzyme is questionable due to several reasons: Only one article describing in vitro activity and only one article describing in vivo activity

High non-enzymatic conversion

Need to light on reaction

Arabidopsis IPT knock-outs have lowered levels of tZ or cZ (Miyawaki et al. 2006)

Side chain of tZ and cZ originates from distinct pathways (Kasahara et al. 2004)

ZI activity in plants

3-days old plants nkat/mgwheat root 0.0319rice stem 0.0441rice root 0.0510Arabidopsis 2 days 0.0243Arabidopsis 5 days 0.2783maize roots 0.0492

Upon salt stress, the activity rises in root, while decreases in shoot.

ZI activity in plantsnkat/mg

2 weeks old roots 1.1723 weeks old roots 1.4183 weeks old roots deseeded 2.032endosperm 5 days after pollination 0.532

activity in maize plants

nkat/mg14 days old 0.14130 days old 0.19353 days old 0.28479 days old 0.117100 days old 0.227

activity in Arabidopsis shoots

Totalproteins

(mg)

Specificactivity

(nkat/mg)

Totalactivity(nkat)

Purification(-fold)

Recovery(%)

crude extract 2272.50 0.023 51.19 1.0 100.020-80% (NH4)2SO4 2139.60 0.012 24.86 0.5 48.6Octyl Sep I 337.84 0.061 20.56 2.7 40.2Octyl Sep II 112.55 0.033 3.67 1.4 7.2High Q 4.10 0.341 1.40 15.2 2.7ConA 1.10 0.622 0.68 27.6 1.3Resource Q 0.16 3.492 0.57 155.0 1.1BioSilect 0.071 6.168 0.44 273.8 0.9HiTrap Blue Gel 0.019 33.130 0.62 1470.8 1.2

Purification of ZmZI

starting material: 3 days old roots of maize – 379 g

Zeatin Cis-Trans Isomerase

glucan exohydrolaseNAD-dep malate dehydrogenase

peptidoglycan bindingnucleoside diphosphate binding

peroxidasexylanase inhibitor

phosphoglycerate kinaseserine protease

phosphoglycerate mutaseaspartate proteaseheat shock protein

FAD-binding proteinalpha-glucosidase; xylosidase

ferredoxin NADP-reductasechitinase

polyamine oxidasenodulin

vicilin; globulinpurple acid phosphatase

lactoylglutathione lyase; glyoxylasebeta-galactosidasealdose reductase

nucleaselecithin:cholesterol acyltransferase

methionine synthaseacyl-CoA binding protein

Properties of ZmZI

Properties of ZmZI

0

2

4

6

8

10

12

0 10 20 30 40 50 60

time [min]

conv.[%]

Thank you for your attention