Involvement of cis-Zeatin, Dihydrozeatin, and Aromatic Cytokinins in Germination and Seedling Establishment of Maize, Oats, and Lucerne
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The aims of this study were to monitor endogenous cytokinin levels during germination and early seedling establishment in oats, maize, and lucerne to determine which cytokinin forms are involved in these processes; to quantify the transfer ribonucleic acid (tRNA)-bound cytokinins; and to measure cytokinin oxidase/dehydrogenase (CKX) activity. Cytokinins were identified using UPLC-MS/MS. The predominant free cytokinins present in the dry seeds were dihydrozeatin-type (DHZ) in lucerne and maize and cZ-type (cis-zeatin) in oats. Upon imbibition, there was a large increase in cZ-type cytokinins in lucerne although the cZ-type cytokinins remained at high levels in oats. In maize, the high concentrations of DHZ-type cytokinins decreased prior to radicle emergence. Four tRNA-bound cytokinins [cis-zeatin riboside (cZR)>N 6-(2-isopentenyl)adenosine (iPR), dihydrozeatin riboside (DHZR), trans-zeatin riboside (tZR)] were detected in low concentrations in all three species investigated. CKX activity was measured using an in vitro radioisotope assay. The order of substrate preference was N 6-(2-isopentenyl)adenine (iP)>trans-zeatin (tZ)>cZ in all three species, with activity fluctuating as germination proceeded. There was a negative correlation between CKX activity and iP concentrations and a positive correlation between CKX activity and O-glucoside levels. As O-glucosides are less resistant to CKX degradation, they may provide a readily available source of cytokinins that can be converted to physiologically active cytokinins required during germination. Aromatic cytokinins made a very small contribution to the total cytokinin pool and increased only slightly during seedling establishment, suggesting that they do not play a major role in germination.
Keywordscis-Zeatin Cytokinin oxidase/dehydrogenase Dihydrozeatin Lucerne Maize Oats tRNA degradation
The National Research Foundation, South Africa is thanked for financial assistance. Hana Martinková and Michaela Glosová are acknowledged for their help with cytokinin analyses and Marie Korecká and Dr. Petre Dobrev for their assistance with CKX determinations. The Ministry of Education, Youth and Sports of the Czech Republic (grants MSM 6198959216 and LC06034), the Centre of the Region Haná for Biotechnological and Agricultural Research (grant ED0007/01/01), and the Czech Science Foundation (grant 506/11/0774) are thanked for financial support.
The authors declare that they have no conflict of interest.
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