Proc Natl Acad Sci U S A 98(18): 10487-10492

Regulation of plant growth by cytokinin

^{Centre for Plant Molecular Biology (ZMBP)/Allgemeine Genetik, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany;}^{Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 135, CZ-16502 Prague 6, Czech Republic; and}^{Palacký University and Institute of Experimental Botany ASCR, Department of Botany, Laboratory of Growth Regulators,}

\overset{̆}{S}

lechtitelů 11, CZ-78371 Olomouc, Czech Republic

^{To whom reprint requests should be addressed. E-mail:}ed.negnibeut-inu.pbmz@gnilleumhcs.samoht.

Communicated by Jozef S. Schell, Max Planck Institute for Plant Breeding Research, Cologne, Germany

Received 2001 Feb 15; Accepted 2001 Jun 15.

Abstract

Cytokinins are a class of plant-specific hormones that play a central role during the cell cycle and influence numerous developmental programs. Because of the lack of biosynthetic and signaling mutants, the regulatory roles of cytokinins are not well understood. We genetically engineered cytokinin oxidase expression in transgenic tobacco plants to reduce their endogenous cytokinin content. Cytokinin-deficient plants developed stunted shoots with smaller apical meristems. The plastochrone was prolonged, and leaf cell production was only 3–4% that of wild type, indicating an absolute requirement of cytokinins for leaf growth. In contrast, root meristems of transgenic plants were enlarged and gave rise to faster growing and more branched roots. These results suggest that cytokinins are an important regulatory factor of plant meristem activity and morphogenesis, with opposing roles in shoots and roots.

Abstract

Cytokinins were discovered during the 1950s because of their ability to induce plant cell division (1). Shortly after their discovery, Skoog and Miller coined the auxin–cytokinin hypothesis of plant morphogenesis (2). The hypothesis predicted that cytokinin, together with auxin, plays an essential role in plant morphogenesis, having a profound influence on the formation of roots and shoots and their relative growth.

Chemically, natural cytokinins are N^{-substituted purine derivatives. Isopentenyladenine (iP), zeatin (Z), and dihydrozeatin (DZ) are the predominant cytokinins found in higher plants. The free bases and their ribosides (iPR, ZR, DZR) are thought to be the biologically active compounds. Glycosidic conjugates play a role in cytokinin transport, protection from degradation, and reversible and irreversible inactivation (}3).

Numerous reports ascribe a stimulatory or inhibitory function to cytokinins in different developmental processes such as root growth and branching, control of apical dominance in the shoot, chloroplast development, and leaf senescence (4). Conclusions about the biological functions of cytokinins have mainly been derived from studies on the consequences of exogenous cytokinin application or endogenously enhanced cytokinin levels (5, 6). Up to now, it has not been possible to address the reverse question: what are the consequences for plant growth and development if the endogenous cytokinin concentration is decreased? Plants with a reduced cytokinin content are expected to yield more precise information about processes cytokinins limit and, therefore, might regulate. Unlike other plant hormones such as abscisic acid, gibberellins, and ethylene, no cytokinin biosynthetic mutants have been isolated (7).

The catabolic enzyme cytokinin oxidase (CKX, ref. 8) plays possibly the principal role in controlling cytokinin levels in plant tissues. CKX activity has been found in a great number of higher plants and in different plant tissues (8). The enzyme is a FAD-containing oxidoreductase that catalyzes the degradation of cytokinins bearing unsaturated isoprenoid side chains. The free bases, iP and Z, and their respective ribosides are the preferred substrates. The reaction products of iP catabolism are adenine and the unsaturated aldehyde 3-methyl-2-butenal (8). Recently, a cytokinin oxidase gene from Zea mays has been isolated (9, 10). The manipulation of CKX gene expression could partially overcome the lack of cytokinin biosynthetic mutants and might be used as a powerful tool to study the relevance of iP- and Z-type cytokinins during the whole life cycle of higher plants. In this article, we report the cloning of four putative CKX genes from Arabidopsis thaliana and the results of their systemic overexpression in transgenic tobacco plants. Our data indicate an important role for cytokinins in plant growth regulation via a differential influence on the number and/or duration of cell division cycles in the root and shoot meristems.

Three independently pooled samples of approximately 100 2-week-old seedlings (2.5 g/sample) were analyzed for each clone. Concentrations are in pmol × g fresh weight^{. iP, N}^-(Δ^{isopentenyl)adenine; iPR, N}^-(Δ^{isopentenyl)adenine riboside; iPRP, N}^-(Δ^{isopentenyl)adenine riboside 5′-monophosphate; ZR, zeatin riboside; ZRP, zeatin riboside 5′-monophosphate; ZOG, zeatin}O-glucoside; ZROG, zeatin riboside O-glucoside.

Acknowledgments

We dedicate this article to C. O. Miller and the late F. S. Skoog, who discovered cytokinins almost 50 years ago and coined the auxin–cytokinin hypothesis of plant growth. We are indebted to K. Lemcke for initial help with gene cloning and sequence analyses, M. Riefler for support with structural gene analysis, M. Lenhard and Y. Stierhof for advise for microscopic analyses, and M. Kamínek for helpful comments on the manuscript. We thank H. Martínková and V. Lacmanová for excellent technical assistance and M. J. Beech and C. Scott-Taggart for proofreading. We acknowledge financial support of the Deutsche Forschungsgemeinschaft (Schm 814/13-1), the Volkswagen-Stiftung (I/72076), the Grant Agency of the Czech Republic (522/00/1346), and the Czech Ministry of Education (MSM 153100008).

Acknowledgments

Abbreviations

CKX	cytokinin oxidase
iP	isopentenyladenine
Z	trans-zeatin
SAM	shoot apical meristem

Abbreviations

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