We used loss-of-function mutants to study three Arabidopsis thaliana sensor histidine kinases, AHK2, AHK3, and CRE1/ AHK4, known to be cytokinin receptors. Mutant seeds had more rapid germination, reduced requirement for light, and decreased far-red light sensitivity, unraveling cytokinin functions in seed germination control. Triple mutant seeds were more than twice as large as wild-type seeds. Genetic analysis indicated a cytokinin-dependent endospermal and/or maternal control of embryo size. Unchanged red light sensitivity of mutant hypocotyl elongation suggests that previously reported modulation of red light signaling by A-type response regulators may not depend on cytokinin. Combined loss of AHK2 and AHK3 led to the most prominent changes during vegetative development. Leaves of ahk2 ahk3 mutants formed fewer cells, had reduced chlorophyll content, and lacked the cytokinin-dependent inhibition of dark-induced chlorophyll loss, indicating a prominent role of AHK2 and, particularly, AHK3 in the control of leaf development. ahk2 ahk3 double mutants developed a strongly enhanced root system through faster growth of the primary root and, more importantly, increased branching. This result supports a negative regulatory role for cytokinin in root growth regulation. Increased cytokinin content of receptor mutants indicates a homeostatic control of steady state cytokinin levels through signaling. Together, the analyses reveal partially redundant functions of the cytokinin receptors and prominent roles for the AHK2/ AHK3 receptor combination in quantitative control of organ growth in plants, with opposite regulatory functions in roots and shoots.

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.

The size and activity of the shoot apical meristem is regulated by transcription factors and low molecular mass signals, including the plant hormone cytokinin. The cytokinin status of the meristem depends on different factors, including metabolic degradation of the hormone, which is catalyzed by cytokinin oxidase/dehydrogenase (CKX) enzymes. Here, we show that CKX3 and CKX5 regulate the activity of the reproductive meristems of Arabidopsis thaliana. CKX3 is expressed in the central WUSCHEL (WUS) domain, while CKX5 shows a broader meristematic expression. ckx3 ckx5 double mutants form larger inflorescence and floral meristems. An increased size of the WUS domain and enhanced primordia formation indicate a dual function for cytokinin in defining the stem cell niche and delaying cellular differentiation. Consistent with this, mutation of a negative regulator gene of cytokinin signaling, ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6, which is expressed at the meristem flanks, caused a further delay of differentiation. Terminal cellular differentiation was also retarded in ckx3 ckx5 flowers, which formed more cells and became larger, corroborating the role of cytokinin in regulating flower organ size. Furthermore, higher activity of the ckx3 ckx5 placenta tissue established supernumerary ovules leading to an increased seed set per silique. Together, the results underpin the important role of cytokinin in reproductive development. The increased cytokinin content caused an ;55% increase in seed yield, highlighting the relevance of sink strength as a yield factor.

While testing purines related to the non-specific protein kinase inhibitors M-dimethylaminopurine and N6-(A'-isopenteny1)adenine as potential inhibitors of the p34'd'z/cyclin B kinase, we discovered a compound with high specificity, 2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine (olomoucine). Kinetic analysis of kinase inhibition reveals that olomoucine behaves as a competitive inhibitor for ATP and as a non-competitive inhibitor for histone H1 (linear inhibition for both substrates). The kinase specificity of this inhibition was investigated for 35 highly purified kinases (including p34"dk4/~y~lin D1, p4Wdk6/cyclin D3, CAMP-dependent and cGMP-dependent kinases, eight protein kinase C isoforms, calmodulin-dependent kinase 11, myosin light-chain kinase, mitogen-activated S6 kinase, casein kinase 2, double-stranded EWA-activated protein kinase, AMPstimulated kinase, eight tyrosine kinases). Most kinases are not significantly inhibited. Only the cell-cycle regulating p34'dc2/cyclin B, p33cdk2/~y~lin A and p33cdk2/~y~lin E kinases, the brain ~3 3 '~~' / p35 kinase and the ERKlMAP-kinase (and its starfish homologue ~44"'~~) are substantially inhibited by olomoucine (I& values are 7, 7, 7, 3 and 25 pM, respectively). The cdk4/cyclin D1 and cdk6/ cyclin D3 kinases are not significantly sensitive to olomoucine (I& values greater than 1 mM and 150 pM, respectively). M-(A2-1sopentenyl)adenine is confirmed as a general kinase inhibitor with IC,, values of 50-100 pM for many kinases. The purine specificity of cyclin-dependent kinase inhibition was investigated: among 81 purine derivatives tested, only C2, N6 and N9-substituted purines exert a strong inhibitory effect on the p34'd'2/cyclin B kinase. An essentially similar sensitivity to this olomoucine family of compounds was observed for the brain-specific cdkYp35 kinase. Structure/activity relationship studies allow speculation on the interactions of olomoucine and its analogues with the kinase catalytic subunit. Olomoucine inhibits in vitro M-phase-promoting factor activity in metaphase-arrested Xenopus egg extracts, inhibits in vitro DNA synthesis in Xenopus interphase egg extracts and inhibits the licensing factor, an essential replication factor ensuring that DNA is replicated only once in each cell cycle. Olomoucine inhibits the starfish oocyte G 2 M transition in vivo. Through its unique selectivity olomoucine provides an anti-mitotic reagent that may preferentially inhibit certain steps of the cell cycle.Phosphorylation of serine, threonine and tyrosine residues by protein kinases represents one of the most common post-translational regulatory modifications of proteins. More than 200 protein kinases have been described, following either purification to homogeneity or molecular cloning [1 -41. As much as 2 -3 % of eukaryotic genes may encode protein kinases. The importance of protein kinases in physiological processes has stimulated an active search for specific inCorrespondence to L. Meijer, Centre National de la Recherche Scientifique, Station Biologique, BP ...

Cyclin-dependent kinases (cdk) control the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. A potent inhibitor of cdks, roscovitine [2-( 1 -ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine], was identified by screening a series of C2,P,N9-substituted adenines on purified cdc2/cyclin B. Roscovitine displays high efficiency and high selectivity (Meijer, L., Borgne, A,, Mulner, O., Chong, J. P. J., Blow, J. J., Inagaki, N., Inagaki, M., Delcros, (1997) Eur: J. Biochem. 243, 527-536). It behaves as a competitive inhibitor for ATP binding to cdc2. We determined the crystal structure of a complex between cdk2 and roscovitine at 0.24-nm (2.4 A) resolution and refined to an Rf,,n,, of 0.18. The purine portion of the inhibitor binds to the adenine binding pocket of cdk2. The position of the benzyl ring group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP-complex structure. Analysis of the position of this benzyl ring explains the specificity of roscovitine in inhibiting cdk2. The structure also reveals that the (R)-stereoisomer of roscovitine is bound to cdk2. The (&isomer is about twice as potent in inhibiting cdc2kyclin B than the (S)-isomer. Results from structure/activity studies and from analysis of the cdk2hoscovitine complex crystal structure should allow the design of even more potent cdk inhibitors.Keywords: cell cycle ; cyclin-dependent kinase; purine ; protein-kinase inhibitor; anti-tumor agent.Protein kinases are involved in essentially all intracellular regulatory pathways. In view of their essential role in the regulation of the cell division cycle, cyclin-dependent kinases (cdk) have been the object of considerable investigation (reviews in Numerous examples of cdk deregulation in human primary tumours and in tumour cell lines have been described recently (reviews in [12, 131). These observations encourage the search for selective chemical inhibitors of cdk proteins and the use of natural inhibitors in potential gene therapy [14, 151. Prompted by the abundance of cdc2kyclin B kinase in starfish oocytes, its relatively easy purification by affinity chromatography on p9c7Ks/ p13'""-Sepharose and the rapid assay of its activity with histone H1 and ["PIATP, a few years ago we set up a simple screening test using purified p34'd"2/cyclin Bcdc13 as a target [16]. F-29682 Roscoff cedex, FranceThe first compound to be identified as a cdc2 inhibitor was 6-dimethylaminopurine (IC50: 120 pM) [17, 181. This compound was initially synthesized as a puromycin analogue and found to inhibit mitosis of sea urchin embryos without inhibiting protein synthesis [ 191. By structural analogy we identified N6-(d2-isopenteny1)adenine as a slightly more potent kinase inhibitor (ICs,,: 55 pM) [16]. Following extensive screening of a series of substituted purines designed for other purposes, olomoucine [2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine] was identified as a potent cdc2 inhibitor (ICso: 7 pM) [20]. ...