It has been nearly fifty years since the recognition of some cytokinins as N-6 substituted adenine compounds with biological activity in plant systems. These compounds play a role in regulation of cell division and senescence, among other processes. However, information about the cytokinin signal transduction pathway has been slow in coming, in part because of the pleiotropic effects of this hormone. The identification of an Arabidopsis cytokinin receptor has changed this situation. This receptor is a histidine kinase, CRE1, also known as AHK4 and WOL. In a review article entitled, “CREam of Cytokinin Signaling: Receptor Identified” published in the July, 2001 issue of Trends in Plant Science (6:281-284), Professor Thomas Schmülling* at the University of Tübingen describes this work and its implications.
To identify cytokinin signal transduction mutants, an amenable bioassay was critical. Researchers at Osaka University in Tatsuo Kakimoto’s laboratory developed such an assay. Wild type Arabidopsishypocotyl explants undergo rapid cell proliferation, turn green and form shoots upon exposure to exogenous cytokinins. A genetic screen utilizing this assay identified the recessive mutant cre1 , which is insensitive to cytokinin in this system even at elevated levels. Positional mapping of the affected gene locus identified a histidine kinase gene on chromosome 2. Two CRE1 gene products were detected due, one that is 1057 amino acids in length and another with 23 additional amino acids at the N-terminus.
CRE1 bears resemblance to histidine kinases of two-component regulatory systems, a type of signal transduction machinery found in prokaryotes, lower eukaryotes and plants. In bacteria, these systems consist of a sensor kinase that activates a separate response regulator to result in regulation of target genes triggered by an environmental signal. In eukaryotes, the sensor kinase domain and receiver domains are found within the same polypeptide. In both cases, transmission of the signal is achieved through a phosphorylation cascade. CRE1 has structural features characteristic of eukaryotic sensor kinases: an extracellular input domain and cytoplasmic kinase transmitter and receiver domains. The 277 amino acid input domain protein is predicted to bind to the ligand, causing autophosphorylation at His-459 of the transmitter domain. The phosphate group is then transferred to Asp-973 of the receiver domain. The critical nature of these residues has been confirmed by determining the biochemical effects of point mutations at these positions.
Biochemical evidence that CRE1 is a cytokinin receptor has come from the use of three different receptor mutant complementation systems. In Saccharomyces cerevisiae, a MAP kinase (MAPK) cascade that activates cell division is itself regulated by a two-component regulatory system. The sensor histidine kinase in this system is SLN1, which transfers a phosphate to the histidine-containing transmitter (Hpt) named YPD1. Upon phosphorylation, YPD1 then transfers the phosphate group to SSK1, the response regulator in this system. Phosphorylated SSK1 does not activate the cell division MAPK cascade and progression through the cell cycle does not occur. In sln1 mutants, SSK1 remains in its constitutively active (unphorophorylated) state, which is ultimately lethal. The strain of sln1 used also was capable of suppressing the cell division MAPK cascade by another gene, PTP2, in a galactose-dependent manner. In other words, the MAPK cascade of the sln1mutant was suppressed in the presence of Gal, resulting in viable cells, but was constitutively active in the absence of Gal, which resulted in lethality. If CRE1 was expressed in the sln1 mutant yeast strain, it could substitute for SLN1 and cause the inactivation of the MAPK pathway – thus allowing growth in the absence of Gal, but only if cytokinins were present in the growth medium. This indicated that CRE1 could complement the S. cerevisiae histidine kinase mutant.
At Nagoya University, researchers in Takeshi Mizuno’s group used a different type of yeast to develop a similar complementation system. Mizuno’s group was interested in testing whether three different Arabidopsis genes that are similar to sensor histidine kinases could complement a Schizosaccharomyces pombe mutant. One of these candidates was AHK4, which is the same as CRE1. In S. pombe, G2 to M cell cycle progression is regulated by a two-component system. Three different sensor histidine kinases, PHK1, 2, or 3, function in this system. In response to a signal, they transfer a phosphate to the Hpt factor Spy1, which phosphorylates and thus negatively regulates Mes4 in a manner very similar to the S. cerevisiae system. Mutants lacking a functional histidine kinase divide earlier and are smaller than wild type cells. Complementation of phk mutant lines with AHK4/CRE1 restored normal cell size and division phenotypes in the presence of cytokinin.
Mizuno’s group also developed the third complementation system using E. coli. E. coli has approximately 40 two-component systems that regulate various processes. The capsular polysaccharide biosynthesis (cps) operon is regulated by one such system. The sensor histidine kinase in this system is RcsC, which acts on the Hpt factor YojN, which in turn acts on the response regulator RcsB. A cps::lacZ reporter in a rcsC mutant background allowed a functional screen for histidine kinase complementation. In the parental strain, the lack of cps operon activation resulted in white colonies on media containing the substrate X-Gal. When this strain was transformed with CRE1, activation of the cps operon allowed the formation of blue colonies on X-Gal-containing media in the presence of cytokinin. The ability of CRE1 to function as a cytokinin-responsive sensor histidine kinase in all three complementation systems provides strong support that it acts as a cytokinin receptor.
Cytokinins have also been found to play a role in vascular development in cell culture experiments. An Arabidopsis mutant affected in this cytokinin receptor provides more detail about how this regulation might occur. The wooden leg (wol) mutant is inhibited in root growth and lacks normal cell divisions during embryonic vascular initial formation. The affected gene, WOL, is identical toCRE1. In wol mutants, less cell division occurs in vascular initial precursor cells after the torpedo stage, resulting in the development of a narrower vascular bundle devoid of metaxylem and phloem cells. Furthermore, no lateral roots are formed in such mutants. Developmental and genetic studies indicate that abnormal root development in wol mutants is due to a lack of proper cell division during embryogenesis, rather than aberrant cell differentiation. Expression patterns of WOL/CRE1 also support a role in vascular tissue formation, as the gene is expressed in the four innermost cells of the globular embryo. These four cells are the precursors of the future vascular tissue. Interestingly, shoot growth and leaf senescence were normal in wol/cre1 mutants, despite the fact thatcre1 mutants are defective in chlorophyll biosynthesis and shoot meristem formation in tissue culture. The author suggests that other receptors may be able to substitute in whole plants, as it is likely that one or more closely related histidine kinase candidate genes are capable of responding to cytokinin. Indeed, two other candidate genes, AHK2 and AHK3, were capable of functioning in the yeast sln1 system described above.
Other components of the signal transduction system remain to be verified, but candidates include five Arabidopsis genes similar to Hpt transmitter proteins (AHP1-AHP5 and approximately 20 genes similar to two-component response regulator proteins (ARR genes). One class of ARR proteins is regulated by cytokinins while another is not, and the author introduces a model for cytokinin signal transduction that involves both classes of ARR gene products.
In conclusion, the author emphasizes the importance of the identification of CRE1 as a cytokinin receptor. The author feels that this work is a significant milestone in cytokinin research, as it lays a foundation for elucidation of cytokinin signal transduction at the molecular level.