Cytokinins: Insights into a Classic Phytohormone

In a review article published in the February issue of Plant Physiology (128:354-362) Dr. Georg Haberer and Joseph. J. Kieber at the Department of Biology, University of North Carolina, critically surveys the most recent advancements made in  the field of cytokinin research.

In the introductory remarks, the authors describe the structure of cytokinins and their role in influencing  various physiological processes such as  seed germination, de-etiolation, chloroplast differentiation, apical dominance, flower and fruit development and  plant pathogen interactions.

The breakdown of tRNA was originally suggested as a possible mechanism for cytokinin synthesis. A total of nine ipt-homologs, designated as AtIPT1 to AtIPT9 have been identified. Of these, two, namely, IPT2 and IPT9 have been shown to encode a supposedly tRNA-ipt  and the remaining were found to be related to the bacterial ipt/tmr gene.  When these seven genes were expressed in E. coli,  the cytokinin-ipi  and zeatin were secreted t thereby confirming  that  they are the genes that take part in cytokinin biosynthesis. Subsequent studies have shown  isopentenyl-adenosine-3-phosphate  (IPTP) and isopentenyl-adenosine-2-phosphate (IPDP) are the product of plant enzymes which are converted to zeatin. Recently, anther  alternative cytokinin biosynthesis pathway has been discovered.  The results of an in vivo experiment in which deuterium labeling was used to compare the output of  zeatin riboside-5-monophosphate (ZMP) and (isopentenyl adenosine mono-phosphate (IPM) in wild type and transgenic plants, revealed a 66-fold increase  of biosynthetic rate of  ZPM compared to  IPM.

Perception and signal transduction:

The site of synthesis  of cytokinins and and their site of action   are spatially separated and as such cytokinins must have a receptor. However, none of cytokinin binding proteins identified so far have proved to be indisputably a receptor. One of the strong candidates is a histidine kinase (CKI1) which was shown to overcome the cytokinin requirement for callus growth and greening. Furthermore, the promoter of cytokinin primary response gene was activated by transient expression of CKI1 in Arabidopsis protoplasts.  Recently, two groups of workers have identified a cytokinin receptor  gene, CRE1 in Arabidopsis.  This gene has been found to encode a histidine kinase protein,  somewhat similar to  the  amino acid sequence of CKI1. Among 19,000 artificially induced  mutants, one was found partially sensitive to cytokinin in tissue culture experiments. Furthermore, cre1 mutant  seedlings showed resistance to cytokinin in a root elongation assay. Results of some recent experiments  show that CRE1 encodes an unusual type of hybrid kinase and that it contains His-kinase domain and two transmembrane regions and an extracellular domain. The authors point out that heterologous expression of CRE1 in yeast has  confirmed    that  cytokinin does bind   directly to CRE1 with high affinity. In spite of the above body of evidence, no definitive conclusion can be drawn whether CKI1 is truly  the kinetin receptor.

Biological role of CRE1: The wooden leg  (wol) mutant gene in Arabidopsis has been shown allelic to cre1. In  the wol mutant vascular bundles are poorly developed in the root and the lower part of the hypocotyl.  Phloem and metaxylem cells are conspicuously absent  in these organs and there are only  a smaller number of xylem cells, constituting the vascular tissue. The authors point out that the few initials arising out of  divisions  in  the wol mutant  are all used up in xylem formation leaving no initials to produce phloem cells. The wol phenotype suggests  that the role   of CRE1/WOL1 is that of a cytokinin receptor. It has also been shown that the wol mutation can cause disruption to the binding of cytokinin to CRE1.

The authors then introduce two closely related  His kinases in Arabidopsis: They are: AHK2 and AHK3 expressed mainly in the aerial parts of wild type.  These two kinases have been found to be remarkably similar (homologous) to the entire sequence of CRE1, including regulator domains in their C-terminus.

Five genes encoding phosphotransfer proteins  (AHP’s) have been reported in Arabidopsis. These proteins act as a bridge to transfer phosphate between Asp residues characterizing   both  receiver domain of the hybrid sensor kinase and the receiver domain of the response regulator. By means of suitable experiments it has been shown that CRE1 targets AHPs  in  the downstream.   Results of some other experiments  indicate that at least some of the AHPs take part in cytokinin signaling. Some recent reports also indicate that some of these AHPs transmit the signal from its site of perception, probably the plasma membrane to the effectors in the nucleus turning on or off the concerned gene(s).

The authors then describe response regulator gene family designated ARRs. There are two types of ARRs: type-A consisting  solely of receiver domain and type-B representing the output domain fused to the receiver. Only  there is production of type A mRNAs in response to cytokinin, indicating that only type-A ARRs may be involved in cytokinin signaling. When the type-A ARR is overexpressed, it has a negative feedback effect on signaling. The presence of multiple ARR1 binding sites in the promoter of  type-A  indicates that the primary targets of cytokinin signaling are regulated by ARR1s. 

Several of  the type-B ARRs act as positive regulators of cytokinin responses. According to the authors, an endogenous chemical is released as a result of overexpression of the type-B ARRs. The authors  propose that the phosphorelay hypothesis  best  explains  how ARRs work. All 10 type -A and all 12 type-B in Arabidopsis share these sites with those discovered in rice, maize suggesting  the functional relevance of these sites. 

The authors sum up their article presenting a model of cytokinin action. 

  • Binding of cytokinin  to CRE1/other  related His Kinase
  • Initiation of phosphorelay
  •  Phosphorylation and activation of  the type-B ARRs
  • Transcription of the Type-A genes which, in case of overexpression, negatively feedback into the pathway 
  • Type-A and Type-B ARRs interact with various molecules (effectors) inside the cell and determine the kind of  biochemical reactions  to occur appropriate to  cytokinin.

In conclusion, the authors express the hope that with the availability of the sophisticated molecular tools,  it will soon be possible to find answers to many unsolved questions  such as the in vivo role of most of the members of the phosphorelay in Arabidopsis and their putative function in cytokinin signaling.

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