Hormonal Signaling II: Cytokinin and Ethylene

1. Hormonal Signaling II: Cytokinin and Ethylene • Cytokinin • 1) overview: Zeatin (a purine derivative) is most abundant natural cytokinin, discovered as a DNA breakdown product that stimulate cell division/growth. It also inhibits aging/senescence, ratio of cytokinin/auxin regulate organogenesis (higher cytokinin favors shoot formation)

2. receptor receptor • 2) Signaling pathways • The receptor is similar to the bacterial two-component proteins • The first study was performed using an activation tagging method: the idea is to activate randomly the genes in the genome by inserting a strong promoter anywhere in the genome and screen for phenotype that is related to cytokinin action. IN this case, shoot production from callus was used (more cytokinin favors shoot formation) to select those callus that can form shoot without cytokinin! • One such gene called CKI1 (CytoKinin Independent) was isolated—when this gene is expressed at high level, the callus can form shoot without cytokinin. The gene encodes a protein with similarity to the histidine kinase and response regulator in bacteria. This is the most • Popular receptor system in bacteria • to detect environmental signals • Such as nutrition, temperature, • And chemotaxis signals.

3. Action in cytokinin response and the fact that is is similar to a bacterial receptor suggests that it may be a receptor of cytokinin. Further evidence came from study of another protein CRE1. The gene was identified by mutant screening—cre1 loss-of-function mutant was not sensitive to cytokinin and its callus does not produce shoot even in the presence of cytokinin! This gene also encode a two component-like receptor very similar to CKI1. In addition, CRE1expressed in yeast cells can bind cytokinin. Further study of function of CRE1 and CKI1 was performed in a protoplast system: express the receptor and a reporter gene in the protoplast and see if the receptor activation can turn the reporter gene on or off. Indeed both CRE1 and CKI1 can serve as a receptor for cytokinin to activate the gene expression.

4. The cytokinin receptor appears to be a fused version of The two component system: the essence of this system is to pass the signal by transferring the phosphate group from a histidine (H) to aspartate (D) (bacteria) (Cytokinin signaling in plants)

5. B) Downstream from the receptor Early response genes of cytokinin turn out to be encoding the response regulator-like proteins. Further study using the protoplast and transgenic plant models showed that these RR proteins participate in cytokinin signaling. Detailed picture is still not clear. The RR proteins are divided into two families: A type and B type that serve as negative and positive regulator of cytokinin response. In other words, B type activate and A type inhibits the cytokinin response. RR gene expression is activated by cytokinin Time course after cytokinin treatment

6. Summary pathway (part I) • Cytokinin binding to receptor that may dimerize • Phosphate transfer from H to D due to activation of the histidine kinase activity • The phosphate transfer to another protein called AHP (for arabidopsis histidine phosphotransfer)

7. Summary pathway (part II) 4. Phosphotransfer to the RR protein, activating RR 5. B-type RR protein activate gene expression 6. A-type RR inhibits gene expression 7. All these events combine into cytokinin response at cellular and whole plant level

8. 2. Ethylene 1) OVERVIEW: gaseous hormone was discovered when growing plants under gas lamp (triple response), synthesis induced by stress and pathogen, causing aging and abscision, fruit ripening, etc Inhibitor of C2H4 synthesis (left) or mutant of C2H4 response delays senescence of the flowers and abscision of leaves (right). Arabidopsis Triple response: Shorter hypocotyl Shorter roots Exaggerated apical hook

9. 2) Signaling • Receptors were identified by genetics and biochemical approaches • The mutant selection: ethylene resistant (or insensitive) and constitutive ethylene response mutants • Etr1—ethylene resistant-1: mutant plants are not responsive to ethylene. See all, but one plant, are sensitive to ethylene with triple response. That one plant is etr1 • The ETR1 gene encode a receptor similar to the • cytokinin receptor, the two-component histidine • kinase/response regulator fused together. • Several other genes are identified in similar • genetic screen or simply by comparing the • Sequence similarity. All of them appear to • have extracellular C2H4 binding domain and • transmembrane domains. Some of them have • both the kinase domain and the receiver domain • but others lack one of the domains (See next page)

10. The ethylene Receptor family • ETR1 is a true ethylene receptor with the following properties • ETR1 wild type protein binds C2H4 when expressed in yeast but etr1 mutant protein lacks such binding activity • Binding of C2H4 to the ETR1 requires a metal cofactor (Cu). • Dimerization may be conducted by disulfide bonds B) Downstream from receptor may be a kinase cascade CTR1—ctr1 mutant is constitutive response mutant—always show triple response even without ethylene

11. What is CTR1 gene? Encoding a protein kinase like Raf1 in animal cells (the kinase that activates MAPK kinase…) CTR1--Raf-like kinase MEK-like kinase MAPK EIN3—ein3 is a ethylene insensitive mutant – the gene encoding a transcription factor in the nucleus.

12. The pathway summary: The RAN1 protein is essential for assemble the Cu cofactor with the receptor for C2H4 binding. IN the absence of C2H4, the receptor would activate the downstream kinase CTR1 that in turn inhibits the response/gene expression. In the presence of C2H4, the receptor is “inhibited” so is the CTR1 kinase. The response is now “on”. The kinase cascade may serve as a negative regulator of the gene expression. What is the evidence that receptor is inhibited by C2H4? And CTR1 is a negative regulator?