1. Plant development regulated by cytokinin sinks
Science 353 (6303),
Speaker: Ming-Chen Tsai
Advisor: Jun-Yi Yang

2. Plant development Plant hormone: Auxin Gibberellin Cytokinin…
Plant hormone plays an important role in plant development.

3. Cytokinin
The cytokinin zeatin is named after the genus of corn, Zea, in which it was discovered.
Promote cell division, or cytokinesis, in plant roots and shoots
Control morphogenesis, integrate environmental cues, and mediate biotic interactions.
Delay leaf senescence.

4. Cytokinin Signal Transduction Pathway
Phosphorelay systems
(also called two
component
signaling systems) are
prevalent in bacteria.
Consist of two conserved proteins: a His kinase sensor and a response regulator protein that are phosphorylated at conserved His and Asp residues, respectively.
Arabidopsis His kinases (AHKs)
Arabidopsis His phosphotransfer proteins (AHPs)
Arabidopsis response regulators (ARRs)

5. Plant Embryo
Arabidopsis heart-stage embryos as a model in which the cytokinin response marks the provascular tissue.

6. To evaluate whether bioactive cytokinins are limited
Cytokinin reporter TCSn::GFP (Two Component signaling Sensor new::green fluorescent protein) ,which reflects the transcriptional activity of type-B response regulators.
GFP signal reflecting the cytokinin signaling output.
Heart-stage embryos subjected to
16 hours of mock treatment,
16 hours of treatment with 10 mM BA,
16 hours of hybridization with AHK4 antisense (as) RNA,
and 16 hours of CKI1expression
Cells of the prospective cotyledons fail to activate cytokinin signaling despite a functional signaling system, and even upon addition of abundant active ligand.

7. Transmembrane proteins implicated in cytokinin translocation
Motivation
Transmembrane proteins implicated in cytokinin translocation

8. Arabidopsis PURINE PERMEASE (PUP) family
Cytokinin transporters guide differential cellular localization of cytokinins
Transcription profile of all PUP family members in seedlings, embryos and mesophyll protoplasts determined by qRT-PCR.

9. PUP14 expression pattern
In PUP14::PUP14-GFP transgenic plants
Failed to respond to cytokinins
In situ hybridization with a
PUP14 antisense probe
The exclusive nature of PUP14 expression and the cytokinin
signaling pattern are compatible with
an inhibitory function of PUP14 in the cytokinin response.

10. Eliminate PUP14 function
Constructed an ethanol-inducible artificial microRNA (amiR)
This result supports a role of PUP14 in confining the cytokinin response.

11. The amiRPUP14-induced phenotypes were complemented.
After 2 days of amiRPUP14 induction
Morphological defects in the prospective cotyledons and the nascent root meristem became apparent.
The amiRPUP14-induced phenotypes were complemented.
Suggests that the inducible amiRPUP14 acts specifically.

12. short-term amiRPUP14 induction
Root and shoot meristem activities were both affected when cytokinin signaling patterns were perturbed by the inducible amiRPUP14.

13. Inflorescence initiation and development
LP, leaf primordia; SAM, shoot apical meristem.
A: Shoot apical meristem producing leaf primordia in helical order.
B: Broadening and flattening of SAM prior to conversion to the reproductive phase.

14. Cytokinin controls the homeostasis of the shoot apical meristem
PUP14 expression in the SAM was inversely correlated with cytokinin signaling output assayed by TCSn::GFP.

15. Induced amiRPUP14 expression in adult plants
Inflorescences and inflorescence stems.
Red dots denote flower primordia
Inducing amiRPUP14 expression in adult plants that were allowed to complete embryogenesis and the early vegetative phase of development undisturbed caused ectopic cytokinin output in the SAM.
PUP14 functions to limit the cytokinin
response domains throughout development to support morphogenesis.

16. The major cytokinin transport form
protoplasts
Uptake experiment：
Using labeled trans zeatin(tZ)
Transient expression of PUP14 in mesophyll protoplast
解決了PUP14細胞上的功能後…要來了解PUP14’s cytokinin transport capacity
(A) PUP14-transfected mesophyll protoplasts.
(C) Competition by indicated substances in PUP14-transfected protoplasts.
tZ riboside, the major cytokinin transport form

17. PUP14’s cytokinin transport capacity
Microsomes
(B) Microsomes derived from GFP-, PUP1-, or PUP14- transfected Nicotiana benthamiana.
(D) Competition in microsomes of 35S::PUP14-transfected N. benthamiana
Energy dependent cytokinin uptake into a microsomal cell-free system excludes the possibility that uptake is dependent on
cytoplasmic metabolization.

18. Control seedlings
amiRPUP14 versus Col0 seedlings.
Seedlings with decreased PUP14 levels exhibited a reduced uptake rate for exogenously added tZ.

19. Compared the effects of differentially targeted cytokinin-degrading
enzymes on the cytokinin signaling response
Transient expression of wild-type CKX2
added
a glycosylphosphatidylinositol (GPI) anchor
caused a
reduction in the cytokinin response
Relative TCSn::LUC induction in protoplasts treated with 10 nM tZ or BA, cotransfected with effector genes, or with addition of cell extracts as indicated, normalized to empty vector control.
These data suggest that apoplastic cytokinins initiate signaling,
whereas cytoplasmic cytokinins are inactive.

20. Model of PUP14 function in cytokinin signaling
PUP14 (red) causes the translocation of apoplastic cytokinins to the cytosol, where they are converted to inactive forms (dotted circles). This results in reduced binding to plasma membrane-localized cytokinin receptors, and consequently reduced signaling activation (green denotes cytokinin signaling activity). PM: plasma membrane.

21. Thanks for listening

23. In Situ Hybridization
針對一段DNA或是RNA做一段跟他互補的序列的probe(探針)，這樣就可以挑出specific的DNA或是RNA序列
可以檢測出特定的序列，很適合做translocation detection

24. Ethanol-inducible artificial microRNA (amiR)
成熟的microRNA形成後, 整合到microRNA miRNA-induced silencing complexes,miRISC中, 然後與靶基因mRNA結合, 能夠與靶基因mRNA完全互補或不完全互補的microRNA可能會導致靶基因的特異性剪切, RISC中的Argonaute(Ago)蛋白能直接切開與microRNA互補的靶mRNA中的磷酸二酯鍵, 形成小片段並直接降解。
Engineering of amiRNAs.