CLE peptide are best studied plant peptide hormone

1. CLAVATA3/EMBRYO SURROUNDING
REGION PEPTIDES -
THE POWER OF MOVING AROUND
RITASREE SARMA

2.  Introduction
 Structure of CLE peptide
Synthesis of CLE peptide
 Function of CLE peptide
 Case study
 Summary
Contents
2

3. Introduction
Multicellular organisms rely on cell to
cell communication for their development,
growth and environmental responses
For this purpose, plants and animals
utilize secreted signaling molecules such as
peptide hormones
The animal peptide hormone insulin was
identified in 1922
Systemin was the first plant peptide
hormone to be identified in1991
3Insulin

4. Fig: Types of Plant Peptide hormone
Plant Peptidome
4

5. CLE Peptides
CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (CLE) family
peptides are peptide hormones that act as mediators of cell-to-cell
communication
CLE gene (CLAVATA3/ESR) was first reported in Zea mays (maize) as an
endosperm-specific gene, ESR (Opsahl-Ferstad et al., 1997)
 CLV3 was first found in Arabidopsis to encode a small extracellular protein
(Fletcher et al., 1999)
 CLV3 and the ESR sequences share a short, conserved region of 14 amino
acids but are otherwise unrelated at the sequence level
5

6. Fig: CLE genes express in various parts of Arabidopsis plant
Jun et al.20106

7. Characteristics
 They are small (<15
KDa)
 Have a putative N-
terminal secretion signal
 possess a conserved
~14-aa CLE domain at or
near their C terminus
Post translationally
modified
CLE Peptides
Structure of CLE and PTM 7

8. Fig: CLV 3 structural analysis
Fig: Analysis of the sugar components of the glycopeptide8

9. Structural Specialties of CLE Peptide
Fig : Proline hydroxylation
Fig: Triarabinosylation of Hydroxyproline
Fig: Maldi Tof MS analysis of CLE peptide
Fig: Energy minimized structure
9
Shinohara et al.2013

10. Synthesis of CLE peptide
10
Betsuyaku et al.2011

11. What they will do??
J of Exp Bot.2016Overview of peptide signaling 11

12. CLE receptors and function
Wang et al.201612

13. CLV3 Peptide
Tucker et al.2014
WUS Pathway
The CLV3 gene encodes a small 96-
amino-acid polypeptide
18-amino-acid secretion signal at the
amino terminus
Mature peptide is post transcriptionally
modified 12-amino acid peptide
Fig: SAM and WUS location
Helps in the maintenance of stem cell
reservoirs in shoot apical meristems
(SAMs)
 A key factor in controlling stem cell
identity is the WUSCHEL (WUS) gene,
which encodes a WOX homeodomain
transcription factor essential for stem cell
maintenance
WUS is expressed in the meristem
organizing center (OC) and promotes CK
responses by repressing A-type ARR gene
expression 13

14. Stem Cell Autofeed back loop
14
C

15. CLAVATA GENE Characteristics
CLV1 – Extracellular polypeptide: 96 amino acids.(Ser/Thr) kinase. Extracellular domain is
leucine-rich repeats (LRRs)
CLV2 – Membrane-bound protein receptor with a protein-binding motif
CLV3 – Membrane-bound protein receptor with a protein-binding motif and Restricted to
L1, L2 of SAM Central Zone
KAPP- The type-2C kinase-associated protein phosphatase
ROP-Ras GTPases are typically associated with cytosolic MAPK cascades
Shoot Development : Genetics
CLAVATA gene mechanism
Signal transduction pathway
15

16. Signaling pathway of CLV3 Jun et al.2015
16

17. ?
 Lateral roots extend horizontally from the
primary root and facilitates the extraction
of nutrients
 CLE peptides and the CLAVATA1leucine-
rich repeat receptor-like kinase is
expressed in the root vasculature
 Regulate emergence of the lateral root
system under N-deficient conditions by
CLE1, 3, 4, and 7
Low NO3
_ High NO3
_
CLE-CLAVATA1 peptide-receptor signaling for lateral root
development
NO-
3
NRT2.X
NO-
3
?
17

18. Expression of CLE peptides in low nitrate
concentration
Phenotypic study
18

19. Location of CLE3 and CLV1
Stages of LR development
19

20. CLE-Root Signal Peptide
 13-amino-acid peptide in which the 7th
hydroxyproline residue is further modified
with three L-arabinose sugars
 CLE-RS peptide is present in roots
 Involve in long distance signaling
 Root-to-shoot-to-root negative feedback
signalling loop
 Up regulated by rhizobia infection and
suppress nodulation in legumes
Structure of CLE-RS
20

21. Fig: Detection of root-derived [Ara3]CLE-
RS2 peptide in shoot xylem sap by LC-MSFig: Nodule number regulation Fig: Receptor for CLE-RS by
photo affinity labelling
21

22. v
Schematic presentation of AON model Cytokinin dependent root nodulation22

23. 23Takahara et al.2015
AON by CLERS1/CLERS2

24. CLE peptides are restricted only to plants?? Answer is…No
Yasuka et al.2016
24

25. 25

26. Fig: Model for role of host
factor on nematode parasitism
Nematode infection
Fig: Effect of GrCEL in plant
26

27. Case Study
27

28.  Stem
Introduction
Cell activities in plants are controlled by intercellular signaling through CLE
(CLV3/ESR) family of peptide hormones
 Chemically synthesized 12 amino-acid CLV3 acts on both tissues and its
overdose diminishes the growth of shoot and root while CLE 25 is responsible
for root shortening
CLE41 peptide called tracheary element differentiation inhibitory factor
(TDIF) promotes the stem cell activity in the vascular meristem without
affecting stem cells in the shoot and root
CLV1,2 and TDR are transmembrane receptors for CLV3 and CLE41/TDIF
Structural basis of the specificity has not been fully understood
28

29. Materials and Methods
 Plant material: Col-0 accession of A.thaliana
 Peptide preparation : Peptide synthetizer (CS136XT)
 Photo affinity labelling: [125I]ASA-KIN in the presence or absence
of various concentrations of competitor peptides
 Bioassay: Plants were germinated and grown in Murashige and
Skoog (MS) medium supplemented with 1% sucrose andpeptide/control
solution and observed root length, SAM, stele and leaf vein in light
microscope
 GUS staining and imaging
29

30. Swapping the residues of CLE peptides
30

31. Effect of CLE peptides
31
Effect of CLE peptides on RAM
Effect of CLE Peptides on root growth

32. Effect of CLE peptides
Fig: Effect of CLE Peptides on SAM growth
Fig: WUS expression study in SAM
32

33. Effect of CLE peptides on stele width
33

34. Receptors for KIN
Fig: Effect of CLE peptides on xylem differentiation
34

35. Receptor for CLE41 peptide
Fig: Photo affinity labelling study for receptor-ligand
interction
35

36. Effect of individual amino acid on activity
36

37. Effect of amino acid sustitution on function and receptor binding
37

38. Conclusion
KIN exerts both CLV3 and CLE 41 activity
N terminus of CLE peptide may not be important for the specificity of CLE
activities
CLV3 requires H11th for its activity and the S11th of CLE41 activity
Bifunctional CLE peptides which have not been identified in nature can be
artificially engineered
Artificial peptides would provide a powerful methodology to use the natural
genetic diversity as a source to mine cryptic bioactivities evolutionarily hidden
in the genome and to engineer artificial cell signalling
38

39. Summary
CLE peptides plans an important role from plant growth and stem cell homeostasis,
in response to environmental stimuli, certain plant-microbe interactions that occur
during legume symbiosis and phytopathogenic nematode infection
But we have only revealed the tip of the iceberg with respect to their signaling
potential including the interaction with receptors and downstream changes and many
hundreds still await analysis
Further understanding of it may provide us with very precise tools to modify plant
architecture and development for crop improvement
39

40. 40