Male female plant communication by ralf mediated signaling final 11

Male and Female Plant Communication
by Rapid Alkalinization Factor Mediated
Signaling
Ritasree Sarma

Dept of Plant Biotechnology UAS.GKVK 2
Do they
communicate ?
Yes..
How??

 Fertilization process in plant
 Regulatory steps involve in fertilization
 Function of receptor like kinases
 Rapid Alkalinisation Factor structure and function
 Role of Rapid Alkalinisation Factor in pollen tube
growth
 Case study
 Summary
Contents

Reproductive parts

Fertilization
Presented in botanical section held in Kiew and were published as
an abstract (Nawaschin, 1898)

How much we understand??
Puzzled scientist in the last 20 years…

Fig:Images of a living ovule after the arrival of
the pollen tube (PT), 10 h after pollination
Fig: Images of a living ovule
after 15h pollination

Chemical attractant…concept was 150 years old
Fig: Amino acid alignment for CRPs
Fig:Expression analysis for CRPs

Fig: Directional growth of the pollen tube toward LURE attractant peptide

She is not my boss! Species specificity..
Higashiyamava et
al.,2015

?male or female factor

Fig: Expression of FER
Fig: FER for pollen tube growth control
Fig: FER structure
Fig: FER kinase activity
Fig: Species specificity of FER

Dept of Plant Biotechnology UAS.GKVK
14

15
Fig: Expression of ANXUR
Fig: Expression of ANXUR in pollen tube burst

P -Signaling
cascade
ANX
FER
Interaction between FER and AUXN

Unknown female
secreted ligand for
FER??
?
Hafidh et al., Biochemical Society Transactions.2014
Missing link in pollen tube perception

Pearce et al.2001.PNAS.98:22
Fig: Mature peptide
Serine protease
Fig: RALF peptide structure
Fig: RALF peptide structure
pH

20
Campbell et al.fronitersc.2017
Fig: Function of RALF motif YISY Fig: RALF peptide group
Fig: RALF peptide gene duplication

Fig: RALF Amino acid analysis
Fig: RALF peptide group
Fig: RALF peptide in different plant parts

23
Haruta et al.,Science.2014
Fig: MS spectra showing abundance of FER peptide
Fig:Binding of FER to RALF
Fig:Function of FER-RALF
Function of RALF peptide

24
Fig: Phosphorylation of S899 residue ofAHA2
Fig: AHA2 protein
Fig: AHA2 protein function

Can we correlate with pollen
tube growth?
Fig: RALF interaction with FER and AHA2

•
26
Fig: SlPRALF expression in pollen tube Fig: SlPRALF effects on elongating pollen tubes

28
Fig: Relative expression of RALF 4/19
Fig: Relative expression of downregulated RALF 4/19
Fig:RALF for pollen tube growth

Fig: Control cell wall composition
Fig: RALF 4/19 on upstream of ANX1/2
Function of RALF

Fig: LRX protein structure
Fig: Role of LRX protein
Fig: LRX interaction with RALF

RALF 4/19
LRX8/9/10
Maintain Cell wall composition
ANX1/2
Pollen tube integrity
Interaction with FER and
female ligand (?)
Reception and burst

Case Study

The guided growth, reception and rupture of the pollen tube are essential
for successful fertilization
FERONIA (FER),a CrRLK1L receptor expressed in the female
gametophyte, is essential for pollen tube reception
ANXUR1 and ANXUR2 (ANX1/2), expressed in the pollen tube prevent
precocious pollen tube rupture
But how they interact for pollen tube rupture is not yet answered
Objective: To find extracellular signalling molecule for male female
plant communication
Introduction

Materials and Methods
Plant material and growth conditions
Arabidopsis thaliana (Columbia-0) was used as the wild type
Arabidopsis were transformed using Agrobacterium-mediated floral dip method
 Phylogenic analysis and transcription assay
The sequences of both CrRLK1L family and RALF family from Arabidopsis
Information Resource
 Transcription profile heat-map based on RNA-seq data of different tissues
 GFP assay and T-DNA induced mutants
To test the binding affinity of RALF peptides with the extracellular domains
(ECDs), Peptide synthesis and in vitro treatment on the pollen tube
All RALF peptides were synthesized by Scilight Biotechnology LLC (Beijing,
China) with a purity higher than 95%.

Fig: Expression pattern of CrRLKs
Fig: Expression pattern of BUPS

36
Fig: Mutant alleles of bups1 and bups2
Fig: Effect on vegetative growth
Fig:Effect on reproductive growth

Effect on pollen tube growth

39
Fig: Expression of RALF4/19
Effect of RALF 4/19 on pollen tube growth

BUPS1/2 and RALF4/19 as functional pair

BUPS interact with ANX1/2

Interaction of BUPS,RALF and ANX

How pollen tube integrity is compromised??
Fig: Expression of RALF 34

44
Role of RALF 34

Competitive binding of RALF 34

FER
 RALF4/19 act
upstream of it
 ANX-BUPS form
heterodimer
 RALF 34 present
in synergid cell
 Competitively
bind with
RALF4/19
 Activate paracrine
signalling
 Pollen tube burst
and sperm release
Summary

FER

48
Just touching a small dot!!
Hafidh et al.,2016

Male female plant communication is a complex process
Regulation occurs at every step from pollen tube
growth to zygotic activation
RALF mediated signalling is important for both pollen
tube integrity and rupture
Yet to understand! Secretomics and interactomics
will definitely lead the way…
Conclusion

THANK YOU

Male female plant communication by ralf mediated signaling final 11

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