1. Proline-rich proteins (PRPs)
Nan Jiang

2. Plant cell wall proteins
Structural proteins
Hydroxyproline-rich glycoproteins (HRGPs)
Proline-rich proteins (PRPs)
Research paper

3. Plant cell wall proteins
Structural proteins: the major part of cell wall proteins
Functions: contribute to the cell wall strength
control cell wall assembly, expansion, hydration, and
permeability
serve as possible nucleation sites for lignification and as sources
of signaling molecules
B. Other proteins: small amount, including: the enzymatic, lipid transfer, signaling,
and defense proteins
Structural proteins
Based on the enrichment in specific amino acids and the presence of repeated sequence motifs, they can be classified into two groups:
(1) the glycine-rich proteins (GRPs)
(2) the hydroxyproline-rich glycoproteins (HRGPs)

4. Hydroxyproline Proline Hydroxyproline
Hydroxyproline: formed within the endoplasmic reticulum through hydroxylation of proline by the enzyme prolyl hydroxylase (PHD).
Proline
Hydroxyproline
α-Ketoglutarate
Succinate
McDonough MA. (2006) Proc Natl Acad Sci U S A 103(26):9814-9

5. Hydroxyproline-rich glycoproteins (HRGPs)
HRGPs: the major components of structural cell wall proteins. They all are
glycosylated and contain hydroxyproline (Hyp).
Protein class
% Protein
% Sugar
Peptide periodicity
Hyp-O-glycosylation
Repetitive units
Proline-rich proteins (PRPs)
80~100
0~20
Highly periodic
Lightly glycosylated
Pro-Hyp-Val-Tyr-Lys motif
Extensins
~45
~55
Periodic
Moderate glycosylated
Ser-Hyp4 motif
Arabinogalactan proteins (AGPs)
1~10
90~99
Least periodic
Highly glycosylated
Ser-Hyp-Hyp-Ara-Pro-Ara-Pro or Ara-Hyp motif

6. The structure of PRPs, Extensins, and AGPs
Buchanan, Gruissem and Jones. (2000) Biochemistry & Molecular Biology of Plants, chapter 2

7. Proline-rich proteins (PRPs)
PRPs contain repeated PPVX(K/T) motifs or its variants.
PRPs can be glycosylated on certain Ser by Ser-α-galactosyltransferase or certain Hyp by Hyp-β-arabinosyltransferase.
PRPs are implicated in the integrity of the cell wall, the structural maintenance of organs and defense reaction to pathogen infection.
The expression of PRP genes is influenced by wounding, endogenous and fungal elicitors, ethylene, drought, and light.

8. Proline-rich proteins (PRPs)
PRPs display tissue- and cell-specific patterns of expression.
Four members (OsPRP ) of OsPRP1 gene family showed expression divergence in spatial specificity.
There are four PRPs in Arabidopsis: AtPRP1, AtPRP2, AtPRP3, and AtPRP4.

9. Distribution of motifs
The classification of Proline-rich proteins (PRPs)
Class
Motif type
Distribution of motifs
Proline rich domains
Genes I
Pentapeptide PPVXK/T (X= H, Y or E)
Tandem
C-terminal
MtPRP2, SbPRP2 II
PPYV
N-terminal
AtPRP1, AtPRP3
III
PPV or PV/IY
KKPCPP (Cys-rich)
Dispersed
AtPRP2, AtPRP4, OsPRP3 IV
PEPK
Whole protein
OsPRP, TaPRP V
PKPE, P(V/E)PPK
OsPRP1.1-4

10. The phylogenetic tree of Proline-rich proteins (PRPs)

11. The approaches to study PRPs
Immunolocalization: to verify the tissue location of PRPs
Battaglia M. (2007) Planta 225(5):
Northern Blot or RT-PCR: identify PRP genes are highly
expressed in which part or developmental stage of plant
Menke U. (2000) Plant Physiol 122(3): 677–686
Gothandam KM. (2010) Plant Mol Biol 72(1-2):125-35
C. Generation of T-DNA overexpression or knockout mutants
Wang R. (2006) J Exp Bot 57(11):
Gothandam KM. (2010) Plant Mol Biol 72(1-2):125-35

12. OsPRP3, a flower specific proline-rich protein of rice, determines extracellular matrix structure of floral organs and its overexpression confers cold-tolerance
Kodiveri Muthukalianan Gothandam • Easwaran Nalini • Sivashanmugam Karthikeyan • Jeong Sheop Shin

13. Expression analysis of OsPRP3
OsActin, rice actin used as a positive control
Expression analysis of OsPRP3
OsPRP3 transcript was accumulated in flower and not in leaf.

14. Expression analysis of OsPRP3
Different developmental stages of flower:
1 young flower; 2 immature flower; 3 mature flower
Expression analysis of OsPRP3
OsPRP3 was highly expressed in mature flower.

15. Generation of overexpression mutants
RB and LB, the right and left border of the T-DNA; Ubi, Ubiquitin promoter; Tnos, nos terminator; hph, hygromycin phosphotransferase.
Generation of overexpression mutants
1–9 Individual overexpressed transgenic plants; WT, wild type leaf.
Leaves from overexpression transgenic plants accumulated the OsPRP3 transcript at higher level.

16. Generation of knockout mutants
OsPRP3 cDNA clone was subcloned into both sides of the GUS linker in antisense and sense orientations; NPTII, Kanamycin resistance gene; HPT, hygromycin resistance gene.
Gus linker, the expression of the trigger dsRNA; 1–10, Individual RNAi transgenic plants; WT, wild type flower.
Flowers from RNAi plants showed either suppression or a complete knockout of the gene transcription.

17. OsPRP3 protein expression among these mutants
Top panel, the immunoblot probed with OsPRP3 specific antibody. Bottom panel, the corresponding SDS–PAGE gel (silver stained). L leaf; F flower
OsPRP3 protein was expressed in the leaf of the overexpression plant, but the proteins was absent in the flower of the knockout plant.

18. Where is the OsPRP3 localized?
A, Immunolocalization of OsPRP3 in the leaves from the overexpression transgenic; B, the wild-type plants. CH Chloroplast; CW cell wall
OsPRP3 localized on cell wall, and it is a cell wall protein.

19. Cold treatment
Phenotype overexpression transgenic and wild type plants grown at 4°C.
Overexpression transgenic plant was more tolerant to the cold stress than the wild type plant.

20. Cold-tolerance
Cold-regulated genes
Expression analysis of cold regulated genes in rice leaves after 0, 1, 2, 3, 7 and 14 days under 4°C cold-stress.
Increased OsPRP3 did not alter transcript levels of these cold inducible genes.

21. Cold-stress assay
Real-time quantitative RT-PCR analysis of OsPRP3 in 4°C cold treated leaves.
OsPRP3 mRNA level in the cold-treated transgenic plants was increased constantly.

22. Structural injuries caused by cold treatment
Upper, cross section of control (untreated); Lower, cold-treated transgenic (overexpression); Right, magnification of mesophyll cells. UE upper epidermis; LE lower epidermis; VB vascular bundle; MC mesophyll cells; CP chloroplast.
Overexpression plant leaves retain the cell wall integrity.

23. Structural injuries caused by cold treatment
Upper, cold treated wild type; Lower, cold treated (RNAi) leaves; Right, magnification of mesophyll cells. Arrows indicates the mesophyll cells that lost their cell wall.
Wild type and RNAi plant leaves lost their cell wall integrity.

24. Characterization of OsPRP3 RNAi transgenic plantsWT
osprp3
Left, spikelet of RNAi plant; Inset box, magnification of an abnormal flower; Right, wild type.

25. Characterization of OsPRP3 RNAi transgenic plantsWT
osprp3
Tetrazolium staining of anther.
The anther of the mutant flower produced non-viable pollen.

26. Characterization of OsPRP3 RNAi transgenic plantsWT
OsPRP3
osprp3
Anther locule at micropsore stage of anther development. ep epidermis; ms microspore; t tapetum.
The anther of the knockout mutant did not contain tapetum.

27. Characterization of OsPRP3 RNAi transgenic plantsWT
OsPRP3
osprp3
Magnification of the interlocking of lemma and palea. le lemma; pa palea; ep epidermis; sl sclerenchyma layer; vb vascular bundle.

28. Characterization of OsPRP3 RNAi transgenic plantsWT
OsPRP3
osprp3
Palea histology showing different cell layers. ep epidermis; sl sclerenchyma layer; ie inner epidermis.
The knockout plant showed a severe reduction in sclerenchyma layer and inner epidermis .

29. Summary OsPRP3 was flower-specific.
OsPRP3 is a cell wall protein of rice flower.
OsPRP3 confers cold tolerance by stabilizing the cell wall integrity.
OsPRP3 plays a crucial role in determining the extracellular matrix structure of anther, palea and lemma.

30. Thanks.