Proline-rich proteins (PRPs)

Nan Jiang

• Plant cell wall proteins

• Structural proteins

• Hydroxyproline-rich glycoproteins (HRGPs)

• Proline-rich proteins (PRPs)

• Research paper

Plant cell wall proteins

A. 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 proteinsBased 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)

HydroxyprolineHydroxyproline: 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

Hydroxyproline-rich glycoproteins (HRGPs)

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

HRGPs: the major components of structural cell wall proteins. They all are glycosylated and contain hydroxyproline (Hyp).

The structure of PRPs, Extensins, and AGPs

PRPs

Extensins

Buchanan, Gruissem and Jones. (2000) Biochemistry & Molecular Biology of Plants, chapter 2

AGPs

Proline-rich proteins (PRPs)

The expression of PRP genes is influenced by wounding, endogenous and fungal elicitors, ethylene, drought, and light.

PRPs can be glycosylated on certain Ser by Ser-α-galactosyltransferase or certain Hyp by Hyp-β-arabinosyltransferase.

PRPs contain repeated PPVX(K/T) motifs or its variants.

PRPs are implicated in the integrity of the cell wall, the structural maintenance of organs and defense reaction to pathogen infection.

Proline-rich proteins (PRPs)

PRPs display tissue- and cell-specific patterns of expression.

There are four PRPs in Arabidopsis: AtPRP1, AtPRP2, AtPRP3, and AtPRP4.

Four members (OsPRP1.1-1.4) of OsPRP1 gene family showed expression divergence in spatial specificity.

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 Tandem N-terminal AtPRP1, AtPRP3

III PPV or PV/IYKKPCPP (Cys-rich)

Dispersed C-terminal AtPRP2, AtPRP4, OsPRP3

IV PEPK Tandem Whole protein OsPRP, TaPRP

V PKPE, P(V/E)PPK Tandem C-terminal OsPRP1.1-4

The classification of Proline-rich proteins (PRPs)

The phylogenetic tree of Proline-rich proteins (PRPs)

The approaches to study PRPs

A. Immunolocalization: to verify the tissue location of PRPsBattaglia M. (2007) Planta 225(5):1121-33

B. 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): 677–686Gothandam KM. (2010) Plant Mol Biol 72(1-2):125-35

C. Generation of T-DNA overexpression or knockout mutantsWang R. (2006) J Exp Bot 57(11):2887-97Gothandam KM. (2010) Plant Mol Biol 72(1-2):125-35

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

OsActin, rice actin used as a positive control

Expression analysis of OsPRP3

OsPRP3 transcript was accumulated in flower and not in leaf.

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.

Leaves from overexpression transgenic plants accumulated the OsPRP3 transcript at higher level.

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.

Gus linker, the expression of the trigger dsRNA; 1–10, Individual RNAi transgenic plants; WT, wild type flower.

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.

Flowers from RNAi plants showed either suppression or a complete knockout of the gene transcription.

Top panel, the immunoblot probed with OsPRP3 specific antibody. Bottom panel, the corresponding SDS–PAGE gel (silver stained). L leaf; F flower

OsPRP3 protein expression among these mutants

OsPRP3 protein was expressed in the leaf of the overexpression plant, but the proteins was absent in the flower of the knockout plant.

A, Immunolocalization of OsPRP3 in the leaves from the overexpression transgenic; B, the wild-type plants. CH Chloroplast; CW cell wall

Where is the OsPRP3 localized?

OsPRP3 localized on cell wall, and it is a cell wall protein.

Phenotype overexpression transgenic and wild type plants grown at 4°C.

Cold treatment

Overexpression transgenic plant was more tolerant to the cold stress than the wild type plant.

Expression analysis of cold regulated genes in rice leaves after 0, 1, 2, 3, 7 and 14 days under 4°C cold-stress.

Cold-tolerance

Cold-regulated genes

Increased OsPRP3 did not alter transcript levels of these cold inducible genes.

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.

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.

Structural injuries caused by cold treatment

Overexpression plant leaves retain the cell wall integrity.

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.

Structural injuries caused by cold treatment

Wild type and RNAi plant leaves lost their cell wall integrity.

Left, spikelet of RNAi plant; Inset box, magnification of an abnormal flower; Right, wild type.

WT osprp3Characterization of OsPRP3 RNAi transgenic plants

Tetrazolium staining of anther.

WT osprp3

Characterization of OsPRP3 RNAi transgenic plants

The anther of the mutant flower produced non-viable pollen.

Anther locule at micropsore stage of anther development. ep epidermis; ms microspore; t tapetum.

WT OsPRP3 osprp3

Characterization of OsPRP3 RNAi transgenic plants

The anther of the knockout mutant did not contain tapetum.

Magnification of the interlocking of lemma and palea. le lemma; pa palea; ep epidermis; sl sclerenchyma layer; vb vascular bundle.

WT OsPRP3 osprp3

Characterization of OsPRP3 RNAi transgenic plants

Palea histology showing different cell layers. ep epidermis; sl sclerenchyma layer; ie inner epidermis.

WT OsPRP3 osprp3Characterization of OsPRP3 RNAi transgenic plants

The knockout plant showed a severe reduction in sclerenchyma layer and inner epidermis.

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.

Thanks.