11th International Gluten Workshop

Isolation and characterization of key factors involved

in glutelin trafficking in rice endosperm cells

Jianmin Wan；Yihua Wang

Rice Research Institute, Nanjing Agricultural University

wanjm@njau.edu.cn

yihuawang@njau.edu.cn

High plants accumulate large amounts of storage proteins in

their seeds.

Storage proteins can be divided into glutelin, prolamin, and

globulin in rice seed.

Glutelins can account for up to 80% of the total seed proteins.

At least 15 members have been isolated, which belongs to 4

subfamiles (A, B, C, D).

glutelin

prolamin

globulin

others60~80%

Glutelin is the major part of seed storage protein in rice

Glutelin precursor

Glutelin acidic

subunit

globulin

Glutelin basic

subunit

prolamin

PBI Prolamin

Glutelin

Globulin

Plant Physiology, April 2002, Vol. 128, pp. 1212–1222

Rice accumulates two types of Protein bodies (PBs)

in endosperm cells.

Glutelin trafficking pathways reported in rice

Screening of SSP mutants (2001-2011)

Materials quantities Mutants Frequencylandraces 400 2 0.50%

T-DNA insertion lines 7000 14 0.20%

N22 60

Co radiation induced lines

（2003）1000 1 0.10%

Nipponbare 60

Co radiation

induced lines（2003）300 1 0.30%

931160

Co radiation induced lines 1500 3 0.20%

N22 60

Co radiation induced lines

（2011）10000 10 0.10%

Total 21000 32 0.15%

9311 MNU-induced lines 800 1 0.13%

1、OsVPE1 is responsible for the maturation of proglutelin in rice

Figure1 An SDS-PAGE and

immunoblot analysis of seed storage

proteins in W379 and Nipponbare rice.

57 kD

40 kD

26 kD

20 kD

16 kD

13 kD

a b

W379 Nip F1

Figure 2. Electron microscopy of

developing rice endosperm. In W379

endosperm, most of the PBII are round

instead of irregularly shaped.

Nip b

PBI

I PBI

I

W379

W379

Figure 3. Fine mapping the mutated gene in

W379. The mutated gene was mapped to the long

arm of chromosome 4

0

2

4

6

8

10

12

6 8 10 12 14 16 18

Relative expression

Nipponbare

W379

Figure 4. Real- time RT-PCR analysis of OsVPE1

expression in Nipponbare and W379 developing

endosperm.

0

2

4

6

8

10

12

14

16

18

20

Nipponbare W379

VPE activity(pmol/min.grain)

Figure 5. Developing W379

endosperm has reduced vacuolar

processing enzyme (VPE) activity.

a

mVPE

proVPE

iVPE

b

iVPE

proVPE

mVPE

1 2 3 4 5 6

1 2 3 4 5 6 7 8

Figure 6. Accumulation of OsVPE1 protein

in Nipponbare and W379 seeds.

The Plant Journal (2009) 58, 606–617

WT gpa1 gpa1 WT

57

40

26

20

16 13

A B C

WT

gpa1

kD

2、OsRab5a/GPA1 (glutelin precursor accumulation) regulates post-

Golgi trafficking of storage proteins

PBII SG

PBI

A PB

I

PBII

SG

gpa1 B D

ER

PBI

PBII

gpa1 C

ER

PBII

PBI

WT WT

ECS

SG

PBI

ECS

E

ER

gpa1

PBII

F *

*

* *

* G

CW

ECS

PBII

gpa1 H gpa1 gpa1

Figure 1．Characterization of the gpa1 mutant. (A) Seed storage protein profile by Coomassie blue

staining of SDS-PAGE gel. (B) Immunoblot analysis of seed storage proteins using antibodies against

glutelin acidic subunits. (C) gpa1 shows a floury endosperm.

Figure 2. The ultrastructural of 12 DAF endosperms between gpa1 and wild-type. (C) and (D) ER structure in the wild-

type (C) and gpa1 (D) (black arrowheads in D indicate the dilated region). (D) and (E) ‘secretory vesicle-like structures’

in gpa1 (white arrows). (E) enlarged view of ECS. (F) ‘The vacuole-like structures’ in gpa1 (asterisks). (G) and (H) The

‘vesicle-filled structures’ in gpa1 (black arrow). SG: starch granules; ECS: extracellular space.

PBII

PBI

CW

ECS

B gpa1

PBII PBI

ER

CW

N22 A

ER

PBII CW

D E SG SG

PBI

CW

C

Figure 3. Immunolocalization of glutelins in endosperm cells. (A) Glutelins accumulate in

PBII in endosperm cells of wild type. (B) Glutelins were secreted to the ECS in gpa1. (C)

Glutelin labeling was seen in the vesicle-like structures in gpa1. (D) No glutelin labeling was

observed in dilated ER in gpa1 (indicated by black arrowheads). (E) Considerate amount of

glutelin was mis-localized to the ‘vesicles-filled structures’.

Figure 4. OsRab5a is

the gene responsible

for the gpa1 mutant

phenotype. (A) High

resolution mapping of

the mutant gene in

gpa1. (B) A 13bp-

deletion in OsRab5a

led to a premature stop

of the protein. Asterisk

indicates the new stop

codon. (C) OsRab5a

protein is absent in the

developing seeds of

gpa1.

Figure 5. OsRab5a binds to GTP in vitro. (A) SDS-PAGE profiles of recombinant protein; (B)

GTP-binding activity of His-tagged OsRab5a protein. Lane 1: prestained protein marker; lane

2: purified WT His-OsRab5a; lane 3-4: pellet of cell lysate expressing pET-30a (lane3) and

WT pET-30a-OsRab5a (lane4); lane 5-6: supernatant of cell lysate expressing pET-30a (lane5)

and WT pET-30a-OsRab5a (lane6).

Figure 6. Expression pattern of OsRab5a. (A) The expression level of OsRab5a in different tissues.

17S RNA was used as an internal control. (B) The expression pattern of OsRab5a in developing

endosperms of N22 and gpa1. GUS activity in leaf blades (C), the stems (D), the nodes (E), the leaf

sheathes (F), the hulls (G), the branches (H) and the growing roots (I). (J) GUS activity in the ovular

vascular trace ends (OVE), ovular vesicular (OV), and lateral stylar vascular traces (LV); (J) and (K),

vertical section of the grain. (L) Cross-section of the grain. Data in A to B are mean ± SD.

Figure 7. Subcellular localization of GFP-OsRab5a in Arabidopsis protoplasts. (A) to (C) Coexpressing

of GFP-OsRab5a and mCherry-KDEL (an ER marker); (D) to (F) Coexpressing of GFP-OsRab5a and

GmMan1–mCherry (a cis-Golgi marker); (G) to (I) Coexpressing of GFP-OsRab5a and ST-mCherry (a

trans-Golgi marker); (J) to (L) Coexpressing of GFP-OsRab5a and RFP-AtVSR2-221 (a PVC marker).

Bars = 10 μm.

Plant Journal，2010, 64, 812-824

Relative gene expression in T3612 to Nipponbare

0

2

4

6

8

10

12

14

BiP

-1

Os0

2g0

115

900

BiP

-2

Os0

6g0

212

900

Hsp

70-

1

Os0

2g0

710

900

Hsp

70-

2

Os0

5g0

591

400

Hsp

90

Os0

6g0

716

700

CN

X

Os0

4g0

402

100

CR

T-1

Os0

7g0

246

200

CR

T-2

Os0

3g0

832

200

NE

F

Os0

9g0

512

700

ER

DJ3

like

Os0

5g0

156

500

Stt3

a

Os0

4g0

675

500

UD

PG

-g-T

Os0

6g0

593

100

Der

lin

Os0

3g0

852

200

Fold

Chan

ge

Fig.1 The phenotype of T3612

Fig.2 OsPDIL1-1 is the responsible gene for the T3612

mutant phenotype

Fig.4 Enzyme activity in the developing

endosperms.

Nipponbare，lane1,3；T3612，lane2,4

图7 内质网胁迫相关基因表达分析

3、The failure to express PDIL1-1 results in a floury endosperm and

an ER-stress response in rice

Fig.3 Complementation test of PDIL1-1. Lane1, A582-6; lane2, A582-9; Lane3, A582-10;

Lane4, A582-11

J. Exp. Bot, 2012, 63,121-130

D E

Fig.5 SDS-PAGE analysis

of total mature grain protein

Fig.6 TEM images of the developing endosperm of

(A) cv. Nipponbare, (B) T3612.

4、 gpa2(glutelin precursor accumulation) seeds accumulate

proglutelins and develop abnormal endosperms

Figure 1. Characterization of the gpa2 mutant. (A) Seed storage protein SDS–PAGE gel. (B) Immunoblot. (C) Grain filling process of wild type and gpa2 seeds. (D) Wild type seeds. (E) gpa2 seeds. (F-I) SEM analysis of the endosperms of wild type (F, H) and gpa2 (G, I). Scale bars = 1 mm in (F) and (G), Scale bars = 50 μm

in (H) and (I).

Weight(g)* Stach(%) Protein(%) Lipid (%) Amylose(%)

Wild type 19.6±0.4 65.60±0.05 11.58±0.09 2.67±0.07 15.64±0.15

gpa2 12.5±0.3 63.67±0.63 12.84±0.12 4.45±0.10 12.78±0.08

Δ -36.2% -2.9% +10.9% +66.7% -18.3%

Table 1 Properties of gpa2 seeds

* weight per 1000 hulled kernels

Abnormal structures accumulated in the gpa2 endosperm cells

Figure 2. Ultrastructure of 12 DAF endosperm cells of gpa2 and wild type. (A) wild-type. (B-E) morphology of PBII, novel structures and cell wall in gpa2. Stars in (C, D) indicated vesicle-like structures. Arrowhead in (E) indicated possible fusion of vesicle-like structures. SG: starch granule; PMB: Paramural body; PM: plasma membrane; G: Golgi; LB: lipid body. Scale bars: 1 μm.

WT gpa2

gpa2

gpa2

gpa2

Abnormal trafficking of glutelin and globulin in gpa2 Seeds

Figure 3. Immunofluorescence microscopic analysis of 12 DAF endosperms of gpa2 seeds. (A, D) wild type. (B, C, E, F) gpa2 mutant. (A-C) Glutelin and prolamin were labeled green and red, respectively. (D-F) Glutelin and globulin were labeled green and red, respectively.

Figure 4. Immunolocalization of glutelin in endosperm cells. (A) Glutelins accumulated in PBII, but not seen in PBI and cell wall in wild type endosperm cells. (B-D) gpa2. Glutelins were appeared inside cell wall and extracellular space (ECS), in addition to PBII (B). Glutelins were localized to vesicle-like structures along the cell wall (C) and PMB (D). Dashed circles in (C, D) outline the vesicles. Scale bars: 1 µm.

WT gpa2

gpa2 gpa2

Figure 5. DVs were secre-ted to form PMBs instead of be transport to PBII in endosperm cells of gpa2 and wild type. (A) wild-type. (B-D) gpa2. Arrowheads in (A 、 B) in-dicated dense vesicles. Arrows in (C、D) indica-ted dense vesicles being secreted to apoplast. St-ars in (C) indicated vesic-le-like structures. Scale bars: 1 μm.

gpa2 Affects Post-Golgi Traffic of Storage Protein

WT gpa2

gpa2 gpa2

Figure 6. The distribution of JIM84 (A and B) and α-TIP (C and D) in the wild type and gpa2. (A, C) wild type. (B, D) gpa2 mutant. (A,B) JIM84 and PBI were labeled green and red, respectively. (C,D) α-TIP and PBI were labeled green and red, respectively.

gpa2 Affects Distribution of Polysaccharides and Pectin Synthesized and

Sorted through Golgi

Figure 7. Polysaccharides and Pectin accumulated in PMBs in gpa2 endosperm cells. (A, C, E, F ) wild type. (B, D, F, H) gpa2 mutant. (A-D) Glutelin and (1-3;1-4)-beta-D-Glucan were labeled green and red, respectively. (E-H) pectin and CW were labeled green and blue, respectively.

Map-based cloning of gpa2

Figure 8. OsVPS9A is the gene responsible for gpa2 mutant phenotype. (A) High-resolution mapping of mutant gene in gpa2. The number of recombinants is indicated below the map. Eight ORFs were predicted in the mapping region. (B) A single nucleotide transversion in OsVPS9A led to a premature stop codon. C) SQ RT-PCR of OsVps9a.(D,E,F) Complementation of gpa2 restored normal phe-notypes. (D) Complemented seeds became lucent, L1, L2, L3 are T2 progeny of thee ind-ependent transgenic lines, (E) the amount of proglu-telin and its subunits in thr-ee complemented lines was comparable to that of wild type, and (F) Ultrastructure of com-plemented endosperm cells appear normal. Scale bar: 5 µm.

OsVPS9A was localized on the Golgi to PVC

Figure 9 . Subcellular localization of GFP-OsVPS9A in the transgenic rice plant.

GFP-OsVPS9A localizes to the limiting membrane of MVBs (A and B, arrowheads). the

TGN (C, open arrowheads), Golgi (A and C, arrows) and Scale bars: 100 nm.

OSVPS9A is a guanine nucleotide exchange factor of RAB5 GTPases in rice

Model of RAB GTPase cycle.

Schematic representation of the primary structures of Homo sapiens RAB5c (HsRAB5c) and Plant (Arabidopsis thaliana & Oryza sativa) RAB5 proteins.

Figure 10. Interactions between OsVPS9a and OsRAB5 members. (A)OsVPS9A and OsRAB5s were fused to DNA binding domain (BD) and activation domain (AD) of GAL4 transcription factor, respectively. The transformants were grown on SDII (-Trp, -Leu) and SDIV (-Trp, -Leu, -His,-Ade) plates. (B) Co-immunoprecipitation of VPS9A-FLAG and GFP–RAB5A, or their mutant forms, co- expressed in Nicotiana benthamiana. VPS9A-FLAG was immunoprecipitated with anti-FlAG beads and precipitants were analysed by IB using anti-GFP antibody.

(A) (B)

OsVPS9A Cooperates with OsRAB5A in Transporting of the proglutelin

from Golgi to PBII

Figure 11. rab5a mutation aggravated the abnormal morphological phenotypes of vps9a.

5、The gpa3 Mutant Has Defects in Vacuolar Targeting of

Storage Protein and Endosperm Development

Figure 1. Phenotypic Characteristics of the Wild Type and gpa3 Mutant

Scale bars = 1 mm in D; 10 μm in E.

Distribution of Glutelin and α-globulin in gpa3 Mutant

Subaleurone cells

In Addition to PBIIs Distribution, Glutelin and α-globulin Deposit in Novel Structures in gpa3 Mutant Subaleurone Cells

WT: A,C, and E; gpa3: B, D, and F. Bars= 10 μm.

The PMB Structure Was Closely Associated with

Cell Wall

The PMBs Are Filled with Callose, Surrounded

by (1, 3; 1, 4)-β-glucan and Pectin

WT: A,C, and E; gpa3: B, D, and F. Bars= 10 μm.

Mistargeting of Storage Proteins Induced the Formation of

the PMB

Bars =1 μm in A, B, C, and D; 500 nm in E to K.

PM, Plasma membrane; CW, Cell wall; Pd, Plasmodesma; DV, Dense vesicle.

Map-based cloning of GPA3 gene

GPA3 Protein Localized to TGN and PVC

Bars = 10 μm

GPA3 Physically Interacts with GPA2

Gene expression

mRNA targeting into the ER subdomain

Protein sorting in the TGN ( GPA1(OsRab5a ), GPA2(OsVPS9a), GPA3

Transportation of Glutelin precursor into the vacuole

Cleavage into mature glutelin OsVPE1(W379)

PDIL1-1 (T3612)

Cis-elements: GCN4, AACA,

ACGT, Prolamin box

Transacting factors: RPBF,

RISBZ1, OSMYB5

ER to Golgi （gpa4 on going）

Summary

Acknowledgements

NSFC

863 project

973 project

China Post-doctor Fundation

Dr. Xiaohua Han (OsPDIL1-1);

Dr. Feng Liu (GPA2);

Dr. Yulong Ren (GPA3)

Dr. Yiqun Bao (college of life science, NAU)

Dr. Liwen Jiang (Chinese University of HongKong )

Thank you！