1784 Proteomics 2013, 13, 1784–1800DOI 10.1002/pmic.201200389

REVIEW

Use of proteomics to understand seed development

in rice

Zhu Yun Deng, Chun Yan Gong and Tai Wang

Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Haidianqu,Beijing, China

Rice is an important cereal crop and has become a model monocot for research into crop biol-ogy. Rice seeds currently feed more than half of the world’s population and the demand for riceseeds is rapidly increasing because of the fast-growing world population. However, the molec-ular mechanisms underlying rice seed development is incompletely understood. Genetic andmolecular studies have developed our understanding of substantial proteins related to rice seeddevelopment. Recent advancements in proteomics have revolutionized the research on seeddevelopment at the single gene or protein level. Proteomic studies in rice seeds have providedthe molecular explanation for cellular and metabolic events as well as environmental stressresponses that occur during embryo and endosperm development. They have also led to thenew identification of a large number of proteins associated with regulating seed developmentsuch as those involved in stress tolerance and RNA metabolism. In the future, proteomics,combined with genetic, cytological, and molecular tools, will help to elucidate the molecularpathways underlying seed development control and help in the development of valuable andpotential strategies for improving yield, quality, and stress tolerance in rice and other cereals.Here, we reviewed recent progress in understanding the mechanisms of seed development inrice with the use of proteomics.

Keywords:

Cereal crop / Monocot / Plant proteomics / Rice / Seed development

Received: August 28, 2012Revised: December 24, 2012

Accepted: January 7, 2013

1 Introduction

Rice seeds represent one of the most important staplefoods for humans, currently feeding more than half of theworld’s population; and the demand for rice seeds is ex-

Correspondence: Professor Tai Wang, Key Laboratory of PlantMolecular Physiology, Institute of Botany, Chinese Academy ofSciences, 20 Nanxincun, Xiangshan, Haidianqu, Beijing 100093,ChinaE-mail: twang@ibcas.ac.cnFax: +0086-10-62594170

Abbreviations: DAF, day after fertilization; DEPs, differentially ex-pressed proteins; DHT, day high temperature; ER, endoplasmicreticulum; GBSS, granule-bound starch synthase; hnRNPs, het-erogeneous nuclear ribonucleoprotein; KH, K-homology; LEAs,late-embryogenesis-abundant proteins; NCBI, National Center forBiotechnology Information; NHT, night high temperature; PCD,programmed cell death; PPDKB, pyruvate orthophosphate diki-nase B; RACK, receptor for activated C-kinase; Rboh, respiratoryburst oxidase homolog; RBPs, RNA-binding proteins; REG, riceembryo globulins; RRM, RNA recognition motif; SOD, superoxidedismutase; ssDNA, single-stranded DNA; SSPs, seed storage pro-teins; TCA, tricarboxylic acid; WT, wild-type

panding rapidly because of the fast-growing world popula-tion [1]. In addition to being consumed as staple food, riceseeds are used to make edible starches, vinegars, syrups,and sweet alcoholic beverages. The bran of rice seeds isa raw material for extracting oil used in the cosmetic andhealthcare industries. Besides the aforementioned practi-cal importance for food security and economic production,two other reasons make rice a good model monocot sys-tem to study seed biology. First, the genome of rice isrelatively smaller but highly conserved as compared withthat of other cereal crops such as maize and barley. Sec-ond, the complete genome sequence of rice is available inpublic databases such as the National Center for Biotech-nology Information (NCBI; http://www.ncbi.nlm.nih.gov/)and the Rice Genome Annotation Project Database (RGAP;http://rice.plantbiology.msu.edu/). Therefore, rice seed biol-ogy has been broadly studied at different levels by use ofhistological, physiological, cytological, biochemical, genetic,and molecular tools. These studies have greatly expanded ourknowledge of rice seed development and clearly demonstratethat (i) seed developmental events are programmed to occur

Colour Online: See the article online to view Table 3 in colour.

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Proteomics 2013, 13, 1784–1800 1785

as a result of expression and activation of different proteinsin distinct seed compartments (i.e. embryo, endosperm, andcaryopsis coat) and even within specific regions (e.g. apicalmeristem), at distinct developmental stages [2, 3]; and (ii)seeds regulate the expression and/or activation of specific pro-teins to respond efficiently to a highly dynamic environment(e.g. temperature, water, and light) during development [4].However, the number of identified proteins active in theseprocesses is limited, and elucidation of these regulatory net-works remains a major challenge.

Proteomics has provided an efficient large-scale solutioncomplementing traditional and genomic approaches for in-vestigating rice seed development. Compared with traditionalmolecular and genetic strategies, proteomics involves globalcomparative studies, allowing for large-scale identificationand expression analysis of proteins active in specific seedcompartments or regions at certain developmental stages.This capability may make it possible for us to answer manyquestions that seem unsolvable at the single gene or proteinlevel and to discover novel development-related proteins thatmay be undetectable by forward or reverse genetic methods(e.g. redundant or lethal proteins involved in rice seed devel-opment but without homologues in other species). As com-pared with genomics and transcriptomics tools such as DNAmicroarray, proteomics focuses directly on proteins, the finalactive agents in developing seeds. Although proteins are thetranslated products of gene transcripts and large-scale anal-ysis of genes or transcripts is relatively easy and efficient,proteomics has irreplaceable superiority. Alternative RNAsplicing and various posttranslational modifications such asphosphorylation, methylation, glycosylation, ubiquitylation,and proteolytical processing may change protein localization,activity, stability, and/or function [5], which may play im-portant roles in seed development. Protein isoforms derivedfrom these processes can be identified and analyzed only byproteomic methods, not genomics or transcriptomics. In ad-dition, quantitative proteomics techniques must be used todirectly measure changes in protein abundance during seeddevelopment and environmental response, because RNA lev-els are not always correlated with the levels of correspondingproteins. Finally, the localization of gene products, providingimportant clues of gene function as well as regulatory net-works activated in different seed regions, can be determinedonly at the protein level. Characterization of proteomes ex-pressed in specific cell types, tissues, regions, or organs ofseeds (called subproteomics) is a powerful approach for un-derstanding the molecular mechanisms underlying seed de-velopment. Due to these advantages, proteomic technologieshave been increasingly used to investigate rice seeds over thepast two decades. Most proteomic studies of rice seeds haveaimed to provide practical or theoretical information helpfulfor improving the quality and yield of rice and other cereals.Proteomic studies relevant to seed development focused onthe important but unanswered issues regarding embryo andendosperm development in rice: (i) metabolic and molecu-lar events that occur at different seed developmental stages,

(ii) the effects of environmental factors on seed developmentand rice quality, and (iii) specific biological processes duringseed development such as storage protein biosynthesis andits regulation (Table 1).

In this review, we first briefly describe the structure anddevelopment process of rice seeds, providing the anatom-ical and cellular basis for proteomic studies related torice seed development. Then, we discuss recent proteomicstudies that have contributed to our understanding ofthe mechanisms underlying seed development in rice, aswell as the proteomic technologies used in these studies(Table 1).

2 Rice seed structure and development

2.1 Seed structure

The rice seed is strictly considered a caryopsis. It is a single-seeded fruit with the seed fused with the fruit coat. Therefore,a mature rice seed or caryopsis comprises three main struc-tural elements: a diploid embryo, a triploid endosperm, and amaternal caryopsis coat (Fig. 1A). The embryo represents thenew generation, residing at a small corner of the caryopsis.Its main portions include scutellum, coleoptile, plumule, epi-blast, and radicle (Fig. 1B). The scutellum is the apical portionof the embryo, acting as a storage organ during seed devel-opment and helping to digest and transport endosperm nu-trients during seed germination. The coleoptile, the radicle,and the plumule containing the shoot apical meristem andthree leaf primordia, are precursor organs of rice seedlings.The coleoptile protects the plumule during germination. Theplumule and the radicle eventually develop into the shootand the seminal root, respectively. The endosperm is the ma-jor organ for storage of nutritional reserves, occupying thebulk of a mature rice seed. It consists of the central starchyendosperm filled with lifeless starchy endosperm cells, analeurone layer surrounding the starchy endosperm, and abasal transfer layer between the embryo and the endosperm.Roughly 90% of the nutrients in the endosperm cells arestarches stored in starch granules, and 5–8% of the nutrientsare storage proteins held in proteoplasts (protein bodies). Incontrast, the aleurone layer mainly accumulates proteins andlipids. The caryopsis coat consists of several layers of the peri-carp (fruit coat), one layer of the testa (seed coat) and one layerof the nucellus, coating the seed and serving as a protectionfor the embryo and the endosperm.

2.2 Seed development

The rice seed initiates its development from double fertil-ization and matures after approximately 20 days of growth.During double fertilization in the embryo sac, one spermnucleus fuses with the nucleus of the egg cell to producethe diploid zygote and the other sperm nucleus fuses with

C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

1786 Z. Y. Deng et al. Proteomics 2013, 13, 1784–1800Ta

ble

1.

Rec

ent

pro

teo

mic

stu

die

sre

late

dto

rice

seed

dev

elo

pm

ent

Focu

sed

issu

esS

tud

ied

Dev

elo

pm

enta

lE

xtra

cted

pro

tein

Sep

arat

ion

Iden

tifi

cati

on

No

.of

iden

tifi

edR

ef.

pro

teo

mes

stag

esfr

acti

on

sm

eth

od

sm

eth

od

ssp

ots

(id

enti

ties

,(p

Iran

ge)

un

iqu

ep

rote

ins)

Wh

ole

seed

develo

pm

en

t

Mo

lecu

lar,

cellu

lar,

and

met

abo

licev

ents

du

rin

gse

edd

evel

op

men

t

Pro

teo

me

chan

ges

inw

ho

lese

eds

du

rin

gth

eco

mp

lete

dev

elo

pm

enta

lp

roce

ss

2,4,

6,8,

10,1

2,14

,16,

18,a

nd

20D

AFa)

Tota

llo

wsa

lt-s

olu

ble

frac

tio

nfr

om

dev

elo

pin

gse

eds

2DE

(4–7

)M

ALD

I-TO

F30

9(3

45,2

27)

[32]

En

do

sp

erm

develo

pm

en

t

Mo

lecu

lar,

cellu

lar,

and

met

abo

licev

ents

du

rin

gst

arch

accu

mu

lati

on

Pro

teo

me

chan

ges

inen

do

sper

ms

du

rin

gth

ep

erio

do

fra

pid

star

chac

cum

ula

tio

n

12,1

5,an

d18

DA

Fa)To

tall

ow

salt

-so

lub

lefr

acti

on

fro

md

evel

op

ing

end

osp

erm

s

2D-D

IGE

(4–7

)M

ALD

I-TO

F29

8(3

17,1

87)

[16]

Mo

lecu

lar,

cellu

lar,

and

met

abo

licre

gu

lati

on

by

ligh

t-d

ark

cycl

e

Diu

rnal

chan

ges

inen

do

sper

mp

rote

om

esat

the

mid

dle

ph

ase

of

end

osp

erm

dev

elo

pm

ent

10D

AFa)

(0,4

,8H

AO

b)

atlig

ht

ph

ase,

12H

AO

b)

atlig

ht-

dar

ktr

ansi

tio

n,1

6an

d20

HA

Ob

)at

dar

kp

has

e)

Tota

llo

wsa

lt-s

olu

ble

frac

tio

nfr

om

dev

elo

pin

gen

do

sper

ms

2D-D

IGE

(4–7

)M

ALD

I-TO

F81

(91,

62)

[33]

Em

bry

od

evelo

pm

en

to

rem

bry

og

en

esis

Mo

lecu

lar

and

met

abo

licev

ents

du

rin

gem

bry

og

enes

is

Pro

teo

mic

com

par

iso

nam

on

gem

bry

os

atd

iffer

ent

dev

elo

p-

men

tals

tag

es,a

sw

ella

sm

atu

rean

dd

ryem

bry

os

5,7,

14,2

1,30

DA

Fa)To

talu

rea-

solu

ble

frac

tio

nfr

om

dev

elo

pin

g,m

atu

re,

and

dry

emb

ryo

s

2DE

(3–1

0)LC

/MS

/MS

242

(275

,192

)[2

8]

Mo

lecu

lar

and

met

abo

licev

ents

du

rin

gem

bry

og

enes

is;

glo

bu

lins

and

thei

rro

les

inem

bry

og

enes

is

Pro

teo

me

chan

ges

inem

bry

os

du

rin

gth

em

idd

lean

dla

ted

evel

op

men

tal

ph

ases

6,12

,18

DA

Fa)To

talu

rea-

solu

ble

frac

tio

nfr

om

dev

elo

pin

gem

bry

os

2DE

(3–1

0)M

ALD

I-TO

F/TO

F53

(55,

23)

[29]

Str

ess

resp

on

se

an

dto

lera

nce

Eff

ects

of

hig

hte

mp

erat

ure

on

rice

qu

alit

yan

dse

edd

evel

op

men

t

Pro

teo

me

chan

ges

inse

eds

by

hig

hte

mp

erat

ure

trea

tmen

td

uri

ng

seed

dev

elo

pm

ent

3,6,

9,12

,15,

and

30D

AFa)

Tota

lure

aan

dtr

ito

n-s

olu

ble

frac

tio

nfr

om

dev

elo

pin

gse

eds

2DE

(3–1

0)LC

/MS

/MS

54(5

4,48

)[2

6]

Diff

eren

cein

effe

cts

by

day

and

nig

ht

hig

hte

mp

erat

ure

du

rin

gse

edd

evel

op

men

t

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teo

me

chan

ges

inse

eds

by

day

or

nig

ht

hig

hte

mp

erat

ure

trea

tmen

td

uri

ng

seed

dev

elo

pm

ent

5,10

,15,

and

20D

AFa)

Tota

lNP

-40

and

low

salt

-so

lub

lefr

acti

on

fro

md

evel

op

ing

seed

s

2DE

(4–7

)M

ALD

I-TO

F;M

ALD

I-TO

F/TO

F;LT

Q-E

SI-

MS

/MS

61(6

1,46

)[2

5]

C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2013, 13, 1784–1800 1787

Ta

ble

1.

Co

nti

nu

ed

Focu

sed

issu

esS

tud

ied

Dev

elo

pm

enta

lE

xtra

cted

pro

tein

Sep

arat

ion

Iden

tifi

cati

on

No

.of

iden

tifi

edR

ef.

pro

teo

mes

stag

esfr

acti

on

sm

eth

od

sm

eth

od

ssp

ots

(id

enti

ties

,(p

Iran

ge)

un

iqu

ep

rote

ins)

Mo

lecu

lar

mec

han

ism

su

nd

erly

ing

abio

tic

stre

ssto

lera

nce

Co

mp

aris

on

of

emb

ryo

pro

teo

mes

amo

ng

stre

ss-t

ole

ran

tan

d-s

ensi

tive

rice

vari

etie

s

Mat

ure

stag

eTo

talu

rea-

solu

ble

frac

tio

nfr

om

mat

ure

and

dry

emb

ryo

s

2DE

(3–1

1)M

ALD

I-TO

F;LC

-MS

/MS

;De

nov

ose

qu

enci

ng

28(2

8,22

)[3

0]

Sp

ecifi

cb

iolo

gic

al

pro

cesses

du

rin

gseed

develo

pm

en

t

Gp

rote

in-m

edia

ted

tran

sdu

ctio

np

ath

way

inem

bry

od

evel

op

men

t

Pro

tein

sre

gu

late

db

yth

e�

sub

un

ito

fa

het

ero

trim

eric

Gp

rote

ind

uri

ng

emb

ryo

gen

esis

14,2

1,28

,35

DA

Fa)

and

ger

min

atio

nTo

talu

rea

and

NP

-40-

solu

ble

frac

tio

nfr

om

dev

elo

pin

gan

dg

erm

inat

ing

emb

ryo

s

2DE

(3.5

–7an

d6–

10);

Cle

vela

nd

pep

tid

em

app

ing

Ed

man

seq

uen

cin

g7

(7,3

)[2

7]

Nu

clea

rp

rote

ins

invo

lved

inse

edd

evel

op

men

t

Nu

clea

rsu

bp

rote

om

eo

fse

eds

atth

em

idd

led

evel

op

men

tal

ph

ase

9D

AFa)

Ph

eno

l-ex

trac

ted

frac

tio

nfr

om

nu

clei

of

dev

elo

pin

gse

eds

1DE

;2D

E(3

–11)

;g

elfr

eeM

ALD

I-TO

F/TO

F;LC

/LC

-MS

/MS

468

(468

,468

)[3

5]

RN

A-b

ind

ing

pro

tein

sse

rvin

gp

rote

ctiv

efu

nct

ion

sd

uri

ng

seed

mat

ura

tio

nan

dd

orm

ancy

RN

A-b

ind

ing

pro

tein

sin

mat

ure

dry

seed

sM

atu

rest

age

Low

salt

-so

lub

lean

dss

DN

A-b

ind

ing

frac

tio

nfr

om

dry

seed

s

2DE

(3–1

0)M

ALD

I-TO

F18

(18,

9)[3

6]

RN

A-b

ind

ing

pro

tein

sim

plic

ated

inse

edd

evel

op

men

t

Cyt

osk

elet

on

-as

soci

ated

RN

A-b

ind

ing

pro

tein

sin

dev

elo

pin

gse

eds

12–1

4D

AFa)

Low

salt

-so

lub

le,

cyto

skel

etal

-en

rich

ed,

and

plo

y(U

)-b

ind

ing

frac

tio

nfr

om

dev

elo

pin

gse

eds

2DE

(6–1

1an

d3–

10)

Q-T

OF

162

(148

RN

A-b

ind

ing

pro

tein

s)

[37]

Pro

lam

ine

mR

NA

-bin

din

gp

rote

ins

rela

ted

tom

RN

Atr

ansp

ort

atio

nan

dlo

caliz

atio

nto

ER

du

rin

gst

ora

ge

pro

tein

syn

thes

is

Cyt

osk

elet

on

-as

soci

ated

pro

lam

ine

mR

NA

-bin

din

gp

rote

ins

ind

evel

op

ing

seed

s

10–1

5D

AFa)

Low

salt

-so

lub

le,

cyto

skel

etal

-en

rich

ed,

and

pro

lam

ine

zip

cod

e-b

ind

ing

frac

tio

nfr

om

dev

elo

pin

gse

eds

1DE

LC–M

S/M

SPr

ola

min

em

RN

A-b

ind

ing

pro

tein

sca

ptu

red

wit

h(1

8)o

rw

ith

ou

t(1

32)

hep

arin

[38,

39]

a)D

AF,

day

afte

rfe

rtili

zati

on

;b)

HA

O,h

ou

rsaf

ter

the

on

set

of

12-h

ligh

tan

d12

-hd

ark

cycl

e.

C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

1788 Z. Y. Deng et al. Proteomics 2013, 13, 1784–1800

Figure 1. Structure of the rice seed and its embryo. (A) Morphol-ogy of a mature seed of rice. EN, endosperm; EM, embryo; scalebar = 1 mm. (B) Longitudinal section of the rice embryo. SC,scutellum; CO, coleoptile; PL, plumule; EP, epiblast; and RA, radi-cle; scale bar = 1 mm. Adapted from [32].

the two polar nuclei of the central cell to create the triploidprimary endosperm nucleus. Afterwards, the zygote andthe primary endosperm nucleus develop relatively indepen-dently to become the embryo and the endosperm, respec-tively. Coincidently, the maternal ovary undergoes concor-dant growth and changes to fit the expanding embryo andendosperm. Finally, the ovary wall and the ovule integumentform the caryopsis coat. Here, we concisely describe, respec-tively, the processes of embryo (Table 2) and endosperm (Ta-ble 3) development. Detailed processes of embryo and en-dosperm development have been documented in previousreviews [2, 6].

2.2.1 Embryo development

The zygote begins its first cell division at about 10 h afterdouble fertilization [7] and continues to undergo rapid celldivision in an unfixed direction, thus resulting in a 25-celledearly globular embryo at 1 day after fertilization (DAF) [2]. Thecell division rate decreases after the 25-celled stage, and themiddle globular embryo reaches 150 cells at 2 DAF [2]. Accom-panied by cell division, early embryonic events that includedetermination of organ differentiation, establishment of or-gan position, and control of embryo size occur before mor-phogenetic changes are visible [8]. In the late globular stage,at 3 DAF, besides increasing cell number from 150 to 800cells, the embryo starts organ differentiation and forms thedorsal-ventral axis, making the embryo an oblong shape [2].At early 4 DAF, the first organogenetic events take place whenthe coleoptile develops from the ventral surface of the embryoand the scutellum is distinguishable by cells with vacuolatedcytoplasm [3, 9]. At late 4 DAF, differentiating shoot apicalmeristem and radical are morphologically recognized belowthe coleoptile [9]. At 5 DAF, the size of the scutellum in-creases obviously and the first leaf primordium emerges [2].Then the second and third leaf primordia become visible at

7 and 8 DAF, respectively. At the same time, the epiblaststretches out and enlarges [2]. At 10 DAF, morphologicaldifferentiation of embryonic organs is accomplished, but vol-ume augmentation of these organs is remarkable [2]. After10 DAF, the embryo increases its size slightly and under-goes rapid maturation [2]. Eventually, the embryo completesthe development process and becomes dormant after 20DAF [2].

2.2.2 Endosperm development

The endosperm grows at a relatively rapid rate as comparedwith the embryo. The primary endosperm nucleus starts itsfirst division of nucleus immediately after double fertiliza-tion without halting, and keeps on dividing nuclei in theabsence of cell-wall formation at a rate faster than the divi-sion of the zygote, thus leading to formation of free nucleiwith dense cytoplasm [7]. These free nuclei distribute to theperiphery of the embryo sac cavity and establish a multin-ucleate layer at 2 DAF [7]. Accompanied by the division ofendosperm nuclei, nuclear DNA content is increased from3 to 6–12 C by endoreduplication [10]. At 3 DAF, the en-dosperm begins to cellularize, and cell walls form initiallyin the region near to the developing embryo and then ex-tend to the entire endosperm layer [7]. Simultaneously, all ofthe cells undergo an inward division, thus generating an en-dosperm with two cell layers [7]. Afterwards, the endospermdivides rapidly to significantly increase the number of celllayers and cell numbers of each layer until peak number ofcells is achieved at 7 DAF [11]. In addition to cell division,many important developmental events occur during this pe-riod. The embryo sac cavity is stuffed with endosperm cellsat 4 DAF [11]. DNA endoreduplication continues in the di-viding endosperm cells, thus leading to increased nuclearDNA content to 12–24 C at 6 DAF [10]. Endosperm cellsbegin to accumulate starch and starch granules become vis-ible in endosperm cells at 4 DAF [11]. The outermost en-dosperm cells differentiate into aleurone cells at 5 DAF. Celldivisions, accompanied by differentiation, leads to the forma-tion of the aleurone layer at 7 DAF [12]. Through increasingcell number, the endosperm grows to its full length at 7 DAF.However, after the cell division stage, further growth of theendosperm continues by enlargement of individual cells. Fi-nally, the endosperm reaches its full width and thickness at12 and 15 DAF, respectively [13]. Along with cell enlarge-ment, the endosperm continues to deposit starches mainlyin endosperm cells and proteins and lipids mainly in aleu-rone cells. The size of starch granules in endosperm cellsincreases and reaches the maximum at 15 DAF [14, 15]. En-dosperm cells keep on endoreduplicating nuclear DNA, andthe DNA content peaks (30 C) at 16 DAF [10]. Ultimately, ma-ture endosperm cells undergo programmed cell death (PCD)at 12–20 DAF [16], leaving only the aleurone layer alive at seedmaturity.

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3 Proteomic techniques used in the studyof seed development in rice

Over the past 20 years, proteomics has become a major toolfor surveying protein profiles in rice seed development, pro-viding useful information at the proteomic level. Proteomicresearches into rice seed development employed the sameexperimental workflow and the same proteomic techniquesas other proteomic studies in plants, especially rice, whichhave been well documented and discussed [17–20]. However,multiple technique improvements, such as extraction andpurification methods for obtaining low-abundance seed pro-teins, have been made in the proteomic studies related torice seeds, which have progressed seed proteomics and led toa deeper understanding of seed development in rice. Here,we describe the extraction, separation, and identification ap-proaches used in the proteomic studies reviewed here andhighlight their technical progresses and improvements.

3.1 Extraction of rice seed proteins with or without

seed storage proteins

Mature rice seeds have a total protein content ranging from5 to 12% [21, 22], most of which is contributed by seed stor-age proteins (SSPs), including glutelins, albumins, globu-lins, and prolamines. The predominant SSPs of rice seedsare glutelins, which constitute 80% or more of the total seedproteins. The remaining three types of SSPs, respectively,account for 1–5% (albumins), 4–15% (globulins), and 2–8%(prolamines) of the total seed proteins [23]. During seed de-velopment, SSPs are first synthesized as precursor proteins,then processed and finally deposited in protein bodies in riceseeds [24]. Therefore, SSPs are highly abundant in both de-veloping and mature seeds.

Most studies of rice seeds used total proteins, includingSSPs, as samples for proteomic analysis. Total proteins werefirst extracted from whole seeds [25, 26], endosperms [27],or embryos [27–30] by use of strong denaturing solutionscontaining 8 M urea and/or detergents and then subjectedto 2DE directly or after further extraction and concentrationby the trichloroacetic acid/acetone method. In addition, sev-eral other studies extracted only soluble proteins to eliminatethe impacts of SSPs on further analysis. These studies usedlow-ionic-strength and neutral pH buffers for protein isola-tion, which minimized the extraction of SSPs because onlyalbumins are water soluble, whereas glutelins, globulins, andprolamines are soluble in alkali or acid, salt, and aqueous al-cohol solution, respectively. For example, through grindingmaterials in a simple extraction buffer with 20 mM Tris-HCl (pH 8.0), 20 mM NaCl, and 1 mM EDTA, followed bytrichloroacetic acid/acetone precipitation, our laboratory hasextracted low salt-soluble total proteins with a few SSPs frommature [31] and developing [32] rice seeds and developingrice endosperms [16, 33].

For subproteomic analysis of rice seeds, the highly abun-dant SSPs substantially mask low-abundance seed proteins,thus affecting the effectiveness of proteomic studies withshotgun or 2DE gel-based approaches [34]. Therefore, selec-tive removal of SSPs from seed protein extracts is a key step insample preparation. However, SSPs cannot be completely re-moved by differential fractionation because protein bodies (inwhich SSPs are stored) and various SSP precursor-containingorganelles (e.g. vesicles, Golgi apparatus, and ER) have dif-ferent sedimentation coefficients [35]. SSPs may be isolatedor removed in light of their physical and chemical proper-ties. For example, acid or alkali treatment can eliminate mostglutelins from seed proteins. However, these methods havenot been commonly used in rice seed proteomics. In a ricenuclear subproteome study, Peng et al. first eliminated starchgranules and parts of SSPs sticking on starch granules by cot-ton filtration after isolating nuclei from developing rice seedsat 9 DAF, then removed the major remaining SSPs (i.e. 38and 55 kD glutelin) by directly cutting out the two proteinbands after 1DE separation of the extracted proteins fromthe isolated nuclei [35]. The resulting 1DE gels had a rela-tively large proportion of nuclear proteins and were groundinto powder and extracted with phenol to restore nuclear pro-teins [35]. Using this two-step removal method, the authorsobtained about tenfold more proteins than in total proteinextracts of rice seeds. More importantly, the obtained pro-teins included many low-abundance transcriptional factors[35]. Affinity chromatography is another powerful methodfor removing SSPs and enriching low-abundance seed pro-teins. Motoki and associates fractionated RNA-binding pro-teins (RBPs) from mature dry rice seeds by single-strandedDNA (ssDNA) affinity column chromatography [36]. Thisone-step affinity purification removed all SSPs detected inthe total protein extracts [36]. Okita’s group applied poly(U)-sepharose affinity columns and streptavidin magnetic beadsbound with biotinylated prolamine mRNAs to isolate gen-eral and prolamine RBPs, respectively, from cytoskeleton-enriched proteins of developing rice seeds [37,38]. To identifygeneral cytosolic RBPs, the authors used differential centrifu-gation and sucrose gradients to remove chromatin and nucleiand enrich cytoskeleton-associated fractions, then poly(U)-sepharose affinity purification to obtain the cytoskeleton andpoly(U)-binding fractions [37]. The resulting proteins wereseparated by 2DE with both pH 6–11 and pH 3–10 nonlinearimmobilized pH gradient strips [37]. After MS and peptideanalysis, 124 proteins with basic net charges and 38 pro-teins with acidic isoelectric points were identified from thepH 6–11 and pH 3–10 2DE gels, respectively [37]. Besides21 SSPs among the 162 identified proteins, 148 cytoplas-mic localized, cytoskeleton, and nucleic acid-binding proteinswere obtained ultimately [37]. To capture prolamine RBPs,the enriched cytoskeleton-associated fractions were incubatedwith streptavidin magnetic beads bound with the biotiny-lated RNA oligos containing the prolamine 5’CDS zipcodesequence [38]. This capture method led to the isolation of 138

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cytoplasmic localized, cytoskeleton, and prolamine mRNA-binding proteins [39].

3.2 Separation and identification of rice

seed proteins

The frequently used separation and identification technolo-gies are widely applied in rice seed proteomic studies. Almostall of the studies we review involved gel-based approaches,most combining 2DE separation and MS identification. Untilnow, gel-free-based quantitative approaches have been rarelyused to analyze rice seed protein extracts.

In an early study comparing seed proteomes between wild-type (WT) rice and the dwarf1 mutant, total embryo, and en-dosperm proteins extracted from developing embryos andendosperms, respectively, were separated on 2DE gels andelectroblotted onto PVDF membranes, then identified by Ed-man sequencing on a gas-phase protein sequencer [27]. Be-cause Edman sequencing is slow and requires large amountsof samples, the identification technique for protein spots ex-cised from 2DE gels has currently switched to MS. Develop-ing rice seed proteins [32], endosperm proteins [40], embryoproteins [29,40,41], and mature seed RBPs [36] have been suc-cessfully identified by MALDI-TOF-MS. Quadrupole-TOF-MS (Q-TOF-MS) is also used in 2DE-based approaches; e.g.developing rice seed RBPs from silver-stained gels were iden-tified by Q-TOF-MS [37].

In addition to the improvement in identification, a moresensitive separation method with high reproducibility and lowdemand for sample amounts, namely 2D-DIGE, has beenrecently used to investigate endosperm proteomes duringstarch accumulation [16] and its day-night cycle [33]. Although1DE has a lower resolution than 2DE, it is quick, simple,and easy to perform, providing a useful complement in someproteomic studies. For RBP isolation, cytoskeleton-associatedprolamine mRNA-binding proteins were separated from de-veloping rice seeds by 1DE before LC-MS/MS identifica-tion [38]. In analyzing rice nuclear proteomes, 1DE was usedto separate storage proteins from seed nuclear proteins. Stor-age protein bands were then excised from the 1DE gel andthe remaining proteins were recovered for gel-free LC/LC-MS/MS by the shotgun method [35].

4 Advances and challenges in proteomicanalysis of rice seed development

4.1 Proteomic studies of different seed

developmental stages

The development of rice seeds is a highly coordinated pro-cess that relies on different sets of proteins expressed at theright time. Proteomics has offered powerful approaches forestablishing a global view of protein expression at differentseed developmental stages, helping to elucidate the control

and coordination mechanism underlying seed development.Through 2DE or 2D-DIGE combined with MS, several stud-ies have compared the proteomes of rice embryos [28, 29],endosperms [16, 33], or whole seeds [32] at different devel-opmental stages, especially during the accumulation of re-serves, which have largely broadened our understanding ofthe molecular regulation and metabolic networks during seeddevelopment.

Owning to the importance of reserve accumulation forboth seed development and yield improvement in rice, ourlaboratory has performed several proteomic investigations toelucidate the molecular and metabolic mechanisms relatedto this process. First, we utilized 2DE and MS to analyzethe differences in whole seed proteomes among eight de-velopmental stages covering the full period of reserve accu-mulation, which led to the identification of 345 differentiallyexpressed proteins (DEPs) [32]. The 345 DEPs were classifiedinto nine functional categories, including metabolism (45%),protein synthesis/destination (20%), defense response, andcell growth/division. The metabolism category was furtherdivided into 11 subcategories containing glycolysis, alcoholicfermentation, tricarboxylic acid (TCA) cycle, starch synthe-sis, amino acid metabolism, etc. Using our newly establishedmethod, called the digital expression profile analysis, we re-vealed that the accumulation of proteins in different cate-gories or subcategories is associated with the cellular, molec-ular, and metabolic events that occur in a timely and or-derly manner during seed development. From 2 to 8 DAF,proteins mediating cell growth, division, and morphogene-sis, such as cytoskeleton proteins (tubulin, actin) and theirassociated proteins (actin-binding protein profilin), showedmaximal accumulation, which supports the rapid increasein seed size at the early developmental phase. Meanwhile,at this phase, proteins related to central carbon metabolism(glycolysis and TCA cycle) showed high expression and thoserelated to alcoholic fermentation showed low expression. Inaddition, the high expression of proteins involved in proteinsynthesis, amino acid metabolism, and proteolysis, such asubiquitin/26S proteasome components, indicates an activeturnover of proteins during the early developmental stages.At 8–12 DAF, when the growth rate of developing seedsslowed, the expression of proteins related to growth was de-creased, with an increase in expression of starch synthesis-related proteins, which demonstrates a transition from cellgrowth to reserve accumulation from this stage. Proteinsimplicated in central carbon metabolism were downregu-lated, whereas those involved in alcoholic fermentation be-came upregulated from this stage and remained upregulateduntil seeds matured. At 12–20 DAF, accompanied by thefast deposition of storage compounds, a large number ofstarch synthesis-related proteins were upregulated; proteinfolding and modification-related proteins showed a similartendency, which indicates the importance of protein fold-ing and modification in the accumulation of starch and stor-age proteins. This proteomic study indicates that the switchfrom central carbon metabolism to alcoholic fermentation is

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essential for starch synthesis and accumulation during seeddevelopment.

Based on the observations that starch accumulation mainlyoccurs in the endosperm between 12 and 18 DAF and moststarch synthesis-related proteins are upregulated also in thisperiod [32], we further examined the cytological, physiolog-ical, and proteomic alterations of developing endosperm at12, 15, and 18 DAF by combining biochemical and cyto-logical detection with 2D-DIGE analysis, the improved 2DEtechnology allowing for better quantitative comparison be-tween samples [16]. The biochemical and cytological detec-tion showed that rice endosperm completed the depositionof starch molecules and the formation of organized starchgranules in the central part at 15 DAF and in nearly the entireportion except the outmost part at 18 DAF. Accompanied bystarch accumulation, reactive oxygen species (ROS), repre-sented by hydrogen peroxide, started to appear in the inner-most part of the endosperm at 12 DAF, disappeared from thecentral part but burst into the remaining parts at 15 DAF, andwere present only in the outermost part at 18 DAF. Lifelessendosperm cells were present in the part where starch gran-ule packaging was accomplished and hydrogen peroxide haddisappeared. These observations demonstrate the emergenceof hydrogen peroxide, the completion of starch accumulationand PCD occur successively in the same endosperm region;whereas all of the three developmental events initiate fromthe inner endosperm cells, then expand toward the periph-ery endosperm cells. ROS including hydrogen peroxide aremainly formed as byproducts of the activity of oxidases suchas respiratory burst oxidase homolog (Rboh). After ROS areproduced, their levels are adjusted by ROS-scavenging en-zymes and the remaining ROS act as key signaling moleculesinducing redox-dependent regulation of diverse molecularor metabolic processes during plant development [42]. Real-time RT-PCR analysis revealed two rice Rboh genes, OsR-bohB and OsRbohH, showed the highest transcriptional levelat 15 DAF, and another Rboh gene, OsRbohD, was greatlyupregulated at 15 DAF, with the peak level at 18 DAF. Thus,upregulation of ROS-producing enzymes is chronologicallyconsistent with the burst of hydrogen peroxide. Proteomicanalysis detected 317 DEPs among the three developmentalstages, which included 22 ROS-scavenging proteins show-ing diverse expression regulation. Among them, thioredoxin(one of the most important oxidoreductases in redox regula-tion), GSH peroxidase (a key ROS scavenger), phospholipidhydroperoxide glutathione peroxidases, and trypanothione-dependent peroxidases showed the highest expression lev-els at 15 DAF. Mondehydroascorbate reductase showed thelowest level at 15 DAF. Four isoforms of superoxide dismu-tase and peroxiredoxin showed peak expression at 18 DAF.Four isoforms of ascorbate peroxidase were downregulatedfrom 12 to 18 DAF, and four of the five identified glyox-alase I isoforms showed opposite expression patterns. In ad-dition, two-thirds of the 317 DEPs are potential targets ofthioredoxin, 70% of which function in metabolism (glycol-ysis, TCA cycle, alcoholic fermentation, starch metabolism,

and amino acid metabolism), protein synthesis/destination(protein synthesis and protein folding), cell structure, andcell growth/division. Taken together, this research revealsthat the ROS-induced redox-mediated pathway is activated inrice endosperms, playing essential roles in the regulation andcoordination of starch accumulation and PCD during seeddevelopment.

The previous two studies have shown the proteomechanges during different developmental days; however,whether and how the developing rice endosperm regulates itsproteome to response to the light-dark rhythm in the same dayis still unclear. Therefore, we further compared the proteomesof 10-day-old endosperms at the light phase, the light-darktransition phase, and the dark phase by use of 2D-DIGE andMS, leading to identification of 91 proteins with significantchanges in expression [33]. The 91 DEPs showed differentlight-dark regulation patterns; however, proteins belongingto a certain functional category or subcategory displayed sim-ilar diurnally changed expression patterns. Enzyme proteinsparticipating in the conversion of leaf-importing sucroses toendosperm starches, such as UDP-Glc pyrophosphorylase,pullulanase, and ADP-glucose pyrophosphorylase large chain2 (a subunit of the first key regulatory enzyme in the starchbiosynthetic pathway), were downregulated in the light cy-cle but upregulated in the dark cycle. This diurnal-regulatedexpression of proteins related to starch synthesis was consis-tent with the following two cytological observations. First, indeveloping endosperms, the levels of sucrose, as opposed toglucose or fructose, were highest at the beginning of the lightcycle, lowered to a plateau in the middle of the light cycle,stable at the plateau the light-dark transition, and reducedrapidly in the dark cycle. Second, growth rings, representingalternating amorphous and semicrystalline regions of starchgranules, were visible in endosperm grown under alternat-ing light-dark cycles but invisible in endosperm grown un-der constant light conditions. These results strongly suggestthat endosperm starches are mainly synthesized and accu-mulated in the dark phase during rice seed development.Furthermore, proteins involved in cell division, such as celldivision control protein 48 homolog E and two isoforms ofDNA repair protein RAD23, and those implicated in proteinsynthesis, such as elongation factor 1-beta and 40S riboso-mal protein S12, showed increased accumulation in the lightphase, with a peak at the light-dark switch and a decreasein the dark phase. ROS-scavenging proteins, including glyox-alase I, superoxide dismutase (SOD) 1 [Cu-Zn], chloroplasticSOD [Cu-Zn] isoform, peroxiredoxin-2E-1, and glutaredoxin-C6, had one expression peak in the light phase and the otherpeak in the dark phase; mitochondrial SOD [Mn] showedone expression peak in the dark phase. Similarly, most pro-teins related to protein folding and proteolysis, amino acidmetabolism, and TCA cycle showed two accumulation peaks,similar to ROS-scavenging proteins. Therefore, the circadianclock control of starch accumulation requires the coordi-nation of diverse cellular and molecular processes that in-clude cell division and enlargement, redox regulation, protein

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synthesis, folding, and proteolysis, TCA cycle, and amino acidmetabolism.

In 2012, two groups published their proteomic studiesof embryo development. Wang’s group analyzed the differ-ence in embryo proteomes among five developmental stages,namely first leaf primordium formation (5 DAF), secondand third leaf primordium formation (7 DAF), morphologicalcompletion (14 DAF), maturation (21 DAF), and desiccation(30 DAF), resulting in identification of 275 DEPs [28]. Liu’sgroup compared the proteomic profiles of embryos at threedevelopmental phases that approximately correspond to thethree middle stages detected by Wang’s group, namely themorphologically changing stage (6 DAF) and two morpho-logically quiescent stages (12 and 18 DAF); and finally theyidentified 53 protein spots showing difference in expressionlevels [29]. Analogous to the proteome of endosperms [16],the largest functional categories of the DEPs in the embryoproteome of different developmental stages were metabolism(30.5%) and protein synthesis/destination (17.8%); however,proteins involved in starch synthesis were much less abun-dant in the embryo than in the endosperm [28], which sug-gests that the regulation of metabolism and protein synthesisis necessary and essential not only for endosperm growth andstarch accumulation but also for embryo development. Thechanges in expression of different proteins were closely as-sociated with the cellular, molecular, and metabolic eventsoccurring during embryo development [28, 29], similar tothe situation in the dynamic proteomes of developing en-dosperms [16] and whole seeds [32]. The two studies of riceembryos identified most enzymes involved in the glycolyticpathway, which displayed high-level expression at early devel-opmental stages, with a peak at 7 DAF, and decreased levelsafterwards [28, 29]. Proteins related to the TCA cycle, suchas aconitase, malate dehydrogenase, and succinate dehydro-genase were upregulated until the peak level was attainedat 14 DAF and were downregulated at later developmentalstages [28]. Moreover, proteins of the above two metabolicpathways were expressed at the lowest level at 30 DAF [28].This expression pattern of proteins involved in central carbonmetabolism (glycolysis and TCA cycle) has also been observedin the proteomic study of rice whole seeds [32], which sug-gests that high metabolic activity maintained by glycolysisand TCA cycle is important for early embryo and endospermdevelopmental events such as cell division and differentia-tion, but reduction in metabolic activity at late developmentalstages might be required for embryo maturation and dor-mancy as well as starch accumulation in the endosperm. Late-embryogenesis-abundant proteins (LEAs), including LEA D-34, LEA group 1, LEA group 3, and LEA domain-containingproteins, showed peak expression at 14 or 21 DAF, which isconsistent with their roles in protecting embryo cells againstwater-deficit stress during seed desiccation [28]. In addition toLEAs, globulin might offer a similar protection function. Liuet al. noted that more than half of the identified differentialspots (38 of 53) corresponded to nine rice embryo globulins(REG) or their isoforms with different molecular weight and

isoelectric point. Among them, 13 spots were identified asglobulin Os03g46100 (REG 100), 11 spots were Os03g57960(REG 960), and ten spots were Os08g03410 (REG 410) [29].During rice embryo development from 6 to 18 DAF, thethree major globulins, namely REG 100, REG 960, andREG 410, showed an increase in the number of isoformsand most of these isoforms showed upregulated expres-sion [29]. Further investigation demonstrated that globu-lin isoforms with different molecular weight were delib-erately and orderly produced by embryogenesis-controlledproteolysis, and the degraded isoforms were associatedwith protein complexes during embryo development, al-though the three major globulins contributed differently tothe formation of protein complexes [29]. Thus, these re-sults suggest that the cleaved REG isoforms might pro-tect embryo proteins by formatting protein complexeswith protected proteins during embryo maturation anddesiccation.

4.2 Proteomic studies of stress response and

tolerance in rice seeds

The development of rice seeds is not only controlled by hered-itary but also shaped by environment. Environmental factorssuch as temperature and water influence the expression ofspecific proteins, thus affecting the metabolism of carbohy-drates and proteins and thereby the yield and quality of riceseeds. The discovery and identification of proteins respond-ing to environmental stresses are the first step toward under-standing the relationship between growth environment andseed development, which is notably valuable for yield andquality improvement of rice by biological engineering [43].Recently, several comparative proteomic investigations haveused 2DE combined with MS and bioinformatics to system-atically characterize rice seed proteins responsive to abioticenvironmental stresses such as high temperature, drought,cold, and high salinity [25, 26, 30].

Through comparing the proteome of developing rice seedsgrown under normal environmental conditions (control) andunder high temperature conditions, two research groups an-alyzed the effect of high temperature on seed proteins re-lated to rice grain quality and on those involved in stressresponse [25, 26]. Lur’s group identified 54 DEPs respond-ing to continuous high temperature during seed develop-ment [26]. Among them, 21 and 14 proteins were asso-ciated with carbohydrate and protein metabolism, respec-tively, nine proteins were involved in stress response, nineproteins played other diverse roles, and one protein hadunknown function (NCBI accession number: AU082974).Zeng’s group was more concerned with the different effectsresulting from day and night high temperature (DHT andNHT) treatments. They identified 61 proteins differentiallyexpressed in treated seeds as compared with controls, in-cluding 23 implicated in carbohydrate metabolism, seven inprotein metabolism, ten in stress tolerance, four in signal

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transduction, 12 in other cellular processes, and five with un-known function [25]. When the 61 identified proteins werecompared between DHT and NHT treatments, nine proteinswere less abundant under NHT than DHT at the early stage ofseed development (5 DAF) but more abundant at the middle(10 and 15 DAF) or late (20 DAF) stages; seven proteinsshowed opposite accumulation pattern to these nine proteins;15 proteins were upregulated under NHT only at 1–3 stages;18 proteins were downregulated under NHT only at 1 or 2stages; 12 proteins showed similar expression levels at everystage [25].

Both groups found rice quality-related proteins fromthe identified DEPs involved in carbohydrate or proteinmetabolism, providing possible molecular mechanisms forthe negative effects of high temperature stress on rice quality.The cooking and taste quality of rice is largely influenced bythe ratio of the two types of starch, amylose, and amylopectin.Granule-bound starch synthase (GBSS) and pullulanase (atype of starch-debranching enzyme) are the key enzymesfor the biosynthesis of amylose [44] and amylopectin [45],respectively. Six isoforms of GBSS were detected in devel-oping rice seeds, but only the high-molecular-weight iso-forms showed an accumulation pattern positively correlatedwith amylose content, which suggests that these large iso-forms are the major enzymes involved in amylose biosyn-thesis [26]. Under high temperature, GBSS expression wasdecreased at the translational level instead of the transcrip-tional level [26]. In contrast, the five pullulanase isoformsin developing rice seeds showed upregulated expression af-ter high temperature treatment for 5, 10, and 15 days, al-though they differed in their responses to DHT and NHT [25].The downregulation of GBSS and the upregulation of pullu-lanase caused by high temperature treatment may lead toreduced amylose content and increased amylopectin content,respectively, in seed starches, thus resulting in poor tasteof edible rice endosperms. Furthermore, high temperaturesuppressed the accumulation of four cytosolic isoforms ofpyruvate orthophosphate dikinase B (PPDKB), and three iso-forms showed higher repression under DHT than NHT at5 and 10 DAF, which is consistent with the rice grown un-der DHT, NHT, and normal conditions displaying orderlydecreasing chalkiness in mature seeds [25]. These results,combined with the observation that mutation in one of thePPDKB isoforms caused the formation of endosperms witha white core [46], indicate that high temperature downregu-lates PPDKB, which in turn leads to seed chalkiness, thusultimately affecting the appearance quality of rice grain. Be-sides PPDKB, the functionally unknown protein (AU082974)with 74% GC content in DNA sequence, might also be relatedto chalkiness induced by high temperature, because this pro-tein was downregulated by high temperature and showedlower abundance in chalky parts than in translucent partsof rice seeds [26]. Moreover, Lur’s group revealed that onetype of storage protein, glutelin, was phosphorylated andglycosylated during seed development and another type ofstorage protein, prolamine, showed increased accumulation

under high temperature [26]. These observations provide newinsight into factors influencing the nutritional quality of ricegrains.

The two studies also identified a number of differentiallyexpressed stress-responsive proteins, most of which were up-regulated by high temperature treatment. Examples includedHSP 70, 3 low-molecular-weight HSPs, 2-Cys peroxiredoxin,two isoforms of 1-Cys peroxiredoxin, and Cu/Zn superoxidedismutase [25, 26]. Upregulation of these stress-responsiveproteins is consistent with their critical roles in protectingseed proteins, nucleic acids, or RNAs against thermal dam-age. However, several stress-responsive proteins were down-regulated or showed complex regulation under high temper-ature. For instance, DHT and NHT treatments decreased theexpression of two glyoxalases I isoforms at 5 and 10 DAFbut increased their expression at 15 and 20 DAF [25]. Ascor-bate peroxidases displayed lowered accumulation under bothNHT and DHT. The expression of thioredoxin H-type wassuppressed by DHT but enhanced by NHT. Thus, stresstolerance in rice seeds may be achieved by more complexmechanisms besides upregulation of a few stress-responsiveproteins.

In addition to the above two researches, Pages et al. de-signed a distinct strategy to study stress tolerance in riceseeds. They compared mature embryo proteomes amongthree varieties sensitive to stress (drought, salt, and cold) andthree tolerant to stress and identified 28 DEPs: 16 implicatedin the metabolism of reserve proteins or starches, six in stressdefense or tolerance, and six in other cellular processes [30].Among the 16 proteins involved in reserve metabolism, theglutelin type-B 2 precursor was much more abundant in theembryo proteome of the drought-tolerant variety than in thatof the drought-sensitive variety, suggesting this protein mightpossibly act as a protector against drought stress besides beinga type of storage protein. The six stress-defense proteins rep-resented four LEA groups, namely, group 1 (embryonic abun-dant protein 1), dehydrins (three isoforms of the water-stressinducible protein Rab21, with different isoelectric point),group 3 (a putative embryonic abundant protein similar toAtLEA3–1), and group 7 (a putative LEA similar to AtLEA7–1). The accumulation of the six LEA proteins was highly vari-able among the six rice varieties, not matching the expectedresults that stress-defense proteins should show consistentupregulation in the stress-tolerant varieties as compared withthe stress-sensitive ones. However, the three identified Rab21isoforms as well as the other four Rab21 isoforms visible onthe 2DE gels were less phosphorylated in the stress-tolerantvarieties than in the sensitive ones. In addition, the embryoproteomes of the three stress-tolerant varieties contained anunphosphorylated Rad21 isoform with an approximate iso-electric point (pI) of 6.8, which was invisible on the 2DE gelsbut detected by Western blot analysis. These results clearly in-dicate that posttranslational modification such as phosphory-lation, rather than regulation of gene expression at the trans-lational level, may contribute more to stress tolerance in riceseeds.

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4.3 Proteomic studies of rice seed mutants

Comparison of proteome between WT and genetic mutantsis an alternative and powerful strategy for screening proteinsaffected by gene mutations. Komatsu et al. applied 2DE toanalyze differences in seed proteomes between the WT andrice dwarf1 (d1) mutants, which showed multifaceted phe-notypes (including dwarfism, small and round seeds, anddark green leaves) caused by mutations in the � subunit of aheterotrimeric G protein [27]. Compared to WT, the d1 mu-tants exhibited no significant differences in the endospermproteome. However, seven embryo proteins, namely 1 ricereceptor for activated C-kinase (RACK) and 6 rice embryoglobulin-2s (REG2s), showed reduced expression. In the WT,RACK and the 6 REG2 proteins were accumulated during em-bryo development and maturation. RACK initiated its degra-dation from the early stage of seed maturation and REG2sbegan to degrade until seed germination. Reduction in levelof RACK and REG2s in the d1 mutant occurred throughoutseed maturation and even during seed germination. Treat-ment with abscisic acid during seed germination increasedthe protein level of RACK in WT rather than the d1 mutant.These results show the linkage between the G-protein sub-unit and its mutation-affected proteins (RACK and REG2s)and suggest that these proteins are involved in embryo de-velopment. This study demonstrates that through comparingseed proteomes between WT and mutants defective in seedphenotypes, comparative proteomics can become an effec-tive approach for identification of new proteins associatedwith certain molecular pathways in seed development.

4.4 Subproteomic studies of mature and developing

rice seeds

Subproteomics focuses on investigating proteomes from acertain tissue or organelle (spatial subproteome) or a sub-set of proteins with specific structural or functional prop-erties (functional subproteome) [47]. Subproteomic analysisinvolves specific fractionation and enrichment proceduresbefore or during protein extraction [47], thus enabling pro-teomic identification of low abundance but often biologicallyimportant proteins. Over the past decade, subproteomics hasbeen increasingly used to address biological questions in seeddevelopment; however, related studies of rice seeds are lim-ited because of the challenges in establishing respective andeffective purification and enrichment methods for differentsamples and applications, as well as the difficulties in re-moving the interference of highly abundant storage starchesand proteins. In this section, we review recent progress inrice seed subproteomics, including one study of nuclear pro-teins in developing seeds [35] and three studies of RBPs, onein mature dry seeds [36] and the other two in developingseeds [37, 38].

Genetic and molecular studies have shown that nuclearproteins such as OsCCS52A [48], Orysa; KRP3 [49], gw5 [50],

MEA/FIS1, FIS2, and FIE/FIS3 [51] play important rolesin endoreduplication, syncytium, cell division, and epige-netic regulation during rice embryo and endosperm develop-ment. Therefore, cataloguing nuclear proteins and establish-ing their posttranslational modification map in developingseeds are highly useful for elucidating mechanisms underly-ing the molecular regulation of seed development. A majorchallenge for nuclear subproteome study in rice seeds is todeplete highly abundant starches and storage proteins. Penget al. removed most starch granules and SSPs from nuclearprotein extracts of developing rice seeds simply by cotton fil-tration and gel excision and recovered the remaining proteinsby phenol extraction (detailed in Section 3.1) [35]. Throughthis nuclei purification, and nuclear protein enrichment andrecovery procedure, they identified 468 proteins, including220 nuclear-localized proteins, from the recovered nuclearfractions. In contrast, only 28 proteins, including 24 storageproteins, were identified from the total protein extracts ofrice seeds at the same developmental stage. The 220 nuclear-localized proteins contained not only chromosome structuralproteins (e.g. all the 24 histones and their variants) but alsolow-abundance nuclear proteins such as X1, X2, bZIP, andbHLH transcription factors. These nuclear proteins mightbe involved in various biological processes important forseed development such as DNA metabolism (29.4%), pro-tein metabolism (19.3%), transcription (19%), cell organiza-tion, and biogenesis (15.2%), cell death (0.5%), and cell cycle(0.5%). This nuclear proteome of developing rice seeds alsocontained 208 hypothetical proteins, among which 86 had nu-clear localization signals, which indicates a large number ofunknown knowledge about nuclear proteins involved in seeddevelopment. In addition, Peng et al. analyzed the MS/MSmass spectral data with Bioworks software to characterizearginine and lysine methylation and acetylation of these seednuclear proteins and found higher than expected frequency ofmodification. In all, 59 proteins contained acetylated aminoacids and 40 had methylated residues. Among them, two pro-teins (Brittle-1 protein and Glutelin type-A 3 precursor) wereboth acetylated and methylated. In this subproteomic study,Peng’s group provided the global characterization of nuclearproteins implicated in rice seed development and gave new in-formation about the posttranslational methylation and acety-lation modification of these seed nuclear proteins, thus offer-ing many new targets for genetic and molecular studies inseed development and its regulation.

RBPs are a large class of nucleic acid-binding proteinsthat have one or more RNA-binding domains such as thewidely spreading RNA recognition motif (RRM) and the K-homology (KH) domain [52]. Extensive studies in yeast andmetazoans have demonstrated that RBPs perform diversemolecular functions in all aspects of RNA metabolism, in-cluding RNA exportation from the nucleus to the cytoplasm,mRNA expression and regulation events that occur in thenucleus (e.g. transcription, splicing, and polyadenylation) orin the cytoplasm (e.g. localization, storage, translation, anddegradation) [52]. Due to the absence of plant derived in vitro

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experimental systems for studying RNA metabolism [52] aswell as the presence of numerous plant-specific RBPs lackingyeast and animal homologues, considerably less informationhas been accumulated on plant RBPs, especially those in-volved in seed development and maturation. However, thenucleic acid-binding activity of RBPs facilitates the purifica-tion and enrichment of RBPs from crude protein extracts,which led to the identification of 11 RBPs in mature riceseeds [36] and 257 RBPs in developing rice seeds [37–39]through proteomic approaches.

Using one-step affinity chromatography with denaturedssDNA cellulose column after extraction of total proteinsfrom mature and dry rice seeds, Kanekatsu et al. effectivelyremoved high-abundance storage proteins and obtained anssDNA-binding fraction [36]. Although the protein recoveryyield was low by use of this affinity purification method andonly 18 proteins were identified from the ssDNA-binding frac-tion after 2DE separation and MS analysis, 11 of the identifiedproteins were RBPs, including one glycine-rich protein, threeribosomal proteins, three proteins with RRM, and four pro-teins with KH domains. By comparing the quantity of the 18ssDNA-binding proteins in dry seeds and geminated seeds,the authors found that during seed germination, the threeRRM-containing RBPs, the glycine-rich RBP and the two non-RBPs with ssDNA-binding motifs showed greatly reducedexpression, whereas the three KH domain-containing RBPsshowed almost no change in expression. Inhibition of seedgermination by abscisic acid or cold treatment suppressed thedownregulated expression of the three RRM RBPs and theglycine-rich RBP rather than the two non-RBPs. Therefore,seed germination was accompanied by the degradation of theRRM-containing RBPs and the glycine-rich RBP, which sug-gests that these RBPs may stabilize long-lived mRNAs storedin mature dry rice seeds for rapid protein synthesis at thebeginning of seed germination.

In addition to the above investigation on mature seedRBPs, Okita’s group has conducted a large amount of re-search on RBPs implicated in RNA sorting within the cyto-plasm during rice seed development. Through genetic, cy-tological, and molecular approaches, the group found thatRBPs in developing rice seeds, such as OsTudor-SN [53, 54],can recognize and bind the cis-acting localization elements(or zipcodes) within the mRNA sequence of storage proteins,forming large macromolecular complexes. These protein-RNA complexes are transported from the nucleus to the cy-toplasm along the cell cytoskeleton and then are targeted andanchored properly to the cortical ER where storage proteinsare synthesized. These findings demonstrate RBPs partici-pate in regulating the biosynthesis of storage proteins duringseed development, by controlling multiple molecular eventsoccurring during mRNA transportation and localization tothe ER. However, considering that mRNA sorting is a multi-step process and the two major types of rice SSPs, namely,glutelins and prolamines, are localized to distinct ER subdo-mains, a large number of RBPs should be involved besidesthe few identified ones. To identify RBPs involved in mRNA

transportation and localization during seed development,Okita et al. first searched for proteins able to bind cytoskeletonas well as nucleic acids in developing rice seeds by combin-ing poly(U)-sepharose affinity purification with 2DE and MS,resulting in the identification of 148 putative cytosolic RBPs(see details in Section 3.1) [37]. The 148 proteins included theknown RBPs involved in storage protein RNA sorting, suchas OsTudor-SN, and 20 RNA-binding domain-containing pro-teins (i.e. nucleolar protein NOP5 and DEAD box RNA heli-case [also identified in the nuclear subproteomic study], KHdomain-containing protein LOC_Os02g57640 [also identifiedin the rice dry seed RBP study] and 17 newly identified pro-teins) that are the most probable RBP candidates for involve-ment in RNA metabolism. Other proteins were speculated toplay roles mainly in protein translation (38 proteins), proteinprocessing, turnover, and transport (23 proteins), cell struc-ture and energy production (15 proteins), signaling and stressresponse (16 proteins), carbohydrate or lipid metabolism(22 proteins) and other processes.

To further identify RBPs involved in transportation andlocalization of prolamine mRNAs, Okita et al utilized the zip-code sequence in the prolamine 5’CDS region to capture pro-teins bound with prolamine mRNAs from the cytoskeleton-enriched fractions of developing rice seeds [38]. A highlycharged polyanionic molecule, heparin, was added in all cap-ture experiments at a high concentration to prevent non-specific binding. Via this purification procedure, 18 dis-tinct proteins were identified after 1DE separation and LC–MS/MS analysis. Among them, 15 proteins, which includedthree members of the heterogeneous nuclear ribonucleopro-tein family (hnRNPs, NCBI accession number: AK105751,AK059225, AK100904), bound specifically to the prolaminezipcode sequence. The remaining three proteins had gen-eral RNA-binding activities, as shown by their associationwith both the 5’CDS zipcode sequence and the controlRNA sequence present in the nonzipcode region of the pro-lamine open reading frame. Further molecular analysis pro-vided strong evidence that one of the three hnRNPs, namelyAK105751, may be involved in prolamine and glutelin mRNAexportation from the nucleus and subsequent localization tothe ER, because this hnRNP (i) can bind with prolamineand glutelin mRNA in vivo, (ii) had a protein expressionpattern similar to that of prolamine and glutelin, and (iii)localized not only in the nucleus but also on the cister-nal ER membranes and cell cytoskeleton. Furthermore, be-cause of the observation that heparin inhibited the bindingof OsTudor-SN (the characterized prolamine and glutelinRBP) to its target RNAs, the prolamine zipcode capture as-says were also performed without heparin, which revealed132 putative prolamine RBPs [38, 39]. Among them, 77 pro-teins were newly identified RBP candidates, except for the12 proteins found in the stringent prolamine zipcode cap-ture experiment and the 43 proteins obtained by poly(U)-sepharose affinity purification [39]. In conclusion, Okita’sgroup obtained 148 cytoskeleton-associated RBPs and 138cytoskeleton-associated prolamine mRNA-binding proteins

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from developing rice seeds through isolation with affinitychromatography followed by identification with proteomicapproaches. They have documented these RBPs and a varietyof related information in the RiceRBP website (http://www.bioinformatics2. wsu. edu/RiceRBP), which provides an elab-orate and comprehensive database of RBPs accessible to thepublic [55].

5 Concluding remarks

In the past decade, proteomic studies have provided manynew and detailed insights into the mechanisms of metabolicand molecular control during embryo and endosperm devel-opment in rice, mostly via combination of protein fraction-ation, 2DE separation and MS-based identification. Globalcomparison of proteome changes among different seed de-velopmental stages results in revealing new aspects of theregulation mechanisms underlying seed development. Al-though genome-wide transcriptional analysis of developingrice seeds have demonstrated the coordinate regulation ofexpression of genes associated with seed growth and reserveaccumulation during seed development [56], there remainsignificant gaps in our understanding of this coordination. Atpresent, large-scale analysis of proteome changes associatedwith rice seed development show that: (i) endosperm growth,embryogenesis, and starch accumulation involve the coor-dination of various metabolic, molecular, and cellular pro-cesses, including the switch from central carbon metabolismto alcoholic fermentation, ROS-induced redox regulation ofPCD, diurnal light-dark control of starch accumulation andcell growth; (ii) the expression pattern of proteins involvedin specific metabolic, molecular, or cellular processes is rel-atively consistent during embryo and endosperm develop-ment, which is highly coordinated with the timing and se-quence of seed developmental events (Tables 2 and 3). Com-paring seed proteomes between stress-treated and -untreatedrice or between stress-tolerant and -sensitive rice varietiesprovides new information about the proteins and mecha-nisms related to the effects of environmental stimulus onrice quality and those related to stress response and toler-ance. Molecular studies have shown that the reduction of ricetaste quality by high temperature might result from modifi-cation of the starch structure by the expressional regulationof enzymes related to starch synthesis, such as GBSS [57].Proteomic studies further reveal that although these starchsynthesis-related enzymes may have several isoforms, somebut not all are involved in high temperature-induced reduc-tion of rice grain quality. For example, six isoforms of GBSSare present in the proteome of developing rice seeds, but onlythe high-molecular-weight isoforms of GBSS are downregu-lated by high temperature. Stress-responsive proteins such asHSPs and LEAs are known to play protective roles under en-vironmental stresses, and upregulation of their expression isan important mechanism for stress tolerance in plants [43].Comparison of embryo proteomes in stress-tolerant and -

sensitive rice varieties offers new and direct evidence thatstress tolerance in rice seeds can result from not only upreg-ulation but also posttranslational modification (e.g. phospho-rylation) of stress-responsive proteins. When 2DE is used asa tool to compare WT and mutant proteomes or to analyzespatial and functional subproteomes, proteomics can revealnovel proteins involved in certain molecular and/or cellularprocesses, which helps resolve complex biological questionsrelated to rice seed development. The typical example of pro-teomic comparison between WT and mutants is the analysisof differences between WT and d1 seed proteomes, which ledto the identification of seven new proteins (i.e. RACK and sixREG2s) involved in G-protein-mediated pathways during em-bryo development. The localization and functional domain ofa protein are closely related to its function. Identification andfunctional analysis of proteins with specific localization or do-mains can provide much information for understanding themolecular mechanisms involved in seed development. Com-pared with the low-efficient single-protein study by geneticand molecular approaches, proteomics provides a possiblestrategy for large-scale identification and analysis of proteinscontaining specific functional domains or proteins locatedin specific tissues, organelles, or regions of rice seeds. How-ever, these proteins are often accumulated at lower levels inrice seeds, causing them invisible in 2DE gels if crude pro-tein extracts are used. Innovative improvement in proteinfractionation and sample preparation has led to the isolationand identification of low-abundance seed proteins, including220 nuclear-localized proteins and 268 proteins containingRNA-binding domains, offering a large number of candidateproteins that may play specific and important roles in riceseed development.

In the near future, the proteomic study of rice seed de-velopment will become more exciting with the technologicaland knowledge advances. The extensive use of the advancedproteomic technologies such as DIGE and iTRAQ 2D-LC-MS/MS and the improvement in technologies themselveswill offer increased accuracy and sensitivity in protein identi-fication and quantification. Further improvement in proteinfractionation and purification techniques for subproteomicstudies will result in the large-scale identification of morelow abundance but biologically important proteins. MALDI-imaging MS, an emerging technique enabling MS imagingof biological molecules in plant tissue sections [58,59], can beused to analyze the spatial distribution patterns of metabo-lites and proteins in developing rice seeds. The application ofMALDI-imaging MS will lead to the identification of proteinmarkers expressed specifically in certain organs, cell types, orregions of rice seeds at certain developmental stages, as wellas provide new insights into the metabolic and regulatory net-works in seed development. Combining proteomic tools withgenetic, biochemical, cytological, and molecular approacheswill allow us to obtain biologically relevant answers regardingthe control mechanisms of embryo and endosperm develop-ment in rice. Benefiting from these advances, proteomicswill predictably improve and update our knowledge of

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mechanisms underlying rice seed development and its regu-lation, providing helpful information for molecular breedingand improvement of quality, yield, and stress tolerance in riceand other cereals.

This work was supported by grants from the National ScienceFoundation of China (grant no. 31000694) and the ChineseMinistry of Science and Technology (grant no. 2012CB910504and 2011CB100202).

The authors have declared no conflict of interest.

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