Physicochemical properties and development of wheat large and small starch granules during endosperm...

ORIGINAL PAPER

Physicochemical properties and development of wheat largeand small starch granules during endosperm development

Cunxu Wei • Jun Zhang • Yifang Chen • Weidong Zhou • Bin Xu •

Youping Wang • Jianmin Chen

Received: 15 December 2009 / Revised: 20 January 2010 / Accepted: 12 February 2010 / Published online: 3 March 2010

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2010

Abstract Wheat mature seeds have large, lenticular

A-type starch granules, and small, spherical B-type and

irregular C-type starch granules. During endosperm

development, large amyloplasts came from proplastid,

divided and increased in number through binary fission

from 4 to 12 days after flowering (DAF). Large starch

granules formed and developed in the large amyloplast.

One large amyloplast had only one large starch granule.

Small amyloplasts came from the protrusion of large

amyloplast envelope, divided and increased in number

through envelope protrusion after 12 DAF. B-type starch

granules formed and developed in small amyloplast from

12 to 18 DAF, C-type starch granules formed and devel-

oped in small amyloplast after 18 DAF. Many B- and

C-type starch granules might form and develop in one

small amyloplast. The amyloplast envelopes were asyn-

chronously degraded and starch granules released into cell

matrix when amyloplasts were full of starch granules.

Apparent amylose contents of large starch granules were

higher than that of small starch granules, and increased

with endosperm development. The swelling powers and

crystallinity of large starch granule were lower than that of

small starch granules, and decreased with endosperm

development. Small starch granules displayed broader

gelatinization temperature ranges than did large starch

granules.

Keywords Wheat � Endosperm development �Starch granule � Physicochemical property

Abbreviations

AAC Apparent amylose content

CLSM Confocal laser scanning microscopy

DAF Days after flowering

DAP Days after pollination

DSC Differential scanning calorimetry

LM Light microscopy

SEM Scanning electron microscopy

TEM Transmission electron microscopy

XRD X-ray powder diffraction

Introduction

Wheat (Triticum aestivum L.) grain is one of the major

cereals consumed by human being. Starch is an important

part of wheat endosperm, not only because starch accounts

for three-quarters of the dry weight of a wheat kernel (Hucl

and Chibbar 1996), but also wheat starch has unique

properties in breadmaking that are not replaceable by other

starches from corn (Zea mays L.), rice (Oryza sativa L.) or

oats (Avena sativa L.), or by noncereal starches (Sahlstrom

et al. 1998). Wheat starch granules have been reported to

have bimodal size distribution of large (A type) and small

(B type) starch granules (Evers 1973; Peng et al. 1999), and

trimodal size distributions of A-, B-, and C-type starch

Communicated by S. Weidner.

C. Wei (&) � J. Zhang � Y. Wang � J. Chen (&)

College of Bioscience and Biotechnology, Yangzhou University,

Yangzhou 225009, China

e-mail: [email protected]

J. Chen

e-mail: [email protected]

Y. Chen � W. Zhou � B. Xu

Center of Measurement, Yangzhou University,

Yangzhou 225009, China

123

Acta Physiol Plant (2010) 32:905–916

DOI 10.1007/s11738-010-0478-x

granule (Bechtel et al. 1990; Raeker et al. 1998; Bechtel

and Wilson 2003).

Formation of large starch granules initiates about 4–

5 days after pollination (DAP), about 4 days later the final

number of large starch granules is achieved, their diameter

size reaches up to 45 lm depending on wheat cultivar and

season (Briarty et al. 1979). Small starch granules are

reported to initiate during 12–16 DAP (Parker 1985), or

16–22 DAP (Briarty et al. 1979). In the history of starch

research in wheat, there have been some disagreements

about the origin of small starch granules. Badenhuizen

(1958) reported that small starch granules arose in mito-

chondria. Buttrose (1963) showed that small granules

formed in vesicles budded off from outgrowths of the

A-type granule-containing amyloplasts. Parker (1985)

observed the presence of narrow protrusions between

B-type granules and the parent amyloplasts using trans-

mission electron microscopy (TEM), but evidence for the

budding off of B-type amyloplasts was lacking. Langeveld

et al. (2000) reported that B-type granules were present in

the protrusions emanating from the A-type granule-

containing amyloplasts using TEM and confocal laser

scanning microscopy (CLSM), and the amyloplasts were

interconnected by these protrusions. The protrusions varied

in length from 2 to 30 lm and ranged in width from 0.5 to

1.5 lm, depending on the presence of B-granules. These

protrusions suggested the presence of a communication

system facilitating the coordination of plastid activities.

Bechtel and Wilson (2003) also reported that amyloplasts

in the endosperm of wheat apparently divided and

increased in number through protrusions; B- and C-type

starch granules formed and developed in the protrusions.

Besides the morphological, size, and origin differences,

wheat large and small starch granules have been reported to

possess different characteristics and properties with regard

to chemical composition (amylose, amylose–lipid com-

plex, and phosphorus contents) (Raeker et al. 1998; Shinde

et al. 2003; Geera et al. 2006), molecular structure

(Sahlstrom et al. 2003; Ao and Jane 2007), relative granule

crystallinity (Vermeylen et al. 2005; Ao and Jane 2007),

granule swelling (Van Hung and Morita 2005), gelatini-

zation properties (Sahlstrom et al. 2003; Vermeylen et al.

2005; Geera et al. 2006; Ao and Jane 2007), pasting

behavior (Sahlstrom et al. 2003; Shinde et al. 2003; Van

Hung and Morita 2005; Geera et al. 2006; Ao and Jane

2007), and reactivity to modifying agents (Van Hung and

Morita 2005). These differences result in the two starch

granule types being utilized differently, both in food and

nonfood uses. Starch with predominantly small starch

granules can be used as a fat substitute, a paper coating,

and a carrier material in cosmetics (Lindeboom et al.

2004), while starch with a high percentage of large

starch granules has applications in the manufacture of

biodegradable plastic film, carbonless copy paper, and

brewing beer (Lindeboom et al. 2004).

Wheat cultivars with predominantly large or small

starch granules would be very useful to the food and

nonfood industries, respectively. To develop these wheat

cultivars, the physiochemical properties and development

of starch granules during wheat kernel development must

be understood. Despite significant efforts to characterize

and differentiate various aspects of large and small starch

granules in wheat mature seed, very little information is

available on the physicochemical properties of starch

granules during endosperm development. In the present

study, we investigated the physicochemical properties and

development of wheat large and small starch granules

during endosperm development.

Materials and methods

Plant materials

Wheat (Triticum aestivum L.) cv. Yangmai 12 was

obtained from Agricultural College of Yangzhou Univer-

sity and grown in the university experimental field,

Yangzhou, Jiangsu, China, during the growing season in

2006 and 2007. Heads were tagged at flowering.

Transmission electron microscopy (TEM)

Kernels were harvested at 2, 4, 6, 8, 10, 12, 15, 18, 21, 24,

30 days after flowering (DAF) for TEM specimen prepa-

ration. Two types of specimens were prepared, one for the

conventional glutaraldehyde–osmium tetroxide (GA–

OsO4) fixation, and the other for the potassium perman-

ganate (KMnO4) fixation. For GA–OsO4 fixation, each of

the harvested grains was transversely cut with a razor blade

in the mid-region of wheat kernel and one or two 1.0- to

1.5-mm-thick slices, which were semicircular in shape,

were used. Each slice was further cut into a number of

tissue blocks of *1–2 mm2, each of which included the

peripheral and central regions of endosperm. Tissue blocks

were then immediately fixed by placing them in a fixative

containing 2.5% glutaraldehyde in 0.1 M phosphate buffer,

pH 7.2, for 2 h at room temperature, then overnight at 4�C.

After three changes of washing (15 min each) with the

phosphate buffer solution, the blocks were postfixed in 1%

OsO4 for 2.5 h at room temperature. The blocks were

washed, and dehydrated through an acetone series from 30

to 100%, and embedded in Spurr’s low-viscosity embed-

ding medium (Spurr 1969). For KMnO4 fixation, tissue

blocks were immediately immersed in a 1.2% KMnO4

solution containing 0.5% NaCl in barbital sodium buffer

for 4 h at 4�C. After KMnO4 fixation, samples were

906 Acta Physiol Plant (2010) 32:905–916

123

washed three times (20 min each) with a 0.5% NaCl

solution at 4�C, and then passed once through 35% ethanol.

Next, the samples were further fixed in 70% ethanol for

12 h at 4�C. After ethanol fixation, the samples were suc-

cessively dehydrated in 80, 90, and 100% ethanol and

substituted with propylene oxide twice at room tempera-

ture, and embedded in Spurr’s low-viscosity embedding

medium. Ultrathin sections in 70 nm thickness were cut

with a diamond knife on a Leica Ultrathin Microtome (EM

UC6, Germany), and post-stained with uranyl acetate and

lead citrate. The sections were visualized and photographed

with Philips Tecnai 12 TEM at 100 kV.

Light microscopy (LM)

The semithin sections in 1 lm thickness were cut with a

glass knife on a Leica Ultrathin Microtome (EM UC6,

Germany), and stained with periodic acid-schiff (PAS)

reagent. The images were captured with a digital camera

that was equipped on the Olympus BH2 light microscope

(Japan).

Isolation of starch granules

Wheat starch granules were isolated from developing

grains at 12, 18, 24, 30 and 40 (maturity) DAF using a

method described by Takeda et al. (1999). The grains were

steeped in 0.2% NaOH at 4�C for 2 days. The softened

seeds were degermed and ground in a mortar with pestle.

The homogenate was squeezed through four layers of

cheesecloth to remove endosperm cell debris, and then

filtered with 100-, 200-, 300-, and 400-mesh sieves, suc-

cessively. The starch was washed with 0.2% NaOH by

centrifugation at 3.500g for 10 min until no biuret reaction

occurred, and washed with water and acetone. Then it was

air-dried.

Separation of large and small starch granules

The wheat starch preparation containing both large and

small starch granules was separated into the two types of

granules by the centrifugation through sucrose as described

by Peng et al. (1999). A 5-mL starch suspension in distilled

water (0.1 g/mL) was laid on the top of 10 mL of 80%

(w/v) sucrose in a 15-mL glass tube and centrifuged at 10g

for 10 min. The supernatant which contained small starch

granules was removed to another tube. The starch pellet

was washed twice in distilled water, suspended in 5 mL of

distilled water, and centrifuged four times in fresh 80%

sucrose solution. The starch pellet constituted the large

starch granule population. The supernatants were pooled

and centrifuged at 3,500g for 5 min, and the resulting

starch pellet comprised the small starch granule population.

Finally, large and small starch granules were washed three

times in distilled water and once in acetone, dried at 40�C,

ground into powder, and then passed through a 100-mesh

sieve.

Scanning electron microscopy (SEM)

Two types of specimens were prepared, one for the whole

grain of mature kernel and the other for the isolated starch

granules. For the whole grain specimens, grains were

fractured in the mid-region of kernel with a razor blade by

applying a slight pressure on the top of the grain. During

fracturing, the efforts were made to produce no physical

contact between the razor blade and the fractured surface of

the internal endosperm tissues. Fractured grains, with the

fractured surface upward, were mounted on the specimen

stub and sputter coated with gold before viewing with a

SEM (XL30 ESEM, Philips, Holland) at 20 kV. For iso-

lated starch granule, starch samples were suspended in

acetone to obtain a 1% suspension. One drop of the starch–

acetone suspension was deposited on the specimen stub and

dried automatically. The samples were coated with gold

powder to avoid charging under the electron bean.

Apparent amylose content (AAC)

AACs were determined by the iodine binding method as

described by Chrastil (1987) with some modification. The

sample starch of 100 mg was suspended in the solution

containing 1 ml anhydrous ethanol and 9 ml 1 M NaOH

and heated in a water bath of 95�C for 30 min. The solution

(0.1 ml) was mixed with 0.5% trichloroacetic acid (5 ml)

to decrease pH of the mixture toward approx. 5.5, and

subsequently 0.01 M I2–KI aqueous solution (0.05 ml) was

added. After immediate mixing, color was allowed to

develop for 30 min before reading the absorbance at

620 nm. Reagent grade of potato amylose was used as a

standard substance for calibration. The experiments were

performed in triplicates.

Swelling power

Swelling powers of isolated starches were determined

according to the procedures as described by McCormick

et al. (1991). The starch–water slurries (0.04 g/mL) were

mixed with a vortex mixer for 10 s and placed in a shaking

water bath at 70�C for 4 min. The contents then underwent

a second mixing for 20 s and a further 6 min in the water

bath. The tubes were then transferred to a boiling water

bath for 10 min followed by 5 min in cold water and

centrifugation at 1,700g for 4 min. The supernatant was

removed carefully with suction and the tubes were

weighed. The sediment weight was calculated and swelling

Acta Physiol Plant (2010) 32:905–916 907

123

power was determined as sediment weight divided by dry

sample weight.

Differential scanning calorimetry (DSC)

A DSC (200-F3, METZSCH, Germany) was used to

examine the thermal properties of starch samples. Isolated

starch (5 mg) was mixed with 10 lL of distilled water and

hermetically sealed in aluminum pans overnight at 4�C.

After equilibrating for 1 h at room temperature, samples

were scanned at a heating rate of 10�C/min from 30 to

110�C. The major parameters of the DSC profile were

described as onset temperature (To), peak temperature (Tp),

final temperature (Tc), and the change of enthalpy during

gelatinization (DH).

X-ray powder diffraction (XRD)

An XRD (D8, Bruker, Germany) was used to examine the

X-ray diffraction patterns and crystalline property of iso-

lated starch. The diffractometer was operated at 200 mA

and 40 kV. Samples were scanned from 2h 4� to 40� with a

step size of 0.02� and a count time of 1 s. The crystalline

degree was calculated with Jade 5.0.

Statistical analysis

Starch granule sizes of isolated starch were analyzed from

the images of SEM images with JEDA-801D morpholog-

ical image analysis system (Jiangsu JEDA Science-Tech-

nology Development Co., Ltd, Nanjing, China). Over 200

starch granules were measured per sample. The numbers of

large and small starch granules were counted from the

semithin section stained with PAS, 20 starchy endosperm

cells at lateral part of kernel were analyzed per sample. The

mean and standard deviation (SD) were performed with

SPSS program version 15.0.

Results and discussion

Classification, morphology, and accumulation of starch

granules during wheat endosperm development

The transversely fractured surface of the mid-region in

mature seeds showed that endosperm cells contained large

starch granules and numerous small starch granules. Large

starch granules appeared to be smooth, devoid of high

levels of associated protein matrix, and surrounded by

many small starch granules. Many small starch granules

were tightly arranged around large starch granules. These

small starch granules were spherical, polygonal or irregular

shape. Small amounts of protein were associated with

adjacent small starch granules (Fig. 1a).

The classification of wheat starch has been debated. The

bimodal size distribution of wheat starch has been reported.

Large A-type starch granules are disc or lenticular in shape

with an average diameter of 10–35 lm and contribute

[70% of the total weight and &3% of the total granule

number of endosperm starch. On the other hand, small

B-type starch granules are roughly spherical or polygonal

in shape, ranging from 1 to 10 lm in diameter. Small

starch granules account for [90% of the total granule

number, but \30% of the total weight of starch in wheat

endosperm (Evers 1973). For trimodal size distribution,

Bechtel et al. (1990) reported the formation of very small

C-type granules (less than 5 lm) that were initiated very

late in grain filling. At maturity, large A-type granules with

Fig. 1 SEM images of the

transversely fractured mid-

region of mature kernel (a),

isolated starch granules (b),

large starch granules (c) and

small starch granules (d).

A A-type starch granule;

B B-type starch granule; C:

C-type starch granule; ArrowC-type starch granule with a

perpendicular ‘cutting’ section

to the long axis at the ends.

(scale bar 10 lm)


123

diameters greater than 15.9 lm, B-type granules with

equivalent diameters between 5.3 and 15.9 lm, and small

C-type granules with equivalent diameters less than

5.3 lm. The total number of starch granules comprised

45.7% C-type granules, 49.5% B-type, and 4.8% A-type.

The C-type granules constituted 3.4%, B-type 45.0%, and

A-type granules 51.6% of the total mass at maturity

(Bechtel et al. 1990). While Raeker et al. (1998) reported

small granules with diameters \2.8 lm, midsize granules

with diameters of 2.8–9.9 lm, and large granules with

diameters [9.9 lm. Volume% distribution of granules

within the three size classes had ranges of 9.7–15.2%

(small), 13.4–27.9% (medium), and 57.9–76.9% (large). In

this paper, the classification of bimodal size distribution of

wheat starch is adapted, the large starch granules refer to

the A-type granule of bimodal or trimodal size distribution

with diameter[10.0 lm, small starch granules refer to the

B-type granule of bimodal size distribution or B- and

C-type granules of trimodal size distribution with diameter

\10.0 lm. In addition, the small starch granules were

classified into B- and C-type granules, B-type granules

refer to small spherical starch, while C-type granules refer

to small polygonal or irregular starch (Fig. 1).

Developing starch granules at 12, 18, 24, 30, and 40

(maturity) DAF were isolated (Fig. 1b). SEM observation

showed that there were no apparent contaminations of large

starch granule fractions by small starch granules (Fig. 1c),

and that of small starch granule fractions by large starch

granule (Fig. 1d). The morphology of large and small

starch granules did not vary at different developing stages

(the SEM images of isolated developing starch granules

were omitted). Large starch granules were disc or lenticular

shape (Fig. 1c), small starch granules showed spherical

B-type granules and irregular-shaped (e.g., bell-like,

polygonal, angular, conical, horn-like) C-type granules

(Fig. 1d). Natively fractured surface of mature seed

showed that irregular-shaped small starch granules were

embedded in the protein matrix, with smaller sizes occu-

pying void spaces between and adjacent to A- and B-type

starch granules (Fig. 1a). The observation suggested that

the irregular-shaped small granules within isolated starches

were not artifacts of excessive mechanical treatment

associated with starch isolation.

An image analysis of purified large and small starch

granule fractions from developing wheat endosperm

showed that the size of large starch granule increased

before 18 DAF, and kept stable after 18 DAF. The size of

small starch granule did not also show significantly vari-

able after 18 DAF (Fig. 2a).

Semithin section from developing endosperm tissues

fixed with GA–OsO4 fixative showed that some large

starch granules were distributed around cell wall at 8 DAF

(Fig. 3a). The size of starch granule increased quickly from

8 to 10 DAF (Fig. 3b). At 12 DAF, the quantity of large

starch granules increased and small starch granule began to

appear (Figs. 2b, 3c). After 12 DAF, the quantity of large

starch granules did not increase, but the quantity of small

starch granules obviously increased with endosperm

developing (Figs. 2b, 3d, e). At 24 DAF, the endosperm

cells were almost filled with large starch granules and small

starch granules, the quantity of the small starch granules

was high (Figs. 2b, 3f)

Amyloplast formation and starch granule development

The development of wheat endosperm includes four stages:

syncytial, cellularization, growth and differentiation, and

maturation (Olsen 2004). Parker (1985) observed that

B-type granules formed within protrusions from A-type

amyloplast, but the protrusions were observed only at one

stage of development. Langeveld et al. (2000) observed the

extensive protrusions and interconnections between amy-

loplasts from wheat subaleurone cells only at early endo-

sperm developing stage using bombardment labeling and

confocal laser scanning microscopy; however, bombard-

ment labeling was not successful after 13 DAF. Bechtel

Fig. 2 Dynamic changes of size and number of starch granule during

endosperm development


123

and Wilson (2003) observed the amyloplast formation and

starch granule development in wheat, and thought all am-

yloplasts in the endosperm divided and increased in num-

ber through protrusions. These results were mainly focused

on early developing endosperm, and samples for TEM

were prepared with the conventional GA–OsO4 fixation

(Parker 1985; Langeveld et al. 2000; Bechtel and Wilson

2003). However, it was difficult to observe the amyloplast

envelope and the formation and development of small

starch granule with GA–OsO4 fixation after 12 DAF

(Parker 1985; Langeveld et al. 2000; Bechtel and Wilson,

2003; Fig. 4). Potassium permanganate is a better fixative

for membrane lipids, though it is poor for protein and

polysaccharide. In addition, its osmotic ability is very high

to cell membrane. Thus, it can rapidly fix the membrane

structure within 1–2 min duration and increase the contrast

of membrane structure. Compared with conventional fixa-

tion, potassium permanganate is a better method for

membrane structure (Luft 1956). The envelope of wheat

endosperm amyloplast was clearly observed with

potassium permanganate fixation in the present study

(Figs. 5, 6).

Previous reports and our data strongly suggested that

large amyloplast came from the proplastid at syncytial and

cellularization stages when the endosperm cells were

dividing. Only one large A-type starch granule formed and

developed in this kind of amyloplast (Fig. 4a). When there

were two starch granules formed in large amyloplast, large

amyloplast divided and increased in number through binary

fission (Figs. 4b, c, 5a) (Briarty et al. 1979). Starch granule

in large amyloplast was quickly developed and increased in

volume to become large A-type starch granule (Fig. 5b).

Large A-type starch granules were a prominent feature of

large amyloplast. The granules were elongated and pos-

sessed a cleft at each end (equatorial groove). A network of

small tubular cristae was located in the groove of the starch

granule (Fig. 4d). The envelope of amyloplast was clear

two-layer membrane; the space between amyloplast enve-

lopes was narrower than that of ER (Fig. 5c). After

12 DAF, the envelope of large amyloplast with large starch

Fig. 3 LM images of the

transversely section mid-region

of developing kernel at 8 DAF

(a), 10 DAF (b), 12 DAF (c),

15 DAF (d), 18DAF (e), and

24 DAF (f). (scale bar 10 lm)


123

granule began to protrude into cell matrix (Fig. 4e). Small

B-type starch granules were observed around large starch

granules (Fig. 4f). The plastids exhibited tubular protru-

sions that extended a considerable distance through the

cytoplasm, some of the protrusions contained small starch

granules (incipient B-type starch granules). Small amy-

loplasts divided and increased in number through protru-

sion because binary fission of plastid division was never

observed (Fig. 5d–g). Double membranes of small amy-

loplast were clearly observed (Fig. 5h). More than one

spherical B-type starch granules formed and developed in

one small amyloplast (Fig. 5g–i). After 18 DAF, some

smaller irregular and polygonal C-type starch granules

appeared, and were clustered together in small amyloplast

(Figs. 4g, h, 6). Some C-type starch granules formed and

developed between large starch granule and the envelope

of large amyloplast (Fig. 6c, d). Some small C-type starch

granule showed the ‘cutting’ shape perpendicular to the

long axis (Fig. 6e–g). At 24 DAF, A-, B-, and C-type

starch granules were clearly observed in the endosperm

cells (Fig. 4i).

Bechtel and Wilson (2003) observed the presence of

plastid protrusion in coenocytic cytoplasm, and thought

that amyloplast division in wheat endosperm tissue only

occurred through protrusions. The subsequent breakup of

the protrusions into individual amyloplasts might represent

a unique form of plastid division in wheat endosperm, and

would be different from the more common type that

involved binary fission. Plastid division and expansion

were mutually independent processes (Kuroiwa et al.

1999); the protrusions might represent a form of inter-

plastid communication required for the initiation of the

Fig. 4 TEM images of developing endosperm cells fixed with GA–

OsO4 fixative. a Endosperm cell at 6 DAF, showing large amyloplast

with A-type starch granules (A), vacuole (V), and mitochondrion (M).

b and c Endosperm cell at 8 DAF, showing two A-type starch

granules in one large amyloplast and large amyloplast divided

through binary fission. d Endosperm cell at 10 DAF, showing large

starch granule in large amyloplast. e Endosperm cell at 12 DAF,

showing the protrusion of the large amyloplast. f Endosperm cell at

15 DAF, showing the appearance of B-type small starch granules (B)

around large starch granules. g and h B-type and C-type starch

granules (C) in endosperm cell at 18 and 21 DAF, showing more than

one C-type granules in one small amyloplast (arrows). i Physiological

mature endosperm cell at 24 DAF, showing the distribution of

A-type, B-type, and C-type starch granules. (scale bar 1 lm)


123

B-type and C-type starch granules (Kohler et al. 1997). The

protrusions may also provide for a different environment

for starch granule initiation and growth. These results may

explain why B-type and C-type starch granules lack

equatorial grooves, and may explain why B-type and

C-type granules have a different composition than A-type

starch. Protrusions may also be the only avenue for division

of wheat amyloplasts because binary fission could not be

completed with a large starch granule present in the plastid

stroma.

Amyloplast fulfilled its function when it was full of

starch granules. When the envelope of amyloplast began to

degrade and disappear, starch granules were released to cell

matrix (Fig. 6e–g). For all amyloplasts, the envelope of

amyloplast was asynchronously degraded. The degradation

of envelope was also asynchronous for one amyloplast.

Some regions of envelope were degraded, and the others

were intact. The envelope of amyloplast did not exist after

30 DAF. There were only starch granules in endosperm.

Many double membrane structures were observed around

starch granules (Fig. 6g, h). The double membrane struc-

ture was similar to the envelope of amyloplast in mor-

phology and size (Figs. 5c, h, 6h). Thus, they were

speculated from degraded envelope of amyloplast.

Physicochemical properties of starch granule in

developing endosperm

AACs of large and small starch granules at 18, 24, 30, and

40 DAF were investigated (Table 1). The amylose content

in large starch granules was considerably higher than that

in small starch granules. Peng et al. (1999) isolated A- and

B-type starch granules from six mature wheat seeds, and

also found that all of the A-type starch granules contained

Fig. 5 TEM images of developing endosperm cells fixed with

KMnO4 fixative. a Endosperm cell at 6 DAF, showing the binary

fission of large amyloplast. b Endosperm cell at 10 DAF, showing

large starch granule (A) in large amyloplast. c Endosperm cell at

10 DAF, showing the two-layer membrane of amyloplast and

endoplasmic reticulum (ER). d Endosperm cell at 12 DAF, showing

many small amyloplasts with B-type starch granules (B) in cytoplasm.

e Endosperm cell at 12 DAF, showing amyloplast protrusion.

f Endosperm cell at 15 DAF, showing the small starch granules

around large starch granules. g Amplification of the square region in

Fig. 5f, showing the many small starch granules in one amyloplast.

h and i Endosperm cell at 15 DAF, showing many small B-type starch

granules in one small amyloplast. (scale bar 1 lm)


123

higher amylose concentrations (30–36%) than did B-type

starch granules (24–28%). Furthermore, the amylose con-

centration in large starch granules increased over the

developmental stage. Though the amylose levels of small

starch granules increased from 24 to 40 DAF, the value for

amylose content in small starch granule was higher at

18 DAF than at 24 and 30 DAF (Table 1). A possible

explanation might be that these starch granules contained

some developing starch granules at 18 DAF. An increase in

amylose content during the early stages of development

has also been reported for the barley (McDonald et al.

1991).

Swelling powers of large and small starch granules at

different developmental stages are shown in Table 1. The

swelling power of large starch granule was significantly

lower than that of small starch granule, and the swelling

powders of small starch granules significantly decreased

with endosperm development, especially from 30 to

40 DAF (Table 1). Chiotelli and Meste (2002) reported

that B-type starch granules were associated with a higher

rate of water absorption, earlier hydration and more

swelling than were A-type granules. The reason for this is

the less crystallized arrangement of the polysaccharide

chains in B-type granules (a higher proportion of

Fig. 6 TEM images of

developing endosperm cells

fixed with KMnO4 fixative.

a Endosperm cell at 18 DAF,

showing many starch granules

in one small amyloplast.

b Endosperm cell at 18 DAF,

showing one small amyloplast

full of two small starch

granules. c Endosperm cell at

21 DAF, showing large and

small starch granules.

d Amplification of the square

region in Fig. 6c, showing the

C-type small starch granule (C)

formation between large starch

granule and large amyloplast

envelope. e and f Endosperm

cell at 24 DAF, showing the

disruption of amyloplast

envelope. g Endosperm cell at

30 DAF, showing many

membrane structures among

small starch granules.

h Amplification of the square

region in Fig. 6g, showing the

two-layer membrane structure.

[scale bar 1 lm a–g, scalebar 0.1 lm (h)]


123

amorphous zones more accessible to water). Greater spe-

cific surface area may also contribute to the higher water

absorption of B-type granules.

Large and small starch granules showed a typical A-type

XRD pattern, with strong reflections at 2h about 15� and

23�, and an unresolved doublet at 17�, 18� 2h (Fig. 7). The

crystallinity of starch granules decreased with develop-

ment, and all the small starch granules possessed greater

crystallinity (19.3–21.1%) than did large starch granules

(17.9–20.5%) (Table 1). The difference in percentage of

crystallinity between large and small starch granules was

attributed to the amylose content of starch. Amylose in the

starch granules is amorphous, small starch granule pos-

sessed low amylose, thus displays high percentage of

crystallinity than did large starch granule. The amylose

content increased with developing, so the crystallinity

decreased.

The gelatinization properties of starch are related to a

variety of factors including the size, proportion and kind of

crystalline organization, and ultrastructure of the starch

granules (Lindeboom et al. 2004). The gelatinization

properties of starch granules are usually measured by DSC.

Gelatinization properties of wheat large and small starch

granules are shown in Table 2. The small starch granules

had broader ranges of gelatinization temperatures (Tc - To)

than did the large starch granules during endosperm

development. No significant difference was found between

the gelatinization temperature and enthalpy changes of the

large and small granule starches (Table 2). These results

were consistent with the observations of Ao and Jane

(2007) and Vermeylen et al. (2005). There was no signif-

icant difference in the gelatinization properties of large and

small starch granules at different development stage, but

the endothermic DH values were decreased with endo-

sperm development (Table 2).

In conclusion, wheat mature seeds have large, lenticular

A-type starch granule, and small, spherical B-type and

irregular C-type starch granules. Wheat large and small

starch granules have significantly different chemical com-

positions and functional properties, and are utilized differ-

ently, both for food and nonfood uses. A wheat cultivar,

Yangmai 12, was used to investigate the development and

physicochemical properties of large and small starch gran-

ules during endosperm development. Large amyloplasts

came from proplastid, divided and increased in number

through binary fission from 4 to 12 DAF. Large starch

granules formed and developed in the large amyloplast. One

large amyloplast had only one large starch granule. Small

amyloplasts came from the protrusion of large amyloplast

envelope, divided and increased in number through enve-

lope protrusion after 12 DAF. Small spherical B-type starch

granules formed and developed in small amyloplast from 12

to 18 DAF, irregular C-type starch granules formed and

developed in small amyloplast after 18 DAF. Many B- and

C-type starch granules might form and develop in one small

amyloplast. The amyloplast envelopes were degraded and

starch granules released into cell matrix when amyloplasts

were full of starch granules. AACs of large starch granules

were higher than that of small starch granules, and increased

Table 1 Amylose contents, swelling powers, and crystallinities of large and small starch granules

Physicochemical properties 18 DAF 24 DAF 30 DAF 40 DAF

Apparent amylose content (%)

Large starch granule 30.3 ± 1.4 31.1 ± 0.78 32.9 ± 0.2 33.2 ± 0.1

Small starch granule 27.9 ± 0.38 24.1 ± 0.38 27.5 ± 0.1 28.5 ± 0.3

Swelling power (g/g)

Large starch granule 14.97 ± 1.38 14.35 ± 1.22 13.90 ± 0.54 13.20 ± 0.77

Small starch granule 24.87 ± 0.50 24.62 ± 2.49 23.57 ± 3.22 16.76 ± 1.80

Relative crystallinity (%)

Large starch granule 20.5 19.2 18.1 17.9

Small starch granule 21.1 21.8 20.2 19.3

Fig. 7 X-ray diffraction profiles of large and small starch granules


123

with endosperm development. The swelling powers of

small starch granules were higher than that of large starch

granules, and decreased with endosperm development.

Small starch granules displayed broader gelatinization

temperature ranges than did large starch granules, but the

gelatinization enthalpy changes showed no significant dif-

ference during development. Despite similar A-type X-ray

patterns, the crystallinity of large starch granule was lower

than that of small starch granules, and decreased with

endosperm development.

Acknowledgments We are grateful to Prof. Dr. Rugen Xu at

Agricultural College, Yangzhou University, Yangzhou, China, for

providing wheat materials for this study. This study was financially

supported by grants from the National Natural Science Foundation of

China (30300215), the Natural Science Foundation of Jiangsu

(BK2009186), the China Postdoctoral Science Foundation

(20090451252), the Natural Science Foundation of the Jiangsu Higher

Education Institutions, and Jiangsu Key Laboratory Program of Plant

Functional Genomics.

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18 DAF

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Small starch granule 59.1 ± 0.1 67.0 ± 0.2 70.0 ± 0.4 10.9 35.8 ± 2.2

24 DAF



30 DAF



40 DAF




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