Apoplasmic assimilates and grain growth of contrasting rice cultivars differing in grain dry mass...

ORIGINAL PAPER

Apoplasmic assimilates and grain growth of contrasting ricecultivars differing in grain dry mass and size

Sandhya Rani Kuanar • Rashmi Panigrahi •

Ekamber Kariali • Pravat Kumar Mohapatra

Received: 17 July 2009 / Accepted: 12 February 2010 / Published online: 2 March 2010

� Springer Science+Business Media B.V. 2010

Abstract Apical dominance in assimilate filling impacts

grain growth in basal spikelets of rice panicle. In this study,

organic materials of the pericarp, apoplasmic space and

endosperm of the apical and basal caryopses, and photo-

synthesis of the flag leaf were measured during early part

of grain development in three types of rice cultivars with

similar phenology, but difference in grain weight and size

in the dry and wet seasons of 2006 and 2007, respectively.

Photosynthetic activity of the flag leaf was consistently low

in small-seeded cultivars. Rates of grain filling and cell

division of endosperm and concentration of assimilates,

starch, proteins and chlorophylls of the caryopsis were

lower, but spikelet ethylene production and peroxidase

activity were higher in a small-seeded cultivar compared to

a big-seeded cultivar. Similar disparities in grain filling and

other attributes were noticed for the inferior basal spikelets

of the panicle compared to the superior apical spikelets,

except the assimilate concentration of the pericarp and

endosperm. Temporal fluctuation in assimilate concentra-

tion of the organs were similar between the cultivars.

Concentration of apoplasmic assimilates mostly exhibited

negative correlation with that of pericarp and endosperm.

Compared to the apical spikelets, correlation was more

negative for the basal spikelets. Conversely, correlation

was positive between the concentration of apoplasmic

assimilates and endosperm cell number and grain weight

of the cultivars. Ethylene released from the spikelets

at anthesis affected growth and cell division rates of

endosperm and enhanced protein and chlorophyll degra-

dation and peroxidase activity of the caryopsis. It was

concluded that variation in spikelet ethylene production

may be responsible for differences in size or weight of

grains among rice cultivars and spikelets at different

locations of the panicle. The concentration of apoplasmic

assimilates could be an indicator for grain filling capacity,

and ethylene regulated the concentration by affecting

pericarp activity for assimilate unloading.

Keywords Rice � Assimilates � Spikelets � Ethylene �Apoplasmic space

Introduction

Seed number and weight are important biological and

agronomical features, which give a measure of repro-

ductive success/grain yield of a plant in an environment.

Theoretically, grain yield is a function of average seed

weight summed over the total number of seeds. In rice,

grain weight is considered a stable varietal character

(Yoshida 1972). But, strong apical dominance results in

considerable variation of single rice grains within a pan-

icle; the poorly or partially filled basal grains become

investment loss on account of poor quality and low

market value (Venkateswarlu et al. 1986a, b; Mohapatra

et al. 1993). The ultimate size or weight of the mature

grain in rice is a function of the rate and duration of grain

growth; both these components vary because of differ-

ences in spatial and temporal location within the panicle

(Xu and Vergara 1986) and genotype (Yoshida 1981).

Inferior grains receive low priority in partitioning of

assimilates within the panicle and such discrimination

limits endosperm growth and grain size (Sikder and Das

S. R. Kuanar � R. Panigrahi � E. Kariali � P. K. Mohapatra (&)

School of Life Science, Sambalpur University, Jyoti Vihar,

Sambalpur 768019, India

e-mail: [email protected]

123

Plant Growth Regul (2010) 61:135–151

DOI 10.1007/s10725-010-9459-8

Gupta 1976; Mohapatra et al. 1993; Peng et al. 1994).

During seed filling, assimilates are mostly supplied as

soluble sugars to the developing seeds (Thorne 1985).

They are symplastically transported from the phloem

terminal of the seed coat to the maternal tissue prior to

their entry into the free space of embryonic apoplast and

ultimate storage in the endosperm (Oparka and Gates

1981a, b; Partick and Offler 1995; Olsen et al. 1999). It is

believed that transfer of solutes through this pathway

occurs by an osmotically driven pressure gradient between

the source leaf and the developing seed (Taiz and Zeiger

2002). In developing rice seeds, sucrose is the major

phloem solute and its supply is dependent on the mem-

brane bound sucrose efflux and influx carriers (Lim et al.

2006). Therefore, unloading from the maternal tissue of

the seed coat and concentration of sugars in the embry-

onic free space (apoplasmic space) confers an important

attribute for grain filling. The solutes of the apoplasmic

solution are involved in the regulation of turgor motivated

sugar unloading from the seed coat as well as uptake of

solutes by the sink tissue (Partick and Offler 1995; Lim

et al. 2006). Thus, slackening of grain filling in an inferior

grain may be consequential to the poor physiological

activity of seed coat (Ishimaru et al. 2003) as well as low

concentration of solutes in the embryonic fluid (Partick

and Offler 1995; Weber et al. 1997). The apoplasmic

solutions of developing seeds of cereals or legumes, have

been obtained by empty ovule technique or perfusion

methods so long (Thorne 1985; Wang and Fisher 1994;

Partick and Offler 1995). These techniques could not be

tested on developing rice kernels, because smallness of the

endosperm cavity existing between nucellar projection and

aleurone layer (Hoshikawa 1984; Matsuo and Hoshikawa

1993) precluded scope for surgical manipulations. In the

present experiment, we measured the carbohydrate con-

centration of the pericarp (caryopsis coat—De Datta

1981) as well as apoplasmic space fluid surrounding the

embryo and endosperm in three categories of indica rice

seeds differing in weight and size during the period of

development by using an equilibration procedure through

matching the apoplasmic concentration of sugars with that

of known concentration of the material in the external

solution. This technique is similar to that used by Gifford

and Thorne (1985). In the process, we intended to deter-

mine the genetic as well as positional variability in con-

centration of solutes in apoplasmic fluids on seed yield

between different rice cultivars in relation to partitioning

of assimilates between the source and sink organs. In our

previous work, ethylene was identified as an inhibitor for

the process of dry matter partitioning (Mohapatra et al.

2000, 2009; Mohapatra and Mohapatra 2005). In the

present endeavour, we have tried to elaborate this concept

further.

Materials and methods

Plant material and culture

Three indica rice (Oryza sativa L.) cultivars, differing in

grain size and weight, Jogesh (large), Udayagiri (medium)

and Sidhanta (small), were grown in pots (330 9

330 9 260 mm) placed in the farmyard of the School of

Life Science, Sambalpur University, India during the dry

season of 2006. The cultivars belonged to early duration

group with similar maturity duration. The soil of the pots

was a sandy clay loam. The soil contained sand 67. 4%, silt

9.3% and clay 23.3%. The initial N, P and K composition

was 10.00, 10.04 and 87.12 kg ha-1, respectively and pH

was 6.5. The pots were arranged in three replicates, each

replicate containing 18 pots and identical numbers of pots

selected at random in each replicate were used for culti-

vation of the three cultivars. Four seedlings were trans-

planted to each pot 30 days after growth in a nursery bed.

Plants were grown at a density of 36 plants/m2. Commer-

cial N, P and K were applied at a ratio of 82.8: 17.46:

33.6 kg ha-1 (1 ha = 10,000 kg soil) in split doses. Half

of each fertiliser was applied to the plants at the time of

transplantation and one quarter each at tillering and

anthesis. The water level in the pots was 5 ± 2 cm at the

surface of soil. The plants were free from any physico-

chemical or biological stresses. A similar experiment was

conducted during the wet season of 2007. Three cultivars

differing in grain size and weight, such as, Ramachandi

(large), Mahanadi (medium) and Ganjeikali (small) were

used in the experiment. The cultivars belonged to the late

duration group with similar maturity duration. The mete-

orological observations during the period of plantation are

given in Fig. 1.

Harvesting

For each cultivar, at least 30 plants were screened for

uniform development by leaf count and height measure-

ment. On each occasion of sampling, three plants were

uprooted carefully from each cultivar and brought to the

laboratory in moist condition inside a thermo-cool box

within 2 min for biochemical studies of the developing

caryopses of spikelets from different parts of the panicle.

The spikelets of the two uppermost primary branches of the

panicle, which reached anthesis on the first day, were

separated from the plant. These spikelets were called the

‘apical’ spikelets. Anthesis was delayed for 5 days in many

spikelets of the basal primary branches; these were the

‘basal’ spikelets. The external appendages of the spikelet

like lemma and palea were removed to isolate the cary-

opsis. The caryopsis was dissected carefully to obtain the

pericarp (caryopsis coat) and endosperm. The sampling

136 Plant Growth Regul (2010) 61:135–151

123

commenced a few days past anthesis and continued at

3 days intervals up to the mid grain filling stage. A final

harvest was done at maturity for obtaining the grain yield.

Endosperm cell number and cell division rate

The caryopsis was fixed in Carnoys solution (absolute

ethanol: glacial acetic acid: chloroform; 9:3:1 v/v/v) for

48 h and then kept in 70%(v/v) aqueous ethanol till cell

number counting. The method for isolation and counting of

the endosperm cells were carried out according to Liang

et al. (2001). The caryopsis was dehulled and transferred to

distilled water in a stepwise fashion from the 70% aqueous

ethanol medium. In each step, the percentage of ethanol

decreased in the medium. The caryopsis was kept in dis-

tilled water for 5–7 h prior to dissection of the endosperm

under a dissecting microscope. The dissected endosperm

was dyed with haematoxylin solution for 24 h, washed

several times with distilled water and then hydrolyzed in

0.1%(w/v) cellulase (Sigma chemicals) solutions (pH 5.0)

at 40�C for 4–6 h and shaked in rotary shaker in average

speed. The isolated endosperm cells were diluted to 2–

10 ml according to the development stage of the endo-

sperm from which 20 ll for each sub-sample was taken to

a R.B.C. counting chamber. The total endosperm cell

numbers were calculated using the following equation.

Cell number=sub sample

¼ Number of endosperm cells� dilution

Area counted � depth of fluid

Cell number=endosperm

¼ Cell no: of sub sample� total volume of sample

Volume of each sub sample

The processes of division of endosperm cells and grain

filling were fitted by the beta growth equation (Yin et al.

2003) for calculation of the absolute growth rates.

W ¼ Wmax 1þ te � t

te � tm

� �t

te

� � tete�tm

with 0� tm� te

Endosperm cell division rate or grain filling rate

(difference in cell number or weight divided by

difference in time) was calculated as the derivative of

above equation

dw

dt¼ cm

te � t

te � tm

� �t

tm

� � tmte�tm

where wmax is the maximum value of w (grain weight or

cell number) which is reached at time te; tm, the time at

which the maximum growth rate is obtained; te, the time at

the end of the growth and cm, the maximum growth rate

which is given by

cm ¼2te � tm

te te � tmð Þtm

te

� � tmte�tm

Wmax

Flag leaf photosynthetic rate and efficiency

Photosynthetic rate of the flag leaf of the main-shoot (Pn) lmol CO2 m-2 s-1 was measured using a PP systems

CIRAS-2 gas analyzer with a clip-on leaf cuvette. All

measurements were made during morning hour (8–10 a.m.)

at ambient air conditions, like C02 concentration within

350–380 ll l-1, air temp. 26–30�C and PAR ranged from

1,000 to 1,400 l mol quanta m-2 s-1 at the ground of the

field to the top of the plants. The Fv/Fm value indicating

the quantum efficiency of PS II system of the flag leaf was

measured with a Handy PEA (Hansatech, UK).

Chemical analyses

The total chlorophyll concentration of the flag leaf and

pericarp was determined according to the method of Arnon

(1949). Fresh plant materials were homogenized with 80%

cold aqueous acetone and the slurry was centrifuged several

times by re-suspending the precipitate with extra acetone.

The pericarp and endosperm of developing caryopsis was

assayed for carbohydrates, proteins and amino acids. For

soluble carbohydrates, the plant material was immersed into

Fig. 1 The meteorological observations during the period of exper-

iments in 2006 (dry season) and 2007 (wet season)

Plant Growth Regul (2010) 61:135–151 137

123

3 ml of boiling 80% aqueous methanol for 5 min. The

extract was removed to a 10 ml volumetric flask. The res-

idue was boiled for a second time with 50% aqueous

methanol and both the extracts were pooled. The volume of

the extract was made up to the mark with distilled water.

Aliquot of the extract was used for estimation of soluble

carbohydrates (Buysee and Merck 1993). The free amino

acids were estimated according to the method of Yemm and

Cocking (1955). The residue after methanolic extraction

was digested with 3% HCl and used for estimation of starch

(Buysee and Merck 1993). For total protein estimation,

fresh plant material was homogenized and centrifuged

with10% TCA. The precipitate was dissolved in 1 N NaOH

for the estimation of proteins (Lowry et al. 1951).

Ethylene estimation was carried out after collecting the

spikelets from the panicle and sealing them airtight inside

a 5 ml vial (Naik and Mohapatra 2000). The vial was

incubated in darkness for 2 h at room temperature. Head-

space gas (1 ml) was drawn into an airtight syringe and

injected into gas chromatograph (Hewlett-Packard Com-

pany model 6890, Palo Alto, USA). The micro-capillary

column was 30 m long and internal diameter was

0.53 mm. It was packed with cross-linked methylesilox-

ane. N2 was the carrier gas; H2 and O2 were used for the

flame ionization detector. The detector and injector tem-

peratures were 150 and 100�C, respectively.

For estimation of soluble carbohydrates in the apoplasmic

space, the pericarp was removed very carefully from each

caryopsis with forceps, and the pericarp as well as the

enclosed endosperm/embryo combines were dipped in

0.2 ml solutions of different milli-molar sucrose concen-

trations ranging from 0.5 to 5.0 mM in small tubes for 5 s.

The solution in the tube was mixed properly by shaking.

Then 0.1 ml of the solution was taken from each test tube for

estimation of soluble carbohydrates (Buysee and Merck

1993). Another set of sucrose solutions like the previous one

was prepared without the addition of the seed components,

which served as a control. Percentage change in absorbance

was calculated; taking the percentage change in absorbance

verses sucrose concentration (mM), a graph was plotted. The

isotonic concentration found out from the graph was taken as

the concentration of sugars in the apoplasmic space fluid.

The short duration of the wash was intended only to capture

the apoplasmic sugars adhering to the outer layer of the

endosperm/embryo and inner layer of the pericarp; and

exclude possibility of facilitated diffusion of solutes (Wang

and Fisher 1995) into and from the dipped structures.

Peroxidase enzyme activity was measured in fresh plant

material homogenized with 0.1 M sodium phosphate buffer

(pH 7.0) and centrifuged at 4�C for 10 min at 7000 9 g.The

supernatant was collected and buffer was added to reach the

mark in a 5 ml volumetric flask. Aliquots of the stock

solution were added to reaction mixture consisting of 0.2 ml Ta

ble

1A

.Y

ield

par

amet

ers

of

the

mai

np

anic

leo

fri

cecu

ltiv

ars,

Jog

esh

,U

day

agir

ian

dS

idh

anta

in2

00

6(d

ryse

aso

n);

B.

Yie

ldp

aram

eter

so

fth

em

ain

pan

icle

of

rice

cult

ivar

s,R

amac

han

di,

Mah

anad

ian

dG

anje

ikal

iin

20

07

(wet

seas

on

)

Cu

ltiv

ars

Pan

icle

dry

wt.

(g)

Pan

icle

len

gth

(cm

)

No

.o

fg

rain

sp

er

pan

icle

10

0g

rain

wt.

(g)

Gra

insi

zeF

resh

gra

in

vo

lum

e(m

l)

Per

cen

tag

eo

fP

erce

nta

ge

of

hig

h

den

sity

gra

ins

Len

gth

(cm

)

Bre

adth

(cm

)

Fil

led

gra

ins

Un

fill

ed

gra

ins

A Jog

esh

3.5

7±

0.1

32

4.2

3±

0.8

71

84

.33

±5

.68

2.7

3±

0.0

11

.05

±0

.00

0.5

5±

0.0

00

.04

3±

0.0

02

69

.53

±3

.47

30

.47

±2

.05

32

.34

±2

.10

Ud

ayag

iri

3.0

1±

0.1

72

3.7

1±

0.5

71

79

.33

±5

.50

2.2

0±

0.1

10

.80

±0

.01

0.4

6±

0.1

00

.03

5±

0.0

01

79

.74

±2

.86

20

.26

±2

.86

20

.18

±4

.20

Sid

han

ta1

.89

±0

.16

21

.50

±0

.50

16

3.3

3±

5.5

01

.68

±0

.01

0.7

0±

0.0

00

.40

±0

.01

0.0

26

±0

.00

16

6.1

2±

3.8

13

3.8

0±

1.8

11

7.8

1±

2.3

6

B Ram

ach

and

i4

.73

±0

.24

27

.40

±0

.52

19

5.3

3±

4.7

22

.84

±0

.03

1.0

8±

0.0

00

.56

±0

.00

0.0

44

±0

.00

17

5.3

3±

3.5

12

4.6

6±

3.5

15

7.3

3±

2.5

1

Mah

anad

i5

.23

±0

.14

23

.66

±0

.57

25

1.3

3±

7.5

02

.50

±0

.04

0.9

0±

0.0

20

.45

±0

.05

0.0

38

±0

.00

28

0.6

6±

2.0

81

9.3

3±

2.0

85

9.3

3±

4.5

0

Gan

jeik

ali

6.0

7±

0.2

23

0.2

0±

0.7

53

73

.6±

9.5

51

.74

±0

.06

0.7

0±

0.0

10

.42

±0

.01

0.0

28

±0

.00

18

2.6

6±

3.7

81

7.3

3±

3.7

86

1.6

6±

1.1

5

Fv

alu

eb

etw

een

the

con

tras

tin

gcu

ltiv

ars

0.0

13

9N

S0

.31

35

NS

0.6

17

6N

S7

3.1

*5

1.7

6*

94

.16

*2

74

**

1.0

22

NS

0.1

03

1N

S0

.35

83

NS

Fv

alu

eb

etw

een

seas

on

s8

.11

5N

S2

.37

7N

S2

.75

0N

S4

.6N

S2

.15

3N

S0

.56

41

NS

12

NS

2.8

2N

S2

.84

3N

S4

0.3

1*

Th

e±

val

ues

ind

icat

est

and

ard

dev

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on

of

the

mea

n(n

=3

).T

he

sig

nifi

can

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st(F

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for

the

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ars

and

bet

wee

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aso

ns

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,*

*si

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nt

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and

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123

Fig. 2 Grain dry matter growth

and change in cell number of

endosperm in different cultivars

of rice during the period of grain

filling in the dry season of 2006.

Vertical bars indicate ±

standard deviation of the mean

(n = 3)

Fig. 3 Temporal changes in the

dry weight and volume of the

developing endosperm of rice

cultivars during the period of

grain filling in the dry season of

2006. Difference in dry weight

between the cultivars was

significant at P B 0.01 level

(df = 2, 8). The difference

between apical and basal

spikelets of the cultivars was


(df = 4) on most of the

sampling occasions. Symbols:

Triangle Sidhanta, RectangleUdayagiri, Rhombus Jogesh,

Closed Apical, Open Basal


123

sodium phosphate buffer, 1 ml 0.01 M pyrogallol and 1 ml

0.01 M H2O2. The reaction was terminated by addition of

1 ml 5% H2SO4 after an incubation period of 5 min at 25�C.

Measuring O.D. at 430 nm determined the concentration of

purpurogallin formed. Enzyme activity was calculated as

change in O.D. per g fresh weight per minutes according to

the method of Chance and Machly (1955).

Results

Morphological features of the panicle on main shoot

The three types of cultivars used in the experiment differed

significantly in grain weight, size and volume in both the

dry and wet seasons, although the differences in grain

number, dry weight, high density grain number and per-

centage of filled grain of the panicle were not always sig-

nificant (Table 1). Compared to Jogesh, grain size, volume

and weight were nearly 20 and 30–40% lower in Udayagiri

and Sidhanta cultivars, respectively, in the dry season.

Almost the same trend was noticed in the grains of the

other cultivars in the wet season. Grain weight, volume and

size of Mahanadi and Ganjeikali were nearly 10–20 and

25–39% smaller, respectively, in comparison to that of

Ramachandi. In the dry season, grain quality differed

between the cultivars; Jogesh possessed 32% of high-

density grains compared to Udayagiri and Sidhanta having

20 and 17% of such grains respectively, in the panicle. The

percentage of high density grains was high in the wet

season, although varietal difference was not significant.

Grain dry mass and endosperm cell number

In the dry season, average dry mass of grains increased

temporally during the period of grain filling in all the

cultivars; the apical grains accumulated larger weight

compared to the basal grains (Fig. 2). Increase of dry mass

continued in the apical grains up to day 22 after anthesis

and stopped thereafter until maturity. Conversely, the basal

grains accumulated dry mass slowly up to the time of

maturity. The variation in endosperm volume and weight

Fig. 4 Correlation between the

concentration of ethylene

released at anthesis and the

highest rates of grain filling and

cell division of the apical and

basal spikelets of the rice

cultivars in the dry season of

2006. Level of significance:

* = P B 0.05. The correlations

were drawn to show that high

ethylene production at anthesis

impacts rates of cell division

and grain filling, when these

activities were at the maximum


123

(Fig. 3) matched the variation in weights of grains among

different cultivars. The endosperm cell number also dif-

fered according to the location of spikelets on the panicle

axis and the variation of cultivars; the larger seeds pos-

sessed more weight and cell number (Fig. 2). In the apical

spikelet, endosperm cell number increased rapidly with

passage of time during the first 10 days after anthesis and

the rate declined thereafter; there was no increase in cell

number during the last 6 days of observation. In contrast,

cell number continued to increase with passage of time in

the basal endosperm until day 19 after anthesis, although

the change in cell number during the last 6 days was much

slower compared to the first 6 days of observation. Endo-

sperm volume and weight were larger in the apical spikelet

compared to the basal spikelet on day 7 after anthesis

(Fig. 3). The difference was maintained on subsequent

days of sampling although the rate of growth did not differ

much between the apical and basal spikelets. During the

period of observation, weight of the endosperm increased

more than two times in both the spikelets, although growth


concentration of soluble

carbohydrates and amino acids

in different parts of caryopsis of

rice cultivars during the period

of grain filling in the dry season

of 2006. Vertical bars indicate

± standard deviation of the

mean (n = 3). Difference



for the pericarp (df = 2, 6) and

endosperm (df = 2, 8).

Difference between apical and

basal spikelets of the cultivars

was significant at P B 0.05

level (df = 4) on most of the

sampling occasions


123

in volume did not increase as high. Compared to cell

number or dry matter accumulation, growth in endosperm

volume was not as prominent; it increased marginally

during the period of observation. The pattern of dry matter

accumulation and cell number of the three contrasting

cultivars in the wet season was similar to that of dry sea-

son. Correlations between ethylene released from spikelets

at anthesis with rates of grain filling and cell division of the

endosperm of apical and basal spikelets at early post

anthesis stage, were negative and significant (P B 0.05) in

the dry season of 2006 (Fig. 4).

Assimilates and starch concentrations of the pericarp

and endosperm

The concentrations of soluble carbohydrates of the pericarp

and endosperm decreased with the passage of time in all

three cultivars during the period of observation between

days 7 and 19 after anthesis in the dry season (Fig. 5). The

concentration was high in Jogesh and low in Sidhanta, and

it remained intermediate between the two for Udayagiri. A

trend similar to this was observed for the temporal fluctu-

ation of amino acid concentration of the pericarp and

endosperm of the cultivars used. The concentration of both

soluble carbohydrates and amino acids of the cultivars

were higher in the basal spikelet compared to apical

spikelet on most of the sampling occasions. The basal

spikelets retained high assimilate concentration for a longer

time compared to the apical spikelets. In contrast to

assimilates, the concentration of starch increased tempo-

rally in all three cultivars (Fig. 6). The rate of increase was

fastest in Jogesh and slowest in Sidhanta. Starch concen-

tration increased faster in the apical spikelets in compari-

son to the basal spikelet. Temporal and spatial variation in

the concentration of assimilates and starch between the

three different types of cultivars grown in the wet season

was similar to that of the dry season.

Protein and chlorophyll concentration of the pericarp

and endosperm

The total protein and chlorophyll concentration of the

pericarp and endosperm of the apical and basal caryopses

increased mostly between days 7 and 13 after anthesis and

declined thereafter in all cultivars (Fig. 7). On all sampling

occasions, the big-seeded cultivars contained a higher


concentration of starch in

different parts of caryopsis of

rice cultivars during the period

of grain filling in the dry season

of 2006. Vertical bars indicate

± standard deviation of the

mean (n = 3). Difference



for the pericarp (df = 2, 6) and

endosperm (df = 2, 8).





sampling occasions


123

concentration of the materials compared to the small see-

ded cultivars. The pericarp and endosperm of the apical

caryopses contained higher concentration of the materials

than that of the basal caryopses. Compared to the endo-

sperm, chlorophyll concentration of the pericarp was

higher although protein concentration did not differ as

much.

Peroxidase activity and ethylene concentration

of the pericarp and endosperm

Peroxidase activity increased with the passage of time in

both pericarp and endosperm of the caryopses of all rice

cultivars (Fig. 8). The activity was higher in the basal

caryopsis compared to the apical caryopsis on most of the

sampling occasions. Cultivar difference in peroxidase

activity was significant; the activity was higher in a small-

seeded cultivar than that of a big-seeded cultivar. The

concentration of ethylene of both apical and basal spikelets

increased dramatically in the pre-anthesis period and cul-

minated at anthesis stage of development in all cultivars

(Fig. 9). The basal spikelets produced consistently more

ethylene compared to the apical spikelets in both the sea-

sons and the differences were significant at 0.01 level on

most of the occasions. Difference in spikelet ethylene

production between the cultivars was small, but it was

significant at 0.01 level on all occasions. The difference

was more evident in the basal spikelets compared to apical


concentration of total protein

and chlorophylls in different

parts of caryopsis of rice



2006. Vertical bars indicate ±


(n = 3). The differences in

chlorophyll and protein

concentrations of the pericarp

(df = 2, 6) and endosperm

(df = 2, 8) of the cultivars were

significant at P B 0.001 and

P B 0.05 levels, respectively.





sampling occasions


123

spikelets. The spikelets of a small-seeded cultivar produced

more ethylene compared to that of a big-seeded cultivar.

Because the concentrations of chlorophylls and protein of

the endosperm were maximum on days 13 and 16,

respectively, correlation curves were fitted between them

and ethylene released from the spikelets at anthesis for

assessment of the impact of the hormone. Similar corre-

lation between the concentration of proteins and chloro-

phylls of the pericarp and ethylene were drawn, when the

temporal changes of the materials were at the peak level.

The correlations were significant and negative (Fig. 10).

However, correlation between ethylene concentration at

anthesis and peroxidase activity of the both pericarp and

endosperm was positive and significant (P B 0.05).

Photosynthetic rate and Fv/Fm ratio of flag leaf

In the dry season, Fv/Fm value of the flag leaf declined

slowly from anthesis during the first 2 weeks and sharply in

the last 2 weeks after anthesis in all three cultivars

(Fig. 11). But, photosynthetic carbon fixation remained

high during the first 2 weeks and declined sharply in the

last 2 weeks after anthesis. Although cultivar differences in

photosynthetic activities were small, they were consistently

higher in a big-seeded cultivar compared to a small-seeded

cultivar.

Soluble carbohydrates concentration of the endosperm

space (apoplasm) fluid

During both the dry and wet seasons, the soluble carbo-

hydrates concentrations of the apoplasmic fluid increased

temporally from days 7 to 13 in all cultivars and declined

thereafter on day 16 (Fig. 12). It was not possible to sep-

arate the pericarp from the embryo/endosperm 16 days

after anthesis and the soluble carbohydrates could not be

estimated from the apoplasmic space thereafter. The con-

centration of the material was higher in a big-seeded cul-

tivar compared to a small-seeded cultivar. Similarly, the

concentration was higher in the caryopsis of superior


peroxidase activity in different

parts of caryopsis of rice



2006. Vertical bars indicate ±


(n = 3). Difference between the

cultivars was significant at

P B 0.001 level for the pericarp

(df = 2, 6) and endosperm

(df = 2, 8). Difference between

apical and basal spikelets of the

cultivars was significant at

P B 0.01 level (df = 4) on

most of the sampling occasions


123

spikelets compared to that of the inferior spikelets of dif-

ferent rice cultivars.

Correlation of apoplasmic soluble carbohydrates

with grain weight, cell number of endosperm

and soluble assimilates and starch concentration

of endosperm and pericarp

The soluble carbohydrates concentration of apoplasmic

space fluid correlated negatively with that of the soluble

carbohydrates and amino acids of pericarp and endosperm

of the caryopsis on apical and basal spikelets, when con-

centration of the materials was studied during the first half

of grain filling period in the three different types of rice

cultivars in both dry and wet seasons (Fig. 13). The neg-

ative correlation was more evident in the basal spikelets of

the cultivars than that of the apical spikelets. However, the

concentration of embryonic space soluble carbohydrates

exhibited significant positive correlation with the starch

concentration of caryopsis components, grain weight and

endosperm cell number of the rice cultivars (Fig. 14).

Discussion

Seed development in a plant is genetically programmed.

The whole process of development is prone to the control

of metabolites, which are provided to the filial tissue by the

maternal organs. It is proposed that the early phase of

embryo growth is maternally controlled, but a switchover

to filial control is necessary for growth during maturation

phase (Weber et al. 2005). Sugar and phytohormone

response pathways navigate responses at the level of

transcription and protein phosphorylation for change over

in the control system (Gibson 2004). In our study, seed

development was followed in rice cultivars belonging to

identical duration groups both in the wet and dry seasons.

The grains of the cultivars differed in size and weight.

Besides, weight, size and volume of grains of the cultivars

also varied with difference in positions on the panicle from

early embryonic stage to the maturation phase (Table 1;

Figs. 2, 3). However, the impact of metabolites and hor-

mone on development were only studied during the

embryonic phase, when seed development was fully under

Fig. 9 Concentrations of

ethylene in apical and basal

spikelets of the rice cultivars

during the period of grain

development in dry season of

2006 and wet season of 2007.

Vertical bars indicate ±


(n = 3). Cultivar difference in

ethylene concentration of the

spikelets was significant at

P B 0.01 level (df = 10). The

difference in ethylene

concentration of the apical and



level on most of the sampling

occasions


123

maternal control (Weber et al. 2005). During this period of

development, significant differences were noticed in the

sugar concentrations of pericarp, apoplasmic space and

endosperm of the caryopses of contrasting rice cultivars

used in the experiment (Figs. 5, 12); the concentration was

low when seed size/weight was small due to genetic vari-

ation. When seed weight was low on account of the unfa-

vourable location of panicle axis (basal spikelet), the

apoplasmic sugar concentration was low, but not that of the

pericarp or endosperm. The salient feature of this investi-

gation was that sugar concentration of the maternal tissue

(pericarp), filial tissue (endosperm) and space between the

two tissues of the developing caryopses fluctuated similarly

depending on difference in the size and weight of grains of

the cultivars. Although, concentration of apoplasmic space

sugars varied positively with the weight or size of grains at

different locations of the panicle, no such relationship

could be found for the pericarp or endosperm. It is known

that considerable diversity exists in seed size and weight of

rice cultivars. But diversity in seed growth or grain filling

has not been studied adequately in relation to assimilate

partitioning between the components of the developing

caryopsis. Similarly, the apical spikelets dominate physi-

ologically over the basal spikelets and produce better

quality grains (Mohapatra et al. 1993); but, the expression

of dominance in assimilate partitioning is not known. In

our study, the concentration of apoplasmic sugars corre-

lated positively with grain growth (Fig. 14), and it was

Fig. 10 Correlation between

concentrations of the total

chlorophylls and protein of the

caryopsis organs and ethylene

of the spikelets when temporal

changes of the materials reached

peak values during the grain

filling period as shown in Fig. 7.

Level of significance

* P B 0.05, ** P B 0.01,

*** P B 0.001


123

proved that irrespective of either the genetic or spatial

variation, final seed weight is dependent on assimilates

provided to it by the maternal tissue during the early part of

endosperm development. The developing endosperm

remains confined within the embryonic space (apoplasmic

space; Oparka and Gates 1981a, b; Thorne 1985; Weber

et al. 2005) and does not have any alternate source of

supply for assimilates. Endosperm cell number increases

rapidly during this period and positional disadvantage on

panicle axis restricting cell multiplication limits seed filling

and weight in an inferior caryopsis (Patel and Mohapatra

1996). The low sink efficiency of such caryopses might

have resulted in accumulation of the unused assimilates in

the pericarp and endosperm (Fig. 5). Additionally, the

premature termination of growth might have paved entry of

water into the hull space at the cost of carbon assimilates,

decreasing grain quality at maturity (Mohapatra et al.

1993). Similarly, low sink efficiency owing to poor endo-

sperm cell division rate in small-seeded cultivars (Fig. 2)

could be responsible for the restricted starch synthesis

(Fig. 6). In the present study, both photosynthetic rate and

efficiency of the flag leaf differed significantly between the

cultivars; they were consistently higher in the heavy-see-

ded cultivars compared to the relatively small-seeded cul-

tivars (Fig. 11). High source activity might have sustained

greater concentration of assimilates in different parts of the

caryopses of the former compared to the latter. Hence,

contribution of source activity as a factor for larger grain

size or weight could be significant as well.

A holistic approach was lacking in the literature for

studying the role of pericarp, apoplasmic space solutes and

endosperm in the seed filling of rice hitherto, and our work

proposed a new insight by revealing their contributions to

the process in consortium. The assimilate concentration of

pericarp, apoplasmic space and endosperm of the caryopses

was higher in a heavy-weight seed compared to a low-

weight seed; but, there was no significant correlation

between the concentration of the materials of one organ

with that of the other irrespective of either genetic variation

Fig. 11 Temporal changes in

the Fv/Fm value of the PS II

system and photosynthetic rate

of the flag leaf during the period

of grain filling of rice cultivars

in the dry season of 2006. The

observations taken for the

cultivars differed significantly at

P B 0.001 level (df = 2, 8)

Fig. 12 Temporal changes in concentration of apoplasmic soluble

carbohydrates in the endosperm space of caryposis of rice cultivars

during the period of grain filling in the dry season of 2006 and wet

season of 2007. aa similar letters indicate no significant difference

between apical and basal spikelet, ab dissimilar letters indicate

significant difference between apical and basal spikelet, pqr letters

indicate significant difference between apical spiklets and xyz letters

indicate significant difference between basal spikelets of three

contrasting rice cultivars


123

or difference in spatial location of spikelets on the panicle

axis (Fig. 13). Since negative correlations were observed

consistently between concentrations of the apoplasmic

space soluble carbohydrates and assimilates of pericarp and

endosperm, and it was more evident in the inferior spik-

elets, association of maternal tissue in restricting supply of

assimilates to the growing endosperm could be established

in the present study. Functional disadvantage of the peri-

carp of an inferior caryopsis could be responsible for poor

unloading and accumulation of the solutes in the organ and

reduction in supply to the apoplasmic pool in the inferior

spikelet resulting in high concentration of the material in

the former and low concentration of the material in the

latter. In the literature too much emphasis was laid on the

role of solutes of the apoplasmic space in turgor driven

flow of assimilates from the source to the developing

endosperm by many authors (Thorne 1985; Wolswinkel

1992; Partick and Offler 1995; Lim et al. 2006). It is

implied that sink activity for the utilization of these solutes

by the endosperm is dependent on the concentration of the

solutes in the apoplasmic fluid. Jenner (1974) proposed that

rate of starch synthesis in wheat endosperm could be

enhanced by raising the concentration of sucrose in the

apparent free space of endosperm which remains in direct

Fig. 13 Relationship between

the apoplasmic soluble

carbohydrates of endosperm

space (X-axis) and the soluble

carbohydrates and free amino

acid concentration of pericarp

and endosperm (Y-axis) of

caryopsis during the first half of

grain filling period in the dry

season of 2006 and wet season

of 2007. * Significant at the 0.05

level of probability (df = 23)

and ns = not significant at the

0.05 level of probability

(df = 23). Symbols: closed dry

season, open wet season,

Rhombus grain size big (Jogesh,

Ramachandi), square grain size

medium (Udayagiri, Mahanadi),

triangle grain size small

(Sidhanta, Ganjeikali)


123

contact with apoplasmic space fluid. This proposition was

reaffirmed in our study with evidence. The concentration of

soluble carbohydrates of the apoplasmic space correlated

positively (P B 0.01) with the starch concentration and cell

number of the endosperm and the grain weight of caryopsis

(Fig. 14). Hence, the concentration of sugars in the apo-

plasmic space could be a reliable indicator for assimilate

filling capacity of the caryopsis of rice. Higher concen-

trations of soluble carbohydrates and free amino acids in

the pericarp compared to the endosperm (Fig. 5) might

have induced turgor mediated mass flow of solutes from

the former to the latter. Unlike grain legumes, transport of

nutrients required for the growth of embryo and endosperm

from the seed coat in cereals is a carrier mediated passive

process (Wang and Fisher 1995; Jenner 1974). Because

membrane transport mechanisms for release of nutrients

from the maternal tissue into the apoplasmic space remain

unclear (Zhang et al. 2007), no plausible explanation could

be given for the existence of a turgor homeostat and its role

in phloem unloading of the seed coat (Partick and Offler

1995).

Further, it could be ascertained in our study that ethyl-

ene is the hormone responsible for decreasing the activity

of pericarp and endosperm. High ethylene production in the

basal spikelets of panicle or in the spikelets of small-seeded

cultivars retarded pericarp development because of its

adverse effect on proteins and chlorophylls (Fig. 10) dur-

ing the pre-storage phase and reduced grain filling

Fig. 14 Relationship of

apoplasmic soluble

carbohydrates of the endosperm

space with starch, grain weight

and cell number of developing

caryopsis in different cultivars

of rice during the first half of

grain filling period in the dry

season of 2006 and wet season

of 2007. *, **, *** = significant

at the 0.05, 0.01 and 0.001

levels of probability,

respectively (df = 23). Symbols

are similar to Fig. 13


123

(Mohapatra and Mohapatra 2006). The concentration of

ethylene produced by the spikelets at anthesis correlated

positively (P B 0.05) with peroxidase activity and nega-

tively with photosynthetic pigments, and protein concen-

trations of the pericarp and endosperm (Fig. 10), and grain

filling rate and endosperm cell division rate (Fig. 4) of the

spikelets in our study. It is possible that, ethylene-induced

senescence of the pericarp and endosperm might be

responsible for depleted supply and the resultant low

concentration of assimilates in the apoplasmic space

(Fig. 12) and reduced sink activity (Fig. 2) of the caryopsis

of the basal spikelets or spikelets of small-seeded cultivars.

It is known that grain filling at the base of panicle decreases

because these spikelets produce more ethylene compared to

the apical spikelets (Mohapatra and Mohapatra 2005; Yang

et al. 2006) and the high concentration of ethylene impacts

activities of starch synthesizing enzymes (Mohapatra et al.

2009) resulting in restricted sink activity of the endosperm

for sugar consumption. In our study, unused assimilates

might have accumulated in the pericarp and endosperm of

the inferior basal spikelets because of poor sink activity of

the organs (Fig. 5). Hence unlike apoplasmic pool sugars,

the concentration of assimilate in these organs could not be

an indicator for sink strength of the rice grain. This infer-

ence is similar to the observation made on wheat grain

(Singh and Jenner 1982). In our study, the concentration of

soluble carbohydrates increased in the apoplasmic space

during the embryonic phase of grain development

(Fig. 12), when cell number increased rapidly in the

endosperm (Fig. 2). The soluble carbohydrates were rap-

idly converted to starch in the endosperm (Fig. 6), as long

as carrier mediated transport of sugars in the pericarp was

not affected by any external stimulus. Strong apical dom-

inance in the panicle grain filling (Xu and Vergara 1986;

Peng et al. 1994) resulted in faster grain filling of the

caryopses of apical spikelets, whereas ethylene induced

senescence of the organs (Fig. 10) limited sink activity

(Fig. 4) of the basal spikelets. Therefore, ethylene pro-

duction should be minimized for the sake of fast-syn-

chronized filling and higher yield potential of rice.

Ethylene has been identified as a promoter of senescence,

but its role as a signal determining progression of bud

development has been undermined so long (Dun et al.

2006). We need to know how this hormone cues hetero-

geneity of spikelet development in a strongly apical dom-

inant cultivar and its suppression increases proportion of

high density grains in agronomic fit cultivars. In our study,

genetic difference in seed size influenced concentration of

soluble carbohydrates of the apoplasmic space during seed

filling (Fig. 12); pericarp of a large-sized seed might have

unloaded more sugar into the apoplast than a small-sized

seed. It is also possible that high ethylene production of

spikelets in small-seeded cultivars might have affected

pericarp activity and impacted assimilate unloading into

the apoplasmic space to the detriment of grain filling.

Acknowledgments The authors thank University Grants Commis-

sion, New Delhi for support under DRS project and Council of Sci-

entific and Industrial Research, New Delhi for support under the

Emeritus Scientist project.

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