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Field Crops Research 123 (2011) 170–182

Contents lists available at ScienceDirect

Field Crops Research

journa l homepage: www.e lsev ier .com/ locate / fc r

re-anthesis non-structural carbohydrate reserve in the stem enhances the sinktrength of inferior spikelets during grain filling of rice

ing Fua, Zuanhua Huanga, Zhiqin Wanga, Jianchnag Yanga,∗, Jianhua Zhangb,∗∗

Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Yangzhou University, Yangzhou, Jiangsu, ChinaDepartment of Biology, Hong Kong Baptist University, Hong Kong, China

r t i c l e i n f o

rticle history:eceived 1 March 2011eceived in revised form 12 May 2011ccepted 14 May 2011

eywords:uper rice (Oryza sativa L.)on-structural carbohydrateink strengthnferior spikeletsrain fillingource–sink relationship

a b s t r a c t

Sink strength plays an important role in grain filling of cereals but how it is related to the pre-anthesisnon-structural carbohydrate (NSC) reserves is not clear. This study investigated if and how an increasein NSC reserves could enhance sink strength, and consequently improve grain filling of later-floweringinferior spikelets (in contrast to the earlier flowering superior spikelets) for rice varieties with large pan-icles. Two “super” rice varieties (the recently bred high-yielding rice) and two New Plant Type (NPT,named in IRRI for the extra-large panicle) rice lines were compared with two elite inbred varieties underfield-grown conditions. Three nitrogen (N) treatments, applied at the stages of panicle initiation, spikeletdifferentiation or both, were adopted with no N application during the mid-season as control. Both superrice and NPT rice showed a greater yield capacity as a result of a larger panicle than the elite inbred rice.However, a lower percentage of filled grains limited the realization of higher yield potential in super riceand especially in NPT rice, due to their lower grain filling rate and the smaller grain weight of their inferiorspikelets. The low grain filling rate and small grain weight of inferior spikelets are mainly attributed toa poor sink strength as a result of small sink size (small number of endosperm cells) and low sink activ-ity, e.g. low activities of sucrose synthase (SuSase) and adenosine diphosphoglucose pyrophosphorylase(AGPase). The amounts of NSC in the stem and NSC per spikelet at the heading time are significantlyand positively correlated with sink strength (number of endosperm cells and activities of SuSase andAGPase), grain filling rate, and grain weight of inferior spikelets. Nitrogen application at the spikeletdifferentiation stage significantly increased, whereas N application at the panicle initiation or at both

panicle initiation and spikelet differentiation stages, significantly reduced, NSC per spikelet at the head-ing time, sink strength, grain filling rate, and grain weight of inferior spikelets in super rice. The resultssuggest that pre-anthesis NSC reserves in the stem are closely associated with the sink strength duringgrain filling of rice, and N application at the spikelet differentiation stage would be a good practice toincrease pre-anthesis NSC reserves, and consequently to enhance sink strength for rice varieties withlarge panicles, such as super rice varieties.

. Introduction

Rice (Oryza sativa L.) is one of the most important crops in the

orld, and the foremost staple food in Asia, providing 35–60% of theietary calories consumed by more than 3 billion people (Fageria,003). It is estimated that, by the year 2025, it will be necessary to

Abbreviations: AGFP, active grain filling period; AGPase, adenosine diphos-hoglucose pyrophosphorylase; DPA, days post-anthesis; EW, endosperm weight;ECDR, mean endosperm cell division rate; MECN, maximum endosperm cell

umber; MGFR, mean grain filling rate; NPT, new plant type; NSC, non-structuralarbohydrate; SuSase, sucrose synthase.∗ Corresponding author. Tel.: +86 514 8797 9317; fax: +86 514 8797 9317.

∗∗ Corresponding author. Tel.: +852 3411 7350; fax: +852 3411 5995.E-mail addresses: jcyang@yzu.edu.cn (J. Yang), jzhang@hkbu.edu.hk (J. Zhang).

378-4290/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.fcr.2011.05.015

© 2011 Elsevier B.V. All rights reserved.

produce about 60% more rice than what is currently produced orto increase the yield more than 1.2% per year (Normile, 2008) tomeet the food needs of a growing world population (Fageria, 2007;Normile, 2008). In the past 10 years, however, the growth of riceyield has dropped below 1% annually worldwide and is virtually nilacross Asia (Normile, 2008). Great efforts should be made to breednew rice varieties with higher yield potential and to improve cropmanagement in order to enhance average farm yields (Peng et al.,2008, 2010).

In rice and other cereal crops, grain yield can be defined asthe product of yield sink capacity and filling efficiency (Kato and

Takeda, 1996). To further increase the yield and break the yieldceiling, breeding efforts have expanded yield sink capacity, themaximum size of sink organs to be harvested, mainly by increas-ing the number of spikelets per panicle (Kato et al., 2007). As a

esearch 123 (2011) 170–182 171

rnsRr2haol12

sS22i2pis

aab(asatsoodi2

ttIpae2rNio

passswsa2eponwpd

Fig. 1. Temperature (A), sunshine hours (B), and precipitation (C) during the grow-ing season of rice in 2007 and 2008 at the experiment site of Yangzhou, Southeast

average air temperature, precipitation, and sunshine hours dur-ing the rice growing season across the two study years measured

J. Fu et al. / Field Crops R

esult, varieties with large panicles or extra-heavy panicle types,amely numerous spikelets per panicle, have become available,uch as the New Plant Type (NPT) rice of the International Riceesearch Institute (IRRI) (Peng et al., 1999), hybrid rice and superice or super hybrid rice in China (Cheng et al., 2007; Peng et al.,008). These cultivars, however, do not frequently exhibit theirigh yield potential due to their poor grain filling, which is mainlyttributed to a slow grain filling rate and many unfilled grainsf the later flowering inferior spikelets (in contrast to the ear-ier flowering superior spikelets) (Ao et al., 2008; Peng et al.,999; Yang et al., 2000; Yang and Zhang, 2010; Zhang et al.,009).

There are many explanations to the poor filling of inferiorpikelets, including source limitation (Murty and Murty, 1982;ikder and Gupta, 1976; Wang, 1981), sink size limitation (Kato,004), unbalance in hormonal levels (Yang et al., 2006; Zhang et al.,009), low activities and/or gene expressions of enzymes involved

n sucrose-to-starch conversion (Ishimaru et al., 2005; Jeng et al.,003; Kato et al., 2007; Wang et al., 2008), and assimilate trans-ortation impediment (Yang et al., 2002a; Yang, 2010). So far, the

ntrinsic factor responsible for variations in grain filling betweenuperior and inferior spikelets remains elusive.

It is generally believed that grain-filling rate in cereals is closelyssociated with sink strength (Liang et al., 2001; Venkateswarlund Visperas, 1987; Yang et al., 2003a). The sink strength cane described as the product of sink size and sink activityVenkateswarlu and Visperas, 1987; Warren, 1972). Sink size is

physical restraint that includes cell number and cell size andink activity is the physiological constraint upon a sink organ’sssimilate import (Ho, 1988). As starch in rice grains contributeso about 90% of the final dry weight of an unpolished grain anducrose is the main transported form of assimilates from sourcergans to sink organs (Cao et al., 1992; Yoshida, 1972), activitiesf enzymes involved in the metabolism of the sucrose-to-starch ineveloping rice endosperm are believed to be linked to sink activ-

ty (Ishimaru et al., 2003; Kato, 1995; Liang et al., 2001; Yang et al.,004).

Although sink strength is generally considered to play an impor-ant role in grain filling, the information is very limited with regardo factors linking to, or a technique that can enhance, sink strength.t has been proposed that crop growth rate during a two-weekeriod preceding full heading in rice is closely associated with pre-nthesis non-structural carbohydrate (NSC) reserves in the stem,ndosperm development, and grain filling of rice (Horie et al.,005), which suggests that pre-anthesis NSC in the stem may beelated to sink strength. However, the evidence that pre-anthesisSC links to sink strength is lack and it is not known if an increase

n pre-anthesis NSC reserves in the stem could improve grain fillingf inferior spikelets.

The objective of this study was to investigate if and howre-anthesis NSC in rice stems are related to sink strength,nd if and how an increase in the NSC reserve could enhanceink strength, and consequently improve grain filling of inferiorpikelets. The endosperm cell number was used as an index of sinkize (Ishimaru et al., 2003; Yang et al., 2002b). Indices of sink activityere the activities of some key enzymes involved in sucrose-to-

tarch conversion in grains, sucrose synthase (SuSase, EC 2.4.1.13)nd adenosine diphosphoglucose pyrophosphorylase (AGPase, EC.7.7.27) (Ishimaru et al., 2005; Kato, 1995; Liang et al., 2001; Yangt al., 2004). Two “super” rice varieties and two NPT lines were com-ared with two elite inbred varieties which show better grain fillingf inferior spikelets under field-grown conditions. Treatments ofitrogen (N) application at different stages during the mid-seasonere conducted to observe if an agronomic practice could increase

re-anthesis NSC reserves, and consequently enhance sink strengthuring grain filling.

China. Data are means of per 10 days from the transplanting of rice. Arrows indicatethe heading time.

2. Materials and methods

2.1. Plant materials and growth conditions

Experiment I: The experiment was conducted at a farm belongingto Yangzhou University, Jiangsu Province, China (32◦30′ N, 119◦25′

E) during the rice growing season (May–October) of 2007, andrepeated in 2008. The soil was a sandy loam [Typic fluvaquents, Eti-sols (U.S. taxonomy)] with 24.6 g kg−1 organic matter, 105 mg kg−1

alkali hydrolysable N, 34.3 mg kg−1 Olsen-P, and 68.4 mg kg−1

exchangeable K. The field capacity soil moisture content, mea-sured after constant drainage rate and made gravimetrically, was0.189 g g−1, and bulk density of the soil was 1.34 g cm−3. The

at a weather station close to the experimental site are shownin Fig. 1.

172 J. Fu et al. / Field Crops Research 123 (2011) 170–182

Table 1Nitrogen (N) application treatments in the field Experiment II.

Treatment Stage and rate (kg N ha−1) of N application

Basal (1 day beforetransplanting)

Tillering (12 daysafter transplanting)

Panicle initiation (33–35days before heading)

Spikelet differentiation (18–20days before heading)

Total

T0 60 48 0 0 10872

036

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Emaabcaawwimdt

2

sdre

T1 60 48T2 60 48T3 60 48

Two super rice (Oryza sativa. L) varieties, two NPT lines, andwo elite check rice varieties were grown in the paddy field. Thewo super rice varieties were Liangyoupeijiu (LYPJ, an indica F1ybrid) and Huaidao 9 (HD 9, a japonica variety). The two NPT linesere IR65600-127-6-2-3 (NPT1) and IR65598-112-2 (NPT2) with

ropical japonica background and were bred in IRRI. The two checkarieties were Yangdao 6 (YD 6, an indica inbred) and Zhendao 88ZD 88, a japonica inbred). The two super rice varieties, especiallyYPJ, are the most popular and used in the largest area in ChinaCheng et al., 2007; Peng et al., 2008). The two check varieties areurrently used in local production because of their relatively higherield and better quality and pest resistance compared with otherarieties. The six varieties/lines have similar growth period rang-ng from 148 to 152 days from sowing to grain maturity. The seedsf two super rice varieties and two check varieties were obtainedrom Yangzhou Seed Company (Yangzhou, Jiangsu, China), and theeeds of two NPT lines were introduced from IRRI. Across the twoears, seedlings were raised in the seedbed with sowing date on0–11 May and transplanted on 9–10 June at a hill spacing of.20 m × 0.20 m with two seedlings per hill. Plot dimension was

n 5 m × 8 m. Each of the varieties had four plots as repetitionsn a complete randomized block design. N (60 kg ha−1 as urea),hosphorus (30 kg ha−1 as single superphosphate) and potassium40 kg ha−1 as KCl) were applied and incorporated before trans-lanting. N as urea was also applied at mid-tillering (48 kg ha−1),anicle initiation (36 kg ha−1), and at 18–20 days before heading36 kg ha−1) as top dressing. The total N application was 180 kg ha−1

n line with the local high-yielding practice. Water, weeds, insects,nd diseases were controlled as required to avoid yield loss. Theeading date (50% plants) for HD 9 and ZD 88 was on 22–24 August,

or NPT1 and NPT 2 on 23–25 August, and for LYPJ and YD 6 on 24–26ugust, and plants were harvested on 10–12 October.

Experiment II: The experiment was conducted at a farm close toxperiment I. The soil was a sandy loam with 21.5 g kg−1 organicatter, 98.4 mg kg−1 alkali hydrolysable N, 32.1 mg kg−1 Olsen-P,

nd 59.5 mg kg−1 exchangeable K. Two super rice varieties of LYPJnd HD 9 were field-grown. From panicle initiation (about 35 daysefore heading), three N treatments were conducted, i.e., N appli-ation at the stages of panicle initiation, spikelet differentiation,nd both panicle initiation and spikelet differentiation, with no Npplication during the mid-season as control. The treatment detailsere shown in Table 1. Plot dimensions were 5 m × 8 m and plotsere separated by an alley 0.5 m wide with plastic film inserted

nto the soil to a depth of 50 cm to form a barrier. Each of the treat-ent had four plots as repetitions in a complete randomized block

esign. Other plant growth conditions and crop managements werehe same as those in the Experiment I.

.2. Sampling and measurements

Two hundred panicles that headed on the same day were cho-

en and tagged for each plot in both experiments. The floweringate and the position of each spikelet on the tagged panicles wereecorded. Eight–ten tagged panicles from each plot were sampledvery 4-day intervals from anthesis to maturity for measuring grain

0 18072 18036 180

weight and enzymatic activity. Half-sampled grains were frozenin liquid nitrogen for 1 min and then stored at −80 ◦C for deter-mining SuSase and AGPase activities. Eighty–one hundred sampledgrains were used for measurements of grain dry weight. These weredried at 70 ◦C to constant weight for 72 h, dehulled and weighed.Four–five tagged panicles from each plot were sampled every 2-dayintervals from anthesis to 30 days post-anthesis (DAP) for observ-ing endosperm cell number. Superior spikelets that flowered on thefirst 2 days within a panicle and inferior ones that flowered on thelast 2 days within a panicle were separated from the sampled pani-cles. The difference in flowering date between superior and inferiorspikelets was 3–5 days within a panicle. Unfertilized spikelets (theovary was not enlarged) were removed before determining grainweight, endosperm cell number, and enzymatic activity. After cut-ting a small hole on the edge of a hull, 10–12 grains either fromthe superior or the inferior spikelets were fixed in Carnoy’s solu-tion (absolute ethanol:glacial acetic acid:chloroform = 9:3:1, v/v)for 48 h, then kept in 70% (v/v) ethanol pending for examination ofendosperm cell number.

The method for isolation and counting of endosperm cells wasmodified from Singh and Jenner (1982). Briefly, fixed grains weredehulled and transferred into 50% (v/v) then 25% (v/v) ethanoland finally into distilled water for 5–7 h prior to dissection of theendosperm. The endosperm was isolated under a dissecting micro-scope, stained with Delafied’s haematoxylin solution for 24–30 h,washed several times with distilled water and then hydrolyzed in0.1% (w/v) cellulase (No. c-2415, Sigma Chemical Co., St Louis, MO,USA) solution (pH 5.0) at 40 ◦C for 4–6 h and oscillated. The isolatedendosperm cells were diluted to 2–10 ml according to the develop-ment stage of the endosperm, from which 8–10 sub-samples (20 �lfor each sub-sample) were taken to a counting chamber (1 cm2

area). Using a light microscope, the endosperm cell number of 10fields of view for each counting chamber was noted. Within 4 DPAfor superior spikelets and 4–8 DPA for inferior ones, the numberof nuclei was counted as endosperm cell number. The total cellnumber per endosperm was calculated according to Liang et al.(2001). Six grains (endosperms) were examined for each variety ortreatment at each measurement.

The division processes of endosperm cells, as well as the pro-cesses of grain filling, were fitted by Richards’ growth equation(Richards, 1959) as described by Zhu et al. (1988):

M(W) = A

(1 + Be−kt)1/N. (1)

Endosperm cell division rate or grain filling rate (R) was calcu-lated as the derivative of Eq. (1):

R = AkBe−kt

N(1 + Be−kt)(N+1)/N, (2)

where M is the cell number and W is the grain weight, A is the

maximum cell number/final grain weight, t is the time after anthe-sis (d), and B, k, and N are coefficients determined by regression.The active grain-filing period (D) was defined as that when W wasfrom 5% (t1) to 95% (t2) of A. The average cell-division/grain-filling

J. Fu et al. / Field Crops Research 123 (2011) 170–182 173

Table 2Grain yield and yield components of rice cultivars in the field Experiment I.a

Year/variety Grain yield (t ha−1) Panicles per m2 Spikelets per panicle Spikelets per m2 (×103) Filled grains (%) Filled grain weight (mg)

2007LYPJ 10.8 a 210 e 248 a 52.0 ab 79.6 b 25.6 bHD 9 10.4 a 214 e 222 b 47.5 b 81.6 b 26.5 aNPT1 8.5 ef 241 d 214 bc 51.5 ab 65.7 d 25.7 bNPT2 8.1 fg 252 bc 207 c 52.1 ab 61.5 de 25.6 bYD 6 9.4 bc 251 bc 157 d 39.4 c 91.4 a 26.4 aZD 88 9.0 cd 306 a 125 e 38.3 c 92.8 a 25.6 b

2008LYPJ 9.7 b 211 e 245 a 51.7 ab 74.2 c 25.1 cHD 9 9.4 bc 218 e 218 bc 47.6 b 78.6 bc 26.2 aNPT1 7.8 g 261 b 209 c 54.5 a 58.7 ef 25.3 bcNPT2 7.2 h 254 bc 206 c 52.3 ab 54.9 f 25.1 cYD 6 9.1 cd 246 cd 156 d 38.4 c 90.2 a 26.2 aZD 88 8.7 de 303 a 126 e 38.2 c 91.0 a 25.5 bc

Analysis of varianceYear 19* 2NS 1NS 1NS 12* 8*

Variety 56* 154* 152* 34* 147* 11*

Year × variety 2NS 2NS <1 <1 <1 <1

a Values of grain yield are means of plants of 20 m2 harvested from four plots of each variety. Values of panicles per square meter, spikelets per panicles, spikelets pers f 40 h

level.c

rB

G

D

atptdbo1wbte

(sAsd

wapa82teretS

st

quare meter, filled-grain percentage, and filled-grain weight are means of plants o* F values significant at the P = 0.05 level. NS means non-significant at the P = 0.05

olumn.

ate (G) during this period was therefore calculated from t1 to t2.oth G and D were calculated as the formulas:

= AK

2(N + 2)(3)

= 2(N + 2)K

. (4)

Activities of SuSase and AGPase in grains were determinedccording to the method described by Yang et al. (2003b). Briefly,he sampled grains were dehulled and were homogenized with aestle in a pre-cooled mortar that contained 8 ml frozen extrac-ion medium: 100 mM HEPES-NaOH (pH 7.6), 8 mM MgCl2, 5 mMithiothreital, 2 mM EDTA, 12.5% (v/v) glycerol, and 5% (w/v) insolu-le polyvinylpyrrolidone 40. After being filtered through four layersf cheesecloth, the homogenate was centrifuged at 12 000 × g for0 min, and the supernatant was used for the enzyme assay. SuSaseas assayed in the cleavage direction and analyzed as described

y Ranwala and Miller (1998). AGPase was determined accordingo the method of Nakamura et al. (1989). The activities of bothnzymes were expressed on the basis of fresh weight (FW).

Total aboveground biomass was measured at the heading time80% panicles emerged). Plants from six hills in each plot wereampled from the third row in order to minimize border effect.ll plant samples were separated into green leaf blades, culm andheath (stem), and panicles. Dry matter of each component wasetermined after drying at 70 ◦C to constant weight and weighed.

The method for extraction of non-structural carbohydrate (NSC)as modified according to the method described by Yoshida et

l. (1976). The sample was dried in an oven and ground into fineowder. In a 15-ml centrifuge tube, 10 ml of 630 g l−1 ethanol wasdded to 100 mg of ground sample and kept in a water bath at0 ◦C for 30 min. The tube was then centrifuged at 365.9 × g for0 min after cooling. The supernatant was collected and the extrac-ion was repeated three times. The alcohol in the supernatant wasvaporated on a water bath at 80 ◦C until most of the alcohol wasemoved and the volume was reduced to about 3 ml. The sugarxtract was then diluted to 25 ml with distilled water. The concen-ration of sugars in the extract was then analyzed as described by

omogyi (1945).

The residue left in the centrifuge tube was dried at 80 ◦C fortarch extraction. 2 ml of distilled water was added to the tube con-aining the dried residue. The tube was then shaken in a boiling

ills from four plots of each variety.Different letters indicate statistical significance at the P = 0.05 level within the same

water bath for 30 min. 2 ml of 9.36 M HClO4 was added to the tubeafter cooling. The solution was shaken for 15 min. The extract wasthen made up to about 10 ml and centrifuged at 365.9 × g for 20 min.The supernatant was collected and 2 ml of 4.68 M HClO4 was addedto the residue. The extraction was repeated as above. The super-natants were combined and made up to 50 ml with distilled water.The starch was analyzed by the method of Pucher et al. (1948).

Plants of 10 hills from each plot were harvested at maturity formeasurement of endosperm and grain weight of fertilized supe-rior and inferior spikelets. Plants from a 5-m2 area in each plot(except those in border rows) were harvested at maturity for thedetermination of grain yield. Yield components: panicles per squaremeter, spikelets per panicle, filled-grain percentage, filled-grainweight, and fertilized grain weight of superior and inferior spikeletswere determined from plants of 10 hills (excluding those in borderrows) from each plot. The filled grain percentage was defined as thenumber of filled grains that sank to the bottom of a breaker filledwith salt solution with specific gravity of 1.06 as a percentage oftotal spikelets (both fertilized and unfertilized spikelets). The fertil-ized grains (excluding unfertilized spikelets) were both filled grains(specific gravity ≥1.06) and unfilled grains (specific gravity <1.06).

2.3. Statistical analysis

Analysis of variance was performed using SAS/STAT statisti-cal analysis package (version 6.12, SAS Institute, Cary, NC, USA).The statistical model used included sources of variation due toreplication, year, variety, and the interaction of year × variety inExperiment I, and due to replication, N treatment, variety, andinteractions of year × variety, year × N treatment, and variety × Ntreatment in Experiment II. Data from each sampling date were ana-lyzed separately. Means were tested by least significant differenceat P = 0.05 (LSD0.05).

3. Results

3.1. Grain yield and yield components

Table 2 shows the grain yield and yield components of trialvarieties and their analysis-of-variance (F values). There existedsignificant differences in grain yield among the three types of vari-

174 J. Fu et al. / Field Crops Research 123 (2011) 170–182

Table 3Parameters of Richards’ growth equation for fitting division processes of endosperm cells of superior and inferior spikelets of rice in the field Experiment I.a

Year/variety Superior spikelets Inferior spikelets

A B K N R2 A B K N R2

2007LYPJ 237.4 24514 1.094 3.320 0.999 155.1 c 1.17 × 106 0.998 5.841 0.988HD 9 250.0 24270 1.101 3.180 0.998 159.6 b 8.09 × 105 0.980 5.450 0.987NPT1 232.0 19072 1.087 3.157 0.999 133.4 e 1.12 × 107 0.989 5.063 0.996NPT2 230.0 23040 1.101 3.191 0.999 129.3 f 1.15 × 107 0.987 5.301 0.997YD 6 234.9 22181 1.098 3.148 0.999 171.2 a 1.52 × 106 1.048 5.648 0.994ZD 88 231.7 17962 1.089 3.091 0.999 172.5 a 1.32 × 106 1.049 5.267 0.994

2008LYPJ 236.2 26056 1.094 3.315 0.999 145.6 d 1.09 × 106 0.979 5.972 0.986HD 9 250.0 23459 1.105 3.179 0.997 151.2 c 7.16 × 105 0.989 5.330 0.987NPT1 234.8 26030 1.096 3.114 0.999 119.5 g 9.43 × 106 0.967 5.160 0.997NPT2 232.8 22355 1.096 3.259 0.999 113.7 h 9.70 × 106 0.965 5.315 0.998

0.9990.994

wth e

et(dioprvoac

2atsigbtw

3i

p

TP

YD 6 239.3 22449 1.097 3.211ZD 88 235.9 24149 1.085 3.129

a A represents the maximum endosperm cell number calculated by Richards’ gro

ties. Generally, the two super rice varieties (LYPJ and HD 9) showedhe highest grain yield, and followed by the two check varietiesYD 6 and ZD 88), and the two NPT lines (NPT1 and NPT 2) pro-uced the lowest yield. A higher grain yield in super rice than that

n check rice was mainly due to a larger sink capacity (total numberf spikelets per m2) as a result of a larger panicle (more spikeletser panicle). However, the filled grain percentage of the two superice varieties was significantly lower than that of the two checkarieties. The lowest grain yield in NPT rice among the three typesf varieties was mainly attributed to the lowest filled grain percent-ge, although the NPT rice produced a larger sink capacity than theheck rice (Table 2).

For a given variety, the grain yield was higher in 2007 than in008 (Table 2). A higher grain yield in the former year was mainlyttributed to a higher filled grain percentage and filled grain weighthan in the latter year, probably due to a better climate (more sun-hine hours) around the heading and at the early grain filling stagen 2007 than in 2008 (Fig. 1). The differences in grain yield and filledrain percentage between the two study years were significant foroth super rice and NPT rice, whereas they were not significant forhe check rice, indicating that the response of grain filling to climateould vary with rice varieties.

.2. Endosperm cell number and grain filling of superior and

nferior spikelets

Parameters of Richards’ growth equation for fitting divisionrocesses of endosperm cells and grain filling of superior and infe-

able 4arameters of Richards’ growth equation for fitting grain filling processes of superior and

Year/variety Superior spikelets

A B K N R2

2007LYPJ 22.68 9854 0.520 2.089 0.99HD 9 23.81 9853 0.521 2.087 0.99NPT1 22.49 9854 0.519 2.088 0.99NPT2 22.29 9857 0.519 2.086 0.99YD 6 22.56 9856 0.518 2.094 0.99ZD 88 22.56 9855 0.516 2.101 0.99

2008LYPJ 22.48 9854 0.521 2.095 0.99HD 9 23.72 9854 0.522 2.082 0.99NPT1 22.51 9855 0.518 2.085 0.99NPT2 22.54 9859 0.516 2.089 0.99YD 6 22.53 9854 0.518 2.095 0.99ZD 88 22.51 9904 0.514 2.089 0.99

a A represents the final growth weight of a grain calculated by Richards’ growth equati

170.2 a 1.73 × 106 1.055 5.855 0.994172.8 a 1.45 × 106 1.051 5.353 0.995

quation (×103 cells per endosperm), and R2 is the determinant coefficient.

rior spikelets were listed in Tables 3 and 4, and the maximumendosperm cell number (MECN), mean endosperm cell division rate(MECDR), endosperm cell weight, and endosperm weight (EW) ofboth superior and inferior spikelets were shown in Table 5. The ear-lier flowered superior spikelets exerted dominance over the laterflowered inferior spikelets in MECN, MECDR, and EW. For superiorspikelets, there was no significant difference in MECN, MECDR, andEW among the six varieties except HD 9 which exhibited a greaterMECN, MECDR, and EW than any other varieties (Table 5). For infe-rior spikelets, significant differences existed among the six varietiesin MECN, MECDR, and EW. In general, the two check rice varietiesshowed the greatest MECN, MECDR, and EW, and followed by thetwo super rice varieties, and the two NPT lines showed the smallest,in good agreement with filled grain percentage (refer to Table 2).For a given variety and for superior spikelets, no significant dif-ference was observed in MECN, MECDR, and EW between the twostudy years. However, inferior spikelets of the super rice and NPTrice showed a greater MECN, MECDR, and EW in 2007 than in 2008(Table 5). For a same kind of spikelets, the difference in endospermcell weight was not significant among the six varieties or betweenthe two study years, suggesting that endosperm cell number playsa dominant role in determination of endosperm weight in rice.

Very similar to the MECDR and EW, the mean grain filling rate(MGFR) and grain weight of superior spikelets were much greater

than those of inferior spikelets (Table 6). Except those of HD 9, theMGFR and grain weight of superior spikelets were not significantamong the varieties or between the two study years for a givenvariety. The MGFR and grain weight of inferior spikelets were the

inferior spikelets of rice in the field Experiment I.a

Inferior spikelets

A B K N R2

2 15.40 76618 0.398 3.105 0.9994 15.95 73078 0.402 3.054 0.9982 12.54 79225 0.396 3.125 0.9972 12.01 85849 0.395 3.129 0.9972 17.95 72223 0.411 3.093 0.9961 17.98 72222 0.410 3.065 0.995

2 14.62 73204 0.394 3.085 0.9974 15.29 73149 0.396 3.042 0.9981 11.09 76058 0.393 3.159 0.9977 10.26 79900 0.391 3.154 0.9961 17.55 72222 0.412 3.051 0.9953 17.55 72222 0.413 3.055 0.996

on (mg per dehulled grain), and R2 is the determinant coefficient.

J. Fu et al. / Field Crops Research 123 (2011) 170–182 175

Table 5The maximum endosperm cell number (MECN, ×103 cells per endosperm), mean endosperm cell division rate (MECDR, ×103 cells per endosperm per day), endosperm cellweight (ECW, ng per cell), and endosperm weight (EW, mg per endosperm) of superior and inferior spikelets of rice in the field Experiment I.a

Year/variety Superior spikelets Inferior spikelets

MECN MECDR ECWb EW MECN MECDR ECW EW

2007LYPJ 239 b 24.41 b 90.67 a 21.67 b 159 c 9.87 e 90.44 a 14.38 cHD 9 251 a 26.57 a 89.68 a 22.51 a 164 b 10.70 c 90.00 a 14.76 bNPT1 234 b 24.45 b 92.39 a 21.62 b 137 e 9.34 f 91.31 a 12.51 eNPT2 229 b 24.39 b 93.84 a 21.49 b 132 f 8.74 g 91.14 a 12.03 fYD 6 237 b 25.05 b 90.21 a 21.38 b 176 a 11.73 b 92.44 a 16.27 aZD 88 233 b 24.78 b 91.29 a 21.27 b 177 a 12.45 a 92.71 a 16.41 a2008LYPJ 238 b 24.31 b 91.01 a 21.66 b 146 d 8.94 g 89.93 a 13.13 dHD 9 252 a 26.67 a 89.09 a 22.45 a 158 c 10.20 d 89.62 a 14.16 cNPT1 238 b 25.16 b 90.09 a 21.44 b 122 g 8.07 h 90.32 a 11.02 gNPT2 232 b 24.26 b 91.76 a 21.29 b 115 h 7.50 i 92.43 a 10.53 hYD 6 241 b 25.19 b 88.34 a 21.29 b 174 a 11.43 b 92.07 a 16.02 aZD 88 236 b 24.95 b 90.17 a 21.28 b 177 a 12.35 a 91.98 a 16.28 a

. ValueV fferen

.

gtEfss2gysafi

3

iiasbsa

TA

f

a The mean cell division rate is calculated according to Richards’ growth equationalues of the endosperm weight are means of plants of 40 hills from each variety. Dib Endosperm cell weight = endosperm weight/maximum endosperm cell number

reatest for the check rice, and the smallest for the NPT rice, andhe intermediate for the super rice, consistent with the MECDR,W, and filled grain percentage (refer to Tables 2 and 5). The dif-erences in MGFR and grain weight of inferior spikelets were notignificant for the check rice between the two study years, but bothuper rice and NPT rice showed a greater MGFR and grain weight in007 than in 2008. There was no significant difference in the activerain filling period among the varieties and between the two studyears for superior spikelets. The active grain filling period of inferiorpikelets was shorter in check rice than in super rice or NPT rice,nd showed no significant difference between the two study yearsor a given variety, suggesting that grain weight of inferior spikeletss mainly determined by the grain filling rate in this experiment.

.3. SuSase and AGPase activities in superior and inferior spikelets

Consistent with the MECDR, EW, MGFR, and grain weight, activ-ties of SuSase and AGPase were higher in superior spikelets than innferior spikelets at 4, 8 and 12 DPA for both super rice and NPT ricend at 4 and 8 DPA for the check rice (Fig. 2). Except ZD 88 which

howed no significant difference in SuSase and AGPase activitiesetween superior and inferior spikelets at mid and late grain fillingtages (16 or 20 DPA and thereafter), other five varieties showedhigher enzymatic activity in inferior spikelets than in superior

able 6ctive grain filling period (AGFP), mean grain filling rate (MGFR) and grain weight of fert

Year/variety Superior spikelets

AGFP (d) MGFR (mg grian−1 d−1) Grain Wt (mg grian−

2007LYPJ 15.73 a 1.442 b 28.2 bHD 9 15.69 a 1.518 a 29.1 aNPT1 15.75 a 1.428 b 28.0 bNPT2 15.75 a 1.416 b 27.8 bYD 6 15.81 a 1.427 b 27.6 bZD 88 15.90 a 1.419 b 27.5 b

2008LYPJ 15.72 a 1.430 b 28.1 bHD 9 15.64 a 1.517 a 29.0 aNPT1 15.77 a 1.427 b 27.8 bNPT2 15.85 a 1.422 b 27.6 bYD 6 15.81 a 1.425 b 27.5 bZD 88 15.91 a 1.415 b 27.1 b

a The active grain filling period and mean grain filling rate are calculated according torom four plots of each variety. Different letters indicate statistical significance at the P = 0

s of the maximum endosperm cell number are means of six observed endosperms.t letters indicate statistical significance at the P = 0.05 level within the same column.

spikelets during this period when enzyme activity was expressedon the basis of fresh weight. At the same measurement time, therewere no significant differences in activities of both enzymes insuperior spikelets among the six varieties or between the two studyyears for a given variety. However, activities of SuSase and AGPasein inferior spikelets showed much difference among the varietiesat the early grain filling stage (4–12 DPA). The two check varietiesshowed the greatest activity of both enzymes in inferior spikelets,and followed by the two super rice varieties, and the two NPT linesshowed the smallest. Activities of SuSase and AGPase in inferiorspikelets were higher in 2007 than in 2008 for super rice or NPTrice, but they showed no significant difference between the twostudy years for check rice (Fig. 2).

3.4. Aboveground biomass and NSC in the stem at the headingtime and in relation with sink strength

Generally, aboveground biomass at the heading time was thegreatest for the two super rice varieties, and was the smallest forthe two check varieties, and was intermediate for the two NPT lines

(Fig. 3A). However, check rice showed the greatest ratio of above-ground biomass to number of spikelets (aboveground biomass perspikelet), followed by super rice, and NPT rice was smallest. Therewas no significant difference in aboveground biomass or in above-

ilized superior and inferior spikelets of rice in the field Experiment I.a

Inferior spikelets

1) AGFP (d) MGFR (mg grian−1d−1) Grain Wt (mg grian−1)

25.65 bc 0.600 c 20.5 c25.14 de 0.634 b 21.4 b25.88 abc 0.484 e 17.8 e25.97 ab 0.462 e 17.1 f24.78 ef 0.724 a 23.2 a24.71 ef 0.728 a 23.4 a

25.81 bc 0.566 d 19.6 d25.46 cd 0.600 c 20.5 c26.25 ab 0.422 f 16.3 g26.36 a 0.389 g 15.7 h24.52 f 0.716 a 22.8 a24.48 f 0.717 a 23.2 a

Richards’ growth equation. Values of grain weight are means of plants of 40 hills.05 level within the same column.

176 J. Fu et al. / Field Crops Research 123 (2011) 170–182

Fig. 2. Activities of sucrose synthase (A, C, E, G, I, and K) and adenosine diphosphoglucose pyrophosphorylase (B, D, F, H, J, and L) in superior and inferior spikelets duringthe grain filling period of rice in the field Experiment I. Vertical bars represent ±S.D. of the mean (n = 4) where these exceed the size of the symbol.

J. Fu et al. / Field Crops Research 123 (2011) 170–182 177

F tural cr e thes

gg

ttttpttpsb

shtifiwgwsbps(

3s

tcig(

ig. 3. Aboveground biomass (A), aboveground biomass per spikelet (B), non-strucice in the field Experiment I. Vertical bars represent ±S.D. of the mean (n = 4) wher

round biomass per spikelet between the two study years for aiven variety (Fig. 3B).

Among the six trial varieties, the super rice variety of HD 9 andwo check varieties of YD 6 and ZD 88 accumulated the most NSC inhe stem (culms and sheaths) at the heading time, and followed byhe super rice variety of LYPJ, and the two NPT lines accumulatedhe least (Fig. 3C). The ratio of NSC to number of spikelets (NSCer spikelet) was the greatest for the two check varieties, and washe smallest for the two NPT lines, and was intermediate for thewo super rice varieties (Fig. 3D). The NSC accumulation and NSCer spikelet were more or greater in 2007 than in 2008 for bothuper rice and NPT rice, but they showed no significant differenceetween the two study years for the check rice (Fig. 3C and D).

Correlation analysis showed that aboveground biomass perpikelet, NSC accumulation in the stem, and NSC per spikelet at theeading time were positively and very significantly correlated withhe sink strength (the maximum endosperm cell number and activ-ties of Susase and AGPase in the grains at 4–12 DPA), rates of grainlling and endosperm cell division, endosperm weight, and graineight of inferior spikelets (r = 0.89**–0.98**, Fig. 4, A2–G4). Above-

round biomass at the heading time was not significantly correlatedith sink strength, endosperm weight, and grain weight of inferior

pikelets (r = −0.16 to −0.45, Fig. 4, A1–G1). Neither abovegroundiomass/NSC accumulation in the stem nor aboveground biomasser spikelet/NSC per spikelet was significantly correlated with theink strength and grain or endosperm weight of superior spikeletsr = −0.082 to 0.459, data not shown).

.5. Effect of mid-season N application on grain yield, NSC in thetem, and sink strength

When compared with the control (T0, no N was applied duringhe mid-season), the treatments of N applied at the stages of pani-

le initiation (T1), spikelet differentiation (T2), and at both paniclenitiation and spikelet differentiation (T3) significantly increasedrain yield for both super rice varieties and in both study yearsTable 7). Increase in grain yield was most for the T2 treatment, fol-

arbohydrate (NSC) in the stem (C), and NSC per spikelet (D) at the heading time ofe exceed the size of the symbol.

lowed by the T3 treatment, and the least for the T1 treatment. BothT1 and T3 treatments significantly increased spikelets per panicle,but significantly reduced filled grain percentage, while the T2 treat-ment significantly increased both spikelets per panicle and filledgrain percentage (Table 7).

All the treatments of N application during the mid-season (T1,T2, and T3) significantly increased aboveground biomass at theheading time, but significantly decreased the ratio of abovegroundbiomass to the number of spikelets (aboveground biomass perspikelet) when compared with the control (Table 8). The increaseor decrease was the most for T1, the least for T2, and intermediatefor T3 treatments. The T1, T2, and T3 treatments also significantlyincreased NSC accumulation in the stem at the heading time, withthe most increase for T2, the least for T1, and the intermediate forT3 treatments (Table 8). Both T1 and T3 treatments significantlyreduced, whereas T2 treatments significantly increased, the ratioof NSC in the stem to the number of spikelets (NSC per spikelet), ingood agreement with the filled grain percentage (refer to Table 7).

Parameters of Richards’ growth equation for fitting divisionprocesses of endosperm cells and grain filling processes of infe-rior spikelets of rice under various nitrogen treatments during themid-season were listed in Table 9. For both super rice varietiesand for both study yeas, the T2 treatment significantly increased,while both T1 and T3 treatments significantly reduced, the maxi-mum endosperm cell number, mean endosperm cell division rate,endosperm weight, mean grain filling rate, grain weight, and activ-ities of SuSase and AGPase of the inferior spikelets at the earlygrain filling stage (4–12 DPA) when compared with the con-trol (Tables 10 and 11). All the N application treatments had nodetectable effect (P > 0.05) on endosperm development, grain fill-ing, and SuSase and AGPase activities of superior spikelets (data notshown).

4. Discussion

Our results showed that the two super rice varieties pro-duced 28–30% and the two NPT lines produced 30–33% more

178 J. Fu et al. / Field Crops Research 123 (2011) 170–182

Fig. 4. Correlations of aboveground biomass (AGB) (A1-G1), AGB per spikelet (A2-G2), non-structural carbohydrate (NSC) in the stem (A3-G3), and NSC per spikelet (A4-G4)at the heading time with the activities of sucrose synthase (SuSase) (A1-A4) and adenosine diphosphoglucose pyrophosphorylase (AGPase) (B1-B4) at the early grain fillingstage, grain filling rate (C1-C4), grain weight (D1-D4), the maximum endosperm cell number (E1-E4), mean endosperm cell division rate (F1-F4), and endosperm weight(G1-G4)) of inferior spikelets of rice in the field Experiment I. Data used for the calculation are from Tables 5 and 6 and Figs. 2 and 3. Activities of SuSase and AGPase aremeans of the determinations at 4, 8 and 12 days post-anthesis. Correlation coefficients (r) are calculated and asterisks (**) represent statistical significance at P = 0.01 (n = 12).

J. Fu et al. / Field Crops Research 123 (2011) 170–182 179

Table 7Grain yield and yield components of rice under various nitrogen (N) treatments during the mid-season in the field Experiment II.a

Year Variety Nitrogen treatment Grain yield (t ha−1) Panicles per m2 Spikelets per panicle Filled grains (%) Filled grain weight (mg)

2007 LYPJ T0 7.66 e 201 a 176 g 80.5 c 25.8 cdT1 8.97 d 203 a 252 a 65.8 e 25.2 eT2 10.45 a 207 a 227 c 84.3 b 25.6 deT3 9.70 b 202 a 243 b 74.7 d 25.4 de

HD 9 T0 7.89 e 204 a 168 h 84.5 b 26.2 bcT1 9.20 cd 206 a 220 d 73.8 d 26.3 bT2 10.26 a 208 a 202 f 87.6 a 26.8 aT3 9.62 bc 205 a 211 e 81.3 c 26.5 ab

2008 LYPJ T0 7.34 e 203 a 174 f 78.8 c 25.7 cdT1 8.39 d 208 a 253 a 63.3 f 25.1 eT2 9.66 a 210 a 222 c 81.5 b 25.6 dT3 9.11 b 206 a 235 b 74.6 d 25.2 e

HD 9 T0 7.26 e 208 a 164 g 82.8 b 26.1 bcT1 8.56 c 206 a 221 c 71.6 e 26.2 bT2 9.65 a 210 a 201 e 85.5 a 26.7 aT3 9.03 bc 206 a 211 d 79.7 c 26.3 ab

Analysis of varianceYear 17* <1 <1 12* <1Variety 2NS <1 36* 21* 14*

Treatment 96* <1 178* 48* 6*

Year × variety <1 <1 <1 <1 <1Year × treatment <1 <1 <1 <1 <1Variety × treatment <1 <1 2NS <1 <1

a T0, T1, T2, and T3 represent no N application during the mid-season, N application at panicle initiation, N application at spikelet differentiation, and N application at bothpanicle initiation and spikelet differentiation, respectively (refer to Table 1). Values of grain yield are means of plants of 20 m2 harvested from four plots of each treatment.V and fi

.05 levs

s(aceobryal(

TTo

btD

alues of panicles per square meter, spikelets per panicles, filled-grain percentage,* F values significance at the P = 0.05 level. NS means non-significant at the P = 0

ame year and the same column.

pikelets per square meter than the two elite check varietiesTable 2), and confirmed earlier reports that both super ricend NPT rice have great yield potential due to their large sinkapacity as a result of numerous spikelets per panicle (Chengt al., 2007; Katsura et al., 2007; Peng et al., 1999, 2008). Webserved, however, that the super rice only increased grain yieldy 6–15%, whereas the NPT rice reduced grain yield by 10–16%,elative to the check rice. The limitation to realization of greatield potential in super rice, especially in NPT rice, was mainly

ttributed to the low filled grain percentage as a result of aow grain filling rate and small grain weight of inferior spikeletsTable 6).

able 8otal number of spikelets, aboveground biomass (AGB), non-structural carbohydrate (NSCf rice under various nitrogen (N) treatments during the mid-season in the field Experim

Year Variety Nitrogen treatment Spikelets per m2 (×103) A

2007 LYPJ T0 35.42 g 1T1 51.08 a 1T2 47.02 c 1T3 49.01 b 1

HD 9 T0 34.27 h 1T1 45.32 d 1T2 42.02 f 1T3 43.26 e 1

2008 LYPJ T0 35.29 g 1T1 52.58 a 1T2 46.70 c 1T3 48.33 b 1

HD 9 T0 34.11 h 1T1 45.53 d 1T2 42.21 f 1T3 43.47 e 1

a T0, T1, T2, and T3 represent no N application during the mid-season, N applicationoth panicle initiation and spikelet differentiation, respectively (refer to Table 1). Valuesreatment. Values of aboveground biomass (AGB) and non-structural carbohydrate (NSC) iifferent letters indicate statistical significance at the P = 0.05 level within the same year

lled-grain weight are means of plants of 40 hills from four plots of each treatment.el. Different letters indicate statistical significance at the P = 0.05 level within the

Either small sink size and/or low sink activity would contributeto a low grain filling rate and small grain weight of inferior spikelets.In rice, the endosperm contributes more than 90% of the final grainweight of a caryopsis (Cao et al., 1992; Murata and Matsushima,1975). Thus, sink size is mainly determined by the number of cellsand the cell size of the endosperm. Our results showed that thedifference in endosperm cell weight was not significant amongrice varieties or between superior and inferior spikelets (Table 5).Endosperm weight and grain weight of inferior spikelets were

closely associated with the maximum endosperm cell number, andgrain filling rate was very consistent with the endosperm cell divi-sion rate for inferior spikelets (Tables 5 and 6). The result suggests

) in stems and sheaths, AGB per spikelet, and NSC per spikelets at the heading timeent II.a

GB (t ha−1) NSC (t ha−1) AGB per spikelet(mg spikelet−1)

NSC per spikelet(mg spikelet−1)

0.54 e 2.25 e 29.76 b 6.35 d3.59 a 2.66 c 26.60 e 5.21 g2.81 cd 3.25 a 27.25 d 6.91 c3.15 b 2.98 b 26.83 e 6.08 e0.67 e 2.42 d 31.13 a 7.06 b3.07 bc 2.65 c 28.84 c 5.85 f2.53 d 3.28 a 29.82 b 7.81 a2.78 cd 2.98 b 29.55 b 6.89 c

0.44 e 2.21 e 29.59 b 6.26 d3.61 a 2.64 c 25.88 c 5.02 g2.79 bc 3.22 a 27.39 d 6.89 b3.06 b 2.86 b 27.02 e 5.92 e0.89 d 2.38 d 31.92 a 6.98 b3.15 b 2.62 c 28.88 c 5.75 f2.59 c 3.19 a 29.83 b 7.56 a2.86 bc 2.83 b 29.59 b 6.51 c

at panicle initiation, N application at spikelet differentiation, and N application atof spikelets per square meter are means of plants of 40 hills from four plots of eachn stems and sheaths are means of plant of 24 hills from four plots of each treatment.and the same column.

180 J. Fu et al. / Field Crops Research 123 (2011) 170–182

Table 9Parameters of Richards’ growth equation for fitting division processes of endosperm cells and grain filling processes of inferior spikelets of rice under various nitrogen (N)treatments during the mid-season in the field Experiment II.a

Year/variety N treatment Endosperm cell division Grain filling

Ab B K N R2 A B K N R2

2007LYPJ T0 158.4 1.06 × 107 0.929 5.013 0.996 15.35 9.75 × 105 0.506 3.805 0.998

T1 144.8 1.27 × 107 0.919 5.342 0.997 12.96 1.04 × 106 0.498 3.882 0.996T2 165.5 1.14 × 107 0.929 5.121 0.996 16.05 9.53 × 105 0.512 3.912 0.995T3 154.6 1.18 × 107 0.916 5.215 0.995 14.58 9.88 × 105 0.501 3.551 0.994

HD 9 T0 162.5 1.03 × 107 0.919 4.896 0.994 15.83 9.26 × 105 0.515 3.882 0.997T1 148.9 1.21 × 107 0.883 5.016 0.997 13.57 1.09 × 106 0.489 3.842 0.993T2 170.6 1.14 × 107 0.903 4.935 0.998 16.64 1.01 × 106 0.511 3.946 0.995T3 159.2 1.18 × 107 0.901 4.989 0.995 15.21 1.05 × 106 0.508 3.783 0.996

2008LYPJ T0 156.9 1.09 × 107 0.922 5.027 0.985 15.15 9.55 × 105 0.498 3.707 0.995

T1 143.6 1.29 × 107 0.905 5.365 0.991 12.67 1.06 × 106 0.487 3.719 0.998T2 164.6 1.17 × 107 0.929 5.135 0.986 15.66 9.67 × 105 0.486 3.563 0.996T3 153.1 1.22 × 107 0.912 5.214 0.997 14.25 9.56 × 105 0.485 3.341 0.994

HD 9 T0 160.9 1.08 × 107 0.916 4.989 0.987 15.45 9.19 × 105 0.501 3.617 0.997T1 148.2 1.25 × 107 0.887 5.115 0.992 13.28 9.53 × 105 0.495 3.861 0.996T2 169.5 1.17 × 107 0.894 4.952 0.989 16.45 9.41 × 105 0.486 3.544 0.995T3 158.8 1.21 × 107 0.891 5.035 0.992 14.78 1.02 × 106 0.478 3.336 0.994

a T0, T1, T2, and T3 represent no N application during the mid-season, N application at panicle initiation, N application at spikelet differentiation, and N application at bothp

) or tg

tmwrs

tba(eiLias

TTi

pRw

anicle initiation and spikelet differentiation, respectively (refer to Table 1).b A represents the maximum endosperm cell number (×103 cells per endosperm

rowth equation, and R2 is the determinant coefficient.

hat cell number of rice endosperm plays a dominant role in deter-ining sink size and grain weight. Slow grain filling and low graineight for inferior spikelets of rice, especially for those of super

ice and NPT rice, would be attributed, at least partly, to their slowink enlargement and small sink size.

It was hypothesized that high levels of enzymes involved inhe breakdown of sucrose in the sink would increase sink activityy lowering the local concentration of sucrose, thereby generatinggradient that allows further unloading of sucrose from phloem

Liang et al., 2001; Ranwala and Miller, 1998). Since SuSase is a mainnzyme that involves in sucrose cleavage in rice grains, its activ-ty is regarded as biochemical markers of sink activity (Kato, 1995;iang et al., 2001; Yang et al., 2003b). On the other hand, AGPase

s considered as a key enzyme involved in starch synthesis, and itsctivity is closely associated with the rate and quantity of starchynthesis (Ahmadi and Baker, 2001; Hurkman et al., 2003; Yang

able 10he mean grain filling rate (MGFR), grain weight, maximum endosperm cell number (MECnferior spikelets of rice under various nitrogen (N) treatments during the mid-season in

Year Variety Nitrogentreatment

MGFR(mg grian−1 d−1)

Grain weight(mg grian−1)

2007 LYPJ T0 0.669 c 21.2 cT1 0.591 e 18.6 fT2 0.695 b 21.8 bT3 0.658 c 20.6 d

HD 9 T0 0.693 b 21.9 bT1 0.614 d 19.5 eT2 0.715 a 22.6 aT3 0.668 c 21.2 c

2008 LYPJ T0 0.661 c 20.9 cT1 0.582 e 18.4 fT2 0.684 b 21.6 bT3 0.647 c 20.4 d

HD 9 T0 0.689 b 21.6 bT1 0.603 d 19.3 eT2 0.721 a 22.4 aT3 0.662 c 20.8 cd

a T0, T1, T2, and T3 represent no N application during the mid-season, N application at panicle initiation and spikelet differentiation, respectively (refer to Table 1). The mean gichards (1959) equation. Values of the maximum endosperm cell number are means ofeight are means of plants of 40 hills from each treatment. Different letters indicate stat

he final growth weight of a grain (mg per dehulled grain) calculated by Richards’

et al., 2004). Our results showed that activities of both SuSase andAGPase at the early grain filling stage were much lower in inferiorspikelets than in superior spikelets, and were significantly lower inthe inferior spikelets of super rice or NPT rice than in those of checkrice (Fig. 2). The results indicate that a low sink activity may alsocontribute to a slow grain filling and low grain weight for inferiorspikelets of rice, especially for those of super rice and NPT rice.

The cause for the poor sink strength of inferior spikelets resultedfrom either small sink size and low sink activity is yet to be under-stood. We observed that the two check varieties that showed ahigher filled grain percentage accumulated more NSC in the stemat the heading time than the two super rice varieties or the twoNPT lines that had a lower filled grain percentage (Fig. 3). Both

NSC accumulation in the stem and NSC per spikelet at the head-ing time were positively and very significantly correlated with thesink strength (the maximum endosperm cell number and activ-

N), mean endosperm cell division rate (MECDR), and endosperm weight of fertilizedthe field Experiment II.a

MECN (×103

cells endosperm−1)MECDR (×103

cells endosperm−1 d−1)Endosperm weight(mg endosperm−1)

162 d 10.49 c 15.64 c148 g 9.06 g 13.22 f169 b 10.79 b 16.09 b158 e 9.81 e 15.02 d166 c 10.83 b 16.16 b152 f 9.37 f 13.95 e174 a 11.11 a 16.88 a163 d 10.26 d 15.45 c

160 d 10.29 c 15.24 d146 g 8.82 g 13.09 g168 b 10.72 ab 16.13 b156 e 9.68 e 14.78 e164 c 10.54 b 15.72 c151 f 9.24 f 13.81 f172 a 10.90 a 16.63 a161 d 10.06 d 15.08 d

anicle initiation, N application at spikelet differentiation, and N application at bothrain filling rate and mean endosperm cell division rate are calculated according tosix observed endosperms. Values of the fertilized grain weight and endosperm cellistical significance at the P = 0.05 level within the same year and the same column.

J. Fu et al. / Field Crops Research 123 (2011) 170–182 181

Table 11Activities of sucrose synthase (SuSase) and adenosine diphosphoglucose pyrophosphorylase (AGPase) in the fertilized inferior spikelets of rice at 4, 8 and 12 days post-anthesis(DPA) under various nitrogen (N) treatments during the mid-season in the field Experiment II.a

Year Variety Nitrogen treatment SuSase (�mol g−1 FW min−1) AGPase (�mol g−1 FW min−1)

4 DPA 8 DPA 12 DPA 4 DPA 8 DPA 12 DPA

2007 LYPJ T0 3.69 d 5.67 c 6.65 c 2.49 e 4.87 c 5.97 cT1 2.54 f 4.41 e 5.34 f 1.85 g 3.51 e 4.29 eT2 4.32 b 6.47 b 7.54 b 2.81 d 5.85 b 6.92 bT3 3.27 e 5.35 d 6.32 d 2.17 f 4.46 d 5.64 d

HD 9 T0 4.35 b 6.38 b 7.43 b 3.45 b 5.94 b 6.85 bT1 3.45 e 5.37 d 5.79 e 2.76 d 3.67 e 5.52 dT2 4.76 a 7.16 a 7.85 a 3.77 a 6.27 a 7.46 aT3 3.98 c 5.84 c 6.36 d 3.14 c 5.05 c 5.93 c

2008 LYPJ T0 3.54 c 5.55 c 6.47 c 2.25 d 4.68 c 5.81 cT1 2.52 f 4.12 e 5.19 f 1.64 f 3.42 f 4.05 eT2 4.28 b 6.21 b 7.35 b 2.67 c 5.67 b 6.86 bT3 2.89 e 5.16 d 6.11 d 1.92 e 4.21 d 5.43 d

HD 9 T0 4.24 b 6.35 b 7.38 b 3.21 b 5.69 b 6.74 bT1 3.21 d 5.17 d 5.65 e 2.44 d 3.53 e 5.38 dT2 4.63 a 7.05 a 7.73 a 3.52 a 6.12 a 7.27 aT3 3.46 c 5.56 c 6.14 d 2.75 c 4.78 c 5.75 c

a on at pp easuP

ioesopafsetsgpgcan

cri2iclwtdpsiatcsdiasar

T0, T1, T2, and T3 represent no N application during the mid-season, N applicatianicle initiation and spikelet differentiation, respectively (refer to Table 1). Each m= 0.05 level within the same year and the same column.

ties of SuSase and AGPase at the early grain filling stage), ratesf grain filling and endosperm cell division, grain weight, andndosperm weight of inferior spikelets (Fig. 4). The results demon-trate that less NSC accumulation in the stem at the heading timer a low ratio of NSC to number of spikelets may account for theoor sink strength of inferior spikelets. The results imply that pre-nthesis NSC reserves in the stem not only are used as assimilatesor grain filling but also play an important role in enhancing sinktrength at the early grain filling stage, and consequently accel-rate endosperm development and grain filling. It is noteworthyhat, although aboveground biomass at the heading time was notignificantly correlated with the sink strength, the ratio of above-round biomass to number of spikelets (aboveground biomasser spikelet) was significantly correlated with sink strength andrain/endosperm weight of inferior spikelets (Fig. 4, A2-G2), indi-ating that an increase in aboveground biomass before heading islso very important to increase sink strength for rice varieties withumerous spikelets per panicle, such as super rice and NPT rice.

Increase in grain yield not only needs to expand yield sinkapacity by increasing the number of spikelets per panicle but alsoequires a high filling efficiency of inferior spikelets by enhanc-ng sink strength (Kato and Takeda, 1996; Kato et al., 2007; Yang,010; Yang and Zhang, 2010). The question arises as to whether

t is possible to increase both the number of spikelets per pani-le and sink strength of inferior spikelets for a rice variety with aarge panicle or an extra-heavy panicle. Our results showed that,

hen compared with the treatment with no N application duringhe mid-season, the treatment with N application at the spikeletifferentiation stage significantly increased the number of spikeletser panicle and NSC per spikelet at the heading time, and enhancedink strength and rates of endosperm cell division and grain fill-ng of inferior spikelets (Tables 7–11). Either the treatment of Npplication at the panicle initiation or the treatment of N applica-ion at both panicle initiation and spikelet differentiation stagesould greatly increase the number of spikelets per panicle, butignificantly reduced NSC per spikelet at the heading time andecreased sink strength, endosperm weight, and grain weight of

nferior spikelets. The results suggest that the practice that N is

pplied at the spikelet differentiation stage could coordinate theource–sink relationship for rice varieties with large panicles, suchs super rice varieties. However, it should be noted that there areeports that increase in grain yield is more under the treatments

anicle initiation, N application at spikelet differentiation, and N application at bothrement has four repetitions. Different letters indicate statistical significance at the

of N application at the panicle initiation or at both panicle initia-tion and spikelet differentiation stages than under the treatment ofN application at the spikelet differentiation stage for rice varietieswith small panicles (Ling, 2007; Xue et al., 2010; Zhu et al., 1988),suggesting that the N management strategy would be different forrice varieties with different panicle types.

It is also noteworthy that the grain yield in Experiment II waslower than that in Experiment I although the yield under the T2treatment (N application at the spikelet differentiation stage) in thesecond experiment was comparable with that in the first Experi-ment (Tables 2 and 7). A probable explanation is that soil fertilityis poorer (lower organic matter and available N, P, K content insoil) in Experiment II than that in Experiment I. A decrease in num-ber of spikelets per panicle resulted from the treatment with no Napplication at the mid-season (T0) and the decreased filled grainpercentage due to heavy application of N at the panicle initiationstage (T1) may also contribute to a lower grain yield in ExperimentII.

5. Conclusion

Both super rice and NPT rice showed a greater yield capacityas a result of a larger panicle than the elite check rice. However, alower filled grain percentage limited the realization of great yieldpotential in super rice, especially in NPT rice, due to their lowergrain filling rate and smaller grain weight of inferior spikelets. Thelow grain filling rate and small grain weight for inferior spikelets ofrice, especially for those of super rice and NPT rice, were mainlyattributed to their small sink size (small number of endospermcells) and low sink activity (low activities of SuSase and AGPaseat the early grain filling stage). Less NSC accumulation in the stemat the heading time and a low ratio of NSC to number of spikeletsaccounted for the poor sink strength of inferior spikelets in bothsuper rice and NPT rice. The practice with N application at thespikelet differentiation stage could increase not only spikelet num-ber per panicle but also the ratio of NSC to number of spikelets,and consequently enhance sink strength and grain filling of infe-rior spikelets in super rice. On the other hand, the practice with N

application at the panicle initiation or at both panicle initiation andspikelet differentiation stages could reduce the ratio of NSC to num-ber of spikelets, sink strength, and grain filling of inferior spikeletsalthough it could greatly increase the number of spikelets per pan-

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cle. Nitrogen application at the spikelet differentiation would be aood practice to increase both sink capacity and sink strength forice varieties with large panicles, such as super rice varieties.

cknowledgements

We are grateful for grants from the National Natural Sci-nce Foundation of China (NSFC-IRRI Joint Research Project1061140457; General Project 31071360), the National Basicesearch Program (973 Program, 2009CB118603), the Naturalcience Foundation of Jiangsu Province (BK2009-005), the Hongong University Grants Committee (AOE/B-07/99), Research Grantsouncil (HKBU 262809) and the Hong Kong Baptist Universitytrategic Development Fund.

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