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Journal of Cereal Science 55 (2012) 331e336

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Journal of Cereal Science

journal homepage: www.elsevier .com/locate/ jcs

Pre-anthesis high temperature acclimation alleviates the negative effects of post-anthesis heat stress on stem stored carbohydrates remobilization and grain starchaccumulation in wheat

Xiao Wang a,c, Jian Cai a, Fulai Liu b, Mei Jin a, Hongxi Yu a, Dong Jiang a,*, Bernd Wollenweber c,Tingbo Dai a, Weixing Cao a

aKey Laboratory of Crop Physiology, Ecology and Production, Ministry of Agriculture/Hi-Tech Key Laboratory of Information Agriculture of Jiangsu Province, Nanjing AgriculturalUniversity, No. 1 Weigang Rd., Nanjing, Jiangsu Province 210095, PR ChinabUniversity of Copenhagen, Faculty of Life Sciences, Department of Agriculture and Ecology, Højbakkegaard Allé 13, DK-2630 Taastrup, DenmarkcAarhus University, Department of Genetics and Biotechnology, Research Centre Flakkebjerg, DK 4200 Slagelse, Denmark

a r t i c l e i n f o

Article history:Received 29 September 2011Received in revised form16 December 2011Accepted 7 January 2012

Keywords:FructansHigh temperature pre-acclimationStarch granulesWheat

Abbreviations: CPA, contribution of post-anthesisdays after anthesis; DM, dry matter; DP, degree offructan fructosyltransferase (EC 2.4.1.100); HPLC, higmatography; PAA, post-anthesis dry matter accumamount of pre-anthesis stored; REP, remobilization effidry matter; SST, sucrose:sucrose fructosyltransferasesoluble carbohydrates.* Corresponding author. Tel./fax: þ86 25 84386575

E-mail address: jiangd@njau.edu.cn (D. Jiang).

0733-5210/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.jcs.2012.01.004

a b s t r a c t

The potential role of pre-anthesis high temperature acclimation in alleviating the negative effects ofpost-anthesis heat stress on stem stored carbohydrate remobilization and grain starch accumulation inwheat was investigated. The treatments included no heat-stress (CC), heat stress at pre-anthesis only(HC), heat at post-anthesis only (CH), and heat stress at both stages (HH). Post-anthesis heat stressdecreased grain starch content, reduced the content of fructans and depressed activities of relatedsynthesis enzymes of sucrose:sucrose fructosyltransferase and fructan:fructan fructosyltransferase.Interestingly, HH plants had significantly higher grain yield than the CH plants. In addition, post-anthesishigh temperature lowered grain starch content and increased percentages of volume, number andsurface area of B-type starch granules in CH and HH than in CC treatment. However, HH plants had muchhigher starch content, and caused less modified B-type starch granule size indicators than the CH plants.Our results indicated that, compared with the non-acclimated plants, the pre-anthesis high temperatureacclimation effectively enhanced carbohydrate remobilization from stems to grains, led to less changedstarch content and starch granule size distribution in grains of wheat under post-anthesis heat stress.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Wheat is one of the world’s most important staple crops, and itsgrain production depends on successful reproductive development.Abiotic stresses such as drought and heat are predicted to occurmore frequently due to climate change (Arnell, 1999). Earlier andrecent evidence has indicated that short periods (3e5 days) of hightemperature (>35 �C) stress during the grain filling stage of wheatoccurs quite often (Semenov and Halford, 2009) which may result

photosynthate to grain; DAA,polymerization; FFT, fructan:h performance liquid chro-ulation; RAP, remobilizationciency of pre-anthesis stored(EC 2.4.1.99); WSC, water-

.

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in significant reductions of both grain yield and quality (Stone andNicolas, 1995).

Carbon metabolism in plants is one of the key physiologicalprocesses determining crop growth, yield and quality; and is verysensitive to abiotic stresses. In cereals like wheat, carbohydratessupporting grain growth during the grain filling stage are derivedfrom two sources, namely the current photo-assimilate and storedcarbohydrate reserves in vegetative organs which can be remobi-lized into the grains (Gebbing et al.,1999). The relative importance ofthe two carbon sources in contributing to the grain filling variesamong genotypes and is often dependent on environmental condi-tions (Blum, 1998). Temporary stored carbohydrates in the stemexist mostly in the form of water-soluble carbohydrates (WSC)(Schnyder, 1993). It is well known that fructans are the main storageforms of WSC in the stem, and they play an important role inassimilate partitioning and possibly also in stress tolerance in plants(Kerepesi et al., 1998). It has been reported that sucrose:sucrosefructosyltransferase (SST; EC 2.4.1.99) and fructan:fructan fructo-syltransferase (FFT; EC 2.4.1.100) (Nelson and Spollen,1987) regulate

X. Wang et al. / Journal of Cereal Science 55 (2012) 331e336332

the accumulation of fructans in thewheat stem. SST is considered tobe the most important enzyme for fructan synthesis since it catal-yses the initial step transferring a fructosyl group from a sucrosedonor to a sucrose acceptor to produce trisaccharide and glucose(Wagner et al., 1986). FFT continues the polymerization to buildgradually longer fructans with trisaccharide as the smallest possibledonor (Henry and Darbyshire, 1980; Simmen et al., 1993).

Starch is a major component of the wheat grain, making up60e70% of its dry weight. An earlier study has indicated thatdecline in starch content in cereal grains is responsible for thedecrease in grain yield under heat stress (Labuschagne et al., 2009).Starch is composed of two types of glucose polymers, viz., amyloseand amylopectin. Amylose is a linear a-1, 4 glucan molecule thatcomprises 25e30% of wheat grain starch. Amylopectin is a muchlarger glucan polymer that is highly branched and comprises70e75% of wheat grain starch. The starch of the wheat grain isdistributed in different classes of granules in the endosperm, i.e., A-,B-, C-type starch granules, with each class having unique physi-ochemical properties (Zhang et al., 2010). It has been reported thathigh temperatures during wheat grain filling stage reduced starchcontent and altered the size distribution of starch granules in themature grains (Hurkman et al., 2003).

Plant reproductive development is extremely sensitive to abioticstresses, so that any environmental perturbations may stronglyaffect final yield and quality (Prasad et al., 2008). We have previ-ously reported that pre-anthesis high temperature acclimation inwinter wheat could greatly alleviate the damage of post-anthesisheat stress on photosynthetic capacity of the flag leaf during thegrain filling stage (Wang et al., 2011). However, to our knowledge,there is little information about the effects of such treatment oncarbohydrate (i.e., fructans) metabolism in the stem and on starchaccumulation and granule size distribution in the grain of winterwheat exposed to heat stress during grain filling. Therefore, theobjective of this studywas to investigatewhether pre-anthesis hightemperature acclimation could also improve grain starch accumu-lation by enhancing stem reserve remobilization to the grains ofwheat plants subjected to post-anthesis heat stress.

2. Materials and methods

2.1. Experiment design

The experimental setup has been detailed elsewhere (Wanget al., 2011). Briefly, wheat plants (Triticum aestivum, L. cv. Yang 9)were grown outdoors in 25 cm diameter by 22 cm in height pots, atthe experimental field of Nanjing Agricultural University, Nanjing,Jiangsu Province, P.R. China. The pot was filled with 7.5 kg of claysoil, which contained 13.1 g kg�1 organic matter, 1.1 g kg�1 total N,72.4 mg kg�1 available N, 41.9 mg kg�1 Olsen-P, 146.2 mg kg�1

available K. Fertilizers (0.9 g N, 0.36 g P2O5, and 0.9 g K2O per pot)were applied before seeding and were completely mixed with thesoil; an additional 0.3 g N per pot was applied at the jointing stage(the Zadoks Stage 32, Zadoks et al., 1974) tomeet the N requirementof the plants. The pots were watered regularly to avoid waterdeficiency. Before anthesis (at the seven-leaf and nine-leaf stages,i.e. the Zadoks Stages 17 and 19, Zadoks et al., 1974), the plants weredivided into two batches. One bath of the plants was subjected tohigh temperature acclimation at a day/night temperature of 32/28 �C in one growth chamber for two days. Another batch wasmoved into another growth chamber at a day/night temperature of24/20 �C as non-acclimated treatment. At 7 DAA, half of both thehigh-temperature acclimated and the non-acclimated plants weretransferred to the growth chamber at day/night temperature of 34/30 �C for seven days heat stress. At the same time, the remainingplants were moved to another chamber with a temperature regime

of 26/22 �C as control. Thereafter, four treatments in total wereestablished; i.e. control (CC), post-anthesis heat stress only (CH),pre-anthesis high-temperature acclimation only (HC), and pre-anthesis high-temperature acclimationþpost-anthesis heat stress(HH). In the growth chamber, the photosynthetic active radiation(PAR) was set at ca. 400 mmolm�2 s�1, with a day/night photope-riod of 14/10 h. All pots were moved to the field after the treat-ments. The experiment was a completely randomized block design,with three replicates for each treatment. Namely, at least threepots for each treatment were used for each harvest or eachmeasurement.

2.2. Accumulation and remobilization of dry matter (DM)

At anthesis and physiological maturity, plants were harvestedand the samples were separated into leaves, stems and spikes atanthesis, and into leaves, stems, chaff and grain at physiologicalmaturity. All plant samples were oven dried at 70 �C to constantweight for DM determination.

For the estimate of DM remobilization, it was assumed that all ofthe dry matter lost from vegetative plant parts was remobilized tothe developing grain, since losses of DM due to respirationwere notdetermined.

The following parameters were calculated according to Tan et al.(2008) as:

Remobilization amount of pre-anthesis stored DM (RAP)¼DMof the vegetative organs at anthesis�DMof vegetative organs atphysiological maturity.Remobilization efficiency of pre-anthesis stored DM(REP)¼ (RAP/DM of the whole plant at anthesis) * 100.Post-anthesis DM accumulation (PAA)¼DM of the whole plantat physiological maturity�DM of the whole plant at anthesis.Contribution of post-anthesis photosynthate to grain(CPA)¼ (PAA/grain yield at physiological maturity) * 100.

2.3. Fructan analysis

Dry stem samples (0.5 g) were homogenized in a mortar andpestle with 5 mL of distilled water, and were incubated at 80 �C for10 min. The homogenate was centrifuged at 4000 g for 10 min andthe supernatant was collected. The pellet was extracted twice withdistilled water. The extracts were flash evaporated. The residue wasreconstitutedwith 1e3 mL distilled water and then filtered througha 0.45 mm filter film for later determination of fructan content.

The content of fructans was analyzed by High PerformanceLiquid Chromatography (HPLC) (Zhang et al., 2011). A cationexchange column (Sugar-PAK�, Millipore Waters, USA) withdouble distilled water adding to 0.1 mM EDTANa2-Ca was used asthe mobile phase. Aliquots of 10 mL sample were injected and theelution rate was set to 0.5 mLmin�1 at 90 �C .The sugars weredetected with a 2414 Refractive Index (RI) detector (Waters Corp.,Milford, MA, USA), the temperature was set at 40 �C. Quantificationwas performed on the peak areas with glucose, fructose andsucrose (SigmaeAldrich Corp., St. Louis, MO, USA) as the externalstandards. The content of total fructans was the sum of degree ofpolymerization (DP)¼ 3 and DP� 4 contents.

2.4. Enzyme extraction and assays

Fresh stem sample (1 g) was ground to fine powder with 5 mL ofice-cold extraction buffer (100 mM sodium acetate buffer, pH 5.0,20 mM EDTA-Na2, 20 mM DTT, 10% PVP). The homogenate wascentrifuged at 10,000 g for 10 min and the supernatant was used ascrude extract. All operations were carried out at 4 �C.

Table 1Effects of pre-anthesis high temperature acclimation on remobilization of pre-anthesis stored dry matter and accumulation of post-anthesis photo-assimilatesunder heat stress during grain filling.

Treatment RAP REP GA CPA PAA

CC 1.34 ab 34.12 ab 1.43 a 70.05 a 1.03 aCH 1.27 ab 29.09 c 0.70 c 42.22 c 0.33 cHC 1.14 b 30.21 bc 1.39 a 74.10 a 1.01 aHH 1.46 a 35.49 a 1.02 b 52.33 b 0.54 b

Note: RAP, Remobilization amount of pre-anthesis stored dry matter (g stem�1);REP, Remobilization efficiency of pre-anthesis stored dry matter (%); GA, Grain drymatter accumulation (g stem�1); CPA, Contribution of post-anthesis assimilate tograin (%); PAA, Post-anthesis assimilates accumulation (g stem�1); CC, Control; HC,Treatment applied heat at pre-anthesis only; CH, Treatment applied heat at post-anthesis only; HH, Treatment applied heat at both stages. For each column,different letters mean significant difference at P< 0.05 analyzed by Duncan’sMultiple Range Test (n¼ 3).

X. Wang et al. / Journal of Cereal Science 55 (2012) 331e336 333

SST and FFT were extracted according to Cairns et al. (1989) andHousley and Volenec (1988), with minor modifications. For SST,1 mL mixture containing 50 mL pH 5.0 100 mM sodium acetatebuffer, 50 mL 2 M sucrose, 100 mL, 10 mg/mL BSA, 150 mL H2O and0.5 mL enzyme extract was incubated at 30 �C for 30 min. Thereaction was stopped by heating to 100 �C for 2 min. Samples werestored at �40 �C. For FFT, the extraction procedure was the same asdescribed for SST, except that the medium was pH 6.0, 100 mMsodium acetate buffer, and the reaction was kept at 30 �C for 24 h.

The assays were modified from Guerrand et al. (1996). Themixture was separated and the sugars were quantified by HPLC onthe Sugar-PAK� column, under the same conditions as for thefructan analysis. The glucose oxidase method was used to estimateSST activity, that is, activity was defined using the differencesbetween glucose content in the sample and in the enzymeextraction. FFT activity was defined as the amount of fructancontent (DP� 4) formed in this assay.

2.5. Starch analysis

The isolation procedure of starch was according to Bechtel andWilson (2003). Briefly, the endosperm was carefully squeezed outof the caryopsis. The endosperm was mixed with buffer solutioncontaining 25 mM tricine, 5 mMmagnesium acetate, 50 mM potas-sium acetate, pH 7.5 at 4 �C. After centrifugation of the homogenatefor 10 min at 3000 g at 4 �C, the pelletswere collected and suspendedin ethanol. The suspension was filtered through a nylon screenwitha pore size of 53 mmandwaswashedwith excess ethanol. The starchsample was then collected by centrifugation (3000 g, 10 min, 4 �C),and resuspended in 0.1 M aqueous NaCl solution containing 10%toluene and at a high speed stirred for 1 h. This step was repeateduntil the toluene layer became clear and no proteinwas present. Thestarch was re-purified by washing three times with water and twicewith ethanol and then dried at 30 �C for 48 h.

Measurements of amylose and amylopectin contents in wheatgrains were determined as described by Jiang et al. (2003). Milledwheat grains (100 mg) were prepared for analysis and 2 mL ofsolution, which was diluted to a volume of 50 mL with distilledwater after incubation at 85 �C for 20 min, reacted with the I2eKIreagent and absorption was scanned with a Helios Gamma spec-trophotometer (Thermo Spectronic, Cambridge, UK) at 460, 550,630 and 740 nm. Total starch content was the sum of amylose andamylopectin.

Starch granule size distribution was measured according toZhang et al. (2010) by a Saturn DigiSizer-5200 (MicromeriticsInstrument Corporation, USA). The standard refractive indices usedwere 1.31 for water and 1.52 for starch. Volume frequency percentand mean diameter of starch granules were then automaticallymeasured using the embedded laser light scattering technologyand summing Mie scattering models. Each measurement wasrepeated three times.

2.6. Statistics

Data were analyzed by the one-way ANOVA using SPSS (SPSSInc., Chicago, IL). Significantly different means of themeasured datawere separated at the 0.05 probability level by the Duncan’sMultiple Range Test.

3. Results

3.1. Dry matter (DM) accumulation and remobilization

Remobilization amount of pre-anthesis stored DM (RAP) fromthe stem to grain was not significantly affected by post-anthesis

heat stress (Table 1). The remobilization efficiency (REP) wassignificantly lowered under CH, while it was similar for HH and CC.Heat stress applied during grain filling (CH and HH) significantlyreduced grain dry matter accumulation (GA), the amount of post-anthesis photo-assimilate accumulation (PAA) and the contribu-tion of post-anthesis photo-assimilate to grain (CPA); however,between the two treatments, CH decreased GA, PAA, and CPA muchmore than HH.

3.2. Yield and yield components

Post-anthesis heat stress reduced the kernel yield, kernels perspike and 1000-kernel weight, as compared with the CC plants(Table 2). However, the HH plants had significantly higher 1000-kernel weight and kernel yield than the CH plants.

3.3. Fructan content and SST, FFT

Post-anthesis heat stress decreased fructan accumulation in therest internodes and the peduncle at 10 and 13 DAA; compared tothe CH plants. HH plants had higher fructan content than CH plantsat 10 DAA (Fig.1). At 10 DAA, fructan content was reduced by 12.89%and 6.4% in the rest internodes and 29.4%, and 18.2% in thepeduncle, respectively, by the CH and HH treatments as comparedwith the CC plants. There was also significant difference in fructancontent between HH and CH plants in the peduncle at 13 DAA,though there was no significant difference in fructan contentbetween HH and CH plants in the rest internodes at 13 DAA.

At 13 DAA, SST and FFT activities were inhibited by 52.5% and58.3% in CH, and by 42.8% and 40.8% in HH, respectively, ascompared with the CC plants. FFT in peduncle showed the sametendency as in the rest internodes. SST activity in the peduncle wassignificantly depressed in the CH plants at both 10 and 13 DAA;while for the HH plants, reduction of SSTactivity was observed onlyat 13 DAA.

3.4. Starch accumulation and starch granule distribution

At maturity, the total starch content in the grain was signifi-cantly lowered by both CH and HH treatments; however, thedecrease was much less in HH than in CH relative to the CC plants(Table 3). The starch yield showed the same tendency as for thetotal starch content in grain, except that HC plants showed highergrain starch content while lower starch yield as compared with theCC plants. The amylose and amylopectin contents were significantlydecreased by post-anthesis heat stress, while the amylopectincontent was much lower in CH than in HH plants, resulting inincreased amylopectin/amylose ratio in the HH plants.

Table 2Effects of pre-anthesis high temperature acclimation on grain yield and yieldcomponents in wheat under heat stress during grain filling.

Treatment Spikes perpot

Kernels perspike

1000-kernelmass (g)

Kernel yield(g pot�1)

CC 17.00 a 23.26 a 38.48 a 15.18 aCH 17.00 a 19.17 b 29.87 d 9.72 dHC 17.33 a 23.50 a 36.00 b 14.58 bHH 16.33 a 20.67 b 32.25 c 10.92 c

Note: CC, Control; HC, Treatment applied heat at pre-anthesis only; CH, Treatmentapplied heat at post-anthesis only; HH, Treatment applied heat at both stages. Foreach column, different letters mean significant difference at P< 0.05 analyzed byDuncan’s Multiple Range Test (n¼ 3).

Table 3Effects of pre-anthesis heat acclimation on starch contents and yield in maturewheat grain under post-anthesis heat stress.

Treatment Total starchcontent (%)

Starch yield(g$pot�1)

Amylosecontent (%)

Amylopectincontent (%)

Amylopectin/Amylose

CC 66.66 b 10.21 a 16.70 a 50.05 b 3.02 bCH 57.50 d 5.65 d 14.43 b 43.05 d 2.98 bHC 68.21 a 9.79 b 17.46 a 51.18 a 3.00 bHH 61.52 c 6.86 c 14.15 b 47.43 c 3.37 a

Note: CC, Control; HC, Treatment applied heat at pre-anthesis only; CH, Treatmentapplied heat at post-anthesis only; HH, Treatment applied heat at both stages. Foreach column, different letters mean significant difference at P< 0.05 analyzed byDuncan’s Multiple Range Test (n¼ 3).

X. Wang et al. / Journal of Cereal Science 55 (2012) 331e336334

Fig. 2 shows the change of amylose and amylopectin contents inthe grain from 10 to 25 DAA under different heat stress treatments.It was clear that, comparedwith the CC plants, amylose content wasunaffected at 10 and 13 DAA by the other treatments; whereas itwas significantly lowered from 15 to 25 DAA by CH and HH treat-ments. A similar trend of change in grain amylopectin content wasobserved from 10 to 25 DAA, as exemplified by much loweredamylopectin content in both CH and HH grains than in CC grains.However, the amylopectin content was consistently higher in HHgrains than in CH grains.

The size distribution of the starch granules was significantlyinfluenced by post-anthesis heat stress (Table 4). Compared withthe CC plants, both CH and HH treatments had significantlyincreased the B-type (<10 mm) and decreased the A-type(10e43 mm) of starch granules in terms of their volume percent-ages, number percentages and surface area percentages at 13 DAA.However, the effect on the size distribution of the starch granules

Fig. 1. Effects of pre-anthesis heat acclimation on the fructan contents, SST activities and FFTstress. Note: CC, Control; HC, Treatment applied heat at pre-anthesis only; CH, Treatment ameans �SE (n ¼ 3).

was more pronounced under CH than under HH treatment(Table 4).

4. Discussion

A large body of evidence has demonstrated that heat stressduring grain filling stage could result in significant grain yield lossin winter wheat crops (Ehdaie and Waines, 2001). In agreementwith this, here it was observed that post-anthesis heat stress (i.e.,CH and HH treatments) had significantly decreased grain yield andyield components compared with the CC and HC plants. Never-theless, it was also noticed that the pre-anthesis high temperatureacclimation treatment, viz. HH, led to significantly higher 1000-kernel mass and kernel yield than the non-acclimated treatment,viz. CH. Consistent with this, we have previously reported that pre-anthesis high temperature acclimation could greatly alleviate theimpacts of post-anthesis heat stress on photosynthesis of wheat

activities in rest internodes (left) and peduncle (right) wheat under post-anthesis heatpplied heat at post-anthesis only; HH, Treatment applied heat at both stages. Data are

Fig. 2. Effects of pre-anthesis heat acclimation on starch contents of amylose and amylopectin accumulation in wheat grain under post-anthesis heat stress. Note: CC, Control; HC,Treatment applied heat at pre-anthesis only; CH, Treatment applied heat at post-anthesis only; HH, Treatment applied heat at both stages. Data are means �SE (n ¼ 3).

X. Wang et al. / Journal of Cereal Science 55 (2012) 331e336 335

flag leaf (Wang et al., 2011). Obviously, a greater photosynthetic ratein the flag leaf will sustain current photo-assimilate supply to thegrains and thus sustaining grain filling. However, as has beenmentioned previously, remobilization of the carbohydrate reservesfrom the stem to the grains may contribute greatly to grain filling inwheat particularly when the plants grown under stressful condi-tions. In the present study, we analyzed the total water solublecarbohydrate including fructan content and its metabolic enzymeactivity in the stem internodes and calculated the amount ofremobilized carbohydrate from the stem to the grains of wheatexposed to different treatments. It was found that, compared withthe non-acclimated wheat plants (CH), pre-anthesis high temper-ature acclimation could promote the remobilization of reservedcarbohydrates in the stems to the grains during post-anthesis heatstress through enhancing the fructan catalyzing enzyme (i.e. SSTand FFT) activities. In agreement with this, Blum (1998) demon-strated that remobilization of stored stem reserves into the growinggrain is an important source of carbon for supporting grain fillingunder heat stress. Here, it was observed that the fructan contentand related enzyme activities in the stem were significantlyreduced under post-anthesis heat stress in both rest internodes andpeduncle. Interestingly, the HH plants had much higher fructancontent as well as the related enzyme activities than did the CHplants. The relatively lower fructan content in the HH stems mighthave indicated an enhancement of the post-anthesis dry matterremobilization from the stem to the grain.

It is known that reductions in starch accumulation in the grainsunder heat stress account for significant losses in the grain yield(Jenner, 1991). In the present study, we found that post-anthesisheat stress decreased the amount of starch deposited in themature grains. It was even more interesting that starch accumu-lation in the grains of HH plants was much more than in those of

Table 4Effects of pre-anthesis heat acclimation on starch granules distribution in wheatgrain at 13DAA (after post-anthesis heat stress).

Treatment Volume (%) Number (%) Surface area (%)

<10 mm 10e43 mm <10 mm 10e43 mm <10 mm 10e43 mm

CC 34.01 c 65.75 a 99.68 b 0.32 a 73.71 c 26.29 aCH 42.75 a 57.25 c 99.78 a 0.22 c 78.51 a 21.49 cHC 34.31 c 65.69 a 99.69 b 0.32 a 74.05 c 25.95 aHH 40.15 b 59.85 b 99.74 b 0.26 b 76.45 b 23.55 b

Note: CC, Control; HC, Treatment applied heat at pre-anthesis only; CH, Treatmentapplied heat at post-anthesis only; HH, Treatment applied heat at both stages. Foreach column, different letters mean significant difference at P< 0.05 analyzed byDuncan’s Multiple Range Test (n¼ 3).

the CH plants, and this coincided with the higher grain yield in theHH than in the CH plants. In addition, HC plants had higher starchcontent than CC, despite a reduction in the starch yield, which maybe due to the lowered individual kernel weight in those plants. Thereduction in starch accumulation caused by post-anthesis heatstress could be associated with the reduction of the soluble starchsynthase enzyme activities, which might have led to a decreasedconversion of sucrose into starch and consequently stored in thegrain (Hurkman et al., 2003).

In addition to affecting the amount of starch deposited in thegrain, post-anthesis heat stress has been shown to modify starchgranule size distribution (Hurkman et al., 2003). It is known thatthe synthesis of A-type granules begins in amyloplasts 4e5 daysafter flowering, while the synthesis of B-type granules begins at12e14 days after anthesis and increases continuously in size andnumber before maturity (Parker, 1985). In the present study, thevolumetric, number and surface area of B-type starch granulesunder post-anthesis heat stress increased significantly, whereasthat of A-type starch granules decreased, compared with the CCplants at 13 DAA. One possible reason explaining this may bethat both the starch accumulation rate and the activities ofrelated enzymes have been significantly increased by post-anthesis heat stress at 13 DAA, when the B-type starch gran-ules were synthesized rapidly (Dai et al., 2009). On the contrary,Hurkman et al. (2003) reported that the proportion of the A-typegranules increased in response to heat stress at maturity. Thediscrepancy between the above experimental findings remainsunexplained.

Themain components of wheat starch granules are amylose andamylopectin. Peng et al. (1999) reported that the amylose contentwas higher in large granules, whereas others have found thatsimilar amylose contents exist in both small and large granules(Myllrinen et al., 1998). Dai et al. (2009) observed that the B-typestarch granules were negatively correlated with the content ofstarch in the grain. Here, it was shown that different A-, B-typestarch granule percentages did not change the amylose contentafter heat treatment, which is consistent with earlier findings byDai et al. (2009) under water deficit in wheat at maturity. Theresults suggested that heat treatment has significantly increasedgrain percentage of B-type starch granules, leading to higher starchcontent, compared with CC.

Collectively, compared to the non-acclimation treatment (CH),the pre-anthesis high temperature acclimation effectivelyenhanced the post-anthesis dry matter remobilization from stemsto the grains. At the same time greater starch content and higherproportion of amylopectin in the starch of the HH grains indicatedan improvement of grain quality by the pre-anthesis high

X. Wang et al. / Journal of Cereal Science 55 (2012) 331e336336

temperature acclimation treatment. However, further investiga-tions are needed in order to interpret the underlying mechanismsthat how the pre-anthesis high temperature acclimation is trig-gered to sustain the grain yield and quality inwheat plants exposedto subsequent post-anthesis heat stress.

Acknowledgements

This work was supported by projects of National NaturalScience Foundation of China (30971734, 31028017 and 31171484),the Research Innovation Project for Graduate Students of JiangsuProvince (CX10B-3112), the Natural Science Foundation of JiangsuProvince (BK2008329), the Specialized Research Fund forthe Doctoral Program of Higher Education (20090097110009),the National Non-profit Program by the Ministry of Agriculture(200903003), and the China Agriculture Research System(CARS-03).

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