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Journal of Plant Physiology 169 (2012) 387– 398
Contents lists available at SciVerse ScienceDirect
Journal of Plant Physiology
jou rn al h o mepage: www.elsev ier .de / jp lph
omparative proteomic study reveals dynamic proteome changes betweenuperhybrid rice LYP9 and its parents at different developmental stages
hunyan Zhanga , Yan Yina , Aihong Zhanga , Qingtao Lua , Xiaogang Wena , Zhen Zhub , Lixin Zhanga ,ongming Lua,∗
Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, ChinaState Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
r t i c l e i n f o
rticle history:eceived 28 September 2011eceived in revised form1 November 2011ccepted 12 November 2011
eywords:eterosishotosynthesisroteomic profilingucrose synthesisybrid rice
a b s t r a c t
Heterosis is a common phenomenon in which the hybrids exhibit superior agronomic performance thaneither inbred parental lines. Although hybrid rice is one of the most successful apotheoses in cropsutilizing heterosis, the molecular mechanisms underlying rice heterosis remain elusive. To gain a bet-ter understanding of the molecular mechanisms of rice heterosis, comparative leaf proteomic analysisbetween a superhybrid rice LYP9 and its parental cultivars 9311 and PA64s at tillering, flowering andgrain-filling stages were carried out. A total of 384 differentially expressed proteins (DP) were detectedand 297 DP were identified, corresponding to 222 unique proteins. As DP were divided into those betweenthe parents (DPPP) and between the hybrid and its parents (DPHP), the comparative results demonstratethat proteins in the categories of photosynthesis, glycolysis, and disease/defense were mainly enriched inDP. Moreover, the number of identified DPHP involved in photosynthesis, glycolysis, and disease/defenseincreased at flowering and grain-filling stages as compared to that at the tillering stage. Most of the
up-regulated DPHP involved in the three categories showed greater expression in LYP9 at flowering andgrain-filling stages than at the tillering stage. In addition, CO2 assimilation rate and apparent quantumyield of photosynthesis also showed a greater increase in LYP9 at flowering and grain-filling stages thanat the tillering stage. These results suggest that the proteins involved in photosynthesis, glycolysis, anddisease/defense as well as their dynamic regulation at different developmental stages may be responsiblefor heterosis in rice.C
ntroduction
Heterosis is a common phenomenon in which the hybridsxhibit superior agronomic performance than both parents suchs biomass production, grain yield, and stress tolerance. Heterosisas been widely exploited in crop breeding, such as rice, maize,nd wheat, which has contributed to the dramatically increasedrop production. Yield advantages as a result of hybrids may rangeetween 25% and 50% depending on the crop (Duvick, 1999).espite the fact that utilization of heterosis worldwide has become
major practice for increasing crop production in agriculture, theolecular mechanisms responsible for this basic biological phe-
omenon are not well understood (Lippman and Zamir, 2007;ochholdinger and Hoecker, 2007; Birchler et al., 2010).
Abbreviations: AGPase, ADP-glucose pyrophosphorylase; PGK, phosphoglycer-te kinase; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase.∗ Corresponding author. Tel.: +86 10 6283 6554; fax: +86 10 6259 5516.
E-mail address: [email protected] (C. Lu).
176-1617/$ – see front matter. Crown Copyright © 2011 Published by Elsevier GmbH. Aoi:10.1016/j.jplph.2011.11.016
rown Copyright © 2011 Published by Elsevier GmbH. All rights reserved.
Several genetic models have been proposed to explain hetero-sis, including dominance, over dominance, and epistasis (Birchleret al., 2003). The dominance model states that deleterious alleles atdifferent loci in the two homozygous parental genomes are com-plemented in the heterozygous hybrid. The hybrid will benefit fromthe complementation of these deleterious alleles and thus exhibita superior phenotype. The over dominance model posits that inter-actions between different alleles occur in the hybrid, leading tothe increase in vigor. Both the dominance and over dominancehypotheses are based on single locus theory and are not connectedwith molecular principles. Thus, these two hypotheses may be inad-equate to address the molecular mechanisms for heterosis (Birchleret al., 2003). Epistasis is classically defined as interactions of supe-rior alleles at different loci from two parents, and the effects mayshow additivity, dominance or over dominance (Yu et al., 1997). Inspite of decades of research, it is widely assumed that the geneticcomponents of heterosis are still obscure (Lippman and Zamir,
2007). Comprehensive assessment of the expression of the locicontributing to heterosis is largely not available due to the lim-ited resolution of classic genetic analysis in tracking multiple loci(Birchler et al., 2003; Lippman and Zamir, 2007).ll rights reserved.
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88 C. Zhang et al. / Journal of Pla
With the development of functional genomics, it is possibleo examine gene expression profiles between the hybrid and itsarents and thus to explore the possible molecular mechanismsor heterosis. Recently, the comparative gene expression profilingetween the hybrid and its parents has been investigated in Ara-idopsis (Wang et al., 2006), maize (Stupar et al., 2008; Thiemannt al., 2010), wheat (Sun et al., 2004; Yao et al., 2005), and riceBao et al., 2005; Zhang et al., 2008; Wei et al., 2009; Song et al.,010). These studies have indicated that the hybridization betweenwo parents can lead to the changes in the expression of differentenes, which may be responsible for heterosis. These studies haveound multiple modes of gene action, including additivity, high-nd low-parent dominance, over dominance, and under dominanceSwanson-Wagner et al., 2006). These studies have also suggestedhe potential association between differential gene expression andeterosis (Bao et al., 2005; Wei et al., 2009). However, no consensusas been reached on heterotic genes responsible for elevating cropields using genomics, particularly transcriptomics. Since heterosiss an environmentally modified quantitative phenotype, genomicnalyses alone will not suffice (Lippman and Zamir, 2007).
Although studies on transcriptome analyses of gene expres-ion have made an understanding for the molecular mechanismsf heterosis in rice, maize, and wheat, the changes on the levelsf gene expression do not necessarily reflect the changes in thebundance of proteins. Therefore, the differential protein expres-ion between the hybrid and its parents will be important for uso understand the possible molecular mechanisms for heterosis. Toain a better understanding of the molecular mechanisms for het-rosis, proteomics as a powerful research tool has been recentlypplied to compare the patterns of protein accumulation in hybridsnd their inbred lines. In wheat, it has been demonstrated that theybridization could cause expression changes not only at mRNA
evels but also at protein abundances by comparing the proteomef wheat root between the F1 hybrid and parents (Song et al.,007). It has also been demonstrated that wheat hybridization canause protein expression differences between the hybrid and itsarents and these differentially expressed proteins were involved
n diverse physiological processes that might be responsible foreterosis (Song et al., 2009). In maize, proteomics has been success-
ully applied for investigating non-additive protein accumulationatterns in maize hybrids during root and embryo developmentsMarcon et al., 2010). In rice, proteomic profiling of embryos from aybrid cultivar and its parents has been investigated and it has been
ound out that most of the storage proteins in hybrid rice embryosxhibited over dominance and stress-induced proteins displayeddditivity (Wang et al., 2008).
Rice is one of the most important stable food crops and has beenroposed as an experimental model for grass species. A superhybridice, LYP9, from a cross between 9311 (Oryza stavia L. ssp. Indica)nd PA64s (mixed background of Indica, Japonica, and Javanica), is auccessful rice cultivar with 20–30% more yield per hectare than itsarent crops (Lu and Zhou, 2000). More importantly, both parentenomes, 9311 and PA64s, have been sequenced, thus providingnvaluable information and available genetic resources for furtherunctional studies (Goff et al., 2002; Yu et al., 2002).
In rice, leaf is the main photosynthetic organ, providing about0–100% of the carbon in mature grains during the grain-fillingeriod with flag leaf as the biggest contributor to yield (Yoshida,981). Extensive comparative studies on transcriptional profiling ofice leaves between the hybrid and its parents have been performedBao et al., 2005; Zhang et al., 2008; Wei et al., 2009; Song et al.,010). However, until now there is no report about the differences
n the proteomic profiling of rice leaves between the hybrid and itsarents.
Previously, we have investigated the transcription profiles ofuperhybrid rice LYP9 and its parents (Wei et al., 2009; Song et al.,
siology 169 (2012) 387– 398
2010). In this study, we report further research into the differencesin proteome profiles of leaves between LYP9 and its parents at tiller-ing, flowering, and grain-filling stages. The proteomic analysis inthis study reveals that the proteins involved in photosynthesis, gly-colysis, and disease/defense as well as their dynamic regulation atdifferent developmental stages may be responsible for heterosis inrice. The molecular insights provided by this study might help tobetter understand the possible molecular networks involved in riceheterosis.
Materials and methods
Plant material
The superhybrid rice LYP9, paternal line 9311 (Oryza sativaL. ssp. Indica), and maternal line PA64s (mixed background ofIndica, Japomica and Javanica) were used in this study. Plants weregrown under natural conditions in a field situated in Beijing, China(39◦54′N, 116◦24′E). The rice seeds were sown in March and theseedlings were transplanted at approximately 30 plants/m2. Threerice lines were maintained in the field according to standard pro-tocols (Yuan, 1997).
Leaf sampling
In this study, rice leaves were collected from three develop-mental stages: tillering stage (seedlings with the third-forth tiller),flowering stage (with 70% opened spikelets), and grain-filling stage(the 10th day after flowering). Fully expanded youngest leaves atthe tillering stage and flag leaves at the flowering and grain-fillingstages were used. The leaves were taken from 20 to 30 plants ofthree plots in the morning (around 9:00 A.M.) for each year. Thecollected leaves were immediately immersed in liquid nitrogen andstored at −80 ◦C until used.
Photosynthetic gas exchange measurements
The CO2 assimilation rate was measured on an attached leaf inthe field by an infrared gas analyzer (Ciras-1, PP system, UK) inthe morning (9:00–10:00 A.M.). Fully expanded youngest leavesat the tillering stage and flag leaves at the flowering and grain-filling stages were used for measurements of CO2 assimilationrate. The parameters of CO2 concentration, flow rate and externalhumidity were set at 360 �L L−1, 400 ml min−1 and 60–70%, respec-tively. The temperature inside the leaf chamber was maintained at25 ◦C. The light-saturated photosynthetic rate (Pmax) was obtainedfrom the light-saturation curves of photosynthesis. A range of lightintensities between 40 and 1400 �mol m−2 s−1 were given, startinghigh and progressing toward 0 by attaching a light source on theleaf chamber window which could avoid the penetration of solarlight. At each light intensity, about 5 min was allowed for leavesto achieve steady-state photosynthesis. The slope of the linear partof the light–response curves of photosynthesis was calculated asapparent quantum yield of photosynthesis (AQY).
Preparation of soluble proteins from rice leaves
The PEG-mediated protein fractionation technique was used onproteins extracted from leaves so that ribulose-1,5-bisphosphatecarboxylase/oxygenase, the most abundant leaf protein, wasenriched in the PEG precipitant (Kim et al., 2001; Zhang et al., 2010).
3–5 independent experiments of protein extraction for the leavescollected from each year (2007, 2008 and 2009) were performed,and protein concentration was measured according to Ramagli(1999).
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C. Zhang et al. / Journal of Pla
wo-dimensional polyacrylamide gel electrophoresis
300 �g (the supernatant fraction) and 355 �g (the pellet frac-ion) proteins were loaded onto an IPG strip holder of 18 cm, pH 4–7PG strip or 18 cm, pH 3–11 NL (GE Healthcare), respectively. Elec-rofocusing was carried out for 80 kV h (the supernatant fraction) or00 kV h (the pellet fraction) using IPGphor system at 20 ◦C. Prioro second dimension electrophoresis, the IPG strips were equili-rated with two equilibration buffers: (i) reducing buffer for 15 min50 mM Tris–HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS,nd 1% (w/v) DTT), and (ii) alkylating buffer for 15 min (50 mMris–HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 2.5%w/v) iodoacetamide). The electrophoresed and equilibrated stripsere loaded on 13.5% polyacrylamide gels with 5% staking gels,
nd electrophoresed using the Hoefer SE 600 Ruby electrophoresisnit with 5 mA per gel for 0.5 h followed by 25 mA per gel until theracking dye front reached the bottom. The proteins were stainedith Coomassie Brilliant Blue G-250.
mage acquisition and analysis
Gel images were acquired by the PowerLook 2100XL (UMAX,aiwan) in a transmission mode. Images were subsequently ana-yzed using ImageMaster TM 2D Platinum software version 5.0,
hich allowed spots detection, quantification, background subtrac-ion, and spots matching among multiple gels. Each sample had 3–5iological repeats for the leaves collected from each year, and onlyroteins that emerged repeatedly at least 3 independent sampleets were selected for further analysis. To compensate for subtleifferences in sample loading, gel staining, and destaining, the vol-me of each spot (i.e. spot abundance) was normalized as relativeolume (RV). RV of each protein spot was obtained by dividing eachpot volume value by the sum of total spot volume values. Because
constant protein quantity was loaded onto gels, the normalizedpot volumes, i.e. RV, corresponded to the relative abundance of theorresponding protein in relation to a constant protein basis.
To compare protein abundance between two independentatasets of each sample directly (PEG-fractionated supernatant inH 4–7 and PEG-fractionated pellet in pH 3–11), a normalizedV was performed (Zhang et al., 2010). The RV of each spot wasultiplied by a correction constant. We observed that the ratio of
rotein content of the PEG-fractionated supernatant and the PEG-ractionated pellet was constant during senescence of flag leaves.herefore, the constants for the PEG-fractionated supernatant andhe PEG-fractionated pellet were calculated using the followingormulae:
(supernatant) = total PEG-fractionated supertotal PEG-fractionated supernatant protein content
nd
(pellet) = total PEG-fractionated pellet ptotal PEG-fractionated supernatant protein content + tot
ryptic digestion, MAIDI-TOF MS and statistical analyses
The in-gel tryptic digestion and MS sample preparation wereased on Jensen et al. (1999). Each spot was excised, then destainednd dehydrated with 50% acetonitrile and 100% acetonitrile, respec-ively. The gel piece was reduced with 10 mM dithiotreitol in00 mM NH4HCO3 at 56 ◦C for 45 min, and then alkylated by 55 mM
odoacetamide in 100 mM NH4CO3 in dark at room temperature
or 30 min. Each gel piece was vacuum dried after being thor-ughly washed with 50 mM NH4HCO3 and 50% acetonitrile for5 min, respectively. The gel piece was rehydrated with digestionuffer (50 mM NH4HCO3, 5 mM CaCl2, and 12.5 ng/�l of sequencingsiology 169 (2012) 387– 398 389
nt protein contental PEG-fractionated pellet protein content
in contentG-fractionated pellet protein content
.
grade trypsin) at 4 ◦C for 45 min, and incubated overnight at 37 ◦C.Finally, 0.2 �l digestion solution was added into MALDI-TOF matrixsolution (10 mg/ml �-cyano-4-hydroxycinnamic acid in 50% (w/v)acetonitrile and 0.5% (w/v) trifluoroacetic acid) and air dried ontoa plate for analysis using MALDI-TOF MS in reflector mode on theAXIMA-CFR plus mass spectrometer (Shimadzu, Tokyo).
Peptide mass finger printings (PMFs) obtained from MAIDI-TOFMS were used to search the NCBInr and SwissProt protein databasesusing the MASCOT program (http://www.matrixscience.com). O.sativa (rice) was chosen as taxonomy. All the peptides wereassumed to be monoisotopic, [M+H]+ (protonated molecularions), with carbamidomethylation of cysteine residues and partialoxidation of methionine residues. Proteins that meet the followingcriteria were accepted as unambiguously identified: allowed 1 or 2missed cleavage; 0.3 Da peptide mass deviation; ≥4 matched pep-tides; ≥10% sequence coverage; protein scores given by MASCOT atP < 0.05 level. The identified proteins that had substantial discrep-ancies between the experimental and theoretical pI and Mw weredeemed acceptable, which may be caused by protein degradation ormodifications. The sub-cellular locations of proteins were predictedby Target P (http://www.cbs.dtu.dk/services/TargetP/), WoLFPSORT (http://wolfpsort.org/) and PSORT (http://psort.hgc.jp/).In addition, the functional information of the iden-tified proteins was extracted from Gene OntologyDatabase (http://www.godatabase.org/cgi-bin/amigo/go.cgi),and the pathway was searched from GenomeNet(http://www.genome.ad.jp/kegg/) and Metacyc Database(http://metacyc.org/).
One-way ANOVA (P < 0.05) was performed to detect the signif-icantly differentially expressed proteins among three genotypes(LYP9 vs 9311, LYP9 vs PA64s, and 9311 vs PA64s) at the tillering,flowering, and grain-filling stages, and twofold change was chosenas an empirical cut-off. Based on the comparisons of protein expres-sion abundance between LYP9 and its parents, the differentiallyexpressed proteins were classified into five expression patterns:H2P (abundance of proteins in F1 hybrid was significantly twofoldhigher than abundance of proteins in two parents), CHP (abundanceof proteins in F1 hybrid was not significantly from that in one par-ent but significantly twofold higher than that in the other parent),B2P (abundance of proteins in F1 hybrid was between two parentsand significantly different from both parents or abundance of pro-teins in F1 hybrid was between two parents and not significantlydifferent from both parents but there were significant differencesin the abundance of proteins between two parents), CLP (abun-dance of proteins in F1 hybrid was not significantly from that inone parent but significantly twofold lower than that in the other
parent), and L2P (abundance of proteins in F1 hybrid was signifi-cantly twofold lower than abundance of proteins in two parents).To clearly describe the proteome changes of the three rice lines,we divided the five expression patterns into three basic categories:up-regulation (H2P, CHP), midparent (B2P), and down-regulation(CLP, L2P).
Quantitative real-time PCR
Total RNA in leaves was extracted using plant RNAtrip withfour biological repeats of each sample. The potential contaminat-ing genomic DNA was treated with DNase I (RNase-free) (TaKaRa
390 C. Zhang et al. / Journal of Plant Physiology 169 (2012) 387– 398
Fig. 1. Representative 2DE gels of leaf proteins in LYP9, 9311 (paternal parent), and PA64s (maternal parent) at tillering, flowering, and grain-filling stages, respectively. (A)The PEG supernatant fraction isolated from leaves of LYP9, 9311, and PA64s at tillering, flowering, and grain-filling stages, respectively. (B) The PEG pellet fraction isolated fromleaves of LYP9, 9311, and PA64s at tillering, flowering, and grain-filling stages, respectively. We also denote some specific protein spots in gels with five different expressionpatterns.
C. Zhang et al. / Journal of Plant Physiology 169 (2012) 387– 398 391
Table 1Number and classification of DP.
Growth stage DPHP
DPPP L/N L/P DPHPU DPO H2P CHP B2P CLP L2P
Tillering stage 121 84 73 43 91 10 87 6 28 3Flowering stage 130 103 117 78 103 26 82 3 64 6Grain-filling stage 107 117 100 78 91 37 91 3 32 6
Total 281 247 231 181 222 61 221 12 104 15
DPPP refers to DP between both parents, DPHP refers to DP between the hybrid and parent. DPHPU denotes the unique portion of DPHP, and DPO denotes the overlap betweenDPPP and DPHP. Based on the comparisons of protein expression abundance between LYP9 and its parents, the differentially expressed proteins were classified into fiveexpression patterns: H2P (abundance of proteins in F1 hybrid was significantly twofold higher than abundance of proteins in two parents), CHP (abundance of proteins in F1hybrid was not significantly from that in one parent but significantly twofold higher than that in the other parent), B2P (abundance of proteins in F1 hybrid was between twoparents and significantly different from both parents or abundance of proteins in F1 hybrid was between two parents and not significantly different from both parents butthere were significant differences in the abundance of proteins between two parents), CLP (abundance of proteins in F1 hybrid was not significantly from that in one parentbut significantly twofold lower than that in the other parent), and L2P (abundance of proteins in F1 hybrid was significantly twofold lower than abundance of proteins intwo parents). To clearly describe the proteome changes of the three rice lines, we divided the five expression patterns into three basic categories: up-regulation (H2P, CHP),m
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idparent (B2P), and down-regulation (CLP, L2P).
iotechnology, Dalian, China). The cDNA was then synthesizedsing oligo(dT)15 primer and Reverse Transcriptase AMV RTaseTaKaRa Biotechnology, Dalian, China) according to the manu-acturers’ recommendation. Quantitative real-time PCR (qPCR)as performed using gene-specific primers (Supplementary Table
1) on Mx3000P® QPCR System with SYBR Premix Ex TaqTM.he relative gene expression level was evaluated using compar-tive cycle threshold (Ct) method. Poly-ubiquitin (Accession No.i|116637291) was used as the reference gene.
esults
solation and 2-DE separation of leaves of LYP9 and its parents
The objective of this study was to identify differentiallyxpressed proteins between LYP9 and its parents at the tillering,owering, and grain-filling stages. Proteins were first separated by
soelectric focusing (IEF) either on a linear gradient ranging fromH 4 to 7 or on a nonlinear gradient ranging from pH 3 to 11.ubsequently, proteins were separated according to their molec-lar masses using SDS-PAGE and then stained with CBB. All proteinpots that were consistently detected in each of the three biolog-cal replicates of a particular cultivar for consecutive three years
ere recorded. In total, about 650 and 430 different protein spotsere detected in the supernatant and pellet fraction for each culti-
ar, respectively. There were no significant differences in the totaletected protein spots between different developmental stages forach cultivar. In addition, there were also no significant differencesn the total detected protein spots between LYP9 and its parents.ig. 1 shows the representative 2-DE gels of each cultivar at threeevelopmental stages.
dentification and functional analysis of differentially expressedroteins between LYP9 and its parents
Differentially expressed proteins (DP) between the parentalines were defined as DPPP and those between the hybrid and itsarents as DPHP. DPPP only denote the differences between two
nbred lines, but DPHP may underlie heterosis because differences inxpression between the hybrid and its parents should underlie theirhenotypic differences (Wei et al., 2009). DPHP can be divided into
classes: those shared by DPPP and DPHP (DPO) and those uniquely
elonging to DPHP (DPHPU). A total of 384 protein spots were identi-ed with significantly different expression (P < 0.05) between LYP9nd its parents, and the numbers of DPO were larger than DPHPUt three developmental stages (Table 1). By comparing DP betweenFig. 2. Functional classification of the number of identified DP in LYP9, 9311 (pater-nal parent), and PA64s (maternal parent) at tillering, flowering, and grain-fillingstages.
the hybrid and its parents, it can be seen that the majority of DPHPwas close to either CHP or CLP. To further understand the function ofDP, these DP were identified by MS. Of them, up to 77.3% (297/384)protein spots were qualified, corresponding to 222 unique proteins(Supplementary Table S2 and Fig. S1).
To further understand the function of DP, these proteinswere classified according to their functional categories accord-ing to the method of Bevan et al. (1998). All identified DPwere classified into 14 functional categories (Fig. 2). The high-est category was proteins involved in energy (34.0%), followed byproteins related to metabolism (17.2%), unclassified (12.8%), dis-ease/defense (8.7%), and unclear classification (8.4%). The othercategories were protein synthesis, transcription, signal trans-duction, protein destination and storage, cell growth/division,transporters, secondary metabolism, and cell structure. Since DPHPmay underlie heterosis, the functional classification of DPHP is fur-ther shown in Table 2. Because DPHP are composed of DPO andDPHPU, the functional categories of DPO and DPHPU were furtheranalyzed. Both of them are enriched in 5 subclasses, includingamino acid metabolism, sugars and polysaccharides metabolism,
glycolysis, photosynthesis, and disease/defense.Since the most important trait of hybrid rice is grain yield,the proteins involved in photosynthesis, glycolysis, and dis-ease/defense were further analyzed (Fig. 3 and Supplementary
392 C. Zhang et al. / Journal of Plant Physiology 169 (2012) 387– 398
Table 2Nonadditive-expressed proteins in LYP9.
Growth stage Number of NAP DPHP Number of NAP in DPHP
Up Down Total a% b% DPHPU c% DPO d%
Tillering stage 34 15 49 4.7 49 36.6 25 58.1 24 26.4Flowering stage 42 34 76 7.3 76 42.0 47 60.3 29 28.2Grain-filling stage 63 15 78 7.5 78 46.2 50 64.1 28 30.8
Total 119 59 178 17.2 178 44.2 112 61.9 73 32.9
a ote th
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% denotes the percentage of NAP in the total protein set (1037), b%, c%, and d% den
able S3). 25, 36, and 38 DPHP involved in photosynthesis were
etected at tillering, flowering, and grain-filling stage, respectivelynd the corresponding up-regulated DPHP were 19, 28, and 34Fig. 3A). It is interesting that the number of both DPHP and up-egulated DPHP increased at the flowering and grain-filling stagesig. 3. Changes in the number of DPHP and up-regulated DPHP involved in photosynthesA64s (maternal parent) at tillering, flowering, and grain-filling stages, respectively. ChD), glycolysis (E), and disease/defense (F) between LYP9 and its parents at tillering, flowxpression abundance between LYP9 and its parents, the differentially expressed proteiybrid was significantly twofold higher than abundance of proteins in two parents), CHP (ignificantly two-fold higher than that in the other parent), B2P (abundance of proteins inr abundance of proteins in F1 hybrid was between two parents and not significantly diffroteins between two parents), CLP (abundance of proteins in F1 hybrid was not significaarent), and L2P (abundance of proteins in F1 hybrid was significantly twofold lower thaf the three rice lines, we divided the five expression patterns into three basic categories
e percentage of NAP in total numbers of DPHP, DPHPU and DPO, respectively.
as compared to that at the tilling stage. For the proteins involved in
glycolysis, 5, 9, and 8 DPHP were detected at the tillering, flower-ing, and grain-filling stages, respectively, and the correspondingup-regulated DPHP were 2, 4, and 7. The number of both DPHPand up-regulated DPHP involved in glycolysis also increased atis (A), glycolysis (B), and disease/defense (C) in LYP9, 9311 (paternal parent), andanges in the expression profiles of up-regulated DPHP involved in photosynthesis
ering, and grain-filling stages, respectively. Based on the comparisons of proteinns were classified into five expression patterns: H2P (abundance of proteins in F1abundance of proteins in F1 hybrid was not significantly from that in one parent but
F1 hybrid was between two parents and significantly different from both parentserent from both parents but there were significant differences in the abundance ofntly from that in one parent but significantly twofold lower than that in the other
n abundance of proteins in two parents). To clearly describe the proteome changes: up-regulation (H2P, CHP), midparent (B2P), and down-regulation (CLP, L2P).
C. Zhang et al. / Journal of Plant Physiology 169 (2012) 387– 398 393
Table 3Functional classification of DPHPU and DPO.
Functional categories Tillering stage Flowering stage Grain-filling stage
DPHPU DPO DPHPU DPO DPHPU DPO
MetabolismAmino acid 0 4 7 5 5 5Nitrogen and sulphur 0 1 0 0 1 0Nucleotides 0 0 1 0 0 1Sugars and polysaccharides 3 4 5 4 2 5Lipid and sterol 1 0 0 1 0 0Cofactor 0 1 0 2 0 2
EnergyGlycolysis 2 3 3 6 4 4TCA pathway 1 3 3 2 0 1Respiration 0 1 1 0 1 0Fermentation 0 0 1 0 0 0Photosynthesis 12 13 15 21 25 13
Cell growth/division 0 2 2 1 1 2Transcription 0 2 2 0 2 2Protein synthesis 0 1 1 2 1 2Protein destination and storage 0 0 1 3 1 1Transporters 0 3 1 3 2 2Cell structure 0 0 0 1 1 0Signal transduction 1 1 2 2 3 2Disease/defense 4 4 6 8 4 6Transposons 0 1 0 2 1 2Secondary metabolism 1 1 0 1 1 0Unclear clasification 2 10 5 8 5 10Unclassified 5 9 8 9 6 9
Total 32 64 64 81 66 69
B
flsDa
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old values denote the known functional categories that have more DP.
owering and grain-filling stages as compared to that at the tillingtage (Fig. 3B). Similarly, the number of both DPHP and up-regulatedPHP involved in disease/defense also increased at the floweringnd grain-filling stages as compared to that at tilling stage (Fig. 3C).
The expression profiles of up-regulated DPHP between LYP9 andts parents in photosynthesis, glycolysis, and disease/defense path-
ays were further analyzed (Fig. 3D and E). It was found out thatp-regulated DPHP involved in photosynthesis, glycolysis, and dis-ase/defense pathways showed much higher expression in LYP9han in its parents. What is more interesting is that the higherxpression of most of up-regulated DPHP in LYP9 than its par-nts was much more pronounced at the flowering and grain-fillingtages as compared to the tillering stage.
onadditive-expressed proteins
It has been demonstrated that the relative level of proteinxpression in maize hybrids exhibits two scenarios (Marcon et al.,010). In the first scenario, protein expression in the hybrid exhibits
cumulative mode which is contributed by each allele from theespective parents. In the other scenario, protein expression devi-tes from the midparental level. The former scenario is additive,ndicating that alleles from both parents may contribute to genexpression in the hybrid, attributable mostly to a cis-regulationechanism. The latter scenario is nonadditive, in which other
egulators probably contribute to an altered expression of theorresponding alleles in the hybrid, attributable mostly to trans-egulation (Birchler et al., 2003). In this study, the modes of thedditive and nonadditive protein accumulation were investigatedn rice hybrid and its parents (Table 3). In comparison with proteinxpression between LYP9 and its parents, 119 up-regulated and 59
own-regulated nonadditive proteins (NAP, protein expression sig-ificantly deviates from the midparent level) were detected. Theetailed information on NAP is listed in Supplementary Table S4.he number of NAP ranged from 49 to 78; they composed 4.7–7.5%of the total proteins and 36.6–46.2% of DPHP identified at threedevelopmental stages.
Gene expression analysis by qPCR and integrated analysis ofproteomic and micorarray data
We selected 31, 34, and 27 DP to investigate their expressionpatterns at mRNA level at tillering, flowering, and grain-fillingstages, respectively (Fig. 4 and Supplementary Table S5). Theseselected DP were mainly based on their putative functions, includ-ing energy and metabolism. About 25.8%, 20.6% and 18.5% of DP attillering, flowering, and grain-filling stages, respectively, showedthe same expression patterns with their corresponding mRNAs. 20of the consistent DP exhibited two expression patterns: CHP (90.0%)and CLP (10.0%), 72 of the inconsistent DP fell into five groups:CLP (43.1%), CHP (31.9%), H2P (12.5%), B2P (11.1%), and L2P (1.4%),whereas three expression patterns were found on transcriptionallevel: CHP (34.6%), B2P (13.5%), H2P (11.5%).
The proteomic data in this study (297 identified DP) were com-pared with micorarray data (2061 DG) that generated from thesame rice hybrid cross (Wei et al., 2009). Totally, 84 protein spotswere matched in both datasets, and only 20 spots showed sameexpression pattern under different growth stage. There were 30proteins corresponding to 24 genes were matched at the samegrowth stage. There were 9 proteins found consistent expressionpatterns in both datasets, and 21 proteins were not (SupplementaryTable S6). Most of the DP in the protein data were observed in threeexpression categories: CHP (46.7%), CLP (30.0%), and H2P (13.3%),whereas most of the DP generated from the micorarray data werein other groups: CLP (37.5%), CHP (16.7%), L2P (12.5%), and B2P(12.5). This discrepancy could be largely attributable to biases in
the different expression profiling technologies applied and incon-sistent correlations of gene expression and protein abundance.The 24 genes were found mainly involved in energy (33.3%), dis-ease/defense (16.7%), amino acid metabolism (12.5%), sugars and
394 C. Zhang et al. / Journal of Plant Physiology 169 (2012) 387– 398
F phot( log2-tm
pp
P
tuotddl2a3r
ig. 4. Gene expression analyzed by qPCR. The genes encoding proteins involved inmaternal parent) at tillering, flowering, and grain-filling stages, respectively. The
ean ± SD of 10–12 independent experiments.
olysaccharides (12.5%), which suggested that these physiologicalrocesses were closely related to hybrid vigor.
hotosynthetic rate between LYP9 and its parents
Above results suggest that many proteins involved in photosyn-hesis were up-regulated in the hybrid. To confirm whether thesep-regulated proteins lead to increased photosynthetic efficiencyr not, photosynthetic characteristics in LYP9 and its parents athree developmental stages were examined. Fig. 5 shows that LYP9isplayed higher photosynthetic efficiency than its parents at threeevelopmental stages. For example, as compared with maternal
ine, LYP9 displayed higher CO2 assimilation rate by 17%, 20%, and
5% at tillering, flowering, and grain-filling stage, respectively. LYP9lso displayed higher apparent quantum yield of photosynthesis by4%, 37%, and 58% at the tillering, flowering, and grain-filling stage,espectively.osynthesis and glycolysis were analyzed in LYP9, 9311 (paternal parent), and PA64sransformed ratio between the hybrid and either parent is plotted. The values are
Discussion
The DP exhibit multiple expression patterns in hybrid rice
Although the hybrid vigor has been exploited in agriculturalcrops, the molecular basis of heterosis is still unclear. In this study,proteomic profiles between a superhybrid rice LYP9 and its par-ents were compared. A total of 384 DP were detected among thethree rice lines at tillering, flowering, and grain-filling stages and297 DP were identified by mass spectrometry. Moreover, the com-parison between proteomic data in this study (297 identified DP)with micorarray data (3926 DG) that generated from the samerice hybrid and its parents at the same developmental stage (Wei
et al., 2009) suggests that the abundance of most of identifiedproteins was not matched with the level of their gene expres-sion (Supplementary Table S6). Based on our proteomic analysis,approximately 18.8–25.6% proteins showed differential expression
C. Zhang et al. / Journal of Plant Physiology 169 (2012) 387– 398 395
F atura( ing, ae etwee
beaa((p(tn(oaegmofodvlp
a(a
ig. 5. (A) The response curves of CO2 assimilation rate to light intensity, (B) light-sAQY) in LYP9, 9311 (paternal parent), and PA64s (maternal parent) at tillering, flowerxperiments. Star indicates that the Pmax or AQY levels have significant difference b
etween LYP9 and its parents among three growth stages, providingvidence that proteins were differentially accumulated in F1 hybridnd inbred lines. These DP may provide new insights into the mech-nisms of heterosis in rice. DP between the hybrid and its parentsDPHP) were categorized into 5 basic categories: over dominanceH2P), under dominance (L2P), dominance (CHP and CLP), and mid-arent (B2P). In the present study, it was found out that dominanceCHP and CLP, 72.8–85.8%) was the largest expression patterns athree different developmental stages, which suggested that domi-ance at protein level could be important for the rice leaf heterosisTable 1). However, the gene expression study demonstrated thatver dominance was the largest expression mode in rice leaf, rootnd panicle (33.1%). Although studies also revealed that dominantxpression was the most prevalent among differentially expressedenes (DGHP) in rice leaf and panicle (81.6–91.8%) as well as in riceature embryo (84.3%), the proportion was different from each
ther (Wang et al., 2008; Wei et al., 2009). These variations reportedrom different studies may be due to the facts that different devel-pmental stages, different tissues or organs may display significantifferences in their degree of heterosis (Melchinger, 1999). Theseariations can be also explained by the fact that the changes on theevel of mRNA may not always indicate the changes on the level ofrotein.
Additive and nonadditive expressions have been proposed for
nother possible genetic model for gene expression in hybridsBirchler et al., 2003). Our previous study through a transcriptomicnalysis has demonstrated that the majority of genes in the riceted CO2 assimilation rate (Pmax), and (C) apparent quantum yield of photosynthesisnd grain-filling stages, respectively. The values are mean ± SD of 10–12 independentn the hybrid and inbred parents (P < 0.05).
hybrid showed additive expression and nonadditively expressedgenes accounted for only 0.5–1.4% of the total discovered genes, butaccounted for 29.6–53.7% of differentially expressed genes betweenthe hybrid and its parents (Wei et al., 2009). In this study, theadditive and nonadditive expression patterns based on our pro-teomic data were analyzed. The results show that the majority ofproteins in the rice hybrid showed additive expression and non-additively expressed proteins accounted for 4.7–7.5% of the totalproteins. In addition, nonadditively expressed proteins constitutedabout 36.6–46.2% of DPHP (Table 3). It has been suggested thattrans-regulators and epigenetic mechanism may be involved innonadditivity (Birchler et al., 2003; Wei et al., 2009). In this study,it was indeed found out that many trans-regulators showed differ-ential expression between the hybrid and its parents, such as RNAexport factor (REF) that deposits on mRNA in a splicing-dependentmanner and targets spliced mRNA for export (Nojima et al., 2007),DNA-directed RNA polymerase II 135 kDa polypeptide involved intranscription of mRNA precursors or HnRNA, and PPR proteinsinvolved in RNA processing (Supplementary Table S2). Epigeneticsas a mechanism regulates gene expression through DNA methyla-tion, histone modification, and chromatin packaging (Wolffe andMatzke, 1999; Jenuwein and Allis, 2001). It has been demonstratedthat the degree of methylation in hybrids is different from thatin inbred lines in rice (Xiong et al., 1999). In addition, it has been
reported that epigenetic regulation of a few regulatory genes (CCA1and LHY) may be responsible for the growth vigor and increasedbiomass in hybrids (Ni et al., 2009). In this study, four transposon
396 C. Zhang et al. / Journal of Plant Physiology 169 (2012) 387– 398
Fig. 6. Expression patterns of differentially expressed proteins involved in the Calvin cycle, glycolysis, and photorespiration pathways in LYP9 at tillering, flowering, andgrain-filling stages, respectively. Up-regulated DPHP represents either higher than both parents or close to higher parent; down-regulated DPHP represents either lower thanb paren
peTm2sisitb
I
gysdooaetbtaApLwCtlwfi
oth parents or close to lower parent; mid-parental DPHP represents between both
roteins and one retrotransposon protein were expressed differ-ntly between LYP9 and its parents (Supplementary Table S1).ransposon proteins play an important role in methylation poly-orphism that involved in epigenetic changes (Lukens and Zhan,
007). Retrotransposon proteins have been proved to activate orilence adjacent genes and induce new patterns of gene expressionn allopolyploidy (Levy and Feldman, 2004). The results in this studyuggest that these proteins related to epigenetics may be involvedn the regulation of heterosis. How these proteins are involved inhe biological pathways which are related to heterosis remains toe investigated further.
ncreased photosynthesis in hybrid rice
Photosynthesis provides energy and organic material for plantrowth and development, and is the basis of formation of cropield. In this study, many proteins involved in photosynthe-is were found to be mostly up-regulated in LYP9 at threeevelopmental stages (Fig. 6 and Fig. S2). For example, mostf enzymes involved in the Calvin cycle, the primary pathwayf carbon fixation in plants, were mostly up-regulated in LYP9t three stages (Fig. 6). These enzymes include phosphoglyc-rate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase,riosephosphate isomerase, transketolase, sedoheptulose-1,7-isphosphatase, and phosphoribulokinase. It should be notedhat ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)ctivase that regulates the activity of Rubisco by hydrolyzingTP, and three PGK that catalyze 3-phosphoglycerate into 1,3-hosphoglycerate were specifically identified as up-regulation inYP9 at grain-filling stage (Fig. 6 and Supplementary Table S2). Itas surprising that the clump of Rubisco, the key enzyme in thealvin cycle, in PEG pellet fraction expressed constantly not only in
hree cultivars but also at three developmental stages. Neverthe-ess, degraded leaf ribulose bisphosphate carboxylase large chainsere detected during rice growth (Supplementary Table S2). Thesendings were consistent with recent studies which suggested that
ts.
Rubisco in rice leaf was in a dynamic balance of synthesis andcatabolism during growth (Zhao et al., 2005) and Rubisco in riceleaf could be more than enough to meet the needs of photosynthesisunder normal conditions (Murchie et al., 2002).
Some proteins involved in the light reaction, an indispens-able part of photosynthesis and providing ATP and NADPH forthe operation of the Calvin cycle, were up-regulated in LYP9 atthree stages (Fig. S2). Among them, some hybrid-specific expressedproteins, e.g. LHCII, LHCI, PsaE, atpE, and atpA, were observed atthe flowering and grain-filling stages (Supplementary Table S2).These up-regulated proteins are believed to improve light absorp-tion, transduction, and photophosphorylation that are favorable forphotosynthesis. These hybrid-specific expressed proteins may con-tribute to yield vigor in F1 hybrid, since it has been proposed thatleaf genes that expressed only in F1 generation were positively cor-related with heterosis in six yield-related traits in wheat (Sun et al.,2004).
Photorespiration is a high-flux pathway that operates alongsidecarbon assimilation in C3 plants, and results in considerable carbonand energy losses, which has been considered a stumbling block toimproving food production (Bauwe et al., 2010). Several enzymes,e.g. phosphoglycolate phosphatase (PGP), glycine hydroxymethyl-transferase, hydroxypyruvate reductase, and glycolate oxidaseinvolved in photorespiration process, were down-regulated in LYP9at three developmental stages (Fig. 6). These results suggest thatthe photorespiration was suppressed in hybrid rice. In addition,we observed that non-photochemical quenching (NPQ) was less inLYP9 than its parents in particular at the grain-filling stage (data notshown), indicating that the dissipation of excess energy was less inLYP9 than its parents due to down-regulation of photorespiration.
The up-regulation of a series of enzymes involved in the carbon-fixation pathway and the proteins involved in the light reaction as
well as suppressed photorespiration may lead to higher efficiencyof photosynthesis in the hybrid. Indeed, it was observed that therewas a greater increase in net CO2 assimilation rate and apparentquantum efficiency of photosynthesis in the hybrid than its parents
nt Phy
(ty
I
ssseirhoaeomctsflflugasria
B
eTaTtelArttt(totifsSoata(ipta
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Fig. 5). These results suggest that an accelerated photosynthesis inhe hybrid leaves may be fundamental factors in improving cropield of the hybrid rice.
mproved carbohydrate metabolism in hybrid rice
The main products of photosynthetic carbon assimilation aretarch and sucrose. The previous study through comparative tran-criptional profiling has demonstrated that the P-value was notignificant in the sucrose and starch pathway in hybrid rice (Songt al., 2010). However, in the present study, proteins involvedn starch and sucrose synthesis pathways were found to be up-egulated greatly in particular during the grain-filling period in theybrid (Fig. 6). For example, we observed that its two protein spotsf ADP-glucose pyrophosphorylase (AGPase) were up-regulatedt grain-filling stage in LYP9. Since AGPase is the most criticalnzyme in starch synthesis (Zeeman et al., 2010), its up-regulationf AGPase in the hybrid suggests that leaves of LYP9 maintainedore vigorous starch metabolism during grain-filling period and
ould provide more supply of substrates for sucrose for export tohe grains and the rest of the plant. In addition, we observed thateveral cytosolic enzymes involved in the sucrose synthesis, e.g.ructose-1,6-bisphosphatase and phosphoglucose isomerase, hadower expression in the hybrid than its parents at the tillering andowering stages, but up-regulated during grain-filling period. Thep-regulation of the enzymes involved in the sucrose synthesis atrain-filling stage suggests that the sucrose synthesis is acceler-ted in LYP9 during grain-filling period. This accelerated sucroseynthesis may be benefit to the grain-filling process in the hybridice. Therefore, improved starch and sucrose synthesis in LYP9 dur-ng grain-filling period will be helpful to the grain-filling processnd the formation of grain yield.
etter antioxidant defense system in hybrid rice
Rice grown under field conditions is often exposed to differentnvironmental stresses, such as high light and high temperature.hese environmental stresses often result in the enhanced gener-tion of reactive oxygen species (ROS) (Noctor and Foyer, 1998).o ensure plant growth, development, and high-yield production,here is a need for better antioxidant defense system to scavengexcess ROS. In the study, many proteins involved in protecting cel-ular components from ROS in vivo, such as Cu/Zn SOD, catalase,PX, DHAR, and 2-cys peroxiredoxin (2-Prx), were found up-egulated in LYP9 at three developmental stages (Fig. S3). Amonghem, APX is one of the most critical enzymes. It has been reportedhat a wheat thylakoid-bound APX mutant exhibits impaired elec-ron transport, photosynthetic activity, and biomass accumulationDanna et al., 2003). 2-Prx mentioned above acts as a constitu-ive protector of photosynthetic apparatus and membranes againstxidative damage (Baier and Dietz, 1999; Dietz et al., 2006), andhree 2-Prxs were found up-regulated in the hybrid at flower-ng and grain-filling stages. Besides, many other enzymes thatunction in protecting cellular components from environmentaltresses were detected up-regulated in LYP9, such as glutathione-transferase that protects cells from a broad spectrum of abi-tic stress (Marrs, 1996), and NADPH thioredoxin reductase with
thioredoxin domain in the C-terminus (NTRC) that was reportedhe Arabidopsis NTRC knock-out mutant showed growth inhibitionnd hypersensitivity to methyl viologen, drought, and salt stressSerrato et al., 2004). The up-regulation of these enzymes involved
n antioxidant defense system suggests that as compared with itsarents, the hybrid had a better antioxidant defense system to pro-ect cells against ROS-induced damage and environmental stressesnd thus ensure the hybrid growth, development, and productivity.siology 169 (2012) 387– 398 397
Dynamic proteome changes between LYP9 and its parents atdifferent developmental stages
As discussed above, increased photosynthesis, improved carbo-hydrate metabolism, and better antioxidant defense system maycontribute to hybrid vigor in rice. Although there are many stud-ies about the mechanisms of heterosis, it is unknown whetherheterosis vigor is dependent on different developmental stages.In this study, proteome profiling between LYP9 and its parentswas compared at the tillering, flowering, and grain-filling stages.Surprisingly, the number of identified DPHP involved in photosyn-thesis, glycolysis, and disease/defense increased at the floweringand grain-filling stages as compared to that at the tillering stage(Fig. 3A–C). More importantly, most of up-regulated proteinsinvolved in photosynthesis, glycolysis, and disease/defense showedgreater expression in LYP9 at the flowering and grain-filling stagesthan at the tillering stage (Fig. 3D–F). In addition, CO2 assimilationrate and apparent quantum yield of photosynthesis also showeda greater increase in LYP9 at the flowering and grain-filling stagesthan at the tillering stage (Fig. 5). The results in this study for thefirst time suggest that the performance of hybrid vigor was depen-dent on different development stages in rice. It should be notedthat at the tillering stage, most of up-regulated proteins involvedin photosynthesis, glycolysis, and disease/defense showed greaterexpression in LYP9 than in its parents (Fig. 3D–F). Thus, the mech-anisms of heterosis at the tillering stage can be also explained byimproved photosynthesis, carbohydrate metabolism, and antioxi-dant defense system in rice hybrid.
Grain yield is one of most important agricultural traits for mostcrops. The aim of the breeding of rice hybrid is to increase rice grainyield. Obviously, the grain-filling process is a critical process for ricegrain yield. Since leaf photosynthesis contributes about 60–100% ofthe carbon in mature grains during the grain-filling period (Yoshida,1981), the greater express of up-regulated proteins involved in pho-tosynthesis, the sucrose synthesis, and the starch synthesis in LYP9at the grain-filling period than at the tillering stage will be help-ful to the grain-filling and may lead to high-yield of rice grain.The results in this study suggest that the dynamic regulation ofthe important physiological processes at different developmentalstages, such as photosynthesis, the sucrose synthesis, and the starchsynthesis, may be one of mechanisms underlying heterosis in rice.
Acknowledgements
We thank Li Wang and Lijie Feng for their assistance in MALDI-TOF MS analysis. This study was supported by the National NaturalScience Foundation of China (30725024), the State Key BasicResearch and Development Plan of China (2009CB118503), andthe Frontier Project of the Knowledge Innovation Engineering ofChinese Academy of Sciences (KSCX2-EW-J-1).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jplph.2011.11.016.
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