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Journal of Plant Physiology 213 (2017) 87–97

Contents lists available at ScienceDirect

Journal of Plant Physiology

journa l homepage: www.e lsev ier .com/ locate / jp lph

haracterization and expression patterns of key C4 photosyntheticathway genes in bread wheat (Triticum aestivum L.) under fieldonditions

aoura Goudia Bachir a, Iqbal Saeed a,c, Quanhao Song a, Tay Zar Linn a, Liang Chen a,in-Gang Hu a,b,∗

State Key Laboratory of Crop Stress Biology for Arid Areas, College of Agronomy, Northwest A&F University, Yangling, Shaanxi, 712100, ChinaInstitute of Water Saving Agriculture in Arid Regions of China, Northwest A&F University, Yangling, Shaanxi, 712100, ChinaNIFA, PO BOX 446, Tarnab, Peshawar, KP, Pakistan

r t i c l e i n f o

rticle history:eceived 31 October 2016eceived in revised form 6 March 2017ccepted 7 March 2017vailable online 9 March 2017

eywords:heat

4 pathwayene expression4 enzyme activityhotosynthetic efficiency

a b s t r a c t

Wheat is a C3 plant with relatively low photosynthetic efficiency and is a potential target for C4 pho-tosynthetic pathway engineering. Here we reported the characterization of four key C4 pathway genesand assessed their expression patterns and enzymatic activities at three growth stages in flag leaves of59 bread wheat genotypes. The C4-like genes homologous to PEPC, NADP-ME, MDH, and PPDK in maizewere identified in the A, B, and D sub-genomes of bread wheat, located on the long arms of chromo-somes 3 and 5 (TaPEPC), short arms of chromosomes 1 and 3 (TaNADP-ME), long arms of chromosomes1 and 7 (TaMDH), and long arms of chromosome 1 (TaPPDK), respectively. All the four C4-like geneswere expressed in the flag leaves at the three growth stages with considerable variations among the 59bread wheat genotypes. Significant differences were observed between the photosynthesis rates (A) ofwheat genotypes with higher expressions of TaPEPC 5, TaNADP-ME 1, and TaMDH 7 at heading and mid-dle grain-filling stages and those with intermediate and low expressions. Our results also indicated that

the four C4 enzymes showed activity in the flag leaves and were obviously different among the 59 wheatgenotypes. The activities of PEPcase and PPDK decreased at anthesis and slightly increased at grain-fillingstage, while NADP-ME and MDH exhibited a decreasing trend at the three stages. The results of the cur-rent study could be very valuable and useful for wheat researchers in improving photosynthetic capacityof wheat.

© 2017 Elsevier GmbH. All rights reserved.

. Introduction

Wheat performs CO2 assimilation through the C3 photosyn-hetic pathway. However, some plants, such as maize (Zea mays

.) and foxtail millet (Setaria italica), possess the C4 photosyn-hesis pathway in addition to the C3 pathway, which provideshem with higher light saturation points, higher carboxylation effi-

Abbreviations: ANOVA, analysis of variance; BYPP, biomass yield plant−1; GYPP,rain yield plant−1; BSC, bundle sheath cell; MC, mesophyll cell; MDH, malate dehy-rogenase; NADH, nicotinamide adenine dinucleotide; NADP, nicotinamide adenineinucleotide phosphate; NADP-ME, NADP-dependent malic enzyme; PEPC, phos-hoenolpyruvate carboxylase; PPDK, pyruvate orthophosphate dikinase.∗ Corresponding author at: State Key Laboratory of Crop Stress Biology for Aridreas, College of Agronomy, Northwest A&F University, Yangling, Shaanxi, 712100,hina.

E-mail addresses: huyingang@nwsuaf.edu.cn, huyingang@126.com (Y.-G. Hu).

ttp://dx.doi.org/10.1016/j.jplph.2017.03.002176-1617/© 2017 Elsevier GmbH. All rights reserved.

ciency, and lower CO2 compensation points in comparison withC3 plants. C4 plants have distinct growth advantages over C3plants, especially under stress conditions of high temperature anddrought (Richard, 2000). The four C4 pathway enzymes, namelyphosphoenolpyruvate carboxylase (PEPC), NADP-dependent malicenzyme (NADP-ME), malate dehydrogenase (MDH), and pyru-vate orthophosphate dikinase (PPDK), play important roles inconcentrating CO2 around the enzyme ribulose 1,5-bisphosphatecarboxylase-oxygenase (Rubisco) in C4 plants (Brown et al., 2005).PEPC catalyzes the formation of oxaloacetate (OAA) from phospho-enolpyruvate (PEP) and HCO3 (O’Leary, 1982). OAA is metabolizedinto malate and then diffuses into the bundle sheath cell (BSC)where it is decarboxylated to provide increased concentrationof CO2 around Rubisco. Finally, the initial substrate of the C4

cycle, PEP, is regenerated in the mesophyll cell (MC) by pyru-vate orthophosphate dikinase (PPDK) (Kajala et al., 2011). Severalreports suggested that the photosynthetic enzymes involved in the

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4 pathway were also present in C3 plants but were less activeRosche et al., 1994; Drincovich et al., 1998; Tsuchida et al., 2001;u et al., 2003). Since Hibberd and Quick (2002) reported over-xpression of PEPC, NADP-ME, and PPDK in cells of stems andetioles surrounding the xylem and phloem in tobacco, a typical3 plant, the CO2-refixation function has been the focus of muchttention. Thus, it is accepted that a C4-like CO2-refixation pathwayccurs in C3 plants. C4-specific photosynthetic genes identified inhe bread wheat genome were preferentially found to be expressedn the photosynthetic pericarp tissue of the bread wheat caryop-is (Rangan et al., 2016). Three isozymes of PEPC and two of PPDKere reported in maize (Kawamura et al., 1992; Sheen, 1999). Wang

t al. (2009) reported two NADP-MDH and two PPDK genes inorghum. Activities of maize C4 PEPC in the flag leaves of trans-enic and untransformed bread wheat plants were reported by Lint al. (2012). Li et al. (2001) reported activities of four C4 pathwayelated enzymes in leaves of soybean cultivars.

One of the avenues being recently explored is the improvementf photosynthetic capacity by engineering the C4 photosyntheticathway in C3 plants to raise the potential yield (Surridge, 2002).

major new project has been initiated (funded by the Bill andelinda Gates Foundation) to transfer C4 characteristics into rice,

ncluding the anatomical specialization required for a 2 cell-type4 mechanism (Kranz anatomy) (Parry et al., 2011).

Several previous studies succeeded in introducing the maize4-specific PEPC cDNA into wheat and obtained transgenic plantsith enhanced photosynthetic capacity (Li et al., 2009; Wu et al.,

011; Hu et al., 2012; Han et al., 2013; Zhang et al., 2014). Ku et al.1999, 2000) obtained transgenic C3 plants with high levels of maize4-specific PEPC gene expression after introducing the respectiveDNA into rice. C4-specific PPDK or NADP-ME was transferred intoice (Fukayama et al., 2001; Jiao et al., 2003; Taniguchi et al., 2008),at (Tolley et al., 2012), Arabidopsis thaliana (Ishimaru et al., 1997;ang et al., 2012), and potato (Gehlen et al., 1996). Although C4

nzymes were transferred into C3 plants, trying to improve the pho-osynthetic efficiency, only few were successful (Zhang et al., 2009).owever it is possible to screen cultivars displaying high expres-

ion and high activity of C4 enzymes among C3 plants to enhancehotosynthetic efficiency of C3 plants.

At present, fewer efforts have been made to understand thendogenous expression of these genes in bread wheat at differentrowth stages. Therefore, in this study, we identified and character-zed the homologues of genes encoding the key C4 photosyntheticathway enzymes and assessed their expression patterns and activ-

ties using one isoform of each of the identified C4-like genes athree growth stages in a panel of 59 winter bread wheat genotypes.

. Materials and methods

.1. Plant materials and growth conditions

The investigation was conducted with a panel of 59 bread wheatenotypes, which were mainly from the major winter wheat pro-uction regions of China and widely used in production. They wereown in October 2014 and 2015 at the experimental farm of North-est A&F University, Shaanxi, China (N 34◦10ı́, E 108◦10ı́). The

ltitude of the area is 525 m, and the climate is semi-humid prone toemi-arid with an average annual temperature of 13◦C and averagennual rain fall of 600 mm. Details on the 59 genotypes are pro-ided in Table S1. Each genotype was sown in three rows of 1.67 mength, with 25 cm between rows and 6.7 cm between plants. The

ag leaves of three plants of each variety were collected at head-

ng (Z55), anthesis (Z67), and middle grain-filling (Z73) stages,espectively. The flag leaves were used for RNA isolation, molecularnalysis, and assays for enzyme activities.

hysiology 213 (2017) 87–97

2.2. Gene identification and sequence analysis

C4-specific PEPC, NADP-ME, MDH, and PPDK genes of maizeand foxtail millet were obtained by database mining (NCBI-blastn).To identify wheat homologues, a “blastn” was run with the genesequences on NCBI database, and homologous sequences of TaPEPC(AJ007705 & Y15897), TaNADP-ME (EU082065 & EU170134),TaMDH (AK333412), and TaPPDK (AK333343 & AF475130) werethen downloaded. Gene-specific primers were designed usingPrimer Premier5.0 and PCR Primer Stats (Stothard, 2000) forfragment amplification and homology-based cloning. The primersequences are shown in Table S2. For gene cloning, PCR prod-ucts separated in 1.0% agarose gel and purified by Universal DNAPurification Kit (Tiangen, Beiging) were cloned into pGEM-T EasyVector and sequenced by Shanghai Biotechnology Co., Ltd. (Shang-hai). Sequencing showed all resultant sequences to be basically thesame as the genes recorded in GenBank, with a similarity of 98%.

To identify homologous sequences of isoforms of TaPEPC,TaNADP-ME, TaMDH, and TaPPDK, corresponding to the threebread wheat sub-genomes (A, B, and D), a BLAST search usingthe obtained cDNA sequences was performed in the URGI data-bank (http://wheat-urgi.versailles.inra.fr/). Using the transcriptcode of the corresponding sequence in URGI, the full-length cDNAsequences of the A, B, and D sub-genomes were then retrievedthrough Search in the wheat database of EnsemblPlants (http://plants.ensembl.org/Triticumaestivum/Info/Index).

The amino acid sequences were deduced from the cDNAsequences of the C4 genes, and their conserved domains wereidentified by a BLAST search in the Conserved Domain database(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Phyloge-netic trees were constructed as a Neighbor-Joining (NJ) tree usingthe Geneious Tree Builder Software 9.1.3 based on the pairwisealignment of C4-like gene protein sequences of wheat, rice, brachy-podium, maize, and foxtail millet.

2.3. RNA extraction and cDNA synthesis

Flag leaf samples were collected from five randomly selectedplants from each of the 59 experimental genotypes at heading(Z55), anthesis (Z67), and middle grain-filling (Z73) growth stages.High quality RNA was extracted using a three-step modified HotPhenol method (Verwoerd et al., 1989; Shinmachi et al., 2010). Ini-tial extraction was carried out in 1 mL (80 ◦C) 1:1 Phenol/Extractionbuffer (0.1 M Tris-HCL, pH 8.0, 0.1 M LiCl, 1% (w/v) SDS, and 10 mMEDTA). Afterwards, two phenol/chloroform/isoamyl (25:24:1)extractions were conducted. DNase treatment was carried outusing DNA-Free Kit (Takara, Dalian, China) according to the man-ufacturer’s instructions. Gel electrophoresis was performed, andabsorbance was measured at 260 and 280 nm to ensure RNAintegrity. RNA was reverse-transcribed for 50 min at 42 ◦C with afinal denaturation step at 95 ◦C for 5 min, using oligo (dT) primersand SuperScriptW II Reverse Transcriptase (Takara, Dalian, China).The reaction was carried out in a total volume of 20 �L.

2.4. Expression analysis of the genes encoding the C4photosynthetic enzymes

Gene expression was performed using quantitative reverse tran-scription polymerase chain reaction (qRT-PCR) to quantify the totalexpression levels of the TaPEPC 5, TaNADP-ME 1, TaMDH 7, and TaP-PDK 1 ̨ cDNAs. Specific primers were designed based on the cDNAsequences and were used to differentiate the total expression of the

C4 genes in flag leaves at heading (Z55), anthesis (Z67), and middlegrain-filling (Z73) stages, respectively. For normalization, TaActinand TaSand genes were used as endogenous controls. Details of theprimers used for expression analysis are listed in Table S2. Gene

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pecificity of each primer was confirmed through BLAST searchesf public databases (http://www.ncbi.nlm.nih.gov/tools/primer-last/).

The reactions were carried out in duplicates in a 96-well platesing the real-time PCR system ABI 7300 (Applied Bio systems,SA). Reaction mixtures contained 10 �L Syber Green PremixxTaq Kit (TaKaRa, Dalian, China), 4 pmol of each primer, 1.5 �L ofDNA as template, and ddH2O up to 20 �L total volume. PCR con-itions were set as follows: 95 ◦C for 30 s followed by 40 cycles of5 ◦C for 5 s, and 60 ◦C for 30 s. Relative gene expressions in differentamples were determined using the formula:

E = (EX )−Ct,X

(ER)−Ct,R

E is the relative expression of the target gene, E is the primerfficiency, Ct value is collected where the fluorescence is abovehe threshold value, X indicates values from the target gene, and

indicates the geometric mean of values from the two referenceenes.

We further evaluated the differential expression in A, B, and Dub-genomes of TaPEPC 5, TaNADP-ME 1, TaMDH 7, and TaPPDK 1˛n twenty genotypes with ten showing high expression and tenhowing low expression of the corresponding C4-like genes. Due toigh homology in the coding sequences of the three copies, genomepecific primers were designed in the 5’and 3′UTR regions, andetails of the primers are listed in Table S2.

.5. Extraction of crude enzymes

Flag leaves of three plants of each genotype were sampledt heading (Z55), anthesis (Z67), and middle grain-filling (Z73)tages and stored at −20 ◦C for measurement of enzyme activities.rozen leaves were ground with liquid nitrogen to fine pow-er in 1 mL extraction buffer using a chilled mortar and pestle.he extraction buffer contained 50 mM Tris–HCl (pH 7.5), 10 mMgCl2, 5 mM dithiothreitol (DTT), 1 mM EDTA, 2% (w/v) insolu-

le polyvinylpolypyrrolidone (PVP), and 10% (w/v) glycerol. Crudextracts were centrifuged at 13 000 x g for 20 min at 4 ◦C, and theupernatants were used immediately to measure enzyme activities.

final enzyme concentration of 5 mg/mL was used. The activitiesf the following enzymes were assessed: PEPCase, NADP-ME, MDH,nd PPDK, using methods described by Ku et al. (1999), Tsuchidat al. (2001), and Gonzalez et al. (1984) with slight modifications.ll measurements were performed at 30 ◦C using Tecan Infinite00 Pro (Tecan, Mannedorf, Switzerland) microplate reader. Theolar extinction coefficient of 6.22 mM cm−1 was used for NADH

nd NADPH.

.6. Assay of PEPcase

Phosphoenolpyruvate carboxylase (PEPcase) activity wasssayed in a mixture containing 50 mM tricine-KOH (pH 8.0),0 mM MgCl2, 10 mM NaHCO3, 0.1 mM EDTA, 0.2 mM NADH, 3 Ualate dehydrogenase (MDH), 20 �L of the enzyme extract, and

istilled water to final reaction volume of 200 �L. The reaction wasnitiated by adding phosphoenolpyruvate to a final concentrationf 2 mM, and the rate of NADH consumption was determined byhe absorbance change at 340 nm. One unit of enzyme activity ishe capacity of the enzyme to catalyze the formation of 1 �mol ofxalacetate min−1.

.7. Assay of NADP-ME

The NADP-ME assay medium contained 50 mM Tris–HCl (pH.5), 1 mM MgCl2, 1 mM MnCl2, 1 mM EDTA, 0.5 mM NADP, 20 �L

hysiology 213 (2017) 87–97 89

of the enzyme extract, and distilled water to final reaction volumeof 200 �L. The reaction was started by adding 5 mM malate, and thereduction of NADP+ was monitored by absorbance at 340 nm. 1 Uof enzyme activity is defined as the amount of enzyme that resultsin the production of 1 �mol of NADPH min−1.

2.8. Assay of MDH

The assay mixture contained 100 mM Tris–HCl (pH 7.5), 1 mMEDTA, 0.2 mM NADH, 20 �L of the enzyme extract, and distilledwater to final reaction volume of 200 �L. Oxaloacetic acid with afinal concentration of 2 mM was added to start the assay, and thechange of absorbance at 340 nm was monitored.

2.9. Assay of PPDK

PPDK assay buffer consisted of 25 mM Tricine-KOH, 10 mMMgCl2, 10 mM NaHCO3, 10 mM DTT, 2 mM sodium pyruvate, 5 mM(NH4)2SO4, 2.5 mM K2HPO4, 1 mM glucose-6-phosphate, 0.2 mMNADH, 2 U NAD-MDH, 50 mM ATP, 0.5 U PEPC, 20 �L of the enzymeextract, and distilled water to final reaction volume of 200 �L. 1 U ofPPDK activity corresponds to 1 �mol of pyruvate converted min−1

at 30 ◦C.

2.10. Phenotypic evaluation

Photosynthetic rate (A) was measured using a portable photo-synthesis system (LICOR LI-6400XT, USA) on fully expanded flagleaves of five randomly selected plants of each of the 59 geno-types in each replication at heading (Z55), anthesis (Z65), andmiddle grain-filling stages (Z73), respectively. Conditions in the leafchamber were as reference CO2 concentration = 400 �mol mol−1,PPFD = 1800 �mol m−2 s−1, relative humidity 50–70%, and blocktemperature = 20 ◦C. Measurements were taken between 9:00 and11:00am in sunny and windless weather.

After harvest, biomass per plant−1 (BMPP) and grain yield perplant−1 (GYPP) were determined using ten replicate plants for eachof the 59 wheat genotypes.

2.11. Statistical analysis

Genotypes were grouped according to gene expressions fol-lowing hierarchical cluster analysis at the three growth stages.Variation of TaPEPC, TaNADP-ME, TaMDH, and TaPPDK expressions,activities, photosynthesis rates, and yield among the three groupswas assessed by analysis of variance (ANOVA) according to thegeneral linear model procedure of Statistical Analysis System (SAS8.1; SAS Institute Inc., Cary, NC, USA). Differences between groupswere separated by Fisher’s protected least significance difference(LSD) test at 0.05 level. Correlation analysis between gene expres-sions and enzyme activities was worked out with the help of SPSSstatistics 20.0 (IBM SPSS Statistics, USA).

3. Results

3.1. Characterization of the C4 pathway related genes in breadwheat

Sequences of PEPC (AJ007705 & Y15897) were isolated byhomology-based cloning with PEPC family genes and were iden-tified as fragments of TaPEPC genes. A BLAST search using theobtained TaPEPC cDNA sequences in the URGI databank revealed

the homologous sequences of TaPEPC (AJ007705) were on the longarms of chromosome 5 and designated as TaPEPC 5, while thoseof TaPEPC (Y15897) were on the long arms of chromosome 3 anddesignated as TaPEPC 3. Copies of each gene corresponding to the

90 D.G. Bachir et al. / Journal of Plant Physiology 213 (2017) 87–97

Table 1C4-like genes identified in bread wheat, chromosomal locations, and variations of deduced amino acids among the three sub-genomes.

Gene Location Total Total AA Difference in AAnucleotide Genome A and B Genome B and D Genome A and D

TaPEPC 5ABDL 2919 972 3 4 53ABDL 2928 975 19 30 17

TaNADP-ME 1ABDS 1713 570 26 22 63ABDS 1947 648 8 12 7

TaMDH 7ABDL 1185 394 11 9 101ABDL 1197 398 10 13 8

TaPPDK 1ABDL 2820 939 11 8 21ABDL 2919 972 3 4 5

A ent ths A’ stan

AtinTmsdetposaha

(1tT1rist

FsA

, B, and D represent the three sub-genomes of bread wheat, and ‘L’ and ‘S’ represize in nucleotides, amino acids, and positions of variations in the AA sequences. ‘A

, B, and D sub-genomes of bread wheat were identified. Then,heir full-length cDNA sequences were obtained through searchn the wheat database of EnsemblPlants. The genes were desig-ated as TaPEPC 5AL, TaPEPC 5BL, and TaPEPC 5DL for TaPEPC 5 andaPEPC 3AL, TaPEPC 3BL, and TaPEPC 3DL for TaPEPC 3 gene. Align-ent between the three cDNA copies of TaPEPC 5 and TaPEPC 3

howed 98% and 97% identical nucleotides, respectively. Theeduced protein sequences were encoded by 10 exons and showedxtensive similarities to PEPC of other plants sequenced so far. Thewo open reading frames encoded a deduced polypeptide com-osed of 972 and 975 amino acids with an expected molecular massf 110.1 kDa. In silico analysis of TaPEPC genes showed a single con-erved domain: PEPCase domain. Alignment of the predicted aminocid sequences of the six TaPEPC sub-genome copies showed highomology in the PEPCase domain region, with 90% identical aminocids.

Similarly, the genomic and cDNA sequences of TaNADP-MEEU082065 & EU170134) located on the short arms of chromosomes

and 3 (TaNADP-ME 1 & TaNADP-ME 3), TaMDH (AK333412) onhe long arms of chromosomes 7 and 1 (TaMDH 7 & TaMDH 1), andaPPDK (AK333343 & AF475130) on the long arm of chromosomes(TaPPDK 1 ̨ &TaPPDK 1ˇ) were obtained. Copies of each gene cor-

esponding to the A, B, and D sub-genomes of bread wheat weredentified and full-length cDNA sequences were obtained. Highimilarity in nucleotide sequences was also observed among each ofhe three sub-genome cDNA copies of the C4-like genes. Compari-

ig. 1. Mean relative expression of A, B, and D sub-genome copies of C4-like genes in 20tages.: TaPEPC 5; B: TaNADP-ME 1; C: TaMDH 7; D: TaPPDK 1˛.

e long and short arms of chromosomes, respectively. The numbers represent geneds for amino acids.

son of the deduced amino acid sequences of the sub-genome copiesof those genes showed high homology in their conserved domainregions, with some genes presenting deletions in their sequences(Table 1).

The polygenetic trees of PEPC, NADP-ME, MDH, and PPDK dis-play the relationships of amino acid sequences of C4-related genesfrom wheat, rice, brachypodium, maize, and foxtail millet. Theresults show that wheat PEPC genes are closely related to brachy-podium and rice PEPC, while wheat NADP-ME genes are morerelated to rice and maize. The wheat sub-genome copies of MDHand PPDK genes are classified in the same group respectively withthe two genes more related to brachypodium MDH and PPDK genes(see Fig. S1).

3.2. Expression patterns of C4-like genes in flag leaves of breadwheat

To evaluate the expression patterns of each of the C4-like genesin flag leaf of bread wheat, we first defined their total expressionlevels using the general primers among a panel of 59 wheat geno-types. Then, we detected the genome-specific expressions of three

copies of each of the C4-like genes in twenty genotypes, with tenfor high expression and ten for low expression of the correspond-ing C4-like genes, to see whether they were consistent with thosedetected by general primers.

selected wheat genotypes at heading (Z55), anthesis (Z67), and grain-filling (Z73)

D.G. Bachir et al. / Journal of Plant Physiology 213 (2017) 87–97 91

Table 2Mean relative total expression of the C4-like genes at three growth stages in flag leaf of 59 wheat genotypes.

Growth stages Mean TaPEPC 5 TaNADP-ME 1 TaMDH 7 TaPPDK 1˛

Heading Mean ± SD 0.795 ± 0.30 0.548 ± 0.27 0.832 ± 0.31 0.644 ± 0.19Minimum 0.28 0.11 0.31 0.28Maximum 1.43 1.20 1.50 1.10

Anthesis Mean ± SD 0.671 ± 0.30 0.478 ± 0.19 0.736 ± 0.30 0.539 ± 0.16Minimum 0.18 0.11 0.18 0.16Maximum 1.56 1.10 1.51 1.10

Grain-filling Mean ± SD 0.832 ± 0.30 0.471 ± 0.18 0.595 ± 0.27 0.759 ± 0.30Minimum 0.17 0.10 0.17 0.23Maximum 1.50 1.00 1.48 1.49

Data are presented as mean ± SD (standard deviation).

Table 3The correlation coefficients between the relative total expression of the C4–like genes and their genome-specific expressions in 20 selected wheat genotypes at three growthstages.

Growth stages Genome-specific expression TaPEPC 5 TaNADP-ME 1 TaMDH 7 TaPPDK 1˛total total total total

Heading A genome 0.344* 0.711** 0.661** 0.846**B genome 0.337* 0.208 0.625** 0.205D genome 0.406** 0.419** 0.703** 0.654**

Anthesis A genome 0.594** 0.778** 0.561** 0.468**B genome 0.747** 0.318* 0.659** 0.642**D genome 0.575** 0.333* 0.907** 0.519**

Grain-filling A genome 0.253* 0.111 0.805** 0.385**B genome 0.325* 0.557** 0.677** 0.312*

0.146 0.802** 0.558**

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Table 4Number of genotypes clustered in the high, intermediate, and low expression groupsof different C4-like genes.

C4-like gene Group I Group II Group III

TaPEPC 5 17 34 8TaNADP-ME 1 12 34 13TaMDH 7 13 33 13TaPPDK 1 ̨ 19 28 12Representative 1,7,11 25, 27, 31, 33, 40, 56 6, 37, 45, 50, 51genotypes

D genome 0.168

and ** indicate correlation significant at the 0.05 and 0.01 level, respectively.

The total expression levels of the C4-like genes in wheat flageaves showed similar expression patterns among the wheat geno-ypes at the three stages, with TaPEPC 5, and TaPPDK 1 ̨ showingigher expression levels at heading and middle grain-filling stageut lower at anthesis stage, while the expression levels of TaNADP-E 1 and TaMDH 7 showed a decreasing trend at the three stages

Table 2). Significant variations were observed in the expressionevels of each of the C4-like genes among the 59 diverse bread

heat varieties at the three stages. As shown in Table 2, TaMDH 7howed the highest expression level at heading (1.50) with a meanxpression of 0.83, followed by TaPEPC 5 (1.43) with a mean of 0.80,hile TaNADP-ME 1 displayed the lowest expression level (0.11)ith a mean of 0.55. At anthesis, TaPEPC 5 displayed the high-

st expression level (1.56) with a mean expression of 0.67, whileaNADP-ME 1 (0.11) showed the lowest expression levels with aean of 0.48.

Genome-specific expression analysis of TaPEPC 5, TaNADP-E 1, TaMDH 7, and TaPPDK 1 ̨ showed similar expression

atterns with variations among the three copies of each of the C4-ike genes (Fig. 1). For TaPEPC 5, TaMDH 7, and TaPPDK 1˛, the Dub-genome copies were expressed evidently higher than the otherwo copies, while in B sub-genome the highest expression levelsere displayed for TaNADP-ME 1. Further correlation analysis indi-

ated that the total expression levels of the C4-like genes detectedith the general primers were positively and highly correlated with

heir genome-specific expressions levels with the genome-specificrimers (Table 3).

.3. Clustering of the 59 wheat genotypes on the expression levelsf the C4-like genes

Based on the expression levels of each of the C4-like genes at thehree growth stages, the 59 wheat genotypes could be clusterednto three groups, i.e. wheat genotypes with high, intermediate,nd low expression of the corresponding C4-like gene. The wheat

Numbers under representative genotypes represent the codes of wheat genotypesas listed in Table S1.

genotypes classified into the three groups were different for vari-ous C4-like genes, and the results for each of the three groups arepresented in Table 4.

In general, significant differences in the expression levels of theC4-like genes were observed among the three groups (P < 0.05) atthe three developmental stages (Table 5, Fig. 2, Fig. S2, S3), whilegreat variation could also be observed between wheat genotypeswithin each group (Fig. 2, Fig. S2, S3). Genotypes Shaan 229 (No.10),Jinan 18(No.17), Mianyang 11(No.20), and Xinong 2000-7 (No.26)displayed the highest expression levels of TaPEPC 5, TaMDH 7,TaNADP-ME 1, and TaPPDK 1˛, respectively, across the three stages.The lowest expression levels of TaPEPC 5, TaMDH 7, TaNADP-ME 1,and TaPPDK 1 ̨ were recorded for Jingwang 9(No.50), Shaanmai150 (No.27), Shaanhan 187 (No.12), and Xifeng 20 (No.42), respec-tively.

3.4. Activities of the C4 pathway enzymes in flag leaves of breadwheat

Activities of the C4 pathway enzymes PEPcase, NADP-ME,MDH, and PPDK were determined in the flag leaves of 59 wheatgenotypes at heading (Z55), anthesis (Z65), and middle grain-filling (Z73) stages, respectively. As shown in Table 6, PEPcase,

92 D.G. Bachir et al. / Journal of Plant Physiology 213 (2017) 87–97

Table 5Mean relative expression of C4-like genes in the three groups of 59 wheat genotypes at heading (Z55), anthesis (Z67) and middle grain-filling (Z73) stages.

C4-like genes Growth stage Grouping of 59 wheat genotypes

Group I Group II Group III

Mean ± SD Range Mean ± SD Range Mean ± SD Range

TaPEPC 5 Heading 1.21 ± 0.10a 1.10–1.43 0.69 ± 0.09b 0.56–0.91 0.38 ± 0.05c 0.30–0.43Anthesis 1.05 ± 0.19a 0.80–1.56 0.58 ± 0.13b 0.48–0.72 0.25 ± 0.05c 0.18–0.30Grain-filling 1.24 ± 0.15a 1.00–1.50 0.73 ± 0.14b 0.60–0.90 0.44 ± 0.11c 0.17–0.49

TaNADP-ME 1 Heading 0.98 ± 0.17a 0.80–1.20 0.51 ± 0.06b 0.50–0.73 0.23 ± 0.09c 0.11–0.33Anthesis 0.78 ± 0.15a 0.70–1.10 0.45 ± 0.07b 0.40–0.60 0.30 ± 0.09c 0.11–0.32Grain-filling 0.73 ± 0.15a 0.60–0.98 0.47 ± 0.06b 0.40–0.50 0.29 ± 0.10c 0.10–0.30

TaMDH 7 Heading 1.31 ± 0.13a 1.13–1.50 0.80 ± 0.11b 0.60–0.93 0.45 ± 0.08c 0.31–0.51Anthesis 1.17 ± 0.21a 0.90–1.51 0.69 ± 0.12b 0.60–0.81 0.41 ± 0.13c 0.17–0.52Grain-filling 0.96 ± 0.27a 0.80–1.47 0.58 ± 0.09b 0.51–0.71 0.28 ± 0.08c 0.17–0.40

TaPPDK 1 ̨ Heading 0.77 ± 0.17a 0.60–1.10 0.64 ± 0.17b 0.30–0.90 0.44 ± 0.07c 0.28–0.51Anthesis 0.64 ± 0.15a 0.52–1.10 0.53 ± 0.14b 0.37–0.73 0.41 ± 0.07c 0.30–0.49Grain-filling 1.12 ± 0.18a 0.89–1.49 0.69 ± 0.08b 0.60–0.83 0.37 ± 0.11c 0.23–0.51

Data are shown as the mean ± SD (standard deviation) of the genotypes in each group.Group I: high expression; Group II: intermediate expression; Group III: low expression. Lowercase letters represent differences significant among the three groups (P < 0.05).

F at genA Groupd

Nwt5M

ig. 2. Relative expression of C4-like genes in flag leaves of three groups of 59 whe: TaPEPC 5; B: TaNADP-ME 1; C: TaMDH 7; D: TaPPDK 1˛. Group I: high expression;ifferences significant among the three groups (P < 0.05).

ADP-ME, MDH, and PPDK exist in the flag leaves of breadheat with different activities among the wheat genotypes at

he three stages. Significant differences were observed among the9 wheat genotypes regarding the activities of PEPcase, NADP-E, MDH, and PPDK, respectively. The activities of PEPcase and

otypes at heading stage (Z55). II: intermediate expression; Group III: low expression. Lowercase letters represent

PPDK started decreasing at anthesis and increased at grain-fillingstage, while NADP-ME and MDH exhibited a decreasing trend

at the three stages. PEPcase showed the highest mean activ-ity at heading (1.018 �mol min−1 mg−1 protein) with a range of0.02–2.414 �mol min−1 mg−1 protein, while PPDK displayed the

D.G. Bachir et al. / Journal of Plant Physiology 213 (2017) 87–97 93

Table 6Mean activities of C4 enzymes in the flag leaves of 59 wheat genotypes at three growth stages.

C4 enzyme Growth stage Mean ± SD Minimun Maximum

PEPcase Heading 1.018 ± 0.81a 0.02 2.414Anthesis 0.589 ± 0.71b 0.006 2.153Grain-filling 0.988 ± 0.79a 0.077 2.764

NADP-ME Heading 0.758 ± 0.80a 0.002 2.666Anthesis 0.672 ± 0.55a 0.032 1.846Grain-filling 0.652 ± 0.48a 0.03 2.250

MDH Heading 0.731 ± 0.67a 0.016 2.238Anthesis 0.616 ± 0.71a 0.026 2.490Grain-filling 0.552 ± 0.61a 0.041 2.473

PPDK Heading 0.521 ± 0.49b 0.005 2.117Anthesis 0.410 ± 0.42b 0.024 2.353Grain-filling 0.888 ± 0.74a 0.014 2.916

Values are mean ± SD (standard deviation)Enzyme activity is expressed as �mol min−1 mg−1 protein.Lowercase letters represent differences significant among the three stages (P < 0.05).

Table 7Mean activities of C4 enzymes in the three groups of 59 wheat genotypes based on the expressions of the C4-like genes.

C4 enzymes Growth stage Grouping of 59 wheat genotypes

Group I Group II Group III

Mean ± SD Range Mean ± SD Range Mean ± SD Range

PEPCase Heading 1.90 ± 0.48a 0.59–2.41 0.80 ± 0.61b 0.04–1.95 0.07 ± 0.05c 0.02–1.17Anthesis 1.51 ± 0.54a 0.50–2.15 0.26 ± 0.34b 0.006–1.95 0.04 ± 0.04b 0.006–0.09Grain-filling 1.93 ± 0.48a 1.16–2.76 0.67 ± 0.57b 0.13–2.33 0.34 ± 1.16b 0.08–0.58

NADP-ME Heading 1.77 ± 0.68a 0.57–2.67 0.64 ± 0.65b 0.002–2.01 0.13 ± 0.13c 0.02–0.42Anthesis 1.53 ± 0.32a 0.77–1.85 0.48 ± 0.39b 0.03–1.51 0.39 ± 0.22b 0.08–0.69Grain-filling 1.25 ± 0.67a 0.07–2.25 0.59 ± 0.21b 0.08–1.08 0.26 ± 0.20c 0.03–0.63

MDH Heading 1.72 ± 0.50a 0.40–2.24 0.60 ± 0.36b 0.07–1.85 0.08 ± 0.05c 0.02–0.19Anthesis 1.81 ± 0.47a 0.93–2.49 0.36 ± 0.28b 0.06–1.37 0.09 ± 0.05c 0.03–0.23Grain-filling 1.46 ± 0.77a 0.34–2.47 0.33 ± 0.17b 0.05–0.82 0.22 ± 0.11b 0.04–0.40

PPDK Heading 0.80 ± 0.65a 0.12–2.12 0.49 ± 0.33b 0.04–1.35 0.16 ± 0.16c 0.005–0.59Anthesis 0.70 ± 0.55a 0.12–2.35 0.33 ± 0.27b 0.03–1.35 0.14 ± 0.11b 0.02–0.31Grain-filling 1.51 ± 0.85a 0.16–2.92 0.72 ± 0.46b 0.27–1.67 0.31 ± 0.29b 0.01–0.89

D up.G ion.L ).

lrMptag((

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ata are shown as the mean ± SD (standard deviation) of the genotypes in each groroup I: high expression; Group II: intermediate expression; Group III: low expressowercase letters represent differences significant among the three groups (P < 0.05

owest mean activity (0.521 �mol min−1 mg−1 protein) with aange of 0.005–2.117 �mol min−1 mg−1 protein. At anthesis, NADP-

E presented the highest mean activity (0.672 �mol min−1 mg−1

rotein) with a range of 0.032–1.046, while PPDK displayedhe lowest mean activity (0.410 �mol min−1 mg−1 protein) with

range of 0.024–2.353 �mol min−1 mg−1 protein. At middlerain-filling stage, PEPCase showed the highest mean activity0.998 �mol min−1 mg−1 protein), while the lowest mean activity0.552 �mol min−1 mg−1 protein) was recorded for MDH.

Classified into three groups based on the expression of the C4-ike pathway genes, the corresponding activities of the enzymeshey encode also showed significant differences among the threeroups (P < 0.05) at heading, anthesis, and middle grain-fillingtages, with variations among genotypes within the groupsTable 7, Figs. 3 , S4 and S5). Wheat genotypes with higher expres-ion levels in group I of TaPEPC 5, TaNADP-ME 1, TaMDH 7, andaPPDK 1 ̨ displayed significantly higher mean activities of PEP-ase, NADP-ME, MDH, and PPDK than those with intermediate and

ow expression levels in group II and group III at the three growthtages. Across the three stages, genotypes Zhoumai 16 (No.25),haanken 81 (No.37), Aifeng 3 (No.58), and Han 6172 (No.39) pre-

ented the highest activities of PEPcase, NADP-ME, MDH, and PPDK,espectively. The lowest activities of PEPcase, NADP-ME, MDH, andPDK were displayed by Xinmai 13 (No.23), Xinyuan 958 (No.36),uo 9908 (No.35), and Chanfeng 1 (No.49), respectively.

3.5. Correlation between C4 enzyme activities and geneexpressions

Positive association between the expression of a gene and theenzyme it encodes is crucial for effective plant development ateach growth stage. In the present study, we analyzed the correla-tions between the expression levels of the C4-like genes and theirenzymatic activities in the 59 wheat genotypes at the three stages(Table 8). Significant and positive correlations were found betweenthe expressions of TaPEPC 5 (0.870, 0.481, 0.547), TaNADP-ME 1(0.775, 0.802, 0.866), TaMDH 7 (0.906, 0.907, 0.847), and TaPPDK 1˛(0.797, 0.680, 0.707), and the corresponding enzyme activities theyencode at heading (Z55), anthesis (Z65), and middle grain-fillingstages (Z73) stages, respectively.

3.6. Expression of C4-like genes and photosynthesis, BMPP, andGYPP

The photosynthesis rate (A) was significantly different amongthe 59 wheat varieties at the three growth stages. Wheat genotypeswith higher expression levels in group I of TaPEPC 5, TaNADP-ME 1,

and TaMDH 7 showed significantly higher mean photosynthesisrates at heading stage than those with intermediate and lowexpression levels in group II and group III, while there were nosignificant differences observed between wheat genotypes with

94 D.G. Bachir et al. / Journal of Plant Physiology 213 (2017) 87–97

Fig. 3. Activities of the C4 enzyme in flag leaves of the 59 wheat genotypes of the three groups at heading stage (Z55).A: PEPCase; B: NADP-ME; C: MDH; D: PPDK. Group I: high expression; Group II: intermediate expression; Group III: low expression. Lowercase letters represent differencessignificant among the three groups (P < 0.05).

Table 8Correlation coefficients between the enzyme activities and relative expressions of C4-like genes at heading, anthesis, and middle grain-filling stages.

C4-like genes Growth stages PEPCase NADP-ME MDH PPDK

TaPEPC 5 Heading 0.870a

Anthesis 0.481a

Grain-filling 0.547a

TaNADP-ME 1 Heading 0.775a

Anthesis 0.802a

Grain-filling 0.866a

TaMDH 7 Heading 0.906a

Anthesis 0.907a

Grain-filling 0.847a

TaPPDK 1˛ Heading 0.797a

Anthesis 0.680a

i(gpwIg

woT

Grain-filling

a Correlation is significant at the 0.01 level.

ntermediate and low expression levels in group II and group IIITable 9). Only wheat genotypes with higher expression levels inroup I of TaPEPC 5 and TaNADP-ME 1 also showed higher meanhotosynthesis rates (A) at middle grain-filling stage than thoseith intermediate and low expression levels in group II and group

II. No other significant differences were observed among the threeroups of TaPPDK 1 ̨ at the three stages.

−1 −1

The biomass plant (BMPP) and grain yield plant (GYPP)ere determined after harvest, and significant differences were

bserved (P < 0.05) among the 59 wheat varieties. As shown inable 10, mean GYPP of wheat genotypes with high expression

0.707a

levels of TaPEPC 5, TaNADP-ME 1, TaMDH 7, and TaPPDK 1 ̨ wassignificantly higher than that of genotypes with intermediate andlow expression levels in group II and group III (P < 0.05), whereasBMPP showed no significant differences among wheat genotypesin the three groups of TaPEPC 5, TaNADP-ME 1, and TaPPDK 1˛.

4. Discussion

Wheat is a typical C3 plant with relatively low photosyntheticefficiency. Interestingly, the wheat genome encodes all the C4 pho-tosynthetic pathway genes, but few efforts have been focused

D.G. Bachir et al. / Journal of Plant Physiology 213 (2017) 87–97 95

Table 9Mean photosynthetic rates in the three groups of 59 wheat genotypes based on the expressions of the C4-like genes at heading, anthesis, and grain-filling stages.

C4-like genes A (�mol m−2 s−1) Grouping of the 59 wheat genotypes

at various stages Group I Group II Group III

TaPEPC 5 Heading 17.66 ± 2.44a 15.64 ± 1.73b 14.73 ± 1.88bAnthesis 10.73 ± 1.45a 9.87 ± 1.52b 8.83 ± 1.17bGrain-filling 11.50 ± 1.38a 10.22 ± 1.42b 9.27 ± 1.97b

TaNADP-ME 1 Heading 17.88 ± 1.86a 15.63 ± 2.24b 15.67 ± 1.61bAnthesis 10.63 ± 1.65a 9.93 ± 1.55a 9.48 ± 1.34aGrain-filling 11.56 ± 1.56a 10.28 ± 1.44b 9.92 ± 1.87b

TaMDH 7 Heading 17.86 ± 2.92a 15.75 ± 1.73b 15.21 ± 1.57bAnthesis 11.10 ± 1.83a 9.81 ± 1.27b 9.26 ± 1.42bGrain-filling 11.17 ± 1.88a 10.31 ± 1.67a 10.14 ± 1.15a

TaPPDK 1˛ Heading 16.52 ± 2.37a 16.22 ± 2.27a 15.16 ± 1.58aAnthesis 10.44 ± 1.61a 9.87 ± 1.66a 9.49 ± 1.01aGrain-filling 10.87 ± 1.75a 10.49 ± 1.65a 9.75 ± 1.28a

Data are shown as the mean ± SD (standard deviation) of the genotypes in each group.Group I: high expression; Group II: intermediate expression; Group III: low expression. Lowercase letters represent differences significant among the three groups (P < 0.05).A: photosynthetic rate (�mol m−2 s−1).

Table 10Mean biomass plant−1 (BMPP) and grain yield plant−1 (GYPP) of the three groups of 59 wheat genotypes based on the expression of the C4-like gene expressions.

C4-like genes Traits Grouping of the 59 wheat genotypes

Group I Group II Group III

TaPEPC 5 BMPP (g) 37.97 ± 6.88a 34.55 ± 5.64a 35.59 ± 5.39aGYPP (g) 14.27 ± 2.61a 12.00 ± 1.74b 11.72 ± 1.77b

TaNADP-ME 1 BMPP (g) 35.63 ± 5.33a 35.83 ± 6.44a 35.34 ± 6.16aGYPP (g) 13.77 ± 2.23a 12.09 ± 2.18b 12.92 ± 2.20ab

TaMDH 7 BMPP (g) 40.22 ± 5.02a 34.47 ± 5.91b 34.23 ± 5.62bGYPP (g) 14.40 ± 2.41a 12.16 ± 2.04b 11.99 ± 1.84b

TaPPDK 1˛ BMPP (g) 36.97 ± 7.31a 35.09 ± 5.60a 35.01 ± 5.09aGYPP (g) 13.57 ± 2.55a 12.33 ± 2.13ab 11.79 ± 1.60b

D up.G ion. Lo

ogsPeCgoCmmtntergte

Mfiebtmtpt

ata are shown as the mean ± SD (standard deviation) of the genotypes in each groroup I: high expression; Group II: intermediate expression; Group III: low express

n their potential functions. Along with the advent of the wholeenome sequences of C4 crops, maize, and foxtail millet, theequences coding the four key C4-specific photosynthetic genes viz.,EPC, NADP-ME, MDH, and PPDK, have been identified (Bennetzent al., 2012; Zhang et al., 2012a,b). A complete set of NAD-ME type4-photosynthetic pathway genes has been identified in the wheatenome (Rangan et al., 2016). In this study, we isolated the isoformsf the homologues encoding the four C4 genes in bread wheat.omparison with C4 genes in C4 crops, including maize and foxtailillet, revealed that wheat PEPC, NADP-ME, MDH, and PPDK sharedany conserved domains and active sites that are responsible for

he structure, activity, and regulation of these enzymes. Phyloge-etic analysis revealed that wheat C4-like genes were more relatedo brachypodium and rice than to maize. High sequence homologyxists between the C4 gene isoforms; however, the untranslatedegions (5′ and 3′ UTR) of the gene provide considerable diver-ence between isoforms. The size of the 5′UTR intron of Arabidopsishaliana EF1˛-A3 gene was reported to positively affect the genexpression and the level of gene expression (Chung et al., 2006).

Considerable differences in the expression of OsPEPC, OsNADP-E, OsPPDK, OsCA, and OsMDH genes in the flag leaf during grain-

lling stage were reported among rice genotypes (Muthusamyt al., 2013). A similar expression level of OsCA was observed inoth rice and C4 species (maize, sorghum, and green foxtail), withhe expression of PEPC NADP-MDH, NADP-ME, and PPDK being

uch lower in rice (Ding et al., 2015). Genes specific for C4 pho-osynthesis were identified in the wheat genome and found to bereferentially expressed in the photosynthetic pericarp tissue ofhe wheat caryopsis (Rangan et al., 2016). In this study, signifi-

wercase letters represent differences significant among the three groups (P < 0.05).

cant differences were found in the expression levels of TaPEPC 5,TaNADP-ME 1, TaMDH 7, and TaPPDK 1 ̨ in wheat flag leaves at thethree key growth stages among the 59 tested wheat genotypes. Theexpression level of TaNADP-ME1 is relatively low compared to otherC4-like genes at the three developmental stages. Moreover, theexpression of three copies in A, B, and D sub-genomes of TaPEPC 5,TaNADP-ME 1, TaMDH 7, and TaPPDK 1 ̨ exhibited similar expres-sion patterns with variations among the three copies of each ofthe C4-like genes. Furthermore, correlation analysis among theexpressions of the C4-like genes in bread wheat indicated that theywere not well coordinated. Taniguchi et al. (2008) reported over-production of the maize C4-specific PEPC and PPDK, the sorghumNADP-MDH, and the rice C3-specific NADP-ME in the mesophyllcells of transgenic rice plants independently or in combination. Themaize C4-specific PEPC gene was introduced into rice, and trans-genic C3 plants with high expression levels of maize C4- specificPEPC gene were obtained (Ku et al., 1999, 2000). Two paralogousNADP-ME2 genes (BrNADP-ME2a and BrNADP-ME2b) shared simi-lar expression profiles and differential expression levels in Brassicarapa (Tao et al., 2016).

It has been reported that the leaves of C3 crops, such as wheat(Hata and Matsuoka, 1987) and rice (Wang et al., 2002), containC4 photosynthetic enzyme systems with relatively low enzymaticactivity. Investigating the activities of the four C4 pathway enzymesin flag leaves, our results revealed the existence of the C4 path-

way enzymes in flag leaves with considerable differences in theactivities of PEPcase, NADP-ME, MDH, and PPDK among the 59tested wheat genotypes at the three major developmental stages.Compared to our results, enzyme activities of PEPC and PPDK in

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6 D.G. Bachir et al. / Journal of P

heat transgenic lines were reported to be 4.3- and 2.1-fold higher,espectively, than in the untransformed control lines (Zhang et al.,014). Maize C4-specific PEPCase activity 1.40-fold greater thanhat of untransformed plants was also reported in the flag leavesf transgenic wheat plants (Lin et al., 2012). Higher activities of C4athway enzymes in both flag leaves and lemmas of super high-ield hybrid rice (Liangyoupeijiu) has been reported by Wang et al.2002). Huang et al. (2013) reported different activities of C4 PEP-ase, NADP-MDH, NADP-ME, and PPDK among soybean cultivars.he activity of PEPcase, NADP-MDH, and PPDK were reported to

ncrease with the ages of flag leaves of super high-yield hybrid ricend maize (Ana-Luz et al., 1994; Yang et al., 2003; Zhang et al.,007).

The cumulative carbon assimilation through the growth sea-on is the primary determinant of crop biomass, and thereforet is predicted that improving photosynthetic capacity in wheatould raise the baseline for yield potential by 50% or more (EL-harkawy et al., 2008). In general, our results revealed that wheatenotypes with higher expression levels in group I of TaPEPC 5,aNADP-ME 1, TaMDH 7, and TaPPDK 1 ̨ displayed higher activitynd grain yield. Over-expressed maize PEPC in transgenic wheatlants led to a higher carboxylation efficiency and photosyntheticate (Hu et al., 2012). Higher expression of maize PEPC and PPDK inransgenic wheat plants with enhanced photosynthetic rates waseported by Zhang et al. (2014). Activity of maize PEPC was reportedo be higher in transgenic wheat lines than in the untransformedheat, with the transgenic lines exhibiting higher yield-related

raits. (Han et al., 2013). Grain yield was reported to be higher inEPC and PPDK transgenic rice than in control plants by 10–30%nd 30–35%, respectively (Ku et al., 2001). Significant correlationsetween PEPCase, NADP-MDH, NADP-ME, and PPDK activities andield of soybeans have been reported (Li et al., 2001; Huang et al.,013).

In the present study, the tested bread wheat genotypes exhib-ted significant differences in the expression patterns and activitiesf the C4 pathway enzymes. To better understand the expressionatterns of the C4 pathway genes in wheat, a comprehensive anal-sis of the expression patterns of all the C4 pathway genes isoformshould be investigated in the future. Although several results con-rm the presence of the C4 pathway in C3 plants, the mechanismf the C4 pathway in C3 plants and its contribution to photosyn-hesis needs further investigations. Investigation on regulations ofhe expressions of those C4-like genes in bread wheat should beonducted, including localization of those enzymes in leaf tissuesnd characterization of the promoter regions.

ompeting financial interest

The authors declare that they have no competing financial inter-sts.

uthor contributions

Y.G.H conceptualized and designed the experiment. D.G.B, I.S,.Q, T.Z.L, and C.L performed the experiment. D.G.B and Y.G.H ana-yzed the data and contributed in the preparation of the manuscript..G.B wrote the paper. All authors read and approved the finalanuscript.

cknowledgments

This work was financially supported by the sub-project of the63 Program (2013AA102902) of the Ministry of Science and Tech-ology and the China 111 Project (B12007), P. R. China.

hysiology 213 (2017) 87–97

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.jplph.2017.03.002.

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