Zaid CHACHAR,Siffat Ullah KHAN,ZHANG Xu-huan,LENG Peng-fei,ZONG Na,ZHAO Jun

Faculty of Maize Functional Genomics,National Key Facility for Crop Gene Resources and Genetic Improvement/Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100080,P.R.China

Abstract Wheat is one of the major food crops in the world.Functional validation of the genes in increasing the grain yield of wheat by genetic engineering is essential for feeding the ever-growing global population.This study investigated the role of ABP7,a bHLH transcription factor from maize involved in kernel development,in regulating grain yield-related traits in transgenic wheat.Molecular characterization showed that transgenic lines HB123 and HB287 contained multicopy integration of ABP7 in the genome with higher transgene expression.At the same time,QB205 was a transgenic event of single copy insertion with no significant difference in ABP7 expression compared to wild-type (WT) plants.Phenotyping under field conditions showed that ABP7 over-expressing transgenic lines HB123 and HB287 exhibited improved grain yield-related traits (e.g.,grain number per spike,grain weight per spike,thousand-grain weight,grain length,and grain width) and increased grain yield per plot,compared to WT plants,whereas line QB205 did not.In addition,total chlorophyll,chlorophyll a,chlorophyll b,and total soluble sugars were largely increased in the flag leaves of both HB123 and HB287 transgenic lines compared to the WT.These results strongly suggest that ABP7 positively regulates yieldrelated traits and plot grain yield in transgenic wheat.Consequently,ABP7 can be utilized in wheat breeding for grain yield improvement.

Keywords: transgenic wheat,ABP7,kernel development,grain weight,grain width

1.Introduction

Wheat (Triticum aestivumL.) is the major food crop in the world,wheat is the most important source of carbohydrate in a majority of countries,and globally,it is the leading source of vegetal protein in human food,having a protein content of about 13%,which is relatively high compared to other major cereals.Wheat,eaten as a whole grain,is also a source of micronutrients and dietary fiber,it contains minerals,vitamins and fats(lipids),and with a small amount of animal or legume protein added is highly nutritious (Lafiandraet al.2014;Shewryet al.2015).It is expected that wheat yield needs to increase by 60% by 2050 to fulfill the food demand of the ever-growing world population (Langridge 2013).This increase is unlikely to be achieved in wheat through conventional breeding due to the limited gene pool available (Reifet al.2005).With the rapid development of genomics,molecular biology,and genetic transformation technology,the genetic engineering of wheat has become one of the effective solutions to addressing this problem (Arauset al.2019).

Several genes have been successfully used to modify wheat for better yield through genetic engineering.Ectopic expression of a modified form of maizeShrunken2gene (Sh2r6hs),which encodes an altered AGP large subunit,under the control ofSh2promoter in transgenic wheat led to a 38% increase on average in seed weight per plant (Smidanskyet al.2002).Transgenic wheat plants expressingHvSUT1encoding barley sucrose transporter driven by Hordein B1 promoter exhibited enhanced thousand-grain weight,grain length,and width,with grain yield increasing by as much as 28%(Saalbachet al.2014).Constitutive expression of TaNFYB4 controlled by ubiquitin promoter in transgenic wheat resulted in a 20-30% increased grain yield (Yadavet al.2015).However,the number of genes that have been functionally evaluated in transgenic wheat is limited,given that many candidate genes determining yield-related traits have been identified (Nadolska-Orczyket al.2017).

A major determinant of grain yield is the grain weight which is positively correlated with grain size,including grain length and width (Zhanget al.2014).It is thus important to identify more grain size-related genes and evaluate their effects on grain yield in wheat through genetic engineering.A previous work has identifiedABP7(Gramene ID: Zm00001d021019) as a G-box binding bHLH transcription activator whose ectopic expression led to increased seed size and weight in transgenicArabidopsisplants (China patent number ZL201210036350.4).In an effort to create new wheat varieties aiming at increasing grain yield,expression vectors ofABP7driven by different promoters were transformed into wheat.This study reports the characterization of these transgenic lines expressingABP7.The objectives were as follows: (1) identification of homozygous transgenic wheat lines harboringABP7,(2) expression analysis ofABP7in transgenic lines,(3) phenotyping yield-related traits of transgenic plants,and (4) physiological analyses of transgenic lines.

2.Materials and methods

2.1.Materials and growth conditions

T1seeds of transgenic wheat lines (Table 1) transformed(done in Dr.Ye Xingguo’s lab,Institute of Crop Sciences(ICS),Chinese Academy of Agricultural Sciences (CAAS))with different constructs,and their respective host lines(CB037 and Fielder) were used as start materials in the present study.Among these lines,HB123 (CB037 background) and QB205 (Fielder background) were obtained by transformation with pCAMBIA3301-Pabp7-ABP7-Tnos in which the expression ofABP7is under the control ofABP7promoter (Pabp7),and HB287 (Fielder background)was obtained by transformation with pCAMBIA3301-Pubi-ABP7-Tnos whereABP7is driven by Ubiquitin promoter(Pubi).

During the winter seasons of 2017 and 2018,the transgenic lines at T1and T3generations were grown in pots filled with 15 kg of prepared soil and 10 plants in each pot in the greenhouse (day/night temperature 25/14°C,16/8 h light/dark photoperiod) at Biotechnology Research Institute (BRI),CAAS,Beijing,China.For phenotyping at T3,30 plants for each transgenic line were grown in three pots,with 10 plants in each pot as a replicate.

During the summer seasons of 2018 and 2019,T2and T4transgenic lines and host lines were planted in the field at Zhangbei Agricultural Station,Hebei Province,China.For phenotyping at T4,the transgenic lines,along with their respective host lines,were planted in three rows,12 plants in each row in a 0.27-m2land area (plot).Fertilizers and irrigation were applied as per requirement.

2.2.Polymerase chain reaction (PCR) assay

For the confirmation ofABP7genes in transgenic lines(T1to T4plants),PCR was performed with gene-specific primers,as shown in Table 2.

Table 1 List of transgenic wheat lines at T1 generation

Table 2 List of primer used for this study

PCR was carried out in a 25-μL reaction volume with 100 ng template DNA,1×PCR buffer with ammonium sulphate (NH4)2SO4,10 pmol L-1of each primer,25 mmol L-1MgCl2,10 mmol L-1of dNTPs mix,1 U ofTaqDNApolymerase and RNAs free water.The PCR profile was optimized at 94°C in the denaturation step,followed by annealing (58°C) and final extension (72°C) in 35 cycles.

The PCR products were run on 0.5 μg mL-1ethidium bromide containing 1.5% agarose gel in 1× TAE buffer at 150 V for 25 min during gel electrophoresis.The product of PCR was visualized under a UV trans-illuminator and photographed through a gel documentation system(Syngene,USA.Inc).The amplicon size of transgenes was confirmed with 1 kb ladder (GenStar,Beijing,China).

2.3.RNA extraction and quantitative real-time PCR(qRT-PCR)

The tissues used to prepare RNA for qRT-PCR include the leaf and kernel grain at the grain-filling stage of transgenic wheat and host lines.Total RNA was prepared using TRIpure Reagent (Galen Biopharm,Beijing) according to the manufacturer’s protocol.cDNA was subsequently synthesized from 1 μg of total RNA using ReverTraAce reverse transcriptase (TaKaRa,Shiga,Japan).Gene expression was analyzed by real-time quantitative PCR with SYBR Green I (TaKaRa) on a 7500 Real-Time PCR System (ABI,Thermo Fisher,Catalog number: 4406985,China).PCRs were carried out in a total volume of 20 μL containing 800 ng of cDNA,1× SYBR Select Master Mix(Applied Biosystems,Thermo Fisher Scientific,Beijing,China) based on AmpliTaq?Fast DNA Polymerase and 400 nmol L-1of each primer,under the following thermal conditions.Holding stage: 95°C,30 s;cycling stage: 95°C,5 s,61°C,20 s,40 cycles.The cycle threshold (CT) values were determined,and the relative fold differences were calculated by the 2-ΔΔCTmethod.Theactingene (Sequence ID: XM_037615630.1) of wheat was used as an internal control for the normalization of template cDNA.The genespecific primers are shown in Table 2.

2.4.Southern blot analysis

PCR-positive plants were further analyzed by Southern blotting.The genomic DNA of each line was extracted using the CTAB method (Doyleet al.1990),digested withEcoRI andHindIII,and fragmented by 0.8% agarose gel electrophoresis.The blot was probed with a 550-bp DNA fragment with the sequence spanning the coding region and 3′UTR ofABP7.The primers used to amplify the probe fragment are shown in Table 2.DIG High Prime DNA labeling and detection Starter Kit II (Roche cat.no.11 585 614 910) were used to label the probe and to detect the hybridization signals on the blot.

2.5.Phenotyping of yield components

Transgenic lines were phenotyped at T3grown in the greenhouse conditions during winter 2018 and at T4grown in the field during summer 2019.The grain traits,including grain number per spike,thousand-grain weight,grain weight per spike,grain length,grain width,and tiller number(T4only),were measured using a SC-G grain appearance image analysis system (Hangzhou Wanshen Detection Technology Co.,Ltd.,Hangzhou,China;http://www.wseen.com/).The data for tiller numbers were collected manually.

2.6.Leaf chlorophyll analysis

Chlorophyll content was measured from the expanded leaves of transgenic and host plants.About 0.5 g of leaves were ground and dissolved in 10 mL of 80%acetone.The extract was centrifuged at 1 500×g for 10 min,and then the supernatant was transferred to the new tube.The absorbance was measured at 645 and 663 nm with a spectrophotometer (LUX.NO-515152316).Total chlorophyll content was calculated using the following equation:

Total chlorophyll (mg g-1FW)=8.02A663+20.2A645(Arnon 1949;Porraet al.1989).

2.7.Total soluble sugar measurement

Total soluble sugar was extracted and measured according to the manufacturer’s instructions (Detection Starter Kit II Solarbio,Beijing,China).Briefly,homogenate of 0.1-0.2 g samples in distilled water was boiled in the water bath for 10 min.After cooling to room temperature,centrifuge it and take the supernatant to distilled water to make a final volume of 10 mL.Then add the reagents according to the menu provided,do the absorbance measurement at 620 nm on a spectrophotometer (LUX.NO-515152316) and make the standard curve.Total soluble sugar content was calculated as follows:

Soluble sugar (mg g FW-1)=(Y×V1)/(W×V1/V2)=10×Y/W where Y,concentration (mg mL-1) based on the standard curve;V1,sample volume,0.04 mL;V2,extraction volume,10 mL;W,sample weight,g.

2.8.Nutrient traits measurement

The contents of protein,starch and wet gluten,and sedimentation value were measured by Foss Infratec 1241 (Denmark).

2.9.Statistical analysis

All analyses were conducted in triplicate using the Statistix 8.1 Program (Analytical Software,Tallahassee,FL,USA).The significance of the differences in data was assessed using ANOVA and analyzed statistically with Graph-Pad Prism 7.0 Software (IBMP Crop,Armonk,NY,USA).The results are presented as the mean±SD.The statistical significance of differences between the means was also analyzed byt-test (P＜0.05).

3.Results

3.1.ldentification of homozygous transgenic wheat lines through PCR analysis

The homozygous transgenic plants harboringABP7were screened and identified from 10 transgenic wheat lines of HB123,15 of HB287,and 7 of QB205 at T3generation through PCR using specific primers (Fig.1;Table 3).The T3seeds of these lines were propagated for further molecular and physiological analysis.

Table 3 List of representative homozygous lines

Fig.1 Identification of homozygous transgenic wheat lines harboring ABP7 by PCR at generation T3.M,1 kb Plus DNA ladder;+,ABP7 plasmid DNA;-,host lines;1-15 samples,individual transgenic plants.

3.2.Southern blotting analysis

To confirm the genome integration ofABP7and to determine its copy number in transgenic wheat lines,this study digested total genomic DNA isolated from leaf tissues of homozygous transgenic wheat lines (T4).The total genomic DNA tested positive forABP7by PCR and the host lines was digested with the restriction enzymesHindIIIand EcoRI,respectively.The digestion products were subjected to Southern blot analysis and hybridized with theABP7probe.The results showed that transgenic lines HB123-1-3-2,HB287-15-25-6,and QB205-1-3-3,respectively,have three,four,and one hybridization band(s),whereas the wild-type (WT) plants showed no hybridization signals (Fig.2).These data confirmed the integration ofABP7into the genome of transgenic wheat lines and indicated that both HB123-1-3-2 and HB287-15-25-6 harbored more than one copy ofABP7in the genome,while QB205-1-3-3 was a transgenic event with single copy insertion.

Fig.2 Southern blot analysis of transgenic wheat lines harboring ABP7.A,schematic drawings of the T-DNA regions of the constructs expressing ABP7,showing the location of probe fragment used for hybridization and the restriction sites of EcorRI and HindIII used for genomic DNA digestion.B,southern hybridization.Genomic DNA of each line was digested with HindIII or EcoRI,fragmented by 0.8% agarose gel electrophoreses,and probed with ABP7.1,2,and 3 represent transgenic lines HB123-1-3-2,HB287-15-25-6 and QB205-1-3-3,respectively.FD and CB indicate host wheat lines Fielder and CB037,respectively;+,ABP7 plasmid as positive control.The molecular weight marker is indicated on the right.

3.3.Enhanced expression of ABP7 in transgenic lines

The expression level ofABP7in transgenic wheat lines was examined using quantitative real-time PCR.Results showed that the transcript level ofABP7was significantly(P＜0.05) enhanced in transgenic lines HB123-1-3-2 and HB287-15-25-6 compared to their corresponding host plants CB037 and Fielder,respectively.The expression ofABP7showed no significant increase in QB205-1-3-3 compared to its host plants (Fig.3),although the level was slightly higher in the flag leaves of transgenic than that of the host.

Fig.3 Expression level of ABP7 in homozygous transgenic wheat lines.Different letters indicate the significant difference at P＜0.05 between transgenic and wild-type plants.HB123,HB287,and QB205 represent HB123-1-3-2,HB287-15-25-6,and QB205-1-3-3,respectively;CB037 and Fielder,host plants.Bars mean SD.Different letters indicate the significant difference at P＜0.05.

3.4.Yield parameters in terms of grain production in transgenic plants under the greenhouse and field conditions

During winter seasons of 2018,transgenic wheat lines(T3) were phenotyped in a greenhouse for several traits,including grain number per spike,thousandgrain weight,and grain weight per spike.The results revealed a significant (P＜0.05) increase in all the traits of transgenic line HB123-1-3-2 compared to host CB037.Similarly,all the traits,except grain number per spike,were significantly (P＜0.05) increased in transgenic line HB287-15-25-6 compared to host Fielder.However,the phenotyping results for transgenic line QB205-1-3-3 at T3indicated that only three traits (i.e.,thousand-grain weights,grain weight per spike,and grain width) were significantly (P＜0.05) increased compared to host Fielder(Fig.4).

Fig.4 Phenotyping yield-related traits grain number per spike (A-C),thousand-grain weight (D-F),and grain weight per spike (G-I)in transgenic wheat lines at T3 and host lines in 2018.Bars mean SD.Different letters on top of the bars indicate the significant difference at P＜0.05 between transgenic and host plants.＊,significant difference at P＜0.05.

The transgenic wheat lines (T4) were phenotyped again in the following year 2019 under field conditions.The traits,including grain number per spike,thousand-grain weight,grain weight per spike,grain length,grain width (Figs.5 and 6),and grain yield per plot,were significantly (P＜0.05)increased in both HB123-1-3-2 and HB287-15-25-6 transgenic lines compared to their respective hosts CB-037 and Fielder.The quality traits of grains,including contents of protein,starch and wet gluten,and sedimentation value,were also measured,but we did not detect any significant difference in these traits between transgenic lines and their respective host lines (Appendix A).

For transgenic line QB205-1-3-3,all the traits tested(Figs.5 and 6) showed no significant difference (P＜0.05)to its host Fielder,although the values of most of the traits were slightly higher in transgenic plants than those in the host.

Fig.5 Phenotyping yield-related traits grain number (A-C),thousand-grain weight (D-F),grain weight per spike (G-I),grain length (J-L),and grain width (M-O) in transgenic wheat lines at T4 and host plants in 2019.Bars mean SD.Different letters on top of the bars indicate the significant difference at P＜0.05 between transgenic and host plants.＊,significant difference at P＜0.05.

Fig.6 Grain phenotypes in transgenic lines HB123-1-3-2 (A),HB287-15-25-6 (B),and QB205-1-3-3 (C) at T4 comparing to their hosts CB037 or Fielder.

3.5.Chlorophyll content in transgenic lines

The chlorophyll content in flag leaves reflects the photosynthetic activity and yield potential of wheat plants(Liet al.2012).To examine if the observed phenotypes are related to any change in chlorophyll in transgenic wheat lines,chlorophyll content was measured in flag leaves of transgenic (T4) and host plants.As shown in(Fig.7),total chlorophyll,chlorophylla,and chlorophyllbwere all significantly (P＜0.05) increased in the flag leaves of both HB123-1-3-2 and HB287-15-25-6 transgenic lines compared to the respective host plants.No significant difference (P＜0.05) in chlorophyll was observed between transgenic line QB205-1-3-3 and its host,although the values were slightly higher in the transgenic line.

Fig.7 Chlorophyll measurement in flag leaves of transgenic lines HB123-1-3-2(A-C),HB287-15-25-6 (D-F) and QB205-1-3-3 (G-I) and host lines.Bars mean SD.Different letters on top of the bars indicate the significant difference at P＜0.05.＊,significant difference at P＜0.05.Photos of flag leaves of transgenic lines HB123-1-3-2 (J),HB287-15-25-6 (K),and QB205-1-3-3 (L) with their respective host lines are also shown.

3.6.Soluble sugars in transgenic wheat lines

Soluble sugars are essential for plant growth and development (Lastdrageret al.2014).To explore the possible relationship between soluble sugar and grain phenotypes observed inABP7transformed transgenic wheat lines,this study measured total soluble sugar content in flag leaves of transgenic and host plants.The results showed that total soluble sugar content was increased more than four times in both HB123-1-3-2 and HB287-15-25-6 transgenic lines compared to respective hosts (Fig.8).In transgenic line QB205-1-3-3,the total soluble sugar content was also significantly (P＜0.05) increased,but to a less extent than that in HB123-1-3-2 and HB287-15-25-6.

Fig.8 Soluble sugar in flag leaves of transgenic lines HB123-1-3-2 (A),HB287-15-25-6 (B),and QB205-1-3-3 (C) and host lines.Bars mean SD.Different letters on top of the bars indicate the significant difference at P＜0.05.＊,significant difference at P＜0.05.

4.Discussion

To evaluate the effect of seed size-relatedABP7in wheat aiming at creating new varieties with increased grain yield,we generated and characterized transgenic wheat lines expressingABP7driven by different promoters.We found thatABP7over-expression in homozygous transgenic wheat lines improved grain yield-related traits,including grain number per spike,grain weight per spike,thousandgrain weight,grain length,and grain width compared to WT plants,leading to increased grain yield per plot.These results strongly suggest thatABP7is functional in promoting grain yield-related traits in wheat and can be utilized in wheat breeding for yield improvement.

This study found that transgenic lines harboring multicopy transgenes,such as HB123-1-3-2 and HB287-15-25-6,showed much higher expression ofABP7and accordingly strong phenotypes of yield-related traits compared to WT plants.This may attribute to the combinatorial effects of multicopyABP7in transgenic plants.In contrast,the transgenic line QB205-1-3-3 with single copy insertion of transgene showed no significant difference inABP7expression and yieldrelated phenotypes compared to WT plants.This might reflect the low/null transcription activity at the site ofABP7insertion in the genome of the transgenic plants.These results suggest that the copy number ofABP7insertion into the wheat genome might be involved in the determination of its expression level and its effect on grain yield-related traits in transgenic wheat.

It remains open of howABP7regulates grain yieldrelated traits in transgenic wheat.Many regulators that control seed size and weight through different signaling pathways have been identified inArabidopsisand crops(Li and Li 2016),including wheat homologues TaGW2 (Suet al.2011;Simmondset al.2016),TaGS5 (Wanget al.2015;Maet al.2016),TaGW7 (Wanget al.2019),TaGW8(Caoet al.2019;Yanet al.2019),and TaDA1 (Liuet al.2020).These regulators affect seed size by influencing cell proliferation and cell expansion in maternal tissues or endosperm growth (Li and Li 2016).As this study observed thatABP7over-expression in transgenic wheat lines increased grain length,grain width,and thousandgrain weight,we speculate thatABP7might function to promote cell proliferation and/or cell expansion.

On the other hand,chlorophyll is an important light-absorbing pigment of plant photosystem,largely contributing to photosynthetic capacity (Liet al.2018),and its content in flag leaf is genetically correlated with the grain yield in wheat (Zhanget al.2009).Total soluble sugars not only function as metabolic resources and structural constituents of cells but also act as signals regulating various processes associated with plant growth and development (Lastdrageret al.2014).In the present study,we found that total chlorophyll,chlorophylla,and chlorophyllbwere largely increased together with more than four times increase of total soluble sugars in the flag leaves of both HB123-1-3-2 and HB287-15-25-6 transgenic lines compared to wild type.These data suggest thatABP7might enhance grain yield-related traits,at least in part,by increasing the photosynthetic capacity and the metabolism of photosynthetic assimilates in transgenic wheat lines.

5.Conclusion

Transgenic wheat lines overexpressingABP7were generated and showed increased grain number per spike,grain weight per spike,thousand-grain weight,grain length,grain width,and grain yield per plot compared to WT plants.These data suggest thatABP7positively regulates grain yield-related traits and plot-grain yield in transgenic wheat.Consequently,ABP7can be integrated as a molecular tool into wheat breeding for high grain yield via genetic engineering.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendixassociated with this paper is available on http://www.ChinaAgriSci.com/V2/En/appendix.htm