Yuling Qiu,Hiqing Chen,Shungxi Zhng,Jing Wng,Lipu Du,Ke Wng,*,Xingguo Ye,*

a Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China

b Crop Research Institute,Ningxia Academy of Agri-Forestry Sciences,Yinchuan 750105,Ningxia,China

Keywords:Wheat Aegilops longissima High-molecular-weight glutenin subunits 1Slx2.3*Bread-making quality

ABSTRACT Wheat bread-making quality can be improved by use of high-molecular-weight glutenin subunits(HMW-GSs)from wild relatives.Aegilops longissima is a close relative of wheat that contains a number of HMW-GS-encoding genes including 1Slx2.3*.In this study,transgenic wheat lines overexpressing 1Slx2.3*were obtained by Agrobacterium-mediated transformation and used to investigate the genetic contribution of 1Slx2.3*to wheat flour-processing quality.The 1Slx2.3*transgene was stably inherited and expressed over generations.Expression of 1Slx2.3*increased the relative expression of 1Dx2 and 1Dy12 and reduced that of 1By18 during grain development.In general,integration of 1Slx2.3*stimulated the accumulation of endogenous HMW-GSs and low-molecular-weight glutenin subunits in wheat kernels,greatly increasing the glutenin:gliadin ratio and resulting in faster formation of protein bodies in the endosperm during grain development.A wheat material with improved flour-making quality was developed in which 1Slx2.3*improved wheat bread-making quality.

1.Introduction

As a staple crop grown globally,common wheat(Triticum aestivum L.,AABBDD,2n=42)has vital importance for human nutrition[1].Wheat has unique end uses among major crops.Its flour can be used to make a variety of foods such as bread,cakes,dumplings,and noodles based on its dough’s specially viscoelastic properties,which are mainly dependent on seed storage proteins composed of glutenins and gliadins[2,3].Glutenins can be further divided into high-molecular-weight glutenin subunits(HMW-GSs)and low-molecular-weight glutenin subunits(LMW-GSs)based on their molecular weight,and are the major determinants of dough strength and extensibility for bread-making purposes[4,5].Although HMW-GSs account for only 5%-10% of the storage proteins in wheat grain,they play a major role in baking quality[6].

Wheat HMW-GSs are encoded by three homoeologous alleles located on the long arms of chromosomes 1A,1B,and 1D at loci Glu-A1,Glu-B1,and Glu-D1,respectively[7,8].Within each locus,there are two closely linked genes encoding one larger x-type(80-88 kDa)and one smaller y-type(67-73 kDa)protein subunit[9].Theoretically,six distinct HMW-GSs should be expressed in wheat,but most wheat cultivars carry only three to five HMWGSs because one or two of the genes encoding the subunits are silenced[10-12].Up to date,no commercial wheat cultivar is known to carry an expressed 1Ay allele[11].

Many HMW-GS genes have been identified in wheat and its related species,such as 1Ax2,1Ax2*,1Ax21,and 1Ay21*at the Glu-A1 locus,1Bx7,1By8,1By9,1Bx13,1By16,1Bx17,1By18,1S1x2.3*,and 1S1y16*at the Glu-B1 locus,and 1Dx2,1Dx5,1Dy10,and 1Dy12 at the Glu-D1 locus[13-16].Although environmental conditions impair wheat processing quality[17,18],the genetic contributions of some HMW-GS alleles to end-use quality have been studied by multiple approaches[10,15,19,20].Overexpressing 1Ax1 and 1Dx5 or 1Dy10 genes improved dough elasticity and processing properties[21,22].Deletion of specific HMW-GS genes,such as 1Ax1 at Glu-A1 locus,1Dx2 and 1Dy12 at Glu-D1 locus,and 1Bx14,1By15,1Bx20,1By20,1Bx7 and 1By9 at Glu-B1 locus,consistently led to inferior flour-processing quality[10,19,20,23,24].Wheat cultivars carrying more HMW-GSs normally show relatively high bread-making quality.The effect of various subunit combinations on end-use quality has also been investigated[24-27].Allelic effects of HMW-GS genes on dough quality have been studied.Some alleles such as 1Ax1 or 1Ax2*at the Glu-A1 locus,1Dx5+1Dy10 at the Glu-D1 locus,and 1Bx7+1By8,1Bx13+1By16,and 1Bx17+1By18 at the Glu-B1 locus,were associated with strong gluten for high bread-making quality,whereas other alleles including the null at the Glu-A1 locus,1Dx2+1Dy12 at the Glu-D1 locus,and 1Bx6+1By8 and 1Bx20+1By20 at the Glu-B1 locus displayed weak effects on gluten and bread-making quality[24-27].

HMW-GS allele variation remains limited in modern wheat cultivars,making it difficult to improve wheat end-use quality.It is desirable to identify new HMW-GS alleles,dissect their genetic effects on end-use quality,and further apply them in wheat breeding for quality improvement.As a hexaploid plant species,wheat contains three homeologous genomes,A,B,and D,that originated from T.urartu(2n=2x=14,AA),Aegilops speltoides(2n=2x=14,SS),and Ae.tauschii(2n=2x=14,DD),respectively[28,29].Numerous alleles controlling important wheat traits have evolved and diverged from the three wild relative species,among which many genes associated with desirable traits have been characterized[30,31].

As the plant genus closest to wheat,Aegilops consists of up to 22 species harboring genomes S,M,C,U,N,and T that carry a wide range of allelic variations,providing valuable gene resources for wheat improvement[4,32].In the Aegilops genus,Ae.longissima(2n=2x=14,SlSl)has desirable characteristics including high seed protein content,resistance to powdery mildew(PM),and drought tolerance[4,33,34].Its component species carry two novel HMWGSs(1Slx2.3*and 1Sly16*)on chromosome 1Sl,which have been isolated and characterized in a wheat-Ae.longissima chromosome substitution line 1Sl(1B),in which 1Slx2.3*is the largest HMWGS among the 1Bx-type subunits,larger than all other HMW-GSs reported to date[4].Generally,HMW-GSs with higher molecular weight show better effects on bread-making quality[6].The substitution line 1Sl(1B)showed higher values of parameters associated with strong dough and greater bread volume than its genetic background material Chinese Spring(CS),indicating that the two subunits 1Slx2.3*and 1Sly16*in the substitution line had a positive effect on bread-making quality[4].

Based on an Ae.longissima accession PI542196 and a set of wheat-Ae.longissima chromosome addition lines,DNA markers specific to each chromosome arm of Ae.longissima have been developed[28],accelerating the construction of the wheat-Ae.longissima chromosome translocation line 1SlL/1BS or the transfer of 1Slx2.3*by cytogenetic techniques.Although a 1SlL/1BS line could be potentially directly used in wheat quality breeding,undesirable genes on Ae.longissima chromosome arm 1SlL will also be simultaneously transferred into wheat and some desirable genes on wheat 1BL will be lost in the translocation line.Wheat materials with only the introgressed 1Slx2.3*gene cannot be developed by this route and the detailed effect of 1Slx2.3*on flour-processing quality cannot be well characterized.It is thus desirable to transfer 1Slx2.3*gene into common wheat by a transgenic approach,for flour-processing quality improvement and functional dissection of the 1Slx2.3*subunit.

Integration of HMW-GS alleles into wheat from related species by genetic transformation for flour quality modification has not been reported.In the present study,the 1Slx2.3*gene was transferred into wheat by Agrobacterium-mediated transformation.Homozygous transgenic lines were produced after detailed detections by rapid detection strip,polymerase chain reaction(PCR),sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDSPAGE),fluorescence in situ hybridization(FISH),and genomic in situ hybridization(GISH).The expression patterns of the 1Slx2.3*and endogenous HMW-GSs in kernels of the newly developed material were determined by quantitative real-time PCR(qPCR).The relative content of glutens(HMW-GSs,LMW-GSs,and gliadins)and glutenin macropolymers(GMPs)were measured,and dynamic changes of protein bodies(PBs)were observed in the new material during grain development.Flour-processing quality parameters relevant to production of bread and sponge cakes from the new material were investigated.The results achieved in this study provide new insights into the application of HMW-GS alleles from wild species in wheat breeding.

2.Materials and methods

2.1.Plant materials and cultivation conditions

The wheat cultivar Westonia,an Ae.longissima accession PI542196,a T.urartu accession TMU38,and Ae.tauschii accession TQ27 were provided by Prof.Yueming Yan,Capital Normal University,Beijing,China and Prof.Fangpu Han,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing,China.Westonia carries five HMW-GS:1Ax2*,1Dx2,1Dy12,1Bx17,and 1By18[35].

Westonia was planted in 20 pots filled with substrate peat moss(Parnumaa,Estonia)and controlled release fertilizer(Ekompany Int.,Netherlands)in a growth chamber under optimal conditions:an air temperature of 24 °C,a photoperiod regime of 16 h light/8 h darkness,a light intensity of 300μmol m-2s-1,and a relative humidity of 45%,for collecting immature kernels for obtaining transgenic plants via genetic transformation.Accessions PI542196,TMU38,and TQ27 were planted in pots for DNA extraction for GISH analysis.A selected transgenic line derived from Westonia as well as the wild-type(WT)Westonia was sown in a greenhouse at the experimental station of the Institute of Crop Sciences(ICS),Chinese Academy of Agricultural Sciences,Beijing,China,in 40 rows with a length of 1.0 m and a row spacing of 20.0 cm for seed propagation for flour-processing quality.

2.2.Vector construction for expressing 1Slx2.3*

The pBINHP-GluT plasmid containing the complete 1Slx2.3*coding sequence(CDS)was kindly provided by Prof.Yueming Yan at Capital Normal University,Beijing,China.The pWMB172 plasmid was constructed previously in our laboratory.The 1Slx2.3*CDS was amplified using the pBINHP-GluT plasmid as a template with a pair of specific primers 1Slx2.3*-A-F and 1Slx2.3*-A-R1(Table S1),and the pWMB172 plasmid was double-digested with SpeI and SmaI at the same time.The products of amplification of the 1Slx2.3*CDS fragment and digested pWMB172 plasmid backbone were purified with a DNA purification and concentration kit(Tianmo Biotech,Beijing,China),and ligated by In-Fusion HD Cloning Kit(Takara Bio USA,Inc.,Mountain View,CA,USA)for 15 min under 50°C and then 5 min in an ice bath in a 10-μL reaction system(containing 2μL In-fushion ligase,25 ng digested pWMB172 plasmid,25 ng amplified 1Slx2.3*CDS fragment,and 5μL ddH2O)to form a new expression vector pWMB184,in which the target gene 1Slx2.3*was driven by a 1Dx5 seed-specific promoter.Finally,pWMB184 was introduced into Agrobacterium strain C58C1 by triparental mating[36].

2.3.Genetic transformation of wheat

Agrobacterium-mediated wheat transformation using immature embryos was performed by the proprietary method described in our previous publication[37].Immature kernels were sampled at 14-15 days post-anthesis(DPA)and sterilized with 70% ethanol for 2 min and 4% sodium hypochlorite for 10 min followed by five rinses with sterile water.Then the immature embryos were carefully isolated from the sterilized wheat kernels and incubated with Agrobacterium strain C58C1 harboring pWMB184.After co-cultivation,the embryo axes with scutella tissues removed were transferred onto WLS-Res,WLS-P5,and WLS-P10 media in order at 24 °C under darkness for primary and embryonic callus induction and selection.The embryonic calli were transferred onto LSZ-P5 and the MSF-P5 media for green shoot regeneration and elongation at a temperature of 24 °C,a photoperiod regime of 16 h light/8 h darkness,a light intensity of 100μmol m-2s-1,and a relative humidity of 45%.Plantlets with healthy roots were transplanted into pots and cultivated in a growth chamber at a temperature of 24°C,a photoperiod regime of 16 h light/8 h darkness,a light intensity of 300μmol m-2s-1,and a humidity of 45%.

2.4.Test strip and PCR detection

An analysis of bar gene expression in putative transgenic wheat plants was conducted using a QuickStix kit(EnviroLogix,Portland,ME,USA)according to the manufacturer’s instructions.A 429-bp PCR product was amplified in the transgenic plants to verify the presence of the bar gene using the primer pair 5′-ACCATCGTCAAC CACTACATCG-3′and 5′-GCTGCCAGAAACCACGTCATG-3′(Table S1),and a 283-bp PCR product was amplified in the transgenic plants to verify the presence of the 1Slx2.3*gene using the primer pair 5′-GGTTAGTCCTCTTTGTGGCGA-3′and 5′-CCAAAATATACTTTGTTG GAGTTGT-3′(Table S1)after extraction of genomic DNA from the leaves of the putative transgenic wheat plants using a NuClean Plant Genomic DNA kit(Cowin Biotech Co.,Ltd.,Taizhou,Jiangsu,China).Each reaction volume(15μL)was composed of 2×Taq Plus Master Mix II（Dye Plus)(Vazyme Biotech Co.,Ltd,Nanjing,Jiangsu,China),3μmol L-1of each primer,5.9μL of ddH2O,and 100 ng of genomic DNA.The PCR program started at 95 °C for 5 min,followed by 35 cycles of 95 °C for 30 s,60 °C for 30 s,and 72 °C for 30 s,with a final extension at 72 °C for 5 min.The PCR products were separated on a 1.0% agarose gel.

2.5.Fluorescence and genomic in situ hybridization

Chromosome slides with root tips of the positive transgenic plants identified by PCR were prepared and the target genes integrated in the transgenic lines were confirmed by FISH and GISH using previously described methods[38,39].FISH was first applied to identify the chromosome integration of 1Slx2.3*using plasmid pWMB184 labeled with Alexa Fluor-488-dUTP(green)as a probe,and multi-GISH was then employed to identify the integrated genome of 1Slx2.3*using the genomic DNAs of T.urartu labeled in green and Ae.tauschii labeled in red as probes,with genomic DNA of Ae.longissima used as blocking DNA.Hybridization procedures followed a previously described method with minor modifications[38].The images were visualized with an epifluorescence Olympus BX61 microscope(Olympus China Inc.,Beijing,China).

2.6.Extraction of seed storage proteins and SDS-PAGE assay

Glutenin was extracted from three mature kernels of each transgenic wheat line and WT for HMW-GS composition analysis by SDS-PAGE using a previously described method[10].The collected wheat seeds were ground in a high-flux tissue grinder and transferred into a 1.5 mL tube.The powder samples were washed sequentially with 1 mL 70% ethanol and 1 mL 55% isopropanol,and glutenin was extracted with an extraction buffer consisting of 50% isopropanol,200 mmol L-1Tris-HCl(pH 8.0),and 1% DTT.For SDS-PAGE,10μL of the extracted glutenin simples were added to 12%PAGE gel lanes for electrophoresis on a Bio-Rad PROTEAN II XL(Bio-Rad Laboratories,Inc.,Hercules,CA,USA)apparatus at 30 mA for 4 h.

2.7.Content determination of glutens and glutenin macropolymers

Glutens and glutenin macropolymers(GMPs)in the newly developed material and its WT were extracted and quantified from three biological replicates by reversed-phase ultra-performance liquid chromatography(RP-UPLC)and size-exclusion ultraperformance liquid chromatography(SE-UPLC),respectively,as described previously[10].RP-UPLC and SE-UPLC were performed on a Thermo DGLC UPLC system with a Waters BEH C18 column(100×2.1 mm,1.7μm)and an Agilent SEC-5 column(500?,4.6×300 mm),respectively,with peak area(mAU*min)was used as the target substances for the measurement of gluten and GMPs contents.

2.8.RNA extraction and reverse transcription and qPCR assays

Immature wheat kernels collected from the new material and its WT at several grain development stages were used for total RNA extraction with the TransZol Up Plus RNA kit(TransGen Biotech,Beijing,China).Complementary DNA(cDNA)was synthesized using a reverse transcription kit(Vazyme Biotech Co.,Ltd.,Nanjing,Jiangsu,China).Quantitative real-time quantitative PCR was employed to evaluate the expression of 1Slx2.3*and endogenous HMW-GSs in the new material and its WT at several stages of grain development as described previously[10].For qPCR,a 20-μL reaction volume contained 10μL 2×RealStar Green Fast Mixture with ROX II(GenStar,Beijing,China),1μL first-strand cDNA,0.6μL primer mix(10μmol L-1)each for six HMW-GS genes(1Slx2.3*,1Ax2*,1Dx2,1Dy12,1Bx17,and 1By18),and 8.4μL ddH2O.All primer sequences used for qPCR are listed in Table S1.Amplification was performed on an ABI PRISM 7500 Real-Time PCR System(ABI,Foster city,CA,USA)with a thermal cycling program of 95°C for 5 min,followed by 40 cycles of amplification(95°C for 5 s,60°C for 30 s,and 72 °C for 30 s).

2.9.Observation of PBs by transmission electron microscopy

Immature grain samples were collected at 7,10,13,16,19,22,25,and 28 DPA from the new material and its WT for transmission electron microscopy(TEM)observation.Preparation of TEM samples was as previously described[10].Images were produced with an H7500 transmission electron microscope(Hitachi,Tokyo,Japan).The diameters of the PBs were measured with ImageJ.

2.10.Flour-processing quality evaluation for bread and sponge cake

Based on a previously described method[27],parameters of dough quality including crude protein content,dough development time and stability time,and mixing tolerance index were determined to evaluate the differences between the new material and its WT with respect to bread-making quality.All the experiments for the determination of these parameters and breadbaking were conducted at the Wheat Quality Laboratory at ICS.Bread-making quality evaluation of wheat flour was based on the rapid-baking test according to the National Standards of China GB/T 35869-2018.

Sponge cake processing was performed at the Academy of State Administration of Grain,Beijing,China.Coarser texture and structure were adapted to evaluate sponge cake quality according to the National Standards of China GB/T 24303-2009 as previously described[10].

2.11.Statistical analysis

Data analysis was performed using SPSS version 20 in Windows(IBM,Armonk,NY,USA).The qPCR results were analyzed by ABI 7500 software(ABI,Foster city,CA,USA)and Microsoft Excel 2016(Microsoft,Redmond,WA,USA).

3.Results

3.1.Development and detection of transgenic wheat plants

Fifty immature embryos of Westonia were infected with Agrobacterium carrying the vector pWMB184 containing two TDNA expression cassettes for bar and 1Slx2.3*,respectively(Fig.1A),and seven putative transgenic plants were obtained by in vitro callus induction and regeneration on selection culture medium for a transformation efficiency of 14%.The putative transgenic plants were first identified by Quickstix strip for bar protein(Fig.1B),and further verified by PCR amplification of bar and 1Slx2.3*genes(Fig.1C,D).The results by Quickstix strip were consistent with the results by PCR,indicating that the 1Slx2.3*was integrated into the genome of the putative transgenic wheat plants.

3.2.Acquisition and identification of homozygous transgenic lines

The transgenic plants in the T2generation were screened with simple FISH and multi-color GISH.In FISH,two transgene signals were clearly observed in the distal regions of two wheat chromosomes(Fig.2A).It was clearly observed that the two chromosomes carrying the integrated gene showed red color by multi-color GISH(Fig.2B),indicating that the T-DNA expression cassette with 1Slx2.3*had been inserted onto the wheat D genome.Finally,two of the transgenic wheat lines were identified as homozygous,and one line T-W1 was selected for further characterization of flourprocessing quality.

In the T3generation,all plants from T-W1 were verified by PCR using the specific DNA marker of 1Slx2.3*and the mature seeds were tested with SDS-PAGE.The specific band of 1Slx2.3*was amplified in all plants(Fig.2C),and the 1Slx2.3*subunit was detected in all tested kernels(Fig.2D),indicating that the target gene had not segregated in this new material and that the results were consistent with those by FISH.

Fig.1.Generation and detection of transgenic wheat plants with the 1Slx2.3*gene encoding a high-molecular-weight glutenin subunit mediated by Agrobacterium.(A)Schematic diagram of expression vector pWMB184.(B)Detection of putative transgenic plants using QuickStix strip for the bar protein.(C)Detection of putative transgenic plants by PCR amplification of the bar gene.(D)Detection of putative transgenic plants by PCR amplification of the 1Slx2.3*gene.WT,wild type of wheat cultivar Westonia;1-10,individual transgenic wheat plants;P,positive control of expression vector pWMB184;M,DL5000 DNA marker.

3.3.Expression profiling of HMW-GSs in new material T-W1 at several grain development stages

The 1Slx2.3*protein was normally expressed in new material TW1(Fig.2D).To test whether the integrated 1Slx2.3*gene was normally expressed and affected the expression of native HMW-GS genes(1Ax2*,1Dx2,1Dy12,1Bx17,and 1By18)in transcriptional level in T-W1,total RNA was extracted and reverse-transcribed to cDNA from immature kernels of T-W1 and WT at several grain development stages.The expression profiles of these HMW-GS genes were then characterized by qPCR in T-W1 and WT.1Slx2.3*was expressed in T-W1 at all stages of grain development,showing highest expression at 22 DPA and an overall U-shaped expression trend(Fig.3).

The expression of the endogenous genes 1Dx2 and 1Dy12 was higher in T-W1 than in WT through almost the entire graindevelopment period(Fig.3),suggesting that the introduction of the foreign 1Slx2.3*gene stimulated the expression of 1Dx2 and 1Dy12 in T-W1.At 10 and 28 DPA,the expression of 1Ax2*was slightly lower in T-W1 than in WT,but slightly higher than in WT at other time(Fig.3).At 16,25,and 28 DPA,the expression of 1Bx17 was slightly lower in T-W1 than in WT,but its expression at other time points was slightly higher than in WT.During all grain development stages,the expression level of 1By18 in T-W1 was only 1/5 of that in WT(Fig.3),indicating that the expression of 1By18 was massively suppressed in T-W1.

3.4.Determination of glutens and GMPs in the new material T-W1

RP-UPLC analysis revealed that 1Slx2.3*was normally expressed in T-W1(Fig.4),results consistent with those by SDS-PAGE(Fig.2).The contents of the four HMW-GSs(1Ax2*,1Dx2,1Dy12,and 1Bx17)in T-W1 were increased relative to those in WT(Table S2;Fig.4).

The contents of 1Ax2*,1Dx2,1Dy12,and 1Bx17 in T-W1 were increased by 24.18%,14.20%,39.03% and 18.64% compared with those in WT,respectively.Because the peaks of 1Slx2.3*and 1By18 were too close for clean separation in T-W1,the relative contents of the two subunits could not be calculated separately.The total content of 1Slx2.3*and 1By18 in T-W1 was significantly higher than the content of 1By18 in WT.Thus,the content of total HMW-GSs was dramatically higher in T-W1 than in WT.

The content of total LMW-GSs was also higher in T-W1 than in WT.Thus,total glutenin content was higher in T-W1 than in WT(Table S2;Fig.4).The contents ofω,α/β,andγ-gliadins in T-W1 were similar to those in WT(Table S2;Fig.4).The ratio between the contents of glutenins to gliadins was thus increased in T-W1 compared with that in WT,and thus more optimal for breadmaking.The SE-UPLC analysis showed that the GMP contents in immature kernels at 22 DPA and mature kernels in T-W1 were similar to those in WT(Table S2;Fig.4).

3.5.Protein body changes during grain development in new material T-W1

In order to dissect the effect of the alien 1Slx2.3*gene on morphological features of PBs,TEM was performed to observe PB changes at multiple grain development stages.The new material T-W1 possessed more and larger PBs than WT at all grain development stages except 7 DPA(Fig.5).The size of PBs increased by about 4.0μm every 3 d from 7 DPA to 25 DPA in T-W1,whereas the corresponding increase was was about 2.5μm in WT,indicating that the accumulation rate of PBs in T-W1 was faster than that in WT,and the overexpression of 1Slx2.3*stimulated the accumulation rate of PBs(Fig.4).The lamellar structure of PBs was formed in T-W1 at 25 DPA,but in WT at 28 DPA(Fig.5).

Fig.2.Identification of homozygous transgenic wheat lines by cytogenetic,molecular,and protein techniques.(A)Test of homozygous transgenic lines by fluorescence in situ hybridization,in which the red arrows indicate integration signals of the 1Slx2.3*gene using the labeled pWMB184 as probe.(B)Test of homozygous transgenic lines by multiple genomic in situ hybridization,in which labeled genomic DNAs of T.urartu and Ae.tauschii in green and red,respectively,were used as probes and the genomic DNA of Ae.longissima was used as blocking DNA,and the red arrows indicate chromosomes carrying the integrated 1Slx2.3*gene.(C)and(D)Test of homozygous transgenic lines by PCR and sodium dodecyl sulfate polyacrylamide gel electrophoresis.WT,wild type of wheat cultivar Westonia;P,positive control of expression vector pWMB184;1-8,individual transgenic plants;M,DL5,000 DNA marker.

Fig.3.Dynamic expression patterns of the target 1Slx2.3*and endogenous HMW-GS genes in new material T-W1 and its genetic background control Westonia revealed by quantitative real-time PCR(qPCR).T7-T28,cDNA samples from new material T-W1 carrying 1Slx2.3*at 7,10,13,16,19,22,25,and 28 days post anthesis(DPA),respectively;W7-W28,cDNA samples from wild-type Westonia at 7,10,13,16,19,22,25,and 28 DPA,respectively.

Fig.4.Content determination of grain storage protein compositions in new material T-W1 and its genetic background control Westonia by reversed-phase ultra-performance liquid chromatography(RP-UPLC)or size-exclusion ultra-performance liquid chromatography(SE-UPLC).(A)Content analysis of glutenin subunits by RP-UPLC.(B)Content analysis of gliadins by RP-UPLC.(C)Content analysis of glutens and glutenin macropolymers(GMPs)by SE-UPLC.(D)Comparison of relative contents of glutenins.(E)Comparison of relative contents of gliadins.(F)Comparison of relative contents of GMPs in immature and mature kernels.

3.6.Bread-and sponge cake-processing quality in the new material T-W1

Except for a negative tolerance index,the key quality parameters,including crude protein content,water absorption,dough formation time,and dough stabilization time,were significantly higher in T-W1 than in WT(Table S3;Fig.6).The bread loaf volume of T-W1 was increased by 12.2% relative to that of WT(Table S3;Fig.6).These values resulted in a bread sensory evaluation score of 93.5 in T-W1,which was also significantly higher than that in WT(85.0).

For sponge cake-processing quality,T-W1 displayed a slightly larger specific volume and a better surface appearance than WT,but a coarser and slightly rougher texture than WT(Fig.6).Transverse slices of cakes made from T-W1 and WT showed no sunken tops with collapse and shrinkage,and few irregular air bubbles(Table S3;Fig.6).T-W1 sponge cakes received a slightly greater sensory evaluation value than those of WT(Table S3).

4.Discussion

HMW-GSs are determinants of wheat end-use quality traits[4,10,24].Owing to the limited variety of HMW-GS alleles in common wheat,it is desirable to identify new subunits in wheat wild relative species.Developing new materials using those newly identified subunits is essential for improving wheat processing quality.It was reported[4]that Ae.longissima carries two potential HMWGS genes(1Slx2.3*and 1Sly16*)located on chromosome 1Sl,which provides wheat breeders with a desirable gene resource.1Slx2.3*contains 941 amino acids(aa),with 102 aa including 8 hexapeptides and 6 nonapeptides in the central repetitive domain(RD)[4].In HMW-GS allelic variants,the variations were mostly in the RD region,which contains different numbers of repeated peptide motifs and is associated with the physical properties of wheat dough[10].A large and regular RD strongly influences dough viscosity and elasticity[10].In the present study,we transferred 1Slx2.3*isolated from Ae.longissima into wheat and obtained homozygous transgenic lines(Figs.1-3).The bread-making quality of the new material with integrated 1Slx2.3*was greatly improved.Our results support the conclusion[10]that numbers of HMW-GSs improve dough properties,suggesting that the dough improvement in the new material might be attributed to the extra 102 aa residue in the 1Slx2.3*RD region.

A recent study[40]revealed that the introgression of 1Ay21*in near-isogenic lines(NILs)increased not only the accumulation of HMW-GSs and other storage proteins,but also the protein biosynthesis abundances.In total,115 differentially accumulated proteins(DAPs)were discovered among normal wheat cultivars and the corresponding introgressed NILs carrying 1Ay21*.It has been proposed[40]that the contribution of 1Ay21*to increasing wheat grain protein content and improving bread-making quality is closely associated with a wider reshaping of the grain proteome network.The finding in the present study that overexpression of 1Slx2.3*affected the expression levels of the other five endogenous HMW-GS genes(1Ax2*,1Bx17,1By18,1Dx2,and 1Dy12),suggests that the integrated 1Slx2.3*may influence the regulation patterns of endogenous HMW-GS genes.The overexpression of 1Slx2.3*in the new material T-W1 greatly increased the expression of 1Dx2 and 1Dy12 and reduced that of 1By18.Integration of foreign genes into wheat may result in expression reduction or silencing of some endogenous genes.In two wheat transgenic lines in which the transgenes 1Ax1 and 1Dx5 were integrated,all endogenous HMW-GS genes were silenced,resulting in poor flour-processing quality with reduced dough mixing time,peak resistance,and sedimentation volumes with reduced gluten strength[41].Expression of HMW-GS is generally regulated by molecular chaperones including protein disulfide isomerase(PDI),binding protein(Bip),and associated transcription factors(TFs)such as DNA binding with one finger(Dof)and storage protein activator(SPA)that are associated with the biosynthesis of wheat storage proteins[42,43].

Transcription-regulatory proteins influence many biological processes in plants,such as cellular morphogenesis,signaling transduction,and environmental stress responses[44].As regulators,molecular chaperones can assist peptides to be folded and transported correctly by recognizing incompletely folded proteins and binding to them,but they are not involved in the formation of the final products.Transcription factors regulate gene expression by binding to plant-specific cis-regulatory elements in gene promoter regions and activating or repressing the transcription of their target genes.Elucidating the reason for the expression changes of the endogenous HMW-GS genes in our new wheat material would benefit from acquisition of the expression profiles of more molecular chaperones and TFs.

Fig.5.Dynamic change observation of protein bodies(PBs)during grain development in new material T-W1 and its genetic background control Westonia by transmission electron microscopy(TEM).(A)Dynamic changes of PBs with the prolongation of the grain-filling stage from 7 to 28 days post anthesis(DPA),in which the red arrows indicate PBs,and all bars represent 10μm except those for 5μm in the six photos at 7,10 and 13 DPA in T-W1 and Westonia.(B)Size statistics of PBs in T-W1 and Westonia during the grain-filling stage from 7 to 25 DPA.

Wheat seed storage proteins,mainly glutenins and gliadins,are genetic determinants of wheat quality traits[2].To characterize the effects of the alien gene 1Slx2.3*on wheat storage proteins,the relative contents of glutenins and gliadins in the new material T-W1 were measured by RP-UPLC.The contents of HMW-GSs(1Ax2*,1Bx17,1Dx2,and 1Dy12)in T-W1 were increased in comparison with those in its WT,a finding consistent with the upregulated expression of the HMW-GSs found by qPCR(Table S2;Figs.3,4).The content of the total glutenins(HMW-GSs and LMW-GSs)was markedly higher in T-W1 than in its WT,while the contents ofω,α/β,andγ-gliadins were similar in the two materials(Table S2;Fig.4),leading to an increased ratio of glutenins/gliadins in T-W1.Generally,a higher ratio of glutenins to gliadins results in greater dough elasticity and strength.The composition and quantity changes of glutenins and gliadins could contribute to physical properties of dough by influencing GMP formation[45].In this study,no marked difference was found in the dynamic change of GMPs at 22 DPA,28 DPA and the mature stage between T-W1 and its genetic background control.

During the grain development period,the expression level of 1By18 in T-W1 was significantly lower than that in its WT(Fig.3),indicating that the expression of 1By18 was massively suppressed in the new material.However,the total content of both subunits 1Slx2.3*and 1By18 in T-W1 was higher than the content of the single submit 1By18 in WT(Fig.4).We postulated that the enriched 1Slx2.3*made the 1By18 also abundant in T-W1,given that the peaks of 1Slx2.3*and 1By18 were close together and could not be cleanly separated in the new material.The abundance of 1By18 appears not to have been affected,even though its encoding gene was almost silenced in the new material.

Wheat storage proteins as secretory proteins form in the ribosome,are preliminarily assembled and folded in endoplasmic reticulum(ER),and then are transported into organelles in PBs[46].Thus,wheat storage proteins accumulate mainly as PBs in endosperm during grain development.Secretion and accumulation of HMW-GSs increase PB formation.However,the relationship between the compositions of glutenins and gliadins and protein biosynthesis or PB development in wheat kernels is not clear.In the present study,the PBs in the endosperm of T-W1 during grain development period were larger in size and more rapidly accumulated than those of WT(Fig.5).These results indicated that overexpression of 1Slx2.3*stimulated the expression,secretion and accelerated of endogenous glutenins and fusion with PB in the endosperm.The observation that grain crude protein content was higher in T-W1 than in WT also provides evidence for explaining these phenomena(Table S3).These changes in grain protein composition might be attributed to the regulation of molecular chaperones and TFs caused by the introduced 1Slx2.3*.

Wheat flour with strong gluten and high protein content is suitable for bread-making,while flour with weak gluten and low protein is suitable for sponge-cake making[5].T-W1 showed superior bread-making quality reflected in its dough formation time,dough stabilization time,and bread loaf volume(Table S3;Fig.6).The improvement of bread-making quality may also be attributed to the increased HMW-GS and LMW-GS contents in T-W1.However,overexpression of 1Slx2.3*did not affect sponge cake-processing quality.

Wheat cultivars usually carry five or fewer HMW-GSs,as the genes at the Glu-A1 locus are normally silenced[10-12].The absence or silencing of other HMW-GSs at Glu-A1 and Glu-D1 loci in various wheat mutants always leads to inferior bread-making quality and even a failure of dough formation[10,19,24].In contrast,a Swedish wheat line W3879 was found to carry expressed 1Ax and 1Ay alleles[47].Recently[14],the 1Ax and 1Ay gene products in W3789 were identified as 1Ax21 and 1Ay21*subunits.The silent 1Ay in cultivar Lincoln was replaced by the expressed 1Ay21*after backcrossing,and the introgression line with 1Ay21*showed an improvement in grain protein content and bread-making quality without negative yield effect[11].These results indicate that more HMW-GSs contribute to better bread-making quality.

In the present study,the introduction of Ae.longissima 1Slx2.3*greatly improved the bread-making quality of the new material T-W1 by improving dough formation and stabilization time and dough elasticity and viscoelasticity.However,the new material contains both genes 1Slx2.3*and bar.The next challenge is to develop transgenic materials carrying only 1Slx2.3*without bar.The resulting marker-free material can be used as hybrid parents for genetic improvement of bread-making quality;and flour from the improved material can also be mixed with inferior flour to improve the bread-making quality of the latter.Given that 1Slx2.3*gene was closely linked with 1Sly16*gene at one locus in wheat-Ae.longissimi 1Sl(1B)substitution line showing good bread-making quality[4,48],transferring 1Slx2.3*and 1Sly16*simultaneously into wheat by genetic transformation may result in a better material than transferring 1Slx2.3*alone.The target gene 1Slx2.3*was shown by FISH and GISH to have integrated into the distal region of the D genome in the new material.The chromosomes into which it integrated await identification.

5.Conclusions

The HMW-GS-encoding gene 1Slx2.3*from Ae.longissima was transferred into wheat by Agrobacterium-mediated transformation and a homozogous new material T-W1 was developed.The overexpression of 1Slx2.3*in the new material greatly stimulated the expression of the endogenous HMW-GSs including 1Dx2 and 1Dy12,and the secretion and accumulation of endogenous glutenins(HMW-GSs and LMW-GSs).The integration of 1Slx2.3*in the new material also led to a marked increase in the ratio of glutenins to gliadins and accelerated the formation and fusion of PBs in the endosperm during grain development.1Slx2.3*improved breadmaking quality by improving some main quality parameters but had no effect on sponge cake-processing quality.The excellent new material T-W1 containing 1Slx2.3*developed in this study has potential use in improving wheat bread-making quality.

CRediT authorship contribution statement

Yuliang Qiu:Methodology,Software,Writing-original draft,Writing-review & editing.Haiqiang Chen:Data curation,Formal analysis,Writing-original draft.Shuangxi Zhang:Project administration,Data curation,Formal analysis.Jing Wang:Investigation,Resources,Validation.Lipu Du:Investigation,Resources,Validation.Ke Wang:Conceptualization,Methodology,Project administration, Supervision.Xingguo Ye:Conceptualization,Methodology,Funding acquisition,Project administration,Supervision,Writing-review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Natural Science Foundation of China(31971945)and the Department of Science and Technology of Ningxia in China(2019BBF02020).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2022.04.001.