Yunyun Zhng ,Yng Co ,Hongying Zheng ,Wenqi Feng ,Jingto Qu ,Fengling Fu ,Wnchen Li ,*,Hoqing Yu ,*

a Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region,Ministry of Agriculture,Maize Research Institute,Sichuan Agricultural University,Chengdu 611130,Sichuan,China

b College of Life Science &Biotechnology,Mianyang Teachers’ College,Mianyang 621000,Sichuan,China

ABSTRACT Improved chilling tolerance is important for maize production.Previous efforts in transgenics and marker-assisted breeding have not achieved practical results.In this study,the antifreeze protein(AnAFP) from the super-xerophyte Ammopiptanthus nanus was aligned to KnS-type dehydrins.Phosphorylation in vitro and subcellular localization showed that AnAFP was phosphorylated by maize casein kinase II and translocated from nucleus to cytoplasm under chilling stress.AnAFP also increased lactate dehydrogenase activity.A parent line of commercial maize hybrids was transformed with the AnAFP gene.Based on thermal asymmetric interlaced PCR,one hemizygous and two homozygous integration sites were identified in one T1 line.Ectopic expression of AnAFP in transgenic lines was confirmed by quantitative real-time PCR,RNA-seq,and Western blotting.After chilling treatment,the transgenic lines showed significantly improved phenotype,higher kernel production,survival rate and biomass,and lower relative electrolyte leakage and superoxide dismutation than the untransformed line.Thus,ectopic expression of AnAFP gene improved chilling tolerance in the transgenic lines,which could be used to apply for further safety assessment for commercial breeding.

Keywords:Antifreeze protein Chilling tolerance Dehydrin Ectopic expression Homozygosity identification

1.Introduction

Maize is one of the most important food crops in the world,owing to its high efficiency of photosynthesis and production.Increasing demand drives the continued growth and expansion of maize production [1,2].However,maize originated in the tropics[3].It is extremely vulnerable to chilling stress,having a higher biological minimum temperature (12 °C) than temperate plants(10 °C) [4,5].Maize growth and development are severely constrained by chilling stress at high latitudes and high altitudes,especially during germination and seedling stages [6,7].The complex genetic basis and incomplete knowledge of maize chilling tolerance have led to inefficient improvement in conventional breeding for the trait[8–10].Biotechnologies including transgenics and marker-assisted selection have been applied to improve maize chilling tolerance.But of many cold-inducible genes and quantitative trait loci that have been identified,only a few have been used in transgenic manipulation or marker-assisted selection [11–18].No commercial maize hybrid has been released as a result of such studies.

Antifreeze proteins(AFPs)are found in polar fishes and insects,and permit their survival under subzero environments by noncolligative effects that depress the freezing point of their blood or hemolymph but do not affect their melting point [19,20].However,the chilling tolerance of transgenic tobacco harboring a fishAFPgene was not significantly improved [21].The non-colligative nature of the heterologous animal AFPs,as well as their expression rate,localization and stability,may not be suitable for the plant cell environment.Plant-derived AFPs have been found in overwintering plants such as carrot (Daucus carota),Antarctic hair grass(Deschampsia antarctica),ryegrass(Lolium perenne),perennial grass(Loliurn perenne),and wheat (Triticum aestivum) [22–26].Their inhibitory effect on ice growth and recrystallization is 10 to 100 times stronger than that of AFPs of fishes and insects,although their thermal hysteresis activity (THA) is lower [25,27,28].It is hypothesized that binding of hyperactive AFPs to ice is facilitated by preordering of water at the ice-binding site of the protein in solution[29].Transgenic plants harboring plant-derivedAFPgenes showed significant improvement of chilling tolerance [22,30].

Ammopiptanthus nanusis a super-xerophyte surviving in mid-Asian deserts in the Tertiary period [31].The AnAFP protein has been separated and evaluated for its contribution to dehydration and cold stress tolerance,although its THA(0.46°C)is not the highest in overwintering plants [32].The purpose of the present study was to transform theAnAFPgene into a commercial maize inbred line and evaluate the effect of its ectopic expression on the chilling tolerance of transgenic lines.

2.Materials and methods

2.1.Sequence alignment of AnAFP

The amino acid sequence of AnAFP (NCBI accession number is GQ200581.1)was used as a query to search the NCBI protein database (https://www.ncbi.nlm.nih.gov/guide/proteins/).Target sequences with functional annotation and high similarity to the query were used for phylogenetic analysis with MEGA7 (https://www.megasoftware.net/).Sequences clustering in the same subfamily with AnAFP were aligned with ClustalW 1.81 (http://clustalw.genome.jp/) to reveal conserved domains and their secondary structure was predicted with PRABI (https://prabi.ibcp.fr/).

2.2.Protective enzyme activity and phosphorylation assay of AnAFP

A pair of specific primers,O1F/O1R(Table S1),was designed and used to amplify the open reading frame(ORF)ofAnAFP.The amplified product was cloned into the prokaryotic expression vector pET28a to generate pET28a-AnAFP,which was used to transformEscherichia colistrain BL21.After resistance screening,ectopic expression of theAnAFPgene was induced by 0.5 mmol L-1isopropyl β-D-thiogalactoside (IPTG) at 37 °C at 220 r min-1for 2 h.The AnAFP protein was purified with a Ni-NTA Sefinose Resin Kit(Sangon Biotech,China).

Lactate dehydrogenase (LDH) activity in the presence of AnAFP was determined with a lactate dehydrogenase assay kit (Solarbio,China) according to the manufacturer’s instructions.LDH (32 μL LDH 0.86 U mL-1) was mixed with 16 μL CuCl2(4.63 μmol L-1,16 μL ddH2O for the control).Then,32 μL purified AnAFP(9.3 μmol L-1)was added to the mixtures(32 μL PBS buffer for blank control)and absorbance at 450 nm was recorded in a spectrophotometer(UV-1800,Shimadzu,China).One unit was defined as the amount of enzyme catalyzing 1 μmol lactate to pyruvate per minute.

The phosphorylation assay was performed as described by Liu et al.[33] with minor modification,reaction mixtures containing 100 U CK II,2 μg AnAFP and 200 μmol L-1ATP (125 μmol L-14,5,6,7-tetrabromo-1H-benzotriazole (TBB,CK II inhibitor) was added to sample for control) were incubated at 30 °C for 2 h.The samples were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis.The phosphorylation of AnAFP was identified by gel mobility shift.

2.3.Bimolecular fluorescence complementation between AnAFP and casein kinase II

The ORF sequences of four CK II β subunits encoding genes includingZmCK IIβ1(NM_001111504),ZmCK IIβ2(NM_001112154),ZmCK IIβ3(NM_001254850) andZmCK IIβ4(XM_008660092) were amplified using specific primers (C1F/C1R,C2F/C2R,C3F/C3R,C4F/C4R,Table S1),and combined in the pSAT6-nEYFP-N1 vector for bimolecular fluorescence complementation assay(BiFC).The ORF sequence ofAnAFPwas amplified using specific primers O2F/O2R (Table S1),and ligated into pSAT6AcEYFP-N1 vector.Epidermal cells of onion bulb were cotransformed with the constructed vectors following Hollender and Liu[34].After dark incubation at 28°C for 16 to 20 h,green fluorescence signal was visualized under a confocal microscope(ZEISS 800,Germany).

2.4.Subcellular localization of AnAFP

The ORF sequence ofAnAFPwas amplified with primers O3F/O3R (Table S1),and cloned into vector pCAMBIA2300-35S-eGFP,generating a AnAFP fusion-enhanced green fluorescence protein(eGFP) gene driven by the 35S promoter (35S-eGFP-AnAFP).After preculture on 1/2 MS medium for 4 h,onion bulb scales were transformed with the35S-eGFP-AnAFPplasmid by biolistic bombardment.Part of the transformed scales were cultured at 28 °C for 12 h and then chilled at 4°C for 12 h on 1/2 MS medium,while the rest were incubated in the same medium without chilling treatment (blank control) and with 125 μmol L-1TBB (negative control)at 28 °C for 24 h.The epidermal cells of transformed bulb scales were examined for eGFP signal under the confocal microscope (ZEISS 800,Germany).The mean fluorescence intensities of the cytoplasm and nucleus were determined with ImageJ software(https://imagej.net).

2.5.Maize transformation

The ORF ofAnAFPwas subcloned into the pTF101.1-Ubi-Tnosplasmid to construct the monocotyledonous expression vector pTF101.1 +Ubi+AnAFP+Tnoswith the herbicide-resistance geneBar(used as a selectable marker) (Fig.S1c).Following Zhang et al.[35],embryonic calli were isolated from an inbred line 18–599 used in many commercial hybrids,transformed usingAgrobacterium tumefaciensEHA105,and screened on glyphosate gradient medium (2,3,and 5 mg L-1for 20 days each),and plants were regenerated.The first leaf from each regenerated plant was used for DNA extraction by the CTAB method.Positive plants were identified by PCR amplification of a 612 bp fragment of theAnAFPgene with specific primers J1F/J1R(Table S1),and bag-selfed to produce progeny generations.

2.6.Identification of integration sites and homozygosity

According to Liu et al.[36],six arbitrary degenerate primers(AD1 to AD6,Table S1) and six nested specific primers (LB1,LB2,LB3,RB1,RB2,and RB3,Table S1) were designed from conserved amino acid sequences of universal protein-encoding genes in eukaryotes and T-DNA sequence,respectively.Thermal asymmetric interlaced PCR (TAIL-PCR) amplification was performed using the improved procedure [37].The products of the third-round amplification were separated by 1.5% agarose gel electrophoresis,cloned into pMD19-T vector(TaKaRa,Japan),sequenced at Sangon Biotech,and aligned against the sequence of the maize genome(https://www.maizegdb.org/),or the constructed expression vector with identity ≥90% and E-value ≤e-100.

Three specific primers(LB-F1,2 and 3,Table S1)were designed from flanking sequences of the maize genome adjacent to the left borders of the three T-DNA integration sites and paired with the LB3 primer for third round TAIL-PCR,respectively.If specific fragments were amplified using LB-F1/LB3,LB-F2/LB3,or LB-F3/LB3,the T1plants were identified as having been transformed by TDNA integration at least one of the two homologous chromosomes(Fig.3b).The specific primers RB-R1,RB-R2,and RB-R3 were designed from the flanking sequences of maize genome adjacent to the right borders of the three T-DNA integration sites,respectively.The primer pairs LB-F1/RB-R1,LB-F2/RB-R2,and LB-F3/RBR3 were used to amplify the fragments between these two flanking sequences for 1 min extension during PCR amplification.If specific fragments were amplified,at least one homologous chromosome harbored no T-DNA,because the T-DNA sequence between the flanking sequences adjacent to the left and right borders was too long (5922 bp) to be amplified during 1 min extension (Fig.3b and Fig.S1c).If no specific fragment was amplified,the T-DNA integration was homozygous on the two homologous chromosomes.

2.7.Amplification of integrated T-DNA sequences

Three pairs of specific primers (LA-F1/LA-R1,LA-F2/LA-R2,and LA-F3/LA-R3,Table S1) were designed from maize genome sequences beyond T-DNA-integrated sites and used to amplify the full-length T-DNA sequences from genomic DNA of T2transgenic lines 1,2,and 3 using long-distance LA Taq (TaKaRa,Japan).By reverse-primer PCR(rpPCR)[38],the primer pairs RB3/LB3,RB3/RB3,and LB3/LB3 (Fig.S1c) were used to amplify the interval sequences between the two repeatedly integrated copies of TDNA sequence from genomic DNA of lines 1,2,and 3.The amplified products were separated by 1.2% agarose gel electrophoresis,cloned into pMD19-T vector (TaKaRa,Japan),and sequenced at Sangon Biotech.

2.8.Real-time quantitative PCR

The T3seedlings of each transgenic line as well as the untransformed line were grown in three nursery pots under a diurnal cycle of 28 °C and illumination of 300 mmol L-1m-2s-1for 14 h with 22°C and dark for 10 h.At the three-leaf stage,the seedlings were subjected to chilling stress of 8 °C under illumination of 300 mol L-1m-2s-1for 14 h with 4°C and dark for 10 h.At 0(control),1,2,3,4,and 5 days,the leaves and roots were collected and used for total RNA extraction with a RNAiso Plus Kit (TaKaRa,Japan).After detection of concentration and integrity,parts of the total-RNA samples of 0 (control) and 1 day of chilling stress were set aside for RNA sequencing.The rest was reverse-transcribed into cDNA with a PrimeScript Ⅱ1st Strand cDNA synthesis Kit(TaKaRa,Japan).

Eight pairs of specific primers were designed with Primer-BLAST software (www.ncbi.nlm.nih.gov/tools/primer-blast),to amplify a 103–217 bp fragment of the integratedAnAFP,ZmGAPDH(internal reference),as well as six endogenous genes flanking the integration sites (Table S1).After being screened for optimal annealing temperatures with temperature-gradient PCR,they were used for real-time quantitative PCR (RT-qPCR) amplification as described by Wang et al.[39].The 2-ΔΔCTmethod in CFX Manager software version 2.0(Bio-Rad,USA)was used to normalize the differential gene expression among internal reference and target genes[40].The statistical significance among three biological replicates was tested with Student’st-test using IBM-SPSS software(http://www-01.ibm.com/software/analytics/spss/).

2.9.Western blotting

Purification of AnAFP by prokaryotic expression was used for preparation of polyclonal antibody at Sangon Biotech.Total protein was extracted from leaf samples of three transgenic lines and untransformed line,as well asA.nanus,with a Plant Total Protein Extraction Kit (Sangon Biotech),separated by polyacrylamide gel electrophoresis (with 200 μg in each well),and transferred onto nitrocellulose membrane (Bio-Rad).After blocking with 5% skim milk at 37°C for 2 h,the membrane was incubated with polyclonal antibody at 4 °C overnight.After rewarming at 37 °C for 1 h,the membrane was incubated with peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) at 37 °C for 1 h.Exposure was visualized as increased chemiluminescence.

2.10.RNA-sequencing

The three replicates of total RNA samples from 0 (control) and 1 day of chilling stress were sent to Mega Genomics Company(China) for paired-end sequencing (PE150) on an Illumina HiSeq sequencing platform.Differentially expressed genes (DEGs)between transgenic lines and untransformed line were detected as described by Mortazavi et al.[41] and Li et al.[42],and predicted for their functional annotation and metabolic pathways by Gene Ontology (GO),respectively.

2.11.Histochemical staining and relative electrical leakage detection

As described by Giannopolitis and Ries [43],one of the bottom leaves was collected on the 0th (control) and 5th days of chilling stress (8 °C 14 h/4 °C 10 h),stained in 0.1 mg mL-1nitrotetrazolium blue chloride (NBT) solution (25 mmol phosphate buffer,pH 7.6) in the dark at 25 °C for 16 h,and decolorized by boiling in fixing solution (ethanol:lactic acid:glycerol=3:1:1) for 10 min.After cooling,the leaves were transferred to fresh fixing solution,held at room temperature overnight,and photographed.

The other bottom leaf samples were cut into pieces of 0.5 × 0.5 cm weighing about 0.6 g,immersed in 6 mL deionized water,placed under vacuum at 0.6 MPa for 10 min,and held at room temperature for 3 h.Electrical leakage (EL1) was measured in an electrical conductivity detector,model DDS-308+(Kangyi,Shanghai).The samples were then boiled for 30 min.After cooling,the electrical leakage (EL2) was measured again.The EL value of deionized water was defined as EL0.Relative electrical leakage(REL)was calculated as (EL1 -EL0)/(EL2 -EL0) × 100% [44].

2.12.Phenotyping

T3seedlings of lines 1 and 2 as well as the untransformed line were grown in the same nursery pot with four replicates under a diurnal cycle of 28 °C and illumination of 300 mmol L-1m-2s-1for 14 h/22 °C and dark for 10 h.At three-leaf stage,the seedlings were subjected to frost stress at -2 °C for 5 h.They were then thawed at 4 °C for 0.5 h,held at room temperature for 24 h,and photographed.

The sprouting seeds of transgenic lines 1 and 2 along with the untransformed line were sown in a randomized block experiment with three replicates on the Qinling Mountain at 1337 m altitude in early spring (March 24,2018).Daily maximum and minimum temperatures were recorded during seedling stage.The survival rate of seedlings,biomass at jointing stage,plant height,ear height,row number per ear,kernel number per row,100-kernel weight,and kernel weight per plant were investigated.Differences between transgenic lines and control were identified by Student’st-test using IBM-SPSS.

3.Results

3.1.AnAFP is homologous to dehydrins

From the similarity search,33 proteins with high similarity to dehydrins were obtained,and used for construction of a phylogenetic tree and identification of conserved domains.As shown in Fig.1,AnAFP shared the same conserved domains and was clustered in the same subclass with eight KnS-type dehydrins.Although AnAFP contained eight repetitive fragments that was homologous to other AFPs,AnAFP also contained one S,K and NLS (Nuclear localization signal) domain possessed by the KnS type dehydrins [29].The secondary structure of the K domain and the eight repetitive fragments were predicted as α helix and β folds,respectively,whereas the other domains and fragments were random coils.

Fig.1.Sequence analysis of AnAFP.(a)Phylogenetic relationship of AnAFP with other dehydrins.(b)Multiple sequence alignment of AnAFP with KnS-type dehydrins.Black triangle,arrow,and straight line represent AnAFP,β-pleated sheet,and repeated amino acid fragments,respectively.K,NLS and S domains are indicated with boxes.

3.2.Protein properties of AnAFP

Dehydrins can reactivate and protect LDH activity inhibited by Cu2+or during stress response [33,45–47].As shown in Fig.2a,the LDH activity inhibited by Cu2+was recovered by AnAFP,suggesting that AnAFP shares a similar function with dehydrins(Fig.2a).The phosphorylation assay showed that AnAFP mobility in gel was markedly lower than that of blank controls (0 U CK II)and negative controls with 125 μmol TBB after co-incubation with CK II and ATP (Fig.2b).The BiFC result showed that the CK II subunits β1,β3,and β4 interacted with AnAFP,whereas β2 did not(Fig.2c).In subcellular localization,fluorescence signal was visualized in both the nucleus and cytoplasm of onion epidermal cells transformed with theeGFP-AnAFPfusion vector under normal condition,in the cytoplasm under chilling stress,and in the nucleus with 125 μmol L-1TBB.As blank control,fluorescence signal was visualized in both the nucleus and cytoplasm of onion epidermal cells transformed with empty vector and incubated under normal,chilling,and TBB inhibition conditions (Fig.2d).The fluorescence intensity in the nucleus and cytoplasm of onion epidermal cells transformed with emptyeGFPvector showed no difference after treatments.However,the fluorescence intensity in the nucleus of onion epidermal cells transformed withAnAFP-eGFPwas lower than that in the cytoplasm after chilling treatment.After TBB application,fluorescence was detected only in the nucleus (Fig.2e).Thus,AnAFP was phosphorylated by CK II and translocated from nucleus to cytoplasm in response to chilling stress.

3.3.Three transgenic events on different homologous chromosomes of one T0 plant

One T0plant was positive line identified among 36 regenerated plants.Southern blotting revealed that this plant carried at least five copies of theAnAFPgene (Fig.S2a).All 13 self-pollinated T1plants were identified as positive without segregation (Fig.S2b).Six specific fragments were amplified in the third round of TAILPCR from these 13 T1plants.Sequencing and alignment showed thatAnAFPgenes were integrated into maize genome at three sites on chromosome 1:Chr 1:161618329–161618351 (site 1),Chr 1:46681215–46681263 (site 2),and Chr 1:288071380–288071385(site 3)(Fig S2;Table S2),and at least two of them were integrated into different homologous chromosomes.The genes adjacent to the integration sites are listed in Table S3.

3.4.Homozygous T1 plants

According to the principle shown in Fig.3a and b,four T1plants(2,5,10,and 13) were identified as homozygous transformants ofAnAFPat integration site 1.Three T1plants(1,7,and 8)were identified as homozygous transformants at site 2.However,nine T1plants(2,3,4,5,7,9,11,12,and 13)were not homozygous at site 3.T1plant 10 was a homozygous transformant of theAnAFPgene at site 1 only,and T1plant 1 and 8 were homozygous transformants at site 2 only (Fig.3).T2plant 7 was identified as a homozygous transformant of theAnAFPgene at site 3 only from the 10 T2progeny of T1plant 4 (Fig.S3).Among these plants,three different lines with only one insertion site were identified as lines 1 (site 1),2(site 2),and 3(site 3),and self-pollinated to produce progeny.Interestingly,two tandem copies of T-DNA were integrated into line 1 and 2 but only one copy into line 3 (Fig.S1).

3.5.Ectopic expression of AnAFP

The results of RT-qPCR confirmed the ectopic expression ofAnAFPin transgenic lines (Fig.4a,b;Table S4).In response to cold stress (8 °C in the day and 4 °C at night),the relative expression levels ofAnAFPin transgenic lines were upregulated compared to control(0 d)(Fig.4a and b).Western blotting revealed clear bands with the same mobility shift between positive control and transgenic lines(Fig.4c),indicating the ectopic synthesis of AnAFP protein.The weaker band of line 2 was consistent with the lower relative expression ofAnAFPin the same line (Fig.4).The relative expressions of endogenous genes adjacent to T-DNA integration sites did not change in line 1 and line 2,but increased more than 10 times in line 3 (Fig.S4a).

Fig.2.Protein properties of AnAFP.(a) Effect of AnAFP on lactate dehydrogenase (LDH) activity.(b) Phosphorylation of AnAFP by CK II.(c) Interaction between AnAFP and ZmCK II β subunits by BiFC.(d) Subcellular localization of AnAFP.(e) Mean fluorescence intensity.A,B,C and D indicate statistical significance at P <0.01.

Fig.3.Homozygosity identification of T-DNA integration in T1 plants.(a) Gene separation model of T0 self-progeny.(b) Matching of primers on chromosomes with T-DNA inserted or not inserted.(c) The result of homozygosity identification of T-DNA integration.LB-F1,LB-F2,and LB-F3,specific primers matching maize genomic sequences beyond T-DNA left border integrated sites;RB-R1,RB-R2,and RB-R3,specific primers matching maize genomic sequences beyond T-DNA right border integrated sites;LB3,matching T-DNA left border;M,DNA marker DL2000;CK-,the untransformed line;CK+,positive control (the T0 plant).

Fig.4.Expression of AnAFP in transgenic lines.(a,b)Relative expression levels of AnAFP in leaf(a)and root(b)of three transgenic lines under chilling stress.(c)Western blot of transgenic lines.(d,e)DEGs between transgenic and untransformed lines under normal(d)and chilling stress(e).CK+,positive control(A.nanus);CK-,untransformed line.Red and black numbers are those of upregulated and downregulated endogenous genes.Values represent means of three biological replicates with error bars indicating standard error of the mean.

As shown in Fig.4d and e,among DEGs from RNA-seq,under the control condition,the expression of 115,253,and 137 endogenous genes was upregulated,that of 180,223,and 296 was downregulated in lines 1,2,and 3,respectively,compared to the untransformed line,and 7 and 14 genes were consistently upregulated or downregulated in all three lines.Under chilling,the expression of 58,122,165 endogenous genes were upregulated,that of 69,267,and 137 endogenous genes was downregulated in lines 1,2,and 3 compared to the untransformed line,and 6 and 16 were consistent in all three lines.Four genes (GRMZM2G100102,GRMZM2G023028,GRMZM2G090028,andGRMZM2G080054) were significantly downregulated in all three lines under control and chilling conditions.

3.6.Significant improvement of chilling tolerance

Because the growth vigor and seed size of line 3 were significantly less than those of the other two lines and untransformed line (Fig.S4b and c),and the expression of flanking endogenous genes adjacent to site 3 was significantly upregulated compared to that in untransformed line (Fig.S4a),lines 1 and 2 were used for phenotyping of chilling tolerance.In the pot experiment,seedlings of untransformed line were frostbitten and wilted,whereas seedlings of lines 1 and 2 kept alive after chilling treatment(Fig.5a and b).The mean survival rates of lines 1 and 2 were significantly higher than those of the untransformed line (Fig.5c).

After 5 days of chilling stress,NBT histochemical staining of leaves of line 1 and 2 was markedly lighter than that of untransformed line (Fig.5d).Likewise,their RELs were significantly lower than that of untransformed line (Fig.5e).These results indicated less accumulation of reactive oxygen species [43],and the protection of the AnAFP protein to the cell membrane [44].

During the seedling stage in the field experiment,four cold valleys were recorded after sowing.The minimum temperatures of these cold valleys were 1.2,3.2,–2.8,and 1.2 °C (Fig.6a),which were far below the biological minimum temperature of maize(12°C).Lines 1 and 2 showed more vigorous growth than untransformed line(Fig.6b).Their survival rate,biomass,plant height,ear height,kernel number per ear,and kernel weight per plant were significantly higher than those of untransformed line (Fig.6,Table 1),indicating the increased chilling tolerance of the transgenic lines.

But it was all no good, and the Princess suffered so much from their remedies that the King was obliged to send out a second proclamation that anyone who undertook to cure the Princess, and who failed to do it, should be hanged up to the nearest tree

4.Discussion

Dehydrins are group 2 of late embryogenesis abundant (LEA)proteins widely present in prokaryotes and eukaryotes [48].They are characterized by a conserved lysine-rich K domain (EKKGIME/DKIKEKLPG),serine-rich S domain (SSSSSED),and Nterminal Y domain(V/TDE/QYGNP).The number and order of these domains define their subclasses as YnSKn,YnKn,SKn,Kn,and KnS[49,50].In response to dehydration stress,the disordered structure of the K domain forms amphipathic α-helices to protect proteins and the membrane system via protein-protein and protein-lipid interactions [51–54].In the present study,AnAFP contained eight β folds,distinguishing it from other dehydrins but in common with plant AFPs (Fig.1) [29].AnAFP was clustered in the same subclass and shared similar conserved domains with eight KnS dehydrins(Fig.1).

KnS-type dehydrins show a mixture of phosphorylation and non-phosphorylation in plant cells,where they are localized in the cytoplasm and nucleus[55,56].The location of KnS-type dehydrins depends on phosphorylation of the S domain by CK II,whereas its non-phosphorylated type is localized only in the nucleus [57].Because of the presence of the NLS domain,AnAFP can enter the nucleus after translation,but be translocated from the nucleus to the cytoplasm in response to chilling stress,owing to phosphorylation by CK II (Fig.2).

Fig.5.Identification of chilling resistance of transgenic lines.(a) Seedlings before cold treatment.(b) Seedlings after–2 °C 5 h treatment.(c) Mean survival rate of four replicates.(d)NBT staining after chilling stress at 8°C 14 h/4°C 10 h for five days.(e)REL of every line after chilling stress at 8°C 14 h/4°C 10 h for 5 days.Values represent means of four biological replicates with error bars indicating standard error of the mean.** indicates statistical significance at P <0.01.

Five copies of theAnAFPgene were shown to have been integrated at three different sites on chromosome 1 (Fig.S2;Table S2).Although multi-site integration of multiple copies of exogenous genes has also been found in other transgenic events mediated byA.tumefaciens[58,59],little attention has been paid to the integration of multiple copies on different homologs of one chromosome.This integration pattern resulted in the positive identification of all T1plants,although the identification was much more difficult.Two repeatedly integrated copies of T-DNA were directly tandem in lines 1 and 2 (Fig.S1).The expression ofAnAFPin line 1 and line 3 was higher than that in line 2(Fig.4a and b),in contrast to other reports of gene silencing in multiple integration[60,61].However,the expression of exogenous genes is also affected by transcription activity of their integrated genomic regions [62,63].The high expression ofAnAFPin line 1 and line 3 might be due to differential transcriptional activity at its integration site.

In conventional procedures of transgenic manipulation,homozygous transgenic lines are identified by specific PCR amplification from the progeny of homozygous plants [64,65].In recent years,competitive PCR,RT-qPCR,next-generation sequencing (NGS) and digital droplet PCR (ddPCR) have been introduced to identify homozygosity of transgenes in segregating populations [66–67].These methods have been seldom used in practice because of their low accuracy,requirement of high-quality DNA,and identification of copy number.We used a new method to identify homozygous plants of three integration sites from the T1plants (Fig.3) at least one generation ahead of the conventional procedures.

In the expression vector pTF101.1-Ubi-AnAFP-Tnos,theAnAFPgene was under the control ofUbiquitin(Fig.S1a).However,its relative expression level was upregulated by chilling stress (Fig.4a and b).Similar results were found in transgenic tobacco with theAFPgene under the control of a constitutive promoter [68,69].The degradation of its transcription product may be prevented by some unknown cold-inducible factors such as cold shock proteins[70,71].The stability of the AFP protein was also increased under cold stress [69].

As described by Thakare et al.[72],most DEGs in transgenic lines were not due to T-DNA integration,but were attributed to the comprehensive influence of expression ofAnAFP(Fig.4d and e).However,the downregulated expressions ofGRMZM2G100102,GRMZM2G023028,GRMZM2G090028,andGRMZM2G080054genes were consistent among the three events and two conditions.Their homologs (AT2G19620.1,AT3G16980.1,AT5G27260.1,andAT2G20180.1) inArabidopsisencode respectively N-MYC downregulated-like 3 (NDL3),DNA-directed RNA polymerases II,IV and V subunit 9B-like (NRPB9A,NRPD9A,and NRPE9A),MYB/SANT-like DNA-binding domain protein,and phytochrome interacting factor 3-like 5 (PIF1 and PIL5).All of these proteins were associated with abiotic tolerance and the expressions of their encoding genes were regulated in negative feedback loops [73–76].The consistently downregulated expression of these four genes in the three transgenic lines under chilling stress may involve similar feedback regulation of overexpression ofAnAFPunder the control of the constitutive promoterUbiquitin.

The seedlings of transgenic lines 1 and 2 were protected by AnAFP protein and survived after freezing treatment in the pot experiment (Fig.5).The improved phenotype of chilling tolerance(Figs.5 and 6;Table 1)may have been due to the binding of AnAFP to ice crystals and inhibition of their growth,as well as to superoxide dismutation(revealed by NBT staining)and membrane protection(detected by REL)(Fig.5d and e)[33,77].In high latitudes and high-altitude regions,the yield potential of maize is heavily constrained by frost-free growing season [1,2,6].Early sowing can increase the growth period of maize to prolong photosynthesis,but it is not favorable for seedling establishment under low temperature in early spring [6].In farming practice,maize is usually sown when temperatures remain above 12 °C to prevent chilling stress under cold spells in early spring.The increased chilling tolerance of transgenic lines 1 and 2(Fig.6;Table 1)would allow earlier sowing.

Fig.6.Chilling tolerance of transgenic lines in field experiment.(a) Daily maximum and minimum temperatures during seedling stage.(b) Seedlings at six-leaf stage.(c)Survival rate at seedling stage.(d)Biomass at jointing stage.CK,untransformed line;Lines 1 and 2:the transgenic lines.Values represent means of three replicates with error bars indicating standard error of the mean.**indicates statistical significance at P<0.01.Red arrows represent four cold valleys during seedling stage,and sequentially were 1.2,3.2,-2.8,and 1.2 .

Table 1 Agronomic traits of transgenic lines.

5.Conclusions

Fig.7.Model for the role of An AFP in increasing maize chilling tolerance.

CRediT authorship contribution statement

Yuanyuan Zhang:Designed the study,performed the experiments,w rote the original draft.Yang Cao:Performed the experiments.Hongying Zheng:Performed the experiments.Wenqi Feng:Performed the experiments.Jingtao Qu:Performed the analysis.Fengling Fu:Performed the analysis.Wanchen Li:Designed the study,w rote the original draft.Haoqiang Yu:Designed the study,review ed and edited the manuscript.

Declaration of com peting interest

The authors declare that they have no know n competing f inancial interests or personal relationships that could have appeared to inf luence the w ork reported in this paper.

Acknow ledgem ent

This w ork w as supported by National Key Science and Technology Special Project (2016ZX08003-004) and Sichuan Science and Technology Program(2018JY0470).We thank Professor Kan Wang at Iow a State University for the kind gift of expression vector p TF101.1,and acknow ledge technical support from the Key Laboratory of Biology and Genetic Improvement of Maize in Southw est Region.

Appendix A.Supplem entary d ata

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.08.011.