Qilong Xu,Li Yang,Dan Kang,Zhenjing Ren,Yunjun Liu*

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

Keywords:

A B S T R A C T The anther cuticle and pollen exine play a critical role in male gametophyte development.The sporopollenin precursors and cuticular lipid monomers are transported to the surface of the microspores and the epidermis by lipid transport proteins(LTPs)and ATP-binding cassette G(ABCG)transporters for the formation of the pollen wall and anther cuticle,respectively.However,the function of ABCG transporters in maize anther development is unclear.Here,we cloned the MS2 gene from the maize male sterile2 mutant using map-based cloning and determined that it encodes an ABCG transporter.MS2 protein was experimentally confirmed to be located on the cell membrane.The quantitative real-time PCR(qRT-PCR)results showed that MS2 was ubiquitously expressed in all vegetative and reproductive tissues,whereas a high transcriptional level of MS2 was observed in anthers,especially at the young microspore stage.Gas chromatography-mass spectrometry(GC–MS)analysis showed decreased accumulation of cutin and wax components in ms2 anthers,indicating that MS2 plays a role in the transport of lipid molecules to anther cuticle and pollen exine.To our knowledge,MS2 is the first reported ABCG transporter gene that participates in anther development in maize.

1.Introduction

Maize(Zea mays)is an important crop that can be used as both food and feed.To increase maize yield,the heterosis of maize hybrids is broadly utilized.The production of maize hybrid seeds using male sterile lines is convenient and cost-saving.Male sterility can be classified into two types:cytoplasmic male sterility(CMS)and genic male sterility(GMS).CMS is usually caused by dysfunction of mitochondria and is divided into T,C and S types according to the molecular mechanism of male sterility[1].CMS materials have been widely used in three-line hybrid seed production.However,CMS materials have some disadvantages,e.g.,the instability of their phenotypes,the scarcity of restorer lines,and the disease susceptibility of hybrid plants produced by crossing with CMS materials,which restrict the application of CMS materials[1].

As an alternative to CMS,GMS materials can also be used to develop hybrid seed production systems.GMS materials are usually generated by the mutation of nuclear genes and can be used for two-line hybrid seed production.Seed production technology(SPT)with the use of GMS materials has been successfully developed[2,3].To date,sixty-two putative GMS genes have been predicted in the maize genome[4],and 20 GMS genes have been cloned[4–7].Some GMS genes influence lipid metabolism,which is essential for the formation of anther cuticle and pollen exine.MaizeMS6021encodes a fatty acyl-CoA reductase,and mutation of this gene leads to degraded tapetum and a severe reduction in lipid derivatives[8].MaizeAPV1converts medium-chain saturated fatty acids to monohydroxylated fatty acids[9].MaizeMS26plays a role in the ω-hydroxylation of C16 and C18 fatty acids in the sporopollenin biosynthesis pathway[10].MaizeIPE1participates in the putative C16/C18 ω-hydroxy fatty acid oxidative pathway[11].ZmMS33plays indispensable roles in anther cuticle and pollen exine development[12].MaizeMS45[13]andMS30[14]participate in the formation of anther cuticle and pollen exine.

Cutin,as the structural skeleton of the cuticle,is composed of fatty acids and their derivatives.The cuticular wax that surrounds the surface of the cutin matrix is mainly composed of C16-C34 fatty acids and their derivatives,including esters,aldehydes,alcohols,ketones and alkanes[15,16].It has been revealed that sporopollenin precursors and cuticular lipid monomers are biosynthesized within the tapetum and then transported to the surface of the microspores and the epidermis by lipid transport proteins(LTPs)and ATP-binding cassette G(ABCG)transporters[17–19].ABCG proteins have been previously divided into the white/brown complex(WBC)and pleiotropic drug resistance(PDR)groups.ABCG transporters allocate sporopollenin precursors to the anther locule for the formation of pollen walls[20].It was reported that ArabidopsisABCG26and its rice orthologOsABCG15function in the transport of polyketides for exine formation[21,22].ABCG transporters also play roles in the development of anther cuticle,and it has been demonstrated thatOsABCG26is important for the export of cuticle precursors from the tapetum to the anther surface[23].In addition to the above ABCG transporters,other ABC transporters related to lipid metabolism and pollen development have also been identified,e.g.,AtABCG11[24],AtABCG9,AtABCG31[25],AtABCG1andAtABCG16[26],andAtABCG32[27]in Arabidopsis,andOsABCG3[28]andOsABCG9[29]in rice;however,the function of ABCG transporters in maize anther development remains unclear.

The maizems2mutant lacks microspore wall formation at the time of microspore collapse[30].Our previous work revealed that numerous genes related to the synthesis of sporopollenin precursors and lipid molecules were altered inms2anthers[31].In this study,theMS2gene was identified by map-based cloning to encode an ABCG transporter functioning in exine formation.The results of this study could improve the understanding of anther development in maize.

2.Materials and methods

2.1.Plant materials

Maize(Zea mays)ms2was obtained from the Maize Stock Center(accession number:917D ms2),and its genetic background was oh43.All materials were grown in the field in Beijing or Sanya,China.Thems2mutant was crossed with inbred lines B73 and PH4CV to generate the F2and BC1F2mapping populations,respectively,for gene isolation.

2.2.Phenotypic analysis of the ms2 mutant

Photographs of mature plants and the tassels of the wild type and mutant were obtained with a Nikon D5600 digital camera(Nikon,Japan).After the inner and outer glumes of spikelets were removed with tweezers,the anthers were photographed with a Leica MZ10F anatomical microscope(Leica,Germany).

2.3.Cloning of the MS2 gene

The mapping population was developed using the F2progenies ofms2×PH4CV and F2or BC1F1progenies ofms2×B73.SSR,SNP,or InDel markers were developed within bin 9.03.TheMS2locus was localized to a 252 kb region.The candidate genes in this region were analyzed in MaizeGDB based on the B73 RefGen_V4 genome sequence.The genome sequences of these candidate genes in thems2mutant and WT were PCR-amplified and sequenced to identify the mutation sites.

2.4.RNA extraction,RT-PCR,and qRT-PCR

Total RNA was isolated with the RNAprep pure Plant Kit(Tiangen Biotech,Beijing)from the anthers,roots,leaves,kernels and spikelets of the maize inbred line B73.Reverse transcription was conducted using TransScript First-Strand cDNA Synthesis(Trans-Gen Biotech,Beijing).The reverse transcription product was used as the template for PCR.qRT-PCR was performed using an ABI 7300 real-time PCR system with Trans ScriptII Top/Tip Green qPCR SuperMix.ZmActinwas used as the internal normalization control.Every sample contained three biological replicates and three technological replicates.Relative expression levels were measured using the 2-ΔΔCt method.

2.5.Construction of the CRISPR/Cas9 vector and maize transformation

The vector pBUE411 was provided by Dr.Qijun Chen from China Agricultural University.sgRNA sequences were designed using the CRISPR-P web-based resource(http://crispr.hzau.edu.cn/CRISPR2/)[32].The PCR fragments amplified from pCBC-MT1T2 using the two pairs of primers were inserted between theBsaI sites of pBUE411[33]to construct the pBUE411-2gR-GA vector.The CRISPR/Cas9 plasmid was transformed into the maize inbred line CAL byAgrobacterium-mediated transformation[34].

2.6.Phylogenetic analysis

The most similar sequences in maize,Arabidopsis and rice were identified via a BLAST search in PLAZA(https://bioinformatics.psb.ugent.be/plaza/versions/plaza_v4_monocots/)using the full-length amino acid sequence ofMS2.The sequences were aligned using the ClustalW tool with default parameters,and a neighbor-joining phylogenetic tree was constructed in MEGA 6.0.6 using the following parameters:Poisson model,complete deletion,and 1000 bootstrap replicates.

2.7.Subcellular localization of MS2

To determine the subcellular localization of MS2,YFP fusion protein expression construct was generated with the 35S promoter.The 2028-bpMS2full-length cDNA without the stop codon was amplified and cloned into the pGWC vector,and p35S::MS2-YFP was obtained after LR reaction.The plasmid was injected into tobacco leaves for transient expression.YFP fluorescence was observed under a fluorescence confocal microscope(Zeiss LSM 700).

2.8.Analysis of anther wax,cutin,and fatty acids

Wild-type and mutant anthers of ～3.5 mm-long anthers (5 biological repeats each), which were at the young microspore stage,were placed in 2 mL centrifuge tubes and stored at -80 °C for use. To extract anther wax, approximately 300 mg of fresh anther samples were immersed in 3 mL of chloroform for 1 min. The chloroform extract was added to 25 μg of nonadecanoic acid as an internal standard and transferred to a new vial. The solvent was evaporated under a mild stream of nitrogen. The residue was derivatized with 100 μL of N-methyl-N (trimethylsilyl) trifluoroethylamine and incubated at 50 °C for 1 h. To extract anther cutin, the remaining anthers were immersed in 3 mL of chloroform:methanol (1:1, v/v), incubated at 50 °C for 30 min, and then shaken at room temperature for 72 h. Anthers were lyophilized and immersed in 1 mL of 1 mol L–1methanol HCl at 80 °C for 2 h, with 25 μg of nonadecanoic acid as an internal standard. After 2 mL of saturated sodium chloride solution was added, the hydrophobic monomer was extracted three times with 1 mL of hexane. The solvent was evaporated, and the remaining samples were derivatized as described above. These derived samples were then analyzed by GC–MS (an Agilent gas chromatograph coupled to an Agilent 5975C quadrupole mass selective detector). To extract total fatty acids, 6 mg of freeze-dried anther material was transesterified in 1 mL of 1 mol L-1methanol HCl (containing 25 μg nonadecanoic acid as an internal standard) at 50 °C for 4 h. After 1.5 mL of 0.9%(w/v) NaCl was added, the hydrophobic monomer was extracted with 1.5 mL of hexane.The organic phase evaporated under a mild stream of nitrogen.The residue was dissolved in 100 μL of hexane and then analyzed by GC–MS.

3.Results

3.1.Fine mapping of the MS2 locus

The maizems2mutant has abnormal development of pollen exine and anther cuticle due to the failed formation of sporopollenin precursors and ubisch bodies,leading to the male sterile phenotype[31].Except for the male sterile phenotype,the vegetative growth of thems2mutant was normal(Fig.1).Thems2mutant was crossed with inbred lines B73 and PH4CV to generate F2populations.The F2plants showed a 3:1 segregation of fertile plants to male sterile plants(Table S1),indicating that the male sterile phenotype is controlled by a single recessive gene.MS2has been predicted to be located in bin 9.03(http://www.maizegdb.org/).To validate this locus,mapping was performed using the insertion/deletion(InDel)or SSR markers(Table S2)that were identified in bin 9.03.TheMS2locus was mapped to a 6.9 Mb region between markers umc1271(93,176,604 bp)and InDel7(100,122,823 bp)using 1344 individual male sterile plants from the F2population ofms2×PH4CV(Fig.2A)and to a 3.5 M region between markers InDel4(92,512,733 bp)and InDel5(96,015,270 bp)using 576 individual male sterile plants from the F2population ofms2×B73(Fig.2B).To further narrow down the region of theMS2locus,2163 BC1F2male sterile plants were used for fine mapping,and theMS2locus was mapped to a 252 kb region between markers InDel10(94,416,521 bp)and SNP1(94,669,331 bp)(Fig.2C),within which four putative genes existed(Fig.2D).

3.2.Cloning of the MS2 gene

To clone the candidateMS2gene,we amplified and sequenced the region containing the four candidate genes from the genomic DNA of B73 and thems2mutant.Only theZm00001d046537gene differed in its coding region,with an insertion of a 1547 bp TIR transposon sequence in the fifth exon in thems2mutant(Fig.2E).To further confirm thatZm00001d046537is theMS2gene,a CRISPR/Cas9 vector againstZm00001d046537was constructed and transformed into the maize inbred line CAL.Three independent homozygous mutants with gene editing at the first exon ofZm00001d046537were generated(Fig.3),and all of them showed the male sterile phenotype.These results confirmed thatZm00001d046537is theMS2gene that controls maize anther development.

3.3.MS2 encodes an ABCG transporter

By searching the MaizeGDB database,we found that theMS2gene belongs to the ABCG transporter family.Using the prediction of protein functional domain software(InterPro),we found an ABC transporter domain and ABC-2 transporter domain in the MS2 protein.To further explore the evolutionary relationship of the MS2 protein,we used BLASTP via Plaza software to screen for homologous proteins of MS2 in maize,rice and Arabidopsis.The phylogenetic tree was constructed based on the amino acid sequences of 10 Arabidopsis proteins,6 rice proteins and 5 maize proteins(Fig.4).Os06g40550,an equivalent of the rice male sterility geneOsABCG15participating in lipid transport during anther growth and pollen wall formation,is located in the same clade as MS2.Furthermore,we found that Arabidopsis AT3G13220,which is on the same evolutionary branch as the MS2 protein,encodes the ABCG26/WBC27 protein.According to the above analysis,MS2,LOC_Os06g40550 and AT3G13220 belong to the same evolutionary branch,and all of them belong to the ABCG-type transporter family.

3.4.MS2 was located in the cell membrane and was highly expressed in anthers

To experimentally investigate the cellular localization of the MS2 protein,the full-length cDNA ofMS2was fused to the Nterminus of YFP,generating the plasmid p35S::MS2-YFP,which was then injected into tobacco leaves for transient expression.Empty vector p35S::YFP was used as a control.Confocal laser scanning microscopy analysis revealed that the YFP signal in p35S::YFP tobacco leaves was located both in the nucleus and on the cell membrane,whereas the YFP signal in p35S::MS2-YFP tobacco leaves was located on the cell membrane(Fig.5A).These results demonstrate that the MS2 protein is located on the cell membrane.

To investigate the spatiotemporal expression pattern ofMS2,quantitative real-time PCR(qRT-PCR)was performed with the RNA samples from various tissues of inbred line B73.The expression ofMS2was detected in all vegetative and reproductive tissues.A high transcriptional level ofMS2was observed in anthers,especially in anthers at the young microspore stage(Fig.5B).These results suggest thatMS2plays an important role in anther development.

3.5.Cutin monomers and wax components were altered in ms2 anthers

The phenotypic defects of thems2anther cuticle and pollen wall indicated that the biosynthesis or transport of anther cuticle precursors might be affected in the absence of theMS2gene.Gas chromatography mass spectrometry(GC–MS)was used to detect the contents of wax,cutin and fatty acids in WT andms2anthers at young microspore stage.Multiple randomly selected anther samples were used to measure the surface area of the anthers by plotting the surface area of each anther against the corresponding weights.The total fatty acid content of wild-type anthers was 875.82 ng mm-2,while that of thems2mutant was significantly lower(724.08 ng mm-2).The level of total wax in thems2mutant anther was 135.47 ng mm-2,whereas that in the wild-type anther was 163.94 ng mm-2(P＜0.05).However,there was no significant difference in the total cutin content between WT andms2anthers(Fig.6).The amounts of some fatty acid monomers,specifically the C14,C16,C18,C18:2,C18:3,C20:0,C20:1 and C28:0 acids,were decreased in thems2mutant compared with those in WT anthers.For the cutin constituents,C18:0 acid,C24:0 acid,C26:0 acid,C18:0 tri-OH acid and C16:0 1,16-diacid were decreased significantly in thems2mutant,while ferulic acid was obviously increased.The wax monomers C21:0 alkanes,C23:0 alkanes,C24:0 alkanes were also reduced significantly in thems2anthers compared with those in the wild type anthers(Fig.6).BecauseMS2encodes an ABCG transporter,the above results indicated thatMS2might participate in lipidic transport,which is necessary for anther development.

4.Discussion

Many maize GMS mutants have been reported;however,only 20 GMS genes have been cloned[4–7].In our previous work[31],we proposed that a mutation in the maizems2mutant affected the synthesis and secretion of sporopollenin precursors from the tapetum to the surface of the microspore and epidermis,leading to complete male sterility.MS2has been predicted to be located in bin 9.03(http://www.maizegdb.org/).In this study,we finemapped theMS2locus to a 252 kb region and confirmed thatZm00001d046537is the candidateMS2gene that encodes an ABCG transporter.We found a 1437-bp TIR transposon sequence inserted in the fifth exon of theMS2gene in the mutant.Interestingly,Zm00001d046537has also been predicted to be a GMS gene in maize according to gene information from Arabidopsis and rice[4].Among the twenty cloned GMS genes in maize,none of them encodes an ABCG transporter genes.To our knowledge,MS2is the first reported ABCG transporter gene that participates in pollen development in maize.

Fig.1.Morphological comparisons between the wild-type and ms2 plants.(A)Wild-type and ms2 plants at the tasseling stage.(B)Panicle of wild-type plants after flowering.(C)Panicle of ms2 plants after flowering.(D)A single tassel branch of wild-type plants after flowering.(E)A single tassel branch of ms2 plants after flowering.(F)A single anther of WT plants after flowering.(G)A single anther of ms2 plants after flowering.Scale bars,5 cm for(B–E),and 5 mm for(F)and(G).

Fig.2.Map-based cloning of the maize MS2 gene.(A,B)Primary mapping of the MS2 gene on maize chromosome 9.(C)Fine mapping of the MS2 gene on maize chromosome 9.(D)The four putative coding genes in the interval.(E)A schematic representation of the exon and intron structure of MS2 and the position of the transposon insertion.

Fig.3.Analysis of the mutation of the target gene and phenotype in homozygous mutants.(A)Analysis of the homozygous mutation at the target sequence of MS2.The target sequence is highlighted in red,the PAM sequence is underlined,mutations with insertions are in bold,and the deleted sequences are shown by hyphens.(B)The phenotype of the mutant anthers.WT,wild-type;mut,mutant.Scale bars,5 cm.

Fig.4.Phylogenetic analysis of MS2 and its homologs in rice and Arabidopsis.(A)Phylogenetic tree of homologous proteins of MS2 in maize,rice,and Arabidopsis.The phylogenetic tree was constructed using MEGA software(version 6.0.6).(B)Alignment of the MS2 protein with OsABCG15 and AtABCG26.

Fig.5.The subcellular location of the MS2 protein and expression profiles of the MS2 gene.(A)The subcellular location of the MS2 protein.The left side is the YFP excitation signal,the middle is the bright field image,and the right side is the confocal signal;p35S::YFP indicates the plasmid in which YFP was controlled by the 35S promoter,and p35S::MS2-YFP indicates the plasmid in which the MS2-YFP fusion gene was controlled by the 35S promoter.Scale bars,10 μm.(B)Expression profiles of MS2 in maize inbred line B73.ZmGapdh was used as an internal control.Data are the average±S.E.of three biological replicates.1.5 mm anthers are before meiosis stage;2.5 mm anthers are at the meiosis stage and tetrad stage;3.5 mm anthers are the young microspore stage;DAP,days after pollination.

Fig.6.Analysis of fatty acids,wax and cutin in WT and ms2 anthers.(A)Total trans fatty acids.(B)Total wax and cutin amounts.(C)Contents of anther trans fatty acid constituents.(D)Contents of anther cutin monomers.(E)Contents of anther wax constituents.Error bars indicate S.E.(n=5).*indicates significant difference at P＜0.05;**indicates significant difference at P＜0.01.

ABCG transporters have been identified to participate in the translocation of the cutin and wax in rice and Arabidopsis[35];some example genes includeAtABCG11,AtABCG12,andAtABCG26[36],OsABCG9[29],OsABCG15[37],andOsABCG26[23].It is predicted that approximately 109 ATP-binding cassette genes exist in the maize genome;however,few of them have been functionally investigated[38].Glossy13,encoding a putative ABC transporter,was reported to be involved in cuticular wax accumulation in maize leaves[39].ABC transporters usually transport lipid molecules across the plasma membrane to the epidermis[20].The location of the MS2 protein on the cell membrane indicates that the MS2 protein might play a role in lipid molecule transport.The mutation ofMS2did not affect aspects of maize development except the male sterility,indicating thatMS2has special roles in anther development,which is also consistent with the results showing that theMS2gene was highly expressed in anthers.Lipids are transported via a conserved regulatory network in plants,in which ABCG transporters are involved.The mutation of ABCG transporters leads to significant changes in the expression of cutin or wax biosynthesis-related genes.It has been reported that the mutation ofOsABCG26reduces the expression of several lipid biosynthesis and modifying genes,includingCYP704B2,CYP703A3,Defective Pollen Wall(DPW)andWax-deficient anther1(WDA1)[23].Our previous work also showed that a series of genes related to the long-chain fatty acid metabolism process,including 3-ketoacyl-COA-synthase,long-chain-alcohol O-fatty-acyltransferase,and long-chain acyl-CoA synthase,were influenced inms2mutants compared with their fertile siblings[31].Consistent with the change in gene expression,some wax components showed decreased accumulation in thems2mutant.

In our previous work[31],we found that the fertile anthers can form round pollen grains with particulate exine and germinal aperture near mature stage.However,thems2microspores were completely collapsed,indicating thatms2anthers have defects in the synthesis and transportation of lipophilic molecules,which are necessary for the development of pollen wall and anther cuticle.In good agreement with this phenotype,we detected decreased accumulation of wax inms2anther.For cutin monomers,we observed that C18:0 acid,C24:0 acid,C26:0 acid,C18:0 tri-OH acid and C16:0 1,16-diacid were significantly decreased in thems2mutant,further substantiating the function ofMS2in the development of anther cuticle.The major component of pollen exine is sporopollenin,which may be composed of aliphatic derivatives such as fatty acids and phenolic compounds[40].Total fatty acids were significantly decreased in thems2mutant,indicating thatMS2may participate in lipidic molecule transport and further affect pollen exine formation.The orthologs ofMS2in Arabidopsis and rice,AtABCG26andOsABCG15,have also been reported to be mainly responsible for the transport of lipidic molecules from tapetal cells to anther locules for pollen exine development.Until now,no direct evidence was obtained to confirm the transport of lipidic molecules by ABCG transporters,as the hydrophobic nature of metabolites makes this kind of assay very difficult.The molecular mechanism of the transport of MS2 on lipophilic molecules needs further investigation.

CRediT authorship contribution statement

Qilong Xu,Li Yang and Dan Kanghad the main responsibility for data collection and analysis,Li Yang and Zhenjing Renrevised the manuscript,andYunjun Liu(the corresponding author)had the overall responsibility for experimental design,project management,and manuscript preparation.

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 Fundamental Research Funds for Central Non-Profit of Institute of Crop Sciences,Chinese Academy of Agricultural Sciences(S2018QY07)and National Major Project for Transgenic Organism Breeding(2016ZX08010-004).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.04.001.