Wei Zhang,Hui Zhi,Sha Tang,Haoshan Zhang,Yi Sui,Guanqing Jia,Chuanyin Wu,Xianmin Diao*

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

Keywords:

A B S T R A C T Male sterility is a common biological phenomenon in plant kingdom and has been used to generate malesterile lines,which are important genetic resources for commercial hybrid seed production.Although increasing numbers of male-sterility genes have been identified in rice(Oryza sativa)and Arabidopsis(Arabidopsis thaliana),few male-sterility-related genes have been characterized in foxtail millet(Setaria italica).In this study,we isolated a male-sterile ethyl methanesulfonate-generated mutant in foxtail millet,no pollen 1(sinp1),which displayed abnormal Ubisch bodies,defective pollen exine and complete male sterility.Using bulk segregation analysis,we cloned SiNP1 and confirmed its function with CRISPR/Cas9 genome editing.SiNP1 encoded a putative glucose-methanol-choline oxidoreductase.Subcellular localization showed that the SiNP1 protein was preferentially localized to the endoplasmic reticulum and was predominantly expressed in panicle.Transcriptome analysis revealed that many genes were differentially expressed in the sinp1 mutant,some of which encoded proteins putatively involved in carbohydrate metabolism,fatty acid biosynthesis,and lipid transport and metabolism,which were closely associated with pollen wall development.Metabolome analysis revealed the disturbance of flavonoids metabolism and fatty acid biosynthesis in the mutant.In conclusion,identification of SiNP1 provides a candidate male-sterility gene for heterosis utilization in foxtail millet and gives further insight into the mechanism of pollen reproduction in plants.

1.Introduction

Male sterility(MS)is a common phenomenon in plants and a valuable trait for the efficient production of hybrid crop seed.Given its importance,MS has been a popular focus of plant research.Based on the fertility gene source,MS can be classified as cytoplasmic male sterility(CMS)and genic male sterility(GMS)[1].Accordingly,there are two major types of hybrid seed production systems for crops,namely,a three-line system based on CMS and a two-line system based on photoperiod/thermosensitive genic male sterility(PTGMS).The male sterile line,the maintainer line and the restorer line are the crucial components for the CMS three-line system,whereas changes in fertility of the PTGMS line in different environments is critical for the PTGMS two-line system[2].In addition,a number of other strategies for hybrid seed production use non-PTGMS genes.For instance,the seed production technology(SPT)system was developed to produce genic male-sterile lines using recessive GMS genes in maize(Zea mays)[3,4]and rice(Oryza sativa)[5,6].In maize,a dominant MS system based onms44[7]andZmMs7[8]have been constructed.

Male reproductive development is a precisely coordinated process which has been classified into 14 stages on the basis of morphological features in rice[9].For higher plants,anther and pollen development are essential for the generation of male gametophytes.Pollen grains develop within anthers and are released from the anther locule after maturation.The pollen wall protects the fragile male gametophytes against harsh environmental factors,such as high temperature,ultraviolet radiation,microbial infection,and drought[10].The typical pollen wall consists of three layers:exine,intine,and tryphine.The exine is divided into the inner layer(tectum)and the outer layer(sexine),and these two layers are connected by the bacula[11,12].The predominant constituent of the exine is sporopollenin,a complex biopolymer of medium-to long-chain fatty acids and aromatic compounds[13,14],which is secreted from tapetal cells[15,16].When synthesized in the tapetum,sporopollenin precursors are released into the anther locule and deposited on the pollen grain surface[15,17].Therefore,sporopollenin plays a pivotal role in tapetum and pollen development.

Recently,based on mutants with defective exine formation in monocotyledonous and dicotyledonous species,a number of anther-or tapetum-specific genes that are mainly involved in fatty acid biosynthesis and metabolism have been identified.For example,in Arabidopsis(Arabidopsis thaliana),the anthers of thems2mutant[18]are brown,shriveled,and contain no pollen grains.MALE STERILITY 2(MS2)is a fatty acyl carrier protein reductase that converts the palmitoyl-acyl carrier protein to palmitoyl alcohol[19],which is involved in the synthesis of sporopollenin and required for exine patterning of pollen grain[19,20].In rice,DEFECTIVE POLLEN WALL(DPW)[21]is the ortholog ofMS2and can rescue male fertility of the Arabidopsisms2mutant.Two cytochrome P450 enzymes(CYP703A and CYP704B)[22,23–25]are considered to generate different types of hydroxylated fatty-acyl monomers for subsequent modification.CYP704B1 in Arabidopsis and CYP704B2 in rice catalyze the ω-hydroxylation of long-chain fatty acids(C16–18),whereas CYP703A2 in Arabidopsis and CYP703A3 in rice hydrolyze mid-chain fatty acids(C10–12).In addition to fatty acid synthesis,another conserved pathway is involved in the production of triketide and tetraketide α-pyrones for sporopollenin biosynthesis.Any defects in the biosynthesis,secretion and transport of lipidic compounds for sporopollenin formation may result in reduced pollen viability and compromised male fertility.

The Glucose-Methanol-Choline Oxidoreductase(GMCO)family was first defined by Caverner in 1992[26].Functional research on the GMCO family in plants has mainly centered on crosstalk between insects and their host plants.A particularly striking example is tobacco,in which glucose oxidase(GOX),a caterpillar primary salivary protein,suppresses induction of nicotine in wounded tobacco leaves[27].GMCO family is also involved in asexual and sexual development in prokaryotic and eukaryotic organisms.InAspergillus nidulans,GmcA is a putative GMCO required for induction of asexual development[28].InDrosophila melanogaster,the glucose dehydrogenase gene(Gld)is only expressed in male adults except as a consequence of mating after eclosion[29],which indicates that the GMCO family is associated with male reproductive development.In Arabidopsis,HOTHEAD(HTH)encodes an enzyme associated with a group of flavin adenine dinucleotide-containing oxidoreductase and is required to limit cellular interactions between contacting epidermal cells during floral development[30].Further study by Kurdyukov et al.[31]shows that HTH is involved in the biosynthesis of long-chain α-,ω-dicarboxylic fatty acids.The geneEDA17is the same locus asHTH.Theeda17mutant exhibits an aberrant embryo sac,which is suggestive of a critical role in female gametophyte development in Arabidopsis[32].In maize,IRREGULAR POLLEN EXINE 1(IPE1)/ZmMs20[33,34]encodes a putative GMCO and is preferentially expressed in tapetum cells.Theipe1mutant exhibits defective pollen exine and is male sterile.The geneOsNP1[5,35]is the ortholog of maizeIPE1and controls male fertility in rice.In theosnp1mutant,the amount of aliphatic cutin monomers is substantially decreased,which indicates thatOsNP1is essential for synthesis of lipidic precursors for formation of the anther cuticle and pollen exine in rice.Nevertheless,knowledge of the GMCO family remains limited in the plant kingdom.

Foxtail millet,an ancient cereal that was domesticated in China,is cultivated worldwide,especially in the arid and semi-arid region of East Asia.The foxtail millet genome is relatively small[36],which allows rapid identification of novel genes by wholegenome-resequencing analysis similar to other model species.Although heterosis has been utilized in foxtail millet,no MS gene has been cloned previously.In this study,we successfully cloned the MS geneSiNP1,which encoded a putative GMCO.Using CRISPR/Cas9 technology,we confirmed the function ofSiNP1.The SiNP1 protein was localized to the endoplasmic reticulum(ER)and was preferentially expressed in the panicle.Cytological observation showed thatSiNP1may be involved in pollen exine formation.Transcriptome and metabolome analyses revealed the disturbance of flavonoids metabolism in the mutant.This work presents a case study on a member of the GMCO family and MS in foxtail millet,and identified a candidate gene for heterosis utilization in foxtail millet.

2.Materials and methods

2.1.Plant materials and growth conditions

The plant materials comprised the foxtail millet male sterile mutant‘sinp1’,the wild type‘Yugu 1’,the BC1F2population,and a near-isogenic line(NIL)population.Thesinp1mutant was derived from‘Yugu 1’by ethyl methanesulfonate mutagenesis.We performed a cross between thesinp1mutant(as the female parent)and‘Yugu 1’(as the male parent),and performed backcrossing for three generations to generate a male-sterile NIL(NIL-A)and a male-fertile NIL(NIL-B).In the BC1F2population,the segregation ratio of male fertility to MS was assessed.All materials were grown in fields at the experimental stations in Beijing or in a greenhouse at 28°C with a 14 h light/10 h dark photoperiod at the Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing.

2.2.Phenotypic characterization of sinp1 mutant

The morphology of whole plants and panicles were recorded with a Canon 500D digital camera.For evaluation of pollen viability,anthers of the NIL-B and the NIL-A lines were crushed with forceps.The pollen grains were released from the anthers and stained with iodine–potassium iodide(I2-KI)solution(0.2% iodine,2%potassium iodide).The spikelets,floral organs and pollen grains were examined with LEICA M165FC and LEICA DM5500B microscopes and photographed.Observation of semi-thin sections and transmission electron microscopy(TEM)were conducted following the methods of Li et al.[37].The samples for TEM and scanning electron microscopy(SEM)were observed and photographed with HITACHI HT7700 and HITACHI TM4000 microscopes,respectively.

2.3.DNA extraction,bulk segregant analysis,and MutMap+analysis

Total genomic DNA was extracted using the cetyltrimethylammonium bromide method.The quality and quantity of DNA were examined by electrophoresis in 1%agarose gels and image lab software(Quantity One,Bio-Rad,CA,USA).Thirty homozygous male sterile and 30 male-fertile individuals from the BC1F2population were selected to construct two DNA bulks for whole-genome resequencing.The illumina NovaSeq 6000 system was used for highthroughput sequencing.Raw sequencing data were uploaded to the BioProject database under the accession number PRJNA679342.The MutMap+analysis followed the method of Fekin et al.[38].

2.4.CRISPR/Cas9 genome editing

For knockout ofSiNP1,we used the pYLCRISPR/Cas9-MH vector system.The pCRISPR-SiNP1 plasmid was constructed as described previously[39]and introduced into embryogenic calli of the wild type Ci846 by transformation mediated withAgrobacterium tumefaciensstrain EHA105.The transgenic plants were screened by PCR amplification.The primers used for vector construction and transgenic plant identification are listed in Table S1.

2.5.Subcellular localization

For subcellular localization analysis,the 1.7 kb coding sequence of SiNP1 was amplified from the cDNA library of wild type‘Yugu 1’spikelets.The fragment was cloned into theBamH I sites of the p16318-hGFP vector to generate the constructp16318-35S::SiNP1-GFP.Foxtail millet protoplasts were isolated as described in previously for Arabidopsis[40].The ER markerp16318-35S::SP-mCherry-HDELwas cotransformed withp16318-35S::SiNP1-GFPusing the polyethylene glycol-mediated method.Transformed protoplasts were incubated in 2.0 mL EP tubes in the dark at 23 °C overnight.Fluorescence was observed with a laser scanning confocal microscope(Zeiss LSM700).The primers used for vectors construction are listed in Table S1.

2.6.RNA extraction,RNA sequencing,and quantitative RT-PCR

Total RNA was extracted with TRIzol Reagent(Invitrogen,Carlsbad,CA,USA)from different tissues including the seedling,root,stem,leaf,and panicle.Four microgram of RNA per sample was used for cDNA synthesis using the PrimeScript II 1st Strand cDNA Synthesis Kit(TaKaRa,Kyoto,Japan).For RNA-sequencing(RNA-seq),we also isolated total RNA from spikelets of the NIL-A and NIL-B lines before anthesis.The illumina NovaSeq 6000 system was used for high-throughput sequencing.Raw sequencing data were uploaded to the BioProject database under the accession number PRJNA678878.Quantitative RT-PCR(qRT-PCR)analysis was performed using the ROCGENE Archimed-X6 qRT-PCR System with 2×Universal STBR Green Fast qPCR Mix(ABclonal,Wuhan,China).SiCULLINwas used as an internal control for normalization.Each experiment comprised three technical replicates.To verify the reliability of the RNA-seq data,we randomly selected nine genes among the differentially expressed genes(DEGs).All primers used for qRTPCR analyses are listed in Table S1.

2.7.UPLC-MS/MS analysis

For sample preparation and extraction,we sampled spikelets of the NIL-A and NIL-B lines before anthesis.The freeze-dried spikelets were crushed using a mixer mill(MM 400,Retsch,Haan,Germany)with zirconia beads for 1.5 min at 30 Hz.A sample(100 mg)of the powder was weighed and extracted overnight at 4 °C with 1.0 mL of 70% aqueous methanol.Following centrifugation at 10,000×gfor 10 min,the extracts were absorbed(CNWBOND Carbon-GCB SPE Cartridge,250 mg,3 mL;ANPEL,Shanghai,China,http://www.anpel.com.cn/cnw)and filtered(SCAA-104,0.22 μm pore size;ANPEL,Shanghai,China,http://www.anpel.com.cn/cnw)in preparation for ultra-performance liquid chromatographytandem mass spectrometry(UPLC-MS/MS)analysis.The Shimpack UFLC SHIMADZU CBM30A and Applied Biosystems 6500 QTRAP systems were used as the analysis platform.Each experiment consisted of three technical replicates.

3.Results

3.1.Identification and phenotypic analysis of the sinp1 mutant

From an ethyl methanesulfonate-induced mutant library for‘Yugu 1’,we isolated a male sterile mutant,which displayed normal vegetative growth but defective reproductive development.We designated this mutantno pollen 1(sinp1),given that mature pollen grains were absent in the locules of advanceddevelopment anthers.We performed a cross betweensinp1(as the female parent)and‘Yugu 1’(as the male parent)and three generations of backcrossing to construct a male-sterile NIL population for phenotypic analysis.When backcrossed with‘Yugu 1’,all F1progeny were fertile and the BC1F2population exhibited a clear 3:1 segregation ratio for male fertility(fertile:sterile=311:89,χ2=1.613,χ20.05=3.84).This result confirmed that a single recessive gene governed thesinp1phenotype.

Similar to the NIL-B line(Fig.1A,G),no defects in vegetative growth and floral morphology of NIL-A were observed(Fig.1B,H).The floret structure comprised one pistil,three stamens,and two lodicules.However,the anthers of NIL-A were smaller than those of NIL-B and were shriveled owing to the absence of pollen grains(Fig.1I–L).The NIL-A plants exhibited complete MS and produced no seed after self-pollination,whereas seed set of NIL-B was normal(Fig.1C–F,M,N).However,the NIL-A plants were able to set seed when pollinated with wild-type pollen,which indicated that pistil development of NIL-A was normal.

Fig.1.Characterization of NIL-A and NIL-B.Plant of(A)NIL-B and(B)NIL-A;panicle of(C)NIL-B and(D)NIL-A;primary branch showing spikelets of(E)NIL-B and(F)NIL-A;floret of(G)NIL-B and(H)NIL-A;anther of(I)NIL-B and(J)NIL-A;pollen grains stained by I2-KI for(K)NIL-B and(L)NIL-A;(M)pollen fertility;(N)seed setting rate.Values are the means and SD.**,P＜0.01.NP,no pollen;NS,no seed.Scale bars,10 cm(A and B),5 cm(C and D),5 mm(E and F),500 μm(G and H),200 μm(I and J),and 100 μm(K and L).

Fig.2.Semi-thin transverse section analysis of NIL-A and NIL-B anthers.Anther were sampled at stages 8b(A and F),9(B and G),10(C and H),11(D and I),and 12(E and G).(A–E)are NIL-B and(F–J)are NIL-A.Scale bars,50 μm.DMsp,degraded microspore;E,epidermis;En,endothecium;Ml,middle layer;MP,mature pollen;Msp,microspore;T,tapetum;Tds,tetrads.

3.2.The sinp1 mutant exhibits abnormal Ubisch body morphogenesis and defective microspore development after meiosis

To elucidate the defects during anther development in thesinp1mutant,cytological observation anther development in the NIL-B and the NIL-A lines was conducted using semi-thin transverse sections.No visible difference was observed between NIL-A and NIL-B anthers before tetrad formation(Fig.2A,F).After uninucleate microspore release from the tetrad,the NIL-B microspores were of normal shape,gradually enlarged,and became vacuolated.The tapetal cells of NIL-B were darkly stained and gradually degenerated owing to programmed cell death(PCD;Fig.2B,C).By contrast,NIL-A microspores failed to vacuolate and were irregularly shaped at stage 10.The tapetal cells of NIL-A exhibited abnormal PCD and were lightly stained(Fig.2G,H).Subsequently,NIL-B microspores became falcate during the first mitosis,and the tapetal cells continued to degenerate and were strip-shaped at stage 11(Fig.2D).In contrast,the NIL-A microspores gradually collapsed(Fig.2I).Finally,at stage 12,the tapetum in NIL-B and NIL-A was completely degraded.Mature pollen grains were observed in the anther locule of NIL-B,whereas the NIL-A microspores had aborted and,instead of pollen grains,only debris were observed in the locule(Fig.2E,J).Notably,the middle layer cells of the anther wall of NIL-A persisted and enlarged unnaturally(Fig.2I,J).These observations indicated thatSiNP1functioned in tapetum degeneration and microspore development.

To characterize thesinp1developmental defects in additional detail,SEM and TEM were employed.Consistent with the observations of semi-thin transverse sections,at early stage 10,microspores of NIL-B became vacuolated with an intact exine,and the tapetal cells contained numerous vacuoles and began to degenerate(Fig.3A,G–I).By contrast,NIL-A microspores failed to enlarge and were abnormal with defective exine,and the tapetal and middle layer cells were swollen(Fig.3D,L–N).Subsequently,NIL-B microspores further enlarged with an increase of volume,the middle layer cells vanished,and the tapetal cells degenerated until they disappeared at stage 12(Fig.3B,C).The NIL-A microspores and tapetal cells collapsed,leaving debris in the locule,and the middle layer cells persisted but showed hypertrophy(Fig.3E,F).The anther cuticle of NIL-A was slightly thinner than that of NILB(Fig.3J,K,O,P).It was noteworthy that the Ubisch bodies were abnormal in NIL-A,whereas the Ubisch bodies of NIL-B were regularly shaped and distributed along the tapetum(Fig.3A,G,H).These results illustrated thatSiNP1was involved in Ubisch body morphogenesis and pollen exine patterning.

3.3.SiNP1 encodes a putative glucose–methanol–choline oxidoreductase

To identifySiNP1, we performed whole-genome resequencing with bulk segregant analysis using the male sterile and normal DNA bulk in the BC1F2population. The male sterile and normal bulk produced 99,282,525 and 105,106,581 raw read pairs, respectively. ＜1% of the raw read pairs were removed after quality control. Trimmed reads were aligned to ‘Yugu 1’ reference sequences. A candidate region at the end of chromosome 9 (from～38.3 to ～42 Mb; Fig. 4A) was identified using the MutMap +method as described previously. Eight candidate single-nucleotide polymorphisms (SNPs) were located in this region (Table S1): three were located in the upstream or down-stream regions, four were in the intergenic regions, and one (Chr. 9: 40,752,153) was in the fourth exon ofSeita.9G347800that caused an amino acid substitution from Gly413(GGC) to Ser (AGC) (Fig. 4A–C; Table S2).Coincidentally,Seita.9G347800is the ortholog ofGRMZM2G434500(IPE1/ZmMs20, maize) andLOC_Os10g38050(OsNP1, rice), both of which have been reported to be involved in MS. We further confirmed the mutation inSiNP1by Sanger sequencing using 160 BC1F2individuals and observed that this SNP cosegregated with the MS phenotype. Therefore, we hypothesized thatSeita.9G347800was the candidate gene forSiNP1.

SiNP1encoded a 581-amino acid protein that contained two predicted transmembrane helices(TMH)at the N terminus(TMH1:4–26,TMH2:46–68,TMHMM Server v.2.0,http://www.cbs.dtu.dk/services/TMHMM/;Figs.4B,S1A)and two predicted GMCO domains at the N terminus(49–322)and C terminus(423–570,SMART,http://smart.embl-heidelberg.de/;Fig.4B,S1B),These findings indicated thatSiNP1belonged to the GMCO family.The amino acid substitution resulted in a structural alternation(SWISS-MODEL,https://swissmodel.expasy.org/;Fig.4D),which suggested that the function of SiNP1 in thesinp1mutant was disrupted.Glycine is a hydrophobic amino acid with-H as the side chain.In contrast,Ser is a polar amino acid that has a–CH2OH side chain with hydrophilic properties.The Gly413Ser substitution thus changed the hydrating properties of this portion of the protein.From BLAST searches of Phytozome(https://phytozome.jgi.doe.gov/)and UniProt(https://www.uniprot.org/),we retrieved 56 homologs from seven diverse plant species including monocotyledonous and dicotyledonous species.Some of the homologs showed preferential expression in the male reproductive organ(Table S3).The amino acid sequences of SiNP1 and its homologs were conserved among different species(Figs.4C,5,S1B).Thus,the conserved expression pattern and amino acid sequence were indicative of a conserved function forSiNP1and its homologs.

Fig.3.Transmission electron and scanning electron micrographs of NIL-A and NIL-B anthers from stages 10 to 12.(A–F)Anthers were sampled at stages 10(A and D),11(B and E),and 12(C and F).(A–C)are from NIL-B and(D–F)are from NIL-A.(G and L)Microspores of NIL-B and NIL-A at stage 10,respectively.(H and I)Exine from NIL-B at stage 10.(M and N)Exine from NIL-A at stage 10.(J and K)Epidermis from NIL-B at stage 12.(O and P)Epidermis from NIL-A at stage 12.Scale bars,5 μm(A–G,I,L,and N)and 1 μm(H,J,K,M,O,and P).AEx,abnormal exine;AUb,abnormal Ubisch body;Ba,bacula;C,cutin;CW,cell wall;DMsp,degraded microspore;E,epidermis;En,endothecium;Ml,middle layer;Msp,microspore;Ne,nexin;T,tapetum;Te,tectum;Ub,Ubisch body.

3.4.SiNP1 determines male fertility in foxtail millet

To further confirm the candidate gene,we created three mutant alleles(sinp1-2,sinp1-3,andsinp1-4)ofSeita.9G347800by CRISPR/Cas9 editing with a 1 bp(T)-insertion,a 1 bp(T)-deletion,and a 1 bp(A)-insertion in the first exon,respectively(Fig.6A),which resulted in premature truncation of the SiNP1 protein.All three alleles resulted in shriveled anthers that lacked pollen grains and complete MS(Fig.6B–E).These findings supported the inference thatSeita.9G347800wasSiNP1and suggested thatSiNP1determined MS in foxtail millet.

3.5.SiNP1 is predominantly expressed in the panicle and is localized to the ER

On the basis of the phenotype analysis,the defects of thesinp1mutant were exhibited in the anther during reproductive development,which suggested thatSiNP1was specifically expressed in the anther.To evaluate this hypothesis,we performed qRT-PCR analysis,which showed thatSiNP1was predominantly expressed in the panicle at the heading stage.No expression was detected in the seedling,root,stem and leaf(Fig.S2).To further validate ifSiNP1has higher expression specifically in pancicle.We also employed RNAseq data of 27 different tissues of foxtail millet(data is available at www.setariamodel.cn).The result showed thatSiNP1showed relative high expression level in young panicle(RPKM=13.67)and flowering panicle(RPKM=21.37),and a very low expression level in other tissue(average RPKM＜0.28;Fig.7A).To investigate the subcellular localization of SiNP1,we generated the constructp16318-35S::SiNP1-GFPand co-introduced it with an ER marker(p16318-35S::SP-mCherry-HDEL)into protoplasts isolated from foxtail millet seedlings.The observations showed that SiNP1-GFP fluorescence merged with the fluorescence of the ER marker(Fig.7B).In contrast,the signal of free GFP alone was dispersed in the cytoplasm and did not merge with the ER marker signal(Fig.7B).These results indicated that SiNP1 was preferentially localized to the ER.

Fig.4.Identification and protein sequence analysis of SiNP1.(A)Identification of sinp1.The top section shows the distribution ofΔSNP-index of SNP sites on the nine chromosomes.The bottom section shows the gene structure and mutation sites of sinp1.The mutation site of SiNP1 in the sinp1בYugu 1’BC1F2 population was verified by sequencing.HM-WT,HT,and HM-MT indicate the homozygous wild-type genotype,the heterozygous genotype,and the homozygous mutant,respectively.(B)Schematic representation of the conserved GMCO and TMH domains in the SiNP1 protein.(C)Comparison of aligned sequences of SiNP1,sinp1,and homologous proteins from other plant species.The blue frame indicates the conserved motif.(D)Comparison of the predicted protein model of SiNP1 between the wild type(WT)and sinp1 mutant.The black frames indicate possible structural alterations.

3.6.Transcriptome analysis of sinp1 spikelets revealed changes in carbohydrate metabolism,fatty acid biosynthesis,and lipid transport and metabolism

To characterize the mechanism ofsinp1MS caused bysinp1,we employed transcript profiling of NIL-A and NIL-B spikelets before anthesis by RNA-seq.Using a cutoff(false discovery rate＜0.01 and fold change≥2),we identified 710 DEGs,consisting of 225 up-regulated genes and 485 down-regulated genes in NIL-A.To verify these results,nine DEGs were randomly selected for qRTPCR analysis,which confirmed the reliability of RNA-seq data(Fig.S2).

A Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis was performed to further characterize the functions of the DEGs in NIL-A.Certain pathways involved in carbohydrate metabolism,fatty acid biosynthesis,and lipid transport and metabolism were enriched(Fig.8A).For instance,in the‘‘Pentose and glucuronate interconversions”pathway,four genes putatively encoding pectate lyase and eleven genes putatively encoding pectinesterase were down-regulated in NIL-A.In the‘‘Starch and sucrose metabolism”pathway,the expression levels of two genes involved in starch synthesis and nine genes involved in sugar transport were decreased in NIL-A(Fig.8B;Table S4).These two pathways have previously been shown to be associated with pollen development and exine formation[41,42].

Among genes putatively involved in fatty acid biosynthesis,and lipid transport and metabolism that were differentially expressed in NIL-A,the majority were down-regulated.For example,the expression levels of five genes(Seita.1G167200,Seita.3G087000,Seita.5G440000,Seita.5G440100,andSeita.9G420600)involved in fatty acid biosynthesis were decreased in NIL-A(by-1.76-,-1.42-,-1.45-,-1.42-and-3.15-fold,respectively;Fig.8B;Table S5).A gene(Seita.3G137500)involved in fatty acid elongation showed decreased expression in NIL-A(-1.26-fold;Fig.8B;Table S5).Among cutin,suberin,and wax biosynthesis-related genes,a gene encoding a caleosin-related protein(Seita.1G310900)was downregulated in NIL-A(-1.60-fold;Fig.8B;Table S5),whereas a second caleosin-related protein-encoding gene(Seita.4G112200)was up-regulated(1.24-fold).GDSL esterases and lipases are associated with MS in maize[43]and rice[44].In NIL-A,the expression levels of nine genes putatively encoding GDSL esterases and lipases was changed(Fig.8B;Table S5).Nine genes that putatively encoded lipid transfer proteins(LTPs)involved in lipid transport and metabolism were enriched(Fig.8B;Table S6).The expression levels of several transcription factors and enzymes were slightly decreased in NIL-A(Tables S7,S8).Fatty acids and their derivatives are the main components of cutin in the anther cuticle and sporopollenin in the pollen exine.These results reflected the metabolic disruption and disturbed sporopollenin biosynthesis in thesinp1mutant.The changes in the transcriptome corresponded with the observation of abnormal Ubisch bodies,defective pollen exine,and thinner anther cuticle in NIL-A.

Fig.5.Phylogenetic analysis of SiNP1 homologs in diverse plant species.Foxtail millet SiNP1(black dot)shows close identity with rice OsNP1 and maize IPE1.Ath,Arabidopsis thaliana;Gm,Glycine max;Mt,Medicago truncatula;Os,Oryza sativa;Sb,Sorghum bicolor;Si,Setaria italica;Zm,Zea mays.

Fig.6.Functional confirmation of SiNP1 by CRISPR/Cas9 genome editing.(A)Identification of sinp1 alleles(sinp1-2,sinp1-3,and sinp1-4).(B–E)Comparison of Ci846 and sinp1-2:pollen grains stained by I2-KI for(B)Ci846 and(D)sinp1-2;floret of(C)Ci846 and(E)sinp1-2.Scale bars,100 μm(B and D)and 1 mm(C and E).

3.7.Metabolome analysis of sinp1 spikelets revealed the disturbance of flavonoids metabolism and fatty acid biosynthesis affected male fertility in sinp1

Fig.7.Expression pattern of SiNP1.(A)Gene expression level of SiNP1 in 27 different tissues.(B)Subcellular localization of SiNP1 protein in foxtail millet protoplasts.Scale bars,5 μm.

To validate the results of the transcriptome analysis and to identify metabolites that were significantly affected in thesinp1mutant,we performed high-throughput quantification of the metabolome of NIL-A and NIL-B spikelets before anthesis using a UPLC-MS/MS system.A total 449 metabolic compounds,including flavonoids,amino acids and their derivatives,fatty acids,nucleotides and their derivatives,and other compounds were detected.Using a cutoff(variable importance in project≥1 and fold change≥2),we identified 17 metabolites that exhibited significantly different contents between NIL-A and NIL-B spikelets(Table S9).Among the 17 metabolites,13 belonged to the flavonoids class,including six up-regulated flavonoids and seven down-regulated flavonoids in NIL-A.For instance,insinp1,isorhamnetin acetyl hexoside and isorhamnetin hexose-malonate are increased by 1.82 and 2.11-fold,respectively(Table S9),while 8-C-hexosyl-luteolin O-hexoside,isoquercitrin(quercetin 3-O-β-Dglucoside),luteolin-7-O-rutinoside and 6-hydroxykaempferol-3,6-O-diglucoside are significantly decreased (by-1.94-,-1.04-,-1.30-,-1.42-,and-3.53-fold,respectively;Table S9).Flavonoids are biosynthesized in the ER and are involved in pollen germination,pollen tube growth,and formation of the pollen wall[45–47].Moreover,the content of fatty acid and lipid also changed insinp1(Table S10),for example,palmitoleic acid,punicic acid,10,12,15-octadecatrienoic acid and 10,12-octadecadienoic acid were decreased by-0.62-,-0.53-,-0.57-,and-0.34-fold insinp1,respectively(Table S10).These results indicated that disturbance of flavonoids metabolism and fatty acid biosynthesis affected male fertility in thesinp1mutant.

4.Discussion

4.1.SiNP1 controls male fertility in foxtail millet by affecting carbohydrate metabolism,fatty acid biosynthesis,and lipid metabolism and transport

Sporopollenin biosynthesis is a complex process essential to pollen exine formation.Fatty acid biosynthesis,and lipid metabolism and transport are critical steps in sporopollenin biosynthesis involved in numerous oxydoreduction reactions.Previous reports have indicated that enzymes in the GMCO family catalyze the oxidation of an alcohol moiety to a corresponding aldehyde[48].In Arabidopsis,HTH,a GMCO enzyme,interferes with cross-linking of cutin polyesters[31].In rice,OsNP1is considered to affect polymerization or assembly of the precursors of cutin polyesters whereas IPE1 oxidizes ω-hydroxy C16/C18 fatty acids to C16/C18 dioic acids and corresponding aldehyde dehydrogenases in maize[35,33].In this study,SiNP1encoded a putative GMCO protein.Based on alignment of the amino acid sequence(Figs.4C,S1B),we determined that SiNP1 and its orthologs are conserved among monocotyledons and dicotyledons(SiNP1 share 92.3% and 90.0%identity with OsNP1 and IPE1,respectively).Furthermore,we confirmed the function ofSiNP1by CRISPR/Cas9 genome editing(Fig.5A–E).Thus,we hypothesize thatSiNP1participates in oxydoreduction reactions during male reproductive development similar to its orthologsOsNP1andIPE1.

Fig.8.Transcriptome analysis of NIL-A and NIL-B.(A)KEGG pathway enrichment of DEGs.(B)Heatmaps of DEGs putatively involved in metabolism of carbohydrates,fatty acid biosynthesis and lipid transport and metabolism.The corresponding genes were listed in Tables S4,S5 and S6 according to the labelled number‘‘1”to‘‘7”in each map.A relative color scheme uses the minimum and maximum values in each row to convert values to colors.

Transcript profiling demonstrated that in NIL-A the expression of genes involved in fatty acid biosynthesis,and lipid metabolism and transport were changed,and the majority were downregulated(Fig.8A,B;Tables S5,S6).These genes encoded proteins such as enoyl-(acyl carrier protein)reductase,beta-ketoacyl synthase,fatty acid desaturase,GDSL-like lipase/acyl hydrolase and a LTP family protein.Remarkably,we identified nine GDSL-like lipase/acyl hydrolase-encoding genes and nine LTP family protein encoding genes in DEGs.We speculate that SiNP1 participates in chemical reactions in coordination with these enzymes.We further determined that the content of fatty acid and lipid also changed insinp1(Table S10).In addition,we detected no significant difference between NIL-A and NIL-B in the expression levels of certain genes previously indicated to be associated with tapetum PCD or participate in sporopollenin biosynthesis[i.e.,UDT1,GAMYB,TDF1,bHLH142,MS188,PTC1,MADS3,TIP3,DTC1,EAT1,TDR,DPW,WDA1,CYP704B2,CYP703A3,ACOS5,PKSA,PKSB,TKPR1,andTKPR2](Tables S7,S8).We hypothesize that there are multiple biosynthetic pathways for sporopollenin biosynthesis.In addition to fatty acid biosynthesis,and lipid transport and metabolism,the expression level of certain genes involved in carbohydrate metabolism also decreased in NIL-A(Fig.8A,B;Table S4).The metabolome analysis also showed that the metabolism of several flavonoids,which are essential constituents of the pollen wall,was altered in NIL-A(Table S9).The abnormal Ubisch bodies,defective pollen exine,transcript profiling and metabolome data in NIL-A indicated that the metabolic disorders and disruption of sporopollenin biosynthesis contributed to the MS phenotype of thesinp1mutant.Further evaluation of the anther metabolome should be performed,but the tiny anther of foxtail millet hinders collection of sufficient sample for subsequent analyses(i.e.,the length of the mature anther of foxtail millet is＜1.0 mm).

4.2.SiNP1 and its homologs perform a partially conserved role in pollen development and diverse functions during plant evolution

Similar to its orthologs in rice[5,35]and maize[33,34],sinp1also caused defective microspore development resulting in pollen abortion and complete male sterility(Fig.1D,F,J,L–N).Cytological observations showed that microspore abortion occurred in the post-meiosis stage when microspores began to be vacuolated and enlarged(Fig.2C,H).Observations with TEM and SEM revealed abnormal Ubisch bodies,defective pollen exine,and a thinner anther cuticle in NIL-A(Fig.3L–P).The ricenp1-4[35]andosnp1[5]mutants both exhibit a whitish anther and severe defects in the anther cuticle.Similar morphological defects are observed in the maizeipe1mutant[33].However,no apparent difference in anther color was observed between NIL-A and NIL-B(Fig.1G,H).In addition,abnormal enlargement of the middle layer cells of the anther wall was observed in NIL-A(Fig.2I,J)whereas the middle layer cells vanish completely in thenp1-4,osnp1,andipe1mutants.SiNP1is predominantly expressed in the panicle,although the expression level is low compared with that of its homologs in rice and maize.In rice,NP1is preferentially expressed in the anther and the relative expression level is high at developmental stages 8b,9,and 10,ranging from 0.6 to 1.0.In maize,IPE1transcripts are only detected in the anther during the early uninucleate microspore stage and the relative expression level is approximately 1.0.The difficulty in collecting sufficient anther tissue of foxtail millet for genetic analyses might account for the low gene expression levels observed in the present study.To further validate the specific expression ofSiNP1in panicle,we employed RNA-seq data of 27 different tissues of foxtail millet and determined thatSiNP1showed relative high expression level in young panicle and flowering panicle(Fig.7A).In three T-DNA lines ofAT1G12570(SALK-085330C,SALK-112758,and SALK-031400),the ortholog ofSiNP1in Arabidopsis,pollen-grain extrusion and anther cuticle development is predominantly normal except for a proportion of mutant pollen grains[33].By contrast,in thesinp1,np1-4,osnp1,andipe1mutants,only debris remain in the anther locule.Eight and sevenNP1-like genes have been identified in Arabidopsis and rice,respectively[30,49].In the present study,BLAST searches of public databases retrieved seven and fiveNP1-like genes in foxtail millet and maize,respectively(Fig.5;Table S3).HTH/EDA17,a homolog ofSiNP1in Arabidopsis,is required to prevent floral organs fusion and female gametophyte development instead of male fertility[30,32].In rice,theoni3/minimutant exhibited abnormal organ fusion in developing shoots and seedling lethality[49,50].These phenotypic differences indicate thatSiNP1and its homologs have acquired diverse functions during plant evolution.

4.3.sinp1 is a potentially valuable gene for heterosis utilization in foxtail millet

Heterosis utilization is an important strategy to increase yield in cereals to ensure food security.Especially in maize and rice,the breeding and large-scale adoption of hybrid seeds have been emphatically successful.In rice,the two major types of MS systems including the CMS line-based three-line system and the PTGMS line-based two-line system.The maintainer line and restorer line are crucial for the CMS three-line system[2].However,for a given rice hybrid,establishment of the CMS line-based three-line system is a time-consuming and laborious process.Furthermore,the limited germplasm resources for fertility restoration is the main bottleneck for application of CMS line-based three-line system[51].In the PTGMS line-based two-line system,the male-sterile line also serves as the maintainer line in a specific environment.Although some drawbacks of the CMS line-based three-line system have been overcome in the PTGMS system,certain other intrinsic problems remain,for example,the stability of MS in unstable climatic conditions,and consequently the purity of hybrid seeds produced using the PTGMS two-line system is vulnerable to unpredictable environmental changes[52].

Non-PTGMS traits,similar to PTGMS traits,are controlled by nuclear recessive genes and male fertility can be restored by any cultivar of normal fertility and are not influenced by environmental changes.To date,more than 100 GMS genes have been identified in plants[53].In addition,substantial effort and several strategies have been invested in developing non-PTGMS lines as female parents for hybrid seed production.For instance,SPT was developed to produce non-PTGMS lines in maize[3,4]and rice[5,6].The SPT maintainer line is generated using a transgenic approach that consists of three components:(1)a wild type gene used for male-fertility restoration,(2)a pollen-inactivating gene such asZmAA1used to inactivate transgenic pollens,and(3)a seed-color marker gene such asRFP,used for sorting seeds of the male-sterile line and the maintainer line.In the present study,thesinp1mutant exhibited stable MS in different environments without exhibiting other defects in agronomic traits,thus it represents ideal germplasm for development of a SPT system in foxtail millet.Recently,a dominant MS system have been constructed successfully in maize that requires tapetum-specific promoters[7,8].SiNP1and its orthologs show a specific expression pattern in the panicle or tapetum,thus providing a valuable promoter for further research(Fig.7;Table S3).Therefore,identification ofSiNP1provides a candidate gene with potential for heterosis utilization in foxtail millet.

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.

CRediT authorship contribution statement

Xianmin Diaosupervised the study.Hui Zhiisolated the mutant line.Wei Zhang and Hui Zhicharacterized the mutant and performed experiments.Chuanyin Wu,Yi Sui,and Haoshan Zhangprovided a foxtail millet transgenic platform.Guanqing Jiaprovided technical assistance.Wei Zhang and Sha Tanganalysed whole data and prepared the manuscript.

Acknowledgments

This study was supported by the National Natural Science Foundation of China(31771807),the China Agriculture Research System(CARS06-13.5-A04),the National Key Research and Development Program of China(2018YFD1000700 and 2018YFD1000701),the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.We thank Robert McKenzie,PhD,from Liwen Bianji,Edanz Editing China(www.liwenbianji.cn/ac),for careful English editing.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.03.018.