New Insights Into Invertase Gene Family and Functional Characterization of Critical Cell Wall Invertase TaCWINV40 For Male Fertility in Wheat (Triticum Aestivum L.)


 Invertase (INV, ec3.2.1.26) irreversibly hydrolyzes sucrose into fructose and glucose, and it is regulated by the environment to affect pollen fertility in some plant species. However, there has been a lack of systematic identification of INV gene family in wheat. In order to reveal the potential influence on the male fertility, a total of 130 wheat INVs that unevenly distributed on 21 chromosomes were systematically identified and analyzed in this study. According to physical and chemical properties, subcellular location, and phylogenetic tree, they were divided into two acidic INV (AINV) subtypes: cell wall group (TaCWINV1-68), vacuole group (TaVINV1-42), and two neutral/alkaline INV (A/NINV) subtypes: cytoplasmic α group (TaA/NINV1-11) and cytoplasmic β group (TaA/NINV12-20). The amplification of A/NINVs is mainly attributed to the polyploidization of wheat, and the multiple duplication events experienced in AINVs revealed their non-dose sensitivity characteristic. The wheat RNA-seq data revealed the tissue specificity of A/NINVs and AINVs, and six spike-specific CWINVs showed significant differential expression between the fertile and sterile anthers of thermo-sensitive male-sterile wheat KTM3315A. TaCWINV40 localized in cell wall was effectively silenced in the fertile KTM3315A, and the malformed pollen grains and non-germinating pollen tubes shed light on its indispensability in the development of wheat anthers. This study will spur the interest on manipulating the novel genetic characteristics of TaCWINVs for the construction and improvement of wheat male sterile materials.


Introduction
Sucrose is the nal product of photosynthesis, which is transported from the source tissue to the nonphotosynthetic tissue (sink tissue) through the phloem (Koch 2004). In non-photosynthetic tissues, sucrose and its hexose products (glucose and fructose) play important roles in primary metabolism and special metabolism (Caretto et al. 2015). They are not only raw materials for many metabolic pathways, but also provide energy and carbon skeletons for the production of organic substances such as amino acids, nucleotides and structural carbohydrates, and can be used as signal molecules to coordinate source-sink relationships and resource utilization (Ruan 2012). Two main enzymes are responsible for introducing sucrose into plant metabolism: sucrose synthase (Susy, ec2.4.1.13) and invertase. Susy is glycosyltransferase that reversibly converts sucrose into UDP-glucose and fructose in the presence of UDP. In contrast, INV is hydrolase that irreversibly hydrolyzes sucrose into glucose and fructose.
INV, also called β-fructofuranosidase, means that the reaction catalyzed by the enzyme is the hydrolysis of the terminal non-reducing β-fructofuranoside residues in β-fructofuranoside (Romero-Gomez et al. 2000). According to subcellular location, INVs can be divided into cell wall invertase (CWINV), vacuolar invertase (VINV) and cytoplasmic invertase (CINV) (Sturm 1999). CWINVs and VINVs have similar conserved amino acid residues, belong to glycoside hydrolase family 32 (GH32), and contain two conserved sequences KNWINDPNGP and MWECXD (Gallagher et al. 2004;Den Ende et al. 2009). CINVs belong to glycoside hydrolase family 100 (GH100) and have almost no homology with CWINVs and VINVs (Lee and Sturm 1996). The different evolutionary origins of CWINVs, VINVs and CINVs are re ected in their biochemical and molecular properties. Insoluble CWINVs are glycosylated proteins bound to the cell wall with molecular weights of 28-64kDa and optimal pH values of 3.5-5.0 (Verhaest et al. 2006).
VINVs are soluble enzymes with molecular weights of about 70kDa and optimal pH values of 5.0-5.5 (Kim et al. 2011). These two are summarized as acid INVs (AINVs). Soluble CINVs are low-level expressed non-glycosylated polypeptides with molecular weights of 54-65kda, usually located in the cytoplasm, mitochondria, plastids and nucleus (Vargas and Salerno 2010). For their optimum pH values are 6.8-9.0, they are recognized as neutral/alkaline INVs (A/NINVs).
A/NINVs were found in all organs at different developmental stages of plants, especially in developing tissues. The presence of NINV in rice and Arabidopsis thaliana is essential for root cell development and reproductive development (Qi et al. 2007; Jia et al. 2008; Martin et al. 2013). The vegetative growth rate of Arabidopsis thaliana plants with two neutral invertase (NINV) subtypes deletion (cinv1/cinv2 mutant) were seriously reduced (Barratt et al. 2009), because insu cient glucose production hinders cellulose biosynthesis and leads to the reduction of anisotropic growth and abnormal arrangement of cellulose (Barnes and Anderson 2018). In addition, A/NINVs were found involved in response to environmental stresses in wheat (Vargas et al. 2007). VINVs located in the vacuole usually perform some functions by adjusting the osmotic pressure, such as participating in the sweet taste regulation of tomatoes and potatoes ), and response to drought and freezing stress (Kim et (Oliver et al. 2005). In wheat, the down-regulated expression of the anther-speci c CWINV gene Ivr1 triggered by water stress was considered to be a sign of pollen development failure (Koonjul et al. 2004). Based on growing evidence, the function researches of CWINVs have moved from theoretical basis to practical applications such as obtaining male sterile plants and increasing plant seed setting rate. In tobacco and Arabidopsis thaliana, anther-speci c RNA interference was used to inhibit the activity of CWINVs, effectively avoiding the supply of carbohydrates in pollen, and obtaining plants with reduced pollen germination ability and seed setting rate (Hirsche et al. 2009;Goetz et al. 2017). In Arabidopsis, rice and maize, the overexpression of CWINVs can promote the carbon distribution in the early stage of grain lling to improve grain yield and quality (Lammens et al. 2008;Wang et al. 2008;Coleman et al. 2009).
Wheat is the most important food crop worldwide, and with the continuous growth of world population and the decrease of cultivated land area, increasing wheat yield is of great signi cance to global food demand security. The utilization of heterosis based on the development of male sterility is a widely recognized strategy to obtain high-yield and high-quality wheat varieties. Three subtypes of INV have been proved to have many important functions, such as regulating plant growth and development, responding to abiotic and biotic stresses, affecting fertility and yield. However, the members of the INV gene family in wheat have not been systematically identi ed and analyzed, and the fertility-related INVs that can be used in the development of male sterile wheat need to be discovered urgently. Therefore, we used the whole reference genome of wheat to identify the INV family comprehensively, and analyzed their physical and chemical properties, phylogenetic evolution, gene structures and duplication events, expression pro les, as well as screened out the candidate INVs related to male fertility by virus induced gene silencing (VIGS) veri cation.

Materials And Methods
Plant materials and treatments KTM3315A was bred by the College of Agronomy of Northwest A&F University (34°29′N, 108°08′E), which is a two-line hybrid breeding material with thermo-sensitive male sterility characteristics (Ye et al. 2017).
When the wheat reaches the stage of pollen development, the wheat was transplanted into ower pots and grown in two incubators with different growth conditions. Under sterile conditions (de ned as AS) in the day and night 14h/10h, the temperature of the incubator is 17℃/15℃, the light is 20000xL/0xL, and the humidity is 50%/40%; under fertile conditions (de ned as AF ) the temperature of the incubator is 22℃/20℃ in the 14h/10h day and night, the light is 2000xL/0xL, and the humidity is 50%/40%. When the wheat began to produce anthers, stain with acetic magenta dye to observe the anther stage. The fertile and sterile anthers in late uninucleate, binucleate, and trinucleate stages (Respectively referred to as Luns, Buns and Tns) were collected in different cryopreservation tubes and they were quickly frozen in liquid nitrogen and stored at -80 ℃ in the refrigerator.
The type of tobacco used in this study is Nicotiana benthamiana, The day and night time in the growth incubator is 16h/8h, the temperature is 25℃/23℃, the light intensity is 15000xL/0xL, and the humidity is 60%/50%.  (Mitchell et al. 2019). Finally, the intersection of the three software identi cation results was nally used as the wheat INV gene family. In order to more accurately determine the subcellular location of TaINVs, the comprehensive prediction results of Plant-mPLoc software (Chou and Shen 2010) and ProtComp 9.0 serves (www.softberry.com) were nally adopted. The ProtParam tool (https://web.expasy.org/protparam/) on the ExPASy website (Gasteiger et al. 2003) was used to obtain the physical and chemical properties, and MapGene2Chrom web (http://mg2c.iask.in/mg2c_v2.0/) was used to draw the chromosome distribution location map of TaINVs.

Phylogenetic tree construction for wheat INVs
Taking into account the genetic relationship, the amino acid sequences of 19 rice INVs, 19 Brachypodium INVs and 130 wheat INVs were used to draw a phylogenetic tree. We performed clustalw alignment of a total of 168 amino acid sequences by MEGA (Kumar et al. 2016) with default setting parameters, and removed the gaps in the alignment result. With the above data, a phylogenetic tree was constructed using neighbor joining (NJ) method by passion model and Bootstrap replication 1000.

Duplication events analysis of TaINVs
To analyze genome-wide replication event, all TaINVs amino acid sequences were blast. Among them, each three TaINVs with a similarity of more than 95% and on different sub-genomes of the same chromosome were de ned as homologous copies. The members involved in segmental duplications and tandem duplications in TaINVs were obtained by Mcsacanx software (Wang et al. 2012). Three standards were recognized as necessary for the identi cation of tandem duplication: sequence similarity greater than 75%; the length of similar fragment greater than 75%; on the same chromosome. The results of multiple sequence alignments that comply with these three standards were recognized as tandem duplications of TaINVs, and KA/KS values of tandem duplications were obtained by KAKS_calculator 2.0 (Guo et al. 2014).

Analysis of gene structures and conserved domains in TaINVs
The gene structure information of TaINVs was extracted from the gff3 le of the wheat reference genome

Expression analysis of TaINVs
To analyze the expression pattern of TaINVs, we obtained the express abundance data of TaINVs in nine different stages and tissues (roots, leaves at seeding, vegetative and reproductive stages; spikes at vegetative stage; spikes and grains at reproductive stage) in Wheat Expression Browser database (Clavijo et al. 2017;Borrill et al. 2016). To have a more intuitive understanding for TaINVs, the TPM (Trans Per Million) expression data was selected to draw a horizontal and vertical clustering heatmap.
In this study, Total RNA was extracted by TriGene reagent (GeneStar) and reverse transcribed into cDNA by StarScript II First-strand cDNA Synthesis Kit-II (GeneStar). We used 2⋅RealStar Green Fast Mixture (GeneStar) reagent to prepare the qRT-PCR reaction system according to the manufacturer's instructions, and detected the ampli cation value of the TaINVs through the default program of the Applied Biosystems 7500 Real-Time PCR System. Housekeeping gene actin was used as an internal reference.
Each sample had three technical replicates and 2 −ΔΔCt method was used to calculate the relative expression of TaINVs. All gene quantitative primers were shown in Table 6.
The veri cation of subcellular localization and cis-elements for TaCWINV40 The following primers were used to clone the CDS sequence of TaCWINV40 with the stop codon removed from the cDNA of fertile anthers: F-CGAGCTCAAGCTTCGAAATGGGGATGGCGTCG, R-CGACTGCAGAATTCGAACTGCCCCAGACGCTTGTTCATCG, in which the underlined part represented the homologous arms. The cloned TaCWINV40 CDS fragment was integrated into pCAMBIA1302 vector with a green uorescence protein (GFP) to construct TaCWINV40: GFP by homologous recombination. Finally, 35S: GFP (negative control) and TaCWINV40: GFP transformed into Agrobacterium tumefaciens strain GV3101 were introduced into 4-week-old N. benthamiana tobaccos, and transgenic tobacco leaves showed uorescent signals after 36h-40h. In addition, a cell wall maker with red uorescence protein (RFP) signals and TaCWINV40: GFP fusion protein were co-injected into tobacco epidermal cells for coexpression. The uorescent signal and bright eld of transgenic leaves were observed and photographed by confocal microscope (Nikon A1, Kanagawa, Japan).
Moreover, through PLACE database (https://www.dna.affrc.go.jp/PLACE/?action=newplace) (Higo et al. 1999), we obtained the cis-elements on TaCWINV40 promoter, and focused on the analysis of elements that combine transcription factors and pollen speci c regulators.

Function validation of TaCWINV40
VIGS (Virus-induced gene silencing) refers to infecting plants with viruses that carrying target gene fragments can induce plant endogenous gene silencing and cause phenotypic changes, which is a popular method in recent years to study the function of target genes based on phenotypic variation (Burchsmith et al. 2004). The Barley Stripe Mosaic Virus-VIGS (BSMV-VIGS) system is commonly used in wheat, and the BSMV vector consists of three components, BSMV-α, BSMV-β, and BSMV-γ. To explore the function of TaCWINV40. The following primers were used to clone the 200bp CDS sequence of TaCWINV40: F: TAGCTAGCTGATTAATTAAGCTGGGCTAACAGGGATG, R: TTGCTAGCTGAGCGGCCGCGAGAAGTCGCCGTAGTCGTAT, in which the underlined part represented the homologous arms. Using NotI and PacI digestion sites in cloned sequence and BSMV-γ vector, the 200bp CDS of TaCWINV40 was inserted into the BSMV-γ vector to generate BSMV: TaCWIN40 by homologous recombination. BSMV: 0 was composed of BSMV-α, BSMV-β, and BSMV-γ. In the same way, the CDS fragment of Phytoene desaturase (PDS) gene was inserted into BSMV-γ and used as positive control (BSMV:PDS), because its silence would cause photobleaching characteristics on the leaves. Recombinant BSMV: TaCWIN40 vector, BSMV: PDS vector and the negative control BSMV-γ vector were mixed with BSMV-α and BSMV-β in equal proportions, respectively. Then added appropriate buffer solution and applied to the ag leaves of wheat plants (Yang et al. 2021).

Fertility identi cation and phenotyping of infected plants
The leaves and anthers of the plants infected were collected to observe the phenotype and analyze the gene expression level. Anthers in Tns were used to identify male fertility by DAPI, I 2 -KI staining, and pollen tube germination. Pollen tube germination was determined by germination medium (20g sucrose, 10g PEG 4000 , 4mg H 3 PO 3 , 4mg Ca(NO 3 ) 2 , 1mg VB 1 , dilute to 100ml with distilled), cultured at 37℃ for 30 minutes. The pictures were obtained with the microscope Olympus SZX10 (Japan). In addition, scanning electron microscope (SEM) and transmission electron microscope (TEM) were used to observe the cell morphology in anthers. The calculation of the seed setting rates adopted the percentage of total number of grains in the total number of orets.

Statistical analyses
Student'st-test in the Statistical Product and Service Solutions (SPSS) software were used to perform all statistical analyses. The differences were examined using Student's t-test and the signi cance level was set at 0.05 (P<0.05).

Identi cation and annotation of INV gene family members in wheat
By aligning all the protein sequences of wheat with the INVs of Arabidopsis (17), rice (19) and Brachypodium distachyon (19), 130 wheat INVs were identi ed and strictly screened (Table S1, Table S2). Twenty of them were evaluated as A/NINVs and belong to the GH100 family, and mainly expressed in cytoplasm and chloroplast. According to the position order, these 20 A/NINVs were named as TaA/NINV1-TaA/NINV20. In addition, 68 INVs were predicted to be expressed in the cell wall and were named TaCWINV1-TaCWINV68, and 42 INVs were named TaVINV1-42 for their subcellular location was vacuole.
By analyzing their physical and chemical properties, it was found that the number of amino acids of A/NINV was 505-653 aa, and the corresponding molecular weight was 56.31-72.8 kDa. 509-670 aa and 56.61-74.97 kDa for VINVs, respectively. However, the amino acid number and molecular weight of CWINVs are smaller than those of the former two, which are 332-657 aa and 37.88-74.94 kDa. The theoretical isoelectric points of these 130 TaINVs span a wide range, from 4.69 to 9.31 (Table S3). By mapping their chromosome distribution, we found that the numbers of TaINVs were unequal in the three A, B and D sub-genomes (A:B:D=52:33:41), but it can be predicted that most genes have three homologous copies because of their uniform location distribution in the three sub-genomes (Fig S1).

Phylogenetic analysis of TaINVs
In order to understand the evolutionary relationship of the wheat INV gene family, 130 TaINVs with 19 rice INVs (OsCINVs) and 19 Brachypodium distachyon INVs (BdINVs) were used to construct a phylogenetic tree for cluster analysis. Compared with the previous studies on rice (Ji et al. 2005), Brachypodium (Wang et al. 2017), the classi cation results of TaINVs were similar. The 130 TaINVs were divided into two major categories: AINVs and A/NINVs. The AINVs group included CWINs and VINs, A/NINVs were divided into two branches including α (TaA/NINV1-11) and β (TaA/NINV12-20) (Fig. 1). In the A/NINVs group, almost every OsINV and BdINV corresponded to three homologous TaINVs from the wheat ABD sub-genome (wheat: 20, rice: 8, Brachypodium: 8). In the AINVs group, the situations are a little more complicated. Compared with rice and Brachypodium distachyon, the number of INVs in wheat was more than three times that of them (wheat: 110, rice: 11, Brachypodium: 11), and a higher level of increase. There were only two VINVs in rice and three VINVs in Brachypodium, however, the number of VINVs in wheat was as high as 42.

The duplication of wheat INV gene family
Gene duplication often occurred in the whole genome, the main reasons were genome-wide duplication, tandem duplication, and segmental duplication (Zhang 2003). Of the 130 TaINVs, 54 genes had one copy on each of the three homologous chromosomes in the three sub-genomes, 43 genes had one copy on each of the two homologous chromosomes in the three sub-genomes (Table S4). By analyzing the segmental duplications, we found that there were 82 pairs of segmental duplications involving with 101 TaINVs (Fig. 2). However, except for the six pairs of segmented duplications produced by AINV from different chromosomes, the rest was duplications between different ABD sub-genomes of the same chromosome. Moreover, 13 TaINVs involved with 22 pairs of tandem duplications were found in the wheat INVs gene family (Table S5), which all occurred in TaAINVs and KA/KS values less than one. Overall, polyploidization of wheat played an important role in the expansion of TaINVs, and the segmental and tandem duplication events partly caused the signi cantly higher number of AINVs in wheat.

The gene structures and motifs of TaINVs
Introns were characteristic of eukaryotes, which were subject to relatively little selective pressure, resulting in rapid changes in the size and order of genes structures (Lecharny et al. 2003;Rogozin et al. 2003) However, the positional correspondence between introns and exons was usually highly conserved among homologous genes, so they were used to classify paralogous genes into subfamily (Park et al. 2008). Obviously, different subtypes of TaINVs had different numbers of introns and exons (Fig. 3). In TaAINVs, the number of exons ranged from two to nine. Except for a few genes, most TaCWINVs contain 5-9 exons, and most TaVINVs contain 3-4 exons. In TaA/NINVs, except for TaA/NINV2 (3 exons) and TaA/NINV18 (7 exons), there were four and six exons in α subgroup and β subgroup, respectively. On the contrary, TaINVs of the same subtype contain relatively uniform intron and exon, for example, all TaAINVs contain a miniexon, which encodes NDP. Furthermore, homologous copies, or paralogs formed by duplication events, had almost the same number and structure of introns and exons.
From the amino acid level, motif as a super secondary structure could facilitate the identi cation of functional differentiation within gene family. This study identi ed 15 conserved motifs in the TaA/NINV and TaAINV groups respectively, and they were completely different (Fig. 3). In TaAINVs, motif1 (βfructosidase motif NDPN), motif6 (RDP) and motif9 (WECP/VD) were essential markers, while motif3 and motif15 were speci c to CWINV, and motif 10 was unique to VINV. In TaA/NINVs, there were nine motifs (motif1-3, 5-9, 12) shared by 20 A/N-INVs, while two motifs (motif11, motif15) were speci c in α subgroup and one motif (motif13) speci c in the β subgroup.
Eight AINVs were speci c expressed in wheat spikes during reproductive stage Compared to identifying members with speci c motifs, the time-and space-speci city characteristics of gene expression always provided straightforward information for the study of gene functions related to the expression position. In order to explore the expression pattern of TaINVs and screen important ones, the RNA-seq data from roots, leaves, spikes, and grains in the different growth stages were analyzed. The results showed that 124 TaINVs were detected in the above tissues (Fig. 4). The tissue speci city of these TaINVs was more obvious than the period speci city, as a result, the expression patterns of TaINVs in the same tissue between different stages were similar. The expression patterns of most TaINVs in vegetative organs and reproductive organs were just opposite. Therefore, the expression pro le of 124 TaINVs could be roughly divided into two categories as Fig. 4, and 63 of them expressed preference during the vegetative period and 61 highly expressed during the reproductive period. Notably, eight wheat AINVs (TaCWINV40, TaCWINV53, TaVVIN27, TaCWINV46, TaCWINV68, TaVVIN7, TaCWINV36, and TaCWINV2) speci cally expressed in spikes.

Six TaINVs differentially expressed in anthers of KTM3315A under different fertility conditions
To further investigate the TaINVs that may be related to wheat male fertility, we performed qRT-PCR on eight wheat spikes-speci c TaINVs and four TaINVs highly expressed in wheat spikes. The sterile and fertile anthers of thermo-sensitive male sterile wheat KTM3315A at three stages (uninucleate, binucleate, trinucleate) were used as materials. The results showed that six of them (TaCWINV2, TaCWINV3, TaCWINV4, TaCWINV41, TaVINV7, and TaVINV27) had no signi cant difference in expression level at each stage of sterile and fertile anthers (Fig. 5). The other six TaINVs (TaCWINV36, TaCWINV40, TaCWINV43, TaCWINV46, TaCWINV53, and TaCWINV68) were not only similar in expression patterns, but also had signi cantly up-regulated expression in the fertile anthers than that of sterile anthers at binucleate stage. Interestingly, three genes, TaCWINV40, TaCWINV46, and TaCWINV53, are orthologous genes of rice OsCWINV2 (LOCOs04g33720) (Fig. 1), which has been revealed to lead male abortion when suppressed by low temperature (Oliver et al. 2005).

Silencing of TaCWINV40 induces a decrease in wheat fertility
Rice OsCWINV2 was con rmed to be a cell wall invertase, which was anther-speci c, mainly by affecting the hexose production and starch formation (Oliver et al. 2005). Here, TaCWINV40 was used as a representative to study whether it had similar functions to OsCWINV2. By fusion expression with green uorescent protein, the subcellular location of TaCWINV40 was con rmed to be the cell wall (Fig. 6A).
The analysis of the promoter region of TaCWINV40 revealed the presence of four important cis-elements (Fig. 6B). POLLEN1LELAT52 and GTGANTG10 have been reported in the promoters of the tomato lat52 gene and tobacco late pollen gene g10, respectively, and as regulatory elements responsible for their pollen speci c activation (Bate and Twell 1998; Rogers et al. 2001). WRKY71OS and MYCCONSENSUSAT were recognition sites of transcription factor WRKY and MYC, which were revealed to be a regulator of cold-induced transcriptome and a transcriptional repressor of the gibberellin signaling pathway, respectively (Zhang et al. 2004;Chinnusamy et al. 2003). These four cis-elements were distributed at least 3 sites on the promoter of TaCWINV40, which illustrated that TaCWINV40 may be regulated by them and then speci cally expressed in pollen and regulated by cold and gibberellin pathway.
To investigate the effect of TaCWINV40 on wheat fertility, VIGS technology was carried out using KTM3315A plants grown in fertile environments (>24℃). The infected plants showed abnormal leaves on about 14 days, and the white spots of positive control plants BSMV: PDS indicated that the barley virus successfully infected the plants and effectively silenced PDS gene (Fig. 7A). The qRT-PCR result showed that the expression of TaCWINV40 in the anthers of BSMV: TaCWINV40 was signi cantly lower than that of the negative control plants BSMV: 0 (Fig. 7G), which indicated that TaCWINV40 had been silenced. Although the anthers of BSMV:TaCWINV40 plants were still cracking and pollen grains were formed (Fig. 7B), the pollen microspores stained with I 2 -KI and DAPI showed sterile characteristics, that is, transparent shrunken vacuoles and two round sperm nuclei. On the contrary, in BSMV: 0 plants, the microspores possessed two spindle-shaped sperm nuclei, and they were all dyed into solid regular circles by I 2 -KI due to fulling of starch (Fig. 7C, D). In addition, the pollen tubes of BSMV: TaCWINV40 germinated extremely low, while which of BSMV: 0 almost germinated (Fig. 7E). SEM and TEM observations of trinucleate microspores support the key to further understanding BSMV: TaCWINV40, from which we observed the sparse arrangement and abnormal secretion of ubisch body, as well as the adhesion of shrinking microspores on the anther wall (Fig. 7F). In mature plants, seed setting rate of BSMV: TaCWINV40 was signi cantly lower than that of BSMV: 0 (Fig. 7H). once again con rmed the indispensable role of TaCWINV40 in wheat anther development and fertility determination.

Discussion
The wheat INV gene family is a big family AINVs are non-dose sensitive genes regulated by the sugar-sensitive mechanism, and they are also genes that further affects sugar metabolism in response to stress through their expression (Qian et al. 2018), the occurrence of tandem duplication can help them evolve an increased copy number to cope with the complex metabolism of the huge wheat genome. More gene copy number means being ready at any time to translate more sucrose invertase, so that one sucrose molecule can be quickly turned into two hexose molecules, and to quickly adjust the cell osmotic pressure to respond to the environment stress. In this study, more than half of the 22 pairs of tandem duplications (14 pairs) came from chromosomes 2 and 6 in wheat, and each of these INVs had at least two homologous copies. For example, TaCWINV7 (Chr: 2A) and TaCWINV8 (Chr: 2A) form a pair of tandem duplication, and they each have two other homologous copies (TaCWINV17 and TaCWINV24, TaCWINV18 and TaCWINV25) on chromosomes 2B and 2D. In short, TaCWINV7 may rst be doubled because of tandem duplication, and then tripled because of wheat polyploidization, so the number of TaAINV increased from the original one to at least six. Therefore, the signi cant expansion of TaAINVs should be the result of deliberate breeding of high-resistant varieties and natural selection of wheat itself.

INV gene family responds to temperature stress
Temperature stress is a kind of abiotic stress that TaINVs often responds to (Ruan et al. 2010). In tomatoes, CWINVs improve the fruit setting rate under long-term moderate heat stress by inhibiting the programmed cell death of non-reactive oxygen species, which may involve enhancing the introduction and catabolism of sucrose, the expression of HSP, and the reaction and biosynthesis of auxin ). In order to protect tomato plants from low temperature stress, cold treatment will inhibit the expression of CWINV inhibitors, so that CWINVs are strictly regulated by their inhibitors at the protein level, and the extracellular glucose and fructose content is maintained at an optimal level (Xu et al. 2017 TaCWINV40 is pivotal to male fertility by affecting the function of tapetum In this study, the cold response cis-element (MYCCONSENSUSAT, Fig. 6b) found in the promoter of TaCWINV40 con rmed its response to low temperature, and the existence of two cis-elements required for pollen speci c expression (GTGANTG10, POLLEN1LELAT52) predicted its expression site. In Arabidopsis, promoter analysis also con rmed the anther-speci c expression pattern of sesame Sicwinv1, and the GUS staining was mainly detected in the anther tapetum (Zhou et al. 2019). The tapetum layer is the innermost layer of the microspore mother cell wall (epidermal layer, endothelial layer, middle layer and tapetum layer) after the anther morphogenesis of higher plants is completed, and its degradation at the right time is a necessary condition for the normal development of pollen (Mascarenhas 1989;Yi et al. 2016). The tapetum layer actively synthesizes proteins, fats, carbohydrates and other substances, and secretes them into the anther chamber to provide nutrients needed for the meiosis of the microspore mother cell and the development of microspores (Pacini et al. 1985). Our previous studies have shown that there is no difference in tapetum morphology between fertile and sterile anthers of KTM3315A at the uninucleate stage, but which in fertile anthers begin to degrade at the binucleate stage and almost completely absent at the trinucleate stage (Meng et al. 2016). Interestingly, in this study, we found that the expression level of TaCWINV40 at the binucleate stage in fertile anthers was higher than that in sterile anthers, and the veri cation experiment showed that the silence of CWINV40 led to the delayed degradation of tapetum and the thinning of ubisch body. Combining the above results, we speculated that CWINV40 may be a key factor in promoting the degradation of the tapetum: when CWINV40 was inhibited or expressed at low level, the tapetum was delayed degradation, which leading to insu cient energy supply of the pollen grains and ultimately male sterility. In fact, the provision of carbohydrates for plant male gametophytes is generally summarized as the mechanism of INV affecting fertility (Oliver et al. 2005).
The tapetum has also been reported to have another function, after the microspore mother cell completes meiosis, the tapetum secretes the β-1,3-glucanase in a timely manner to decompose the corpus callose that wraps the tetrad and release the microspores (Pacini et al. 1985). Under the control of the tapetumspeci c promoter Osg6B, the introduced β-1,3-glucanase gene can prematurely dissolve the callosum layer of the pollen tetrad wall and cause male sterility (Tsuchiya et al. 1995). In this study, scanning electron microscope and transmission electron microscope showed an intuitive result, that is, after TaCWINV40 was silenced, abnormal microspores adhered to the inner side of anther wall, and most of the microspores showed abnormal adhesion to each other, just like the phenotype that the tapetum fails to secrete β-1,3-glucanase. In addition, gibberellin was reported to induce β-1,3-glucanase genes upregulation (Rinne et al. 2011), but a transcriptional repressor of the gibberellin signaling pathway (WRKY71OS) was found in the promoter region of TaCWINV40. Therefore, we speculated that TaCWINV40 was an inducement of β-1,3-glucanase co-existing in the tapetum, and its down-regulation affected the synthesis of β-1,3-glucanase and caused abnormal adhesion of microspores. This nding is of great signi cance for guiding breeding practice of hybrid wheat.      Self-setting rate of BSMV: 0 and BSMV: TaCWINV40. Each data had three repetitions. Analysis of signi cance of differences was identi ed by Students't text (* p < 0.05, ** p < 0.01).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.