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TaWI12 may be involved in pistillody and leaf cracking in wheat

BMC Plant Biology volume 25, Article number: 123 (2025) Cite this article

Abstract

TaWI12 is a member of the wound-induced (WI) protein family, which has been implicated in plant stress responses and developmental processes. Wheat (Triticum aestivum L.) is a crucial staple crop upon which human sustenance relies. Consequently, investigating the developmental mechanisms of pistils and stamens in wheat is profoundly significant for enhancing wheat characteristics and boosting productivity. In this study, we cloned TaWI12, from common wheat and observed a significant resemblance among the three homoeologs of TaWI12. The open reading frames (ORFs) of TaWI12-4A, TaWI12-4B and TaWI12-4D were 408 bp, 417 bp and 417 bp, respectively, and encoded 135, 138 and 138 amino acids, respectively. The phylogenetic tree revealed a high degree of homology between the protein sequences of TaWI12 and the wound-induced proteins of Hordeum vulgare (KAI4994568) and Aegilops tauschii (XP_020196548). To clarify the characteristics and functions of TaWI12 homoeologs, we obtained transgenic positive plants of Arabidopsis thaliana and observed significant filament shortening and decrease. Simultaneously, we used the CRISPR/Cas9 system to generate mutant plants via the modification of three homoeologs of TaWI12 in wheat. We noticed two distinct phenotypic differences in the knockout mutant. First, we observed the different degrees of homologous conversion of stamens to pistils in the single mutant TaWI12-4D. Second, we observed leaf cracking in both the single mutant TaWI12-4A and the double mutants TaWI12-4A and TaWI12-4D. Our findings further revealed that TaWI12 plays an important role in flower development, which is important for revealing the molecular mechanisms of pistil and stamen development in wheat and has important application value for high-yield wheat breeding.

Highlights

TaWI12 overexpression in Arabidopsis caused filament shortening and decreasing.

1 bp insertion or deletion in upstream of ATG increased the expression of TaWI12 and caused varying degrees of pistillody traits in wheat.

Utilizing CRISPR/Cas9 technology to modify TaWI12 in the coding region caused varying degrees of leaf cracking in wheat.

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Introduction

Allohexaploid bread wheat (AABBDD) is a monocot and belongs to the order Poales [1]. It is widely regarded as the primary source of plant-based protein in human nutrition and contains an abundance of essential nutrients, including proteins, fats, carbohydrates, dietary fibers, sugars, vitamins, and micronutrients [2]. The versatility of wheat flour is evident in its capacity to craft a variety of delicious dishes, including bread, cakes, dumplings, and noodles, satisfying people’s culinary preferences admirably [3]. Therefore, as one of the foremost food crops worldwide, wheat plays a vital role in ensuring food and nutritional security. According to the United Nations [4], the global population is projected to increase by 30% by 2050, with developing nations expected to face greater food demands than their developed counterparts [5]. Meeting future requirements for food security becomes imperative, necessitating the enhancement of wheat yield-related attributes to address challenges such as population growth, climate change, and the diminishing availability of arable land [6].

Mutants are a form of beneficial germplasm for plant breeding and genetics [7]. In its natural state, common wheat flowers are characterized by one pistil, three stamens and three types of vegetative organs, namely two lodicules, one palea and one lemma. The pistil undergoes development into a single grain following fertilization [8]. Abnormal pistil mutants serve as valuable resources for increasing wheat yield and are categorized into two types [9]. Type I is multi-pistil, which is characterized by the presence of three normal stamens and three fertile pistils, including three-pistil mutants (TP) [10], the three pistils in one floret mutant (12TP) [11], multi-ovary II (DUOII) [12], multi-ovary (MOV) wheat [13], and trigrain wheat [14]. Type II is pistillody, exhibiting varying degrees of homologous transformation of stamens into pistils or pistil-like structures, such as homologous transformation sterility-1 (HTS-1) [15]. Phenotypes with increased pistils or carpels can also be found in rice [16], Arabidopsis [17], maize [18], and tomato [19]. HTS-1 is a plant exhibiting pistillody traits that were identified during the cultivation process of the three-pistil near-isogenic line CSTP [15]. Genetic analysis revealed that this trait is governed by two recessive genes. One of these genes is likely located on the 2D chromosome, and is closely linked to the Pis1 gene, which is responsible for regulating the three-pistil trait. The second recessive gene, hts-1, is located within a 7.2 Mb interval on the 4A chromosome [20]. Through RNA-Seq analysis, we discovered a gene called TaWI12 that is activated in response to wounds. This gene is located inside the hts-1 localization interval. This gene is expressed at significantly elevated levels in pistillody stamens (PS). Consequently, we hypothesize that the overexpression of the TaWI12 gene is crucial in the development of pistillody characteristics in HTS-1.

Wound-induced gene families, including the win1 and win2 genes, which are arranged in a closely linked tandem array, were first identified in potato. When potato plants undergo wounding, these two genes show differential organ-specific expression [21]. A study conducted by Yen et al. [22] revealed that the protein WI12, which is produced in response to wounds, tends to accumulate mostly in the cell wall of injured Mesembryanthemum crystallinum mesophyll cells. This finding indicates its function in enhancing the composition of the cell wall after injury. Research in soybeans [23], strawberries [24], and rice [25] revealed that WI12 is involved in the control of biological stress in plants. This engagement is believed to be linked to modifications in the cell wall at the site of wounding and the enhancement of resistance against plant diseases and insect pests. Furthermore, WI12 is implicated in abiotic stress. In rice [26] and tea plants [27], WI12 may play a role in regulating cold stress. In Arabidopsis, WI12 (SAG20) participates in leaf senescence under ethylene treatment [28]. Despite these advances, there is currently no direct evidence linking WI12 to the development of flower organs. Interestingly, in Mesembryanthemum crystallinum, WI12 actively participates in reinforcing the cell wall composition at the wound site. Additionally, WI12 is highly expressed in petals, styles, placenta, and seeds [22]. These observations suggest that WI12 may have functions beyond wound responses and stress resistance, potentially influencing the development of reproductive structures in plants. Understanding whether WI12 plays a role in flower morphogenesis is critical, as floral organ development directly impacts reproductive success and, consequently, crop yield. Linking the expression and function of TaWI12 to floral and reproductive development in wheat could provide insights into mechanisms that regulate key yield traits.

On the basis of the above research, this study further analyzed the function of TaWI12 in pistil and stamen development in wheat through gene overexpression in Arabidopsis and CRISPR/Cas9 gene editing in wheat. The outcomes of this investigation aim to elucidate the function of TaWI12 in regulating wheat flower development, contribute to a deeper understanding of WI12 family proteins in overseeing plant development, and potentially establish a new theoretical foundation for enhancing wheat resistance and increasing wheat yield through molecular breeding techniques.

Materials and methods

Plant materials

In this study, Seeds of bread wheat (Triticum aestivum L.) Chinses Spring (CS), Chuanmai 28 (CM28) and Fielder were obtained from Sichuan Agricultural University. HTS-1, Chuanmai 28 TP (CM28TP), and Fielder were employed as the plant materials. HTS-1 was selected from the hybrid progeny isogenic of T. aestivum cv. CS and three-pistil wheat (TP), whose stamens transformed into pistils or pistil-like structures [15, 20]. CM28TP was selected from the hybrid progeny isogenic of T. aestivum cv. CM28 and TP; it has three healthy stamens and three fertile pistils [29]. The seeds of HTS-1 and CM28TP were sown in the experimental field of China West Normal University, Nanchong, Sichuan, China (30°49’N, 106°3’E), in October 2020. Fielder, T0 and T1 generation transgenic plants subsequently were grown in pots (10 cm × 20 cm) within a greenhouse under conditions of 16 h of light and 8 h of darkness, maintaining a temperature of 22 °C and a relative humidity of 70% [30]. In accordance with the classification standard of Waddington et al. [31] and considering observations from scanning electron microscopy, young spikes of HTS-1, CM28TP, Fielder, and homozygous Fielder plants edited via CRISPR-Cas9 at the stylar canal closing stage (Developmental score W5.5, spike length 5–6 mm, where pistillody traits began to form), the normal stamens (FS) of Fielder, pistillody stamens (EDPS) and pistils (EDP) of edited homozygous plants at the heading stage were manually dissected for RNA extraction. Arabidopsis WT (Col-0, Colombia) and transgenic lines were grown under greenhouse conditions (16-h light/8-h dark cycle, 22 °C, and 70% relative humidity) [32], Arabidopsis thaliana was obtained from Arabidopsis Biological Resource Center (ABRC). The leaves of Arabidopsis (including Col-0 and transgenic lines) were dissected for DNA and RNA extraction.

DNA, RNA extraction and cDNA synthesis

Total DNA was extracted from the leaves of Arabidopsis (including Col-0 and transgenic lines) via the LABGENE™ Plant DNA Isolation Kit (Chengdu Labbio Biotechnology, Chengdu, China), following the manufacturer’s instructions. Total RNA was extracted from young spikes of HTS-1, CM28TP, Fielder, homozygous Fielder plants edited via CRISPR-Cas9, EDP, and EDPS of transgenic wheat, FS of Fielder, and leaves of Arabidopsis (including Col-0 and transgenic lines) via the Eastep® Super Total RNA Extraction Kit (Promega, Shanghai, China), following the manufacturer’s instructions. The integrity and purity of the DNA and RNA were confirmed through 1% RNase-free agarose gel electrophoresis. The DNA and RNA concentrations were calculated via a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, MA, USA). First-strand cDNA was synthesized from total RNA according to the instructions of the PrimeScript TM RT reagent kit with gDNA Eraser (Takara, Dalian, China).

Cloning of TaWI12

The ORFs sequence of TaWI12 was cloned from the young spike cDNA of HTS-1 and CM28TP. Since common wheat contains A, B, and D genomes, the specific primers for cloning TaWI12 from the A (TracesCS4A02G115600), B (TracesCS4B02G188400), and D (TracesCS4D02G189800) genomes were designed with Primer Premier 5.0 (Table 1).

Table 1 Sequences of primers used in study
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PCR amplification was conducted via the TaKaRa EX Taq® DNA polymerase (Takara, Dalian, China) and reactions consisting of 3.0 µL of 10× EX Taq Buffer, 2.4 µL of dNTP Mixture, 0.36 µL of each primer, 1.0 µL of template cDNA and a certain amount of ddH2O in a final reaction volume of 25 µL. A T-100 Thermal Cycler (Bio-Rad, Singapore, USA) was used to perform PCR amplification according to the following procedure: predenaturation at 95℃ for 5 min, 35 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 32 s, and extension at 72 °C for 60 s; and a final extension at 72 °C for 7 min. After the PCR, 1.5 µL of 10 × loading buffer was added to the PCR product, and the amplified products were electrophoresed on 1.0% agarose gels and then visualized with a Gel Doc 2000 system (Bio-Rad, Singapore, USA). The target DNA bands were recovered and purified from the gels via the E.Z.N.A® Gel Extraction Kit (Omega, Georgia, USA). The purified PCR products were subsequently cloned and inserted into the pMD-19T vector (Takara, Dalian, China) and transformed into E.coli DH5α competent cells (Takara, Dalian, China) according to manufacturer’s instructions. The transformants were screened on LB agar plates containing ampicillin (100 µg. mL− 1) (Meilunbio, Dalian, China). Clones with inserts were identified via blue/white colony selection. Positive clones were subjected to further screening through PCR, and 10 clones from each sample were chosen for sequencing by Sangon Biotech (Sangon, Shanghai, China). Multiple sequence alignment was conducted via DNAMAN 9.0 software.

Bioinformatics analysis of TaWI12 in wheat

The expression specificity of TaWI12 in tissues was examined via WheatOmics 1.0 (http://202.194.139.32). The Expasy online tool (https://web.expasy.org/protparam/) was used to analyze the physicochemical properties of the proteins, including their molecular weights, theoretical isoelectric points, and hydrophobicity. The secondary structure analysis was accomplished by Novopro and Prabi (https://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html/).Tertiary structure analysis of the protein was performed via the online software SWISS-MODL (https://swissmodel.expasy.org/interactive/). The conserved domain of the TaWI12 protein was predicted via NCBI Conserved Domain Search (CDD, NCBI Conserved Domain Search (nih.gov)). Homologous genes of TaWI12 were searched via BLAST from the NCBI database (BLAST: Basic Local Alignment Search Tool (nih.gov)), and phylogenetic evolutionary trees were built via the maximum likelihood method in MEGA7.0 software.

Overexpression analysis of TaWI12 in Arabidopsis

The entire coding sequence (CDS) of TaWI12 (TracesCS4A02G115600) was amplified from the cDNA from the spike of CM28TP as a template with a pair of specific primers TaWI12-OE (Table 1). The entry clone was obtained via the PentrTM/D-TOPO™ cloning Kit (HuaYueYang, Beijing, China), mixed with E.coli DH5α competent cells, cultivated on LB with kanamycin (50 µg.mL− 1) (Meilunbio, Dalian, China), and sequenced by Sangon Biotech(Sangon Biotech, Shanghai, China). The recombinant vector D35S::TaWI12, driven by the CaMV35S promoter, was constructed as follows: 1 µL of entry clone, 1 µL of Destination vector pC1300s (BioVector NTCC Inc., Beijing, China), and 6 µL of TE Buffer (pH 8.0), followed by incubation at 25 °C for 1 ~ 2 h; then, 2 µL of LR clonase II enzyme mixture and 1 µL of proteinase K were added, followed by incubation at 37 °C for 10 min. Subsequently, the LR reaction was mixed with E.coli DH5α competent cells, which were cultivated on LB with kanamycin (50 µg.mL− 1) and hygromycin (50 µg.mL− 1) (Meilunbio, Dalian, China), and sequenced by Sangon Biotech(Sangon, Shanghai, China). The recombinant vector D35S:: TaWI12 was subsequently transformed into Agrobacterium GV3101 via the liquid nitrogen freeze-thaw method [33]. This mixture was subsequently transformed into Arabidopsis thaliana (Col-0) via the floral dip method [34]. Transgenic plants from the T0, T1, and T2 generations were screened on kanamycin-containing medium (50 µg.mL− 1), and successful integration of the transgene was confirmed by PCR. The expression of TaWI12 was verified by qRT-PCR, with AtGAPDH (At1g13440) serving as an internal reference [35]. The primers used for amplifying the target gene and AtGAPDH are listed in Table 1. Relative quantification of gene expression was performed via the 2−△△Ct method [36]. The error bars represent the standard deviation (SD). Significant differences were statistically analyzed by one-way ANOVA using SPSS 26.0 and GraphPad 8.0 software. All experiments included at least three biological and two technical replicates. Two T2 seedlings from each transgenic line were grown for further screening of homozygous lines by evaluating the segregation ratio of hygromycin resistance in their progeny at the germination stage. Lines where nearly all progeny seeds (approximately 100%) displayed hygromycin resistance were designated as homozygous. Homozygous T2 lines were subsequently self-pollinated to produce T3 seeds, which were used for phenotypic analysis.

Generation of TaWI12 CRISPR/Cas9 mutants in wheat

Using the homogeneous gene sequences of TaWI12 on chromosomes A, B, and D (TraesCS4A02G115600, TraesCS4B02G188400, and TraesCS4D02G189800), we designed 23 bp target points (sgRNA sequences) containing PAM sites (5’-NGG-3’) in the exon and promoter regions. sgRNA1 is located on the exons of the TaWI12-4A and TaWI12-4B genes, and sgRNA2 is located on the TaWI12-4D promoter (Fig. 1A). A schematic diagram of the structure of the dual-target editing vector is shown in Fig. 1B. Following genetic transformation of Agrobacterium species [37], seedlings of transgenic plants were generated. To screen for edit-positive plants, the vector-specific primers TaU3-F and CeHind-R were used for PCR detection of T0 generation transgenic wheat. Specific mutation analysis of the T0 generation was conducted on the selected CRISPR/Cas9-positive plants via TA cloning and sequencing of the TaWI12 gene. The T1 generation was obtained via self-fertilization of the T0 generation. The TaWI12 gene from T1 generation plants was subsequently cloned, and 10 positive clones were selected for sequencing. If the 10 clones exhibited the same mutation site, we considered that we obtained homozygous CRISPR/Cas9 mutant plants.

Fig. 1
figure 1

Schematic diagram of gene editing. (A). Schematic diagram of the gene structure of the target site and sgRNA sequence. Note: The green letters and red letters represent the PAM sequences and the sgRNA sequences, respectively. (B). Structure of the dual-target editing vector

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qRT-PCR was used to analyze the expression of TaWI12 in young spikes of Fielder (including both wild-type and edited homozygous plants), EDP, EDPS, and FS with β-Actin (AB181991) used as the internal reference [38]. The primers for the target gene and β-Actin are provided in Table 1.

Phenotypic identification

Ten individual plants were randomly selected from each homozygous edited wheat line and transgenic Arabidopsis line for phenotypic analysis. The morphological structures of floral organs in wheat and Arabidopsis were observed via a SZX9-3122 stereomicroscope (Olympus, Tokyo, Japan), and images of the plants, spikes and leaves were acquired via an EOS 80D camera (Canon, China).

Results

Isolation and sequence analysis of homoeolog genes of TaWI12 on chromosomes 4A, 4B and 4D

The cDNA derived from reverse transcription of total RNA extracted from young spikes of CM28TP and HTS-1 served as a template for PCR amplification. Three homologous genes of TaWI12, namely TaWI12-4 A, TaWI12-4B, and TaWI12-4D, were extracted from chromosomes 4A, 4B, and 4D, respectively. The ORFs of the three homologous genes were 408 bp, 417 bp and 417 bp (Fig. 2A), encoding 135,138, and 138 amino acid residues, respectively. The amino acid sequence similarity was 95.17% (Fig. 2B). Additionally, the sequences of the three homologous genes are identical in both CM28TP and HTS-1.

We identified potential orthologs of TaWI12 by comparing its amino acid sequence with those of various plant species via NCBI. A phylogenetic tree of the WI protein was subsequently constructed through the application of the maximum likelihood method to a multiple alignment of amino acid sequences. Within the phylogenetic tree, the 16 WI proteins were categorized into two clusters. Notably, TaWI12-4A, TaWI12-4B, TaWI12-4D and several putative orthologs from Hordeum vulgare, Aegilops tauschil subsp. strangulate, Brachypodium distachyon, Lolium perenne, and Lolium rigidum were grouped together in one cluster (Fig. 2C). Further analysis through BLAST revealed that the amino acid sequence of TaWI12 exhibited 96.3% identity with HvWI (Hordeum vulgare, XP_020196548) and 93.48% identity with AsWI (Aegilops tauschil subsp. Strangulate, KAI4994568).

Fig. 2
figure 2

PCR amplification, amino acid sequence comparison, and phylogenetic analysis of TaWI12. (A). PCR amplification of the TaWI12 gene. M: DL2000 marker; 1–3: Amplification results of TaWI12-4A, TaWI12-4B and TaWI12-4D in CM28TP; 4–6: Amplification results of TaWI12-4A, TaWI12-4B and TaWI12-4D gene in HTS-1. (B). Amino acid sequence comparison of the TaWI12. (C). Maximum likelihood tree of the TaWI12 protein from various plant species, with sequences retrieved from the NCBI protein database

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Bioinformatics analysis of the TaWI12 protein

The computations of various physical and chemical parameters, including the number of amino acids, molecular weight, theoretical pI, total number of negatively charged residues (Asp + Glu), total number of positively charged residues (Arg + Lys), formula, instability index (II), aliphatic index and grand average of hydropathicity (GRAVY), for the TaWI12-4A, TaWI12-4B, and TaWI12-4D proteins are listed in Table 2.

Table 2 Computations of various physical and chemical parameters of TaWI12
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All three proteins were categorized as unstable and assumed to be hydrophobic proteins (Fig. 3A). The ratios of the secondary structure compositions of the TaWI12-4A, TaWI12-4B, and TaWI12-4D proteins are listed in Table 3, and the secondary structures of which are shown in Fig. 3B.

Table 3 Secondary structure of the TaWI12 protein
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The tertiary structures of the TaWI12-4A, TaWI12-4B, and TaWI12-4D proteins are shown in Fig. 3C. As shown in Fig. 3C, the tertiary structures of the TaWI12-4B and TaWI12-4D proteins were highly similar. The prediction of conserved structural domains verified that the TaWI12-4A, TaWI12-4B and TaWI12-4D proteins belong to the WI12 superfamily (Fig. 3D).

Fig. 3
figure 3

Bioinformatics analysis of the TaWI12 protein. (A). Hydrophobicity profile of the TaWI12-4A, TaWI12-4B, and TaWI12-4D proteins. Note: The x-axis represents the sequence of amino acids encoding the protein, and the y-axis represents the hydrophilic and hydrophobic values. A value of 0 indicates a hydrophobic region, whereas a value of 0 indicates a hydrophilic region. (B). The predicted secondary proteins of TaWI12-4A, TaWI12-4B, and TaWI12-4D. Note: The red, blue, and black parts represent the alpha helix, β-extended strand, and random coil, respectively. (C). Tertiary structure prediction of the TaWI12-4A, TaWI12-4B, and TaWI12-4D proteins. (D). Conserved domain prediction of the TaWI12-4A, TaWI12-4B, and TaWI12-4D proteins

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Phenotypic analysis of TaWI12 overexpressing Arabidopsis

To investigate the potential role of TaWI12, an overexpression vector was constructed and introduced into Arabidopsis through the floral dip method [34]. Screening of transgenic plants was conducted using hygromycin (50 µg.mL− 1), and positive lines were further confirmed via PCR analysis (Fig. 4A). Among the 15 T1 generation plants tested, 7 individuals (plants 1, 2, 6, 7, 8, 12, and 15) were confirmed as positive transgenic lines. The transcript levels of the TaWI12 gene were assessed via qRT-PCR. The results revealed a significant increase in the expression levels in the transgenic plants compared with those in the wild type (Col-0) plants (Fig. 4B). The flower phenotypes of homozygosity T3 generation transgenic plants before and during pollination were subsequently examined via stereomicroscopy, revealing noticeable anther filament shortening and a number of filaments varying from 2 to 6 compared with those of Col-0 plants (Fig. 4C). These observations suggest that overexpression of TaWI12 in Arabidopsis induces notable alterations in stamen development, providing insights into the potential roles of this gene. However, we emphasize that these findings primarily reflect the effects of TaWI12 overexpression in the Arabidopsis system and may not fully represent its function in wheat, given the differences in developmental contexts and genetic backgrounds.

Fig. 4
figure 4

Differences in the inflorescence phenotypes of Col-0 and transgenic Arabidopsis lines. (A). 15 independent transgenic lines of the TaWI12 gene were detected via PCR. (B). TaWI12 expression levels in 1, 2, 6, 7, 8, 12, and 15 leaves of the TaWI12 transgenic lines. AtGAPDH was used as the reference gene. (C). Filament growth of transgenic Arabidopsis − 1, -2, -6, -7, -8, -12, and − 15 before and during pollination in the T3 generation. Notes: The error bars represent the standard deviation (SD). Different letters indicate significant differences(P < 0.05), and the same letters indicate no significant differences(P > 0.05)

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Phenotypic analysis of TaWI12 in wheat edited by CRISPR/Cas9

To investigate the functional characterization of homologs of TaWI12 in hexaploidy wheat, we generated TaWI12 knockout mutants via the CRISPR-Cas9 technique in the wheat cultivar Fielder background. One construct, carrying both sgRNA1 and sgRNA2, was introduced into the WT via Agrobacterium-mediated transformation. The TaWI12 genes in the A, B, and D subgenomes were validated through sequencing in the T0 generation. Eventually, we successfully obtained 4 edited plants from TaWI12-4 A and 4 edited plants from TaWI12-4D. However, no edited sites were detected in TaWI12-4B or in the coding region or promoter region.

We subsequently self-pollinated these T0-edited plants to establish homozygous TaWI12 knockout lines. Following the sequencing of editing sites in the T1 generation plants, we successfully obtained homozygous editing materials on chromosomes A, D and AD (Fig. 5). On chromosome A, there are two edited homozygotes: ED10 and ED16. ED10 exhibited a 3 bp deletion, whereas ED16 featured a 5 bp deletion at the PAM site (Fig. 5A). For chromosome D, we also obtained two edited homozygotes: ED3 and ED20. ED3 displayed a 1 bp insertion, and ED20 had a 1 bp deletion at the PAM site (Fig. 5B). Furthermore, we also obtained two edited plants that were homozygous for chromosomes A and D: ED6 and ED15. ED6 displayed a 1 bp deletion on chromosome 4 A and a 1 bp insertion on chromosome 4D. ED15 had a 1 bp deletion on the 4 A chromosome and a 4 bp deletion on the 4D chromosome (Fig. 5C).

Fig. 5
figure 5

Genotyping of TaWI12 knockout lines generated via wheat gene editing in the T1 generation. (A). Editing on chromosome A. (B). Editing on chromosome D. (C). Editing chromosomes A and D. Note: The green letters, the red letters, the blue letters, and the dashed lines represent the PAM sequences, the sgRNA sequences, the nucleotide insertions, and the nucleotide deletions, respectively

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Phenotypic analysis was carried out on homozygous editing materials on chromosomes A, D and AD, including comparison of the morphological characteristics of pistils, stamens and leaves between T1 knockout mutant plants and Fielder. Typically, Fielder’s florets exhibit one pistil and three stamens, resulting in the production of one seed per floret. In contrast, the florets of ED3 and ED20 presented pistillody traits, with the most extreme manifestation being the homologous transformation of all three stamens into pistils; however, only a single seed was produced instead of the expected 2 to 4 seeds for each floret. Moreover, the florets of ED10, ED16, ED6, and ED15 did not exhibit pistillody (Fig. 6A-C). The expression of the TaWI12 gene in pistillody stamens (EDPS) of ED20 and ED3 was analyzed via qRT-PCR. The expression levels of TaWI12 in EDPS and EDP were significantly greater than those in FS, with an increase of more than 100-fold (Fig. 6D). We further analyzed the expression levels of the TaWI12 gene in the spikes of edited plants. The results revealed that TaWI12 was highly expressed in the spikes of ED3 and ED20, but the expression levels in the spikes of Fielder, ED10, ED16, ED6 and ED15 were slightly lower (Fig. 6F). Consequently, we hypothesize that editing of the promoter region did not inhibit the expression of the TaWI12 gene but rather increased its expression of both ED20 and ED3. Upon further examination, we discovered a 1 bp deletion in ED20 and a 1 bp insertion in ED3. These mutations occurred at the fourth base upstream of the PAM site in both gene editing lines (Fig. 5B). Consequently, we postulate that the deletion and insertion of 1 bp in the promoter region led to alterations in promoter activity, ultimately enhancing downstream gene expression. This may contribute to the development of pistillody stamen traits. In addition, we observed that the leaves of edited homozygous plants, including ED10, ED16, ED6, and ED15, exhibited varying degrees of cracking compared to those of Fielder. Notably, this cracking was not limited to the flag leaves; nearly all leaves of each mutant plant displayed this phenomenon. However, there was no leaf cracking in the edited homozygous plants at ED3 and ED20 (Fig. 6E). Therefore, we speculate that TaWI12 may be involved in regulating pistillody and leaf cracking simultaneously.

Fig. 6
figure 6

Phenotype and expression analysis of Fielder and TaWI12 knockout lines in the T1 generation. (A). Plant phenotypes of Fielder and transgenic lines. (B). Pistil and stamen phenotypes of Fielder and transgenic lines. (C). Spike phenotypes of Fielder and transgenic lines. Note: P represents pistil; PS represents pistillody stamen; S represents stamen. (D). Leaf phenotypes of Fielder and transgenic lines. Note: A represents homozygous editing on chromosome A; D represents homozygous editing on chromosome D; A and D represents homozygous editing on chromosomes A and D. The arrows represent leaf cracking. (E). Expression of pistils and stamens in Fielder and transgenic lines. Note: FS represents a normal stamen; EDPS represents a pistillody stamen; EDP represents a pistil. (F). Expression of spikes in Fielder and transgenic lines. The error bars represent the standard deviation (SD). Different letters indicate significant differences(P < 0.05), and the same letters indicate no significant differences(P > 0.05)

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Discussion

Wheat is a globally significant food crop that is extensively cultivated worldwide [39]. Multi-pistil wheat stands out as a valuable and rare resource for wheat breeding and the investigation of wheat flower development [40]. In this study, TaWI12 was isolated from HTS-1 and CM28TP, and its sequences were compared and analyzed. The results revealed no disparity in TaWI12 between HTS-1 and CM28TP, suggesting that the phenomenon of pistillody stamens might not be attributed to sequence differences but potentially linked to the differential expression of TaWI12. Bioinformatic analysis of TaWI12 revealed that the protein is a member of the WI12 family.

The TaWI12 gene may play a vital role in response to wounds

Plant cells are encapsulated by their rigid cell walls, which is a considerable challenge for wound healing in plant tissue. Cell-specific auxin accumulation occurs near plant wounds, and this increase in local auxin balances the rate of wound-induced cell expansion and restorative division in a dose-dependent manner [41]. Plants often transmit external signals into cells through interconnected signaling pathways, eventually activating defense responses by upregulating a range of defense-related genes in response to environmental stress [42]. In Populus, the expression of wound-inducible cDNA (win4) was examined in wounded leaves [43]. In maize, GRMZM2G010909 encodes wound-induced protein 1, the expression of which is induced in response to mechanical wounding or pathogen attack [44]. In rubber trees, the dominance of hevein proteins is similar to that of wound-induced proteins. Hevein mRNA is frequently upregulated in leaves, stems, and latex derived from tapping stems in response to wounding or exogenous application of stress-related hormones such as ABA and ethylene [45].

While research on the WI12 family is limited, existing studies indicate that WI12 family members are activated in plants in response to wounds, diseases and insect pests [23,24,25]. In this study, we observed a distinct leaf cracking phenotype in plants where TaWI12 was edited using CRISPR/Cas9. This phenomenon was evident in both homozygous mutants of the A genome and double homozygous mutants of the A and D genomes. We hypothesize that this phenotype may result from tissue damage caused by mechanical stress or pest/pathogen activity. In the absence of functional TaWI12, the damaged tissues might be unable to heal properly, leading to the observed cracking. This hypothesis aligns with the potential role of TaWI12 suggested by prior studies. However, direct evidence linking the loss of TaWI12 function to impaired tissue repair is currently lacking. Further studies are needed to explore the molecular mechanisms underlying this phenotype and to validate this proposed connection.

Overexpression of TaWI12 could be the key factor contributing to the development of pistillody traits

Currently, there are few studies on the association between WI12 and floral development. In Mesembryanthemum crystallinum, WI12 is highly expressed in petals, styles, placenta, and seeds [22]. In rice, LOC_Os05g27590, which is associated with abiotic stress and cell wall defense, encodes the wound induced protein WI12 and might participate in the cross-talk between pollination and stress responses [46]. Moreover, during the development and germination of rice pollen, 474 transcripts related to defense/stress response, including the wound-induced protein WI12, were preferentially expressed in the pollen. These findings suggest that the capacity to handle abiotic and biotic stresses developed during pollen maturation may be critical for successful fertilization [8]. In this study, overexpression of the wheat TaWI12 gene in Arabidopsis revealed clear phenotypic alterations. Specifically, transgenic plants displayed shortened filaments relative to the stigma and variability in filament number, ranging from 2 to 6, compared to the wild type. These findings indicate that TaWI12 may influence stamen development, potentially causing abnormalities that disrupt floral organ structure. However, we recognize that these results primarily reflect the role of TaWI12 within the Arabidopsis system and may not directly represent its function in wheat due to the differing developmental and regulatory contexts between these species. The overexpression experiments provide valuable insights into the potential effects of TaWI12, but further studies in wheat are essential to validate its native role. To this end, our CRISPR/Cas9-mediated editing of TaWI12 in wheat demonstrated that specific promoter modifications, such as a 1 bp deletion or insertion upstream of the PAM site in the TaWI12-4D promoter, led to pistillody traits. Interestingly, this deletion or insertion did not lead to a reduction in or inactivation of TaWI12 expression; instead, it resulted in the upregulation of TaWI12 expression in floret organs. We speculate that the deletion or insertion of the 4th base upstream of the editing site (PAM structure) in the promoter of TaWI12 on the 4D chromosome enhances promoter activity, thereby inducing the upregulation of TaWI12. However, in the pistillody edited plants, each floret did not produce 2 to 4 seeds, which we speculated might have been caused by an incomplete pistillody. As a next step, we intend to overexpress this gene in wheat to further validate our hypothesis.

Conclusion

In this study, we successfully cloned TaWI12 from common wheat, and analyzed how the TaWI12 gene affects wheat development at the molecular level. To further explore the regulatory mechanism of the TaWI12 gene, we generated transgenic Arabidopsis and wheat plants. In TaWI12 knockout mutants, we observed different degrees of homologous conversion of stamens to pistils, as well as the occurrence of leaf cracking. We speculate that TaWI12 may be involved in response to wounds in wheat in addition to the development of pistils and stamens. We subsequently observed the healing response of WT and genetically edited plants to wounds by artificially wounding wheat leaves. In addition, we observed obvious filament shortening and decreasing in transgenic Arabidopsis. In further studies, an overexpression vector could be constructed for transformation into wheat to better study the function of TaWI12. In summary, the results of this study hold significant theoretical value for research on leaf cracking, pistil and stamen development, and offer important application for high-yield breeding of wheat.

Data availability

The nucleotide sequence and protein sequence of the TaWI12 gene on chromosomes A, B, and D (TraesCS4A02G115600, TraesCS4B02G188400, and TraesCS4D02G189800) were obtained through the WheatOmics 1.0 website (http://202.194.139.32) and the website is open to all researchers. All supporting data for this manuscript are included in the manuscript and its additional files.

Abbreviations

HTS-1:

Homologous transformation sterility-1

TP:

Three-pistil mutants

FS:

The normal stamens of Fielder

EDPS:

Pistillody stamens of edited homozygous plants

EDP:

Pistils of edited homozygous plants

ORFs:

Open reading frames

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Funding

This study was supported by the National Natural Science Foundation of China (Grant No. 32272066, 32060456), and the Natural Science Foundation of Sichuan Province (Grant No. 2025ZNSFSC0156).

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Authors and Affiliations

  1. Triticeae Research Institute, Sichuan Agricultural University, Chengdu, 611130, China

    Yuhuan Guo & Yonghong Zhou

  2. Key Laboratory of Southwest China Wildlife Resource Conservation (ministry of education), College of Life Science, China West Normal University, Nanchong, 637002, China

    Yuhuan Guo, Yan Zhang, Yuhao Li, Yichao Wu, Mingli Liao & Zaijun Yang

  3. School of Agricultural Science, Xichang University, Xichang, 615000, China

    Zhengsong Peng

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Contributions

Yuhuan Guo, and Yan Zhang: Investigation, Data curation, Formal analysis, Writing – original draft, Writing – review & editing, Visualization. Yuhao Li: Investigation, Data curation, Formal analysis. Yichao Wu and Mingli Liao: Validation, Writing – review & editing. Zhengsong Peng: Resources. Yonghong Zhou: Writing – review & editing. Zaijun Yang: Writing – review & editing, Funding acquisition.

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Guo, Y., Zhang, Y., Li, Y. et al. TaWI12 may be involved in pistillody and leaf cracking in wheat. BMC Plant Biol 25, 123 (2025). https://doi.org/10.1186/s12870-025-06133-5

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