TaWI12 may be involved in pistillody and leaf cracking in wheat

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.

Keywords: TaWI12; Filament shortening; Flower development; Pistillody; Wheat.

Fig. 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

Fig. 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

Fig. 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

Fig. 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)

Fig. 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

Fig. 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)