WPA1 encodes a vWA domain protein that regulates wheat plant architecture

Abstract

Plant height, spike, leaf, stem and grain morphologies are key components of plant architecture and related to wheat yield. A wheat (Triticum aestivum L.) mutant, wpa1, displaying temperature-dependent pleiotropic developmental anomalies, was isolated. The WPA1 gene, encoding a von Willebrand factor type A (vWA) domain protein, was located on chromosome arm 7DS and isolated by map-based cloning. The functionality of WPA1 was validated by multiple independent EMS-induced mutants and gene editing. Phylogenetic analysis revealed that WPA1 is monocotyledon-specific in higher plants. The identification of WPA1 provides opportunity to study the temperature regulated wheat development and grain yield.

Headnote

ABSTRACT

Plant height, spike, leaf, stem and grain morphologies are key components of plant architecture and related to wheat yield. A wheat (Triticum aestivum L.) mutant, wpa1, displaying temperature-dependent pleiotropic developmental anomalies, was isolated. The WPA1 gene, encoding a von Willebrand factor type A (vWA) domain protein, was located on chromosome arm 7DS and isolated by map-based cloning. The functionality of WPA1 was validated by multiple independent EMS-induced mutants and gene editing. Phylogenetic analysis revealed that WPA1 is monocotyledon-specific in higher plants. The identification of WPA1 provides opportunity to study the temperature regulated wheat development and grain yield.

Keywords:

Wheat plant architecture

Map-based cloning

vWA

Environmental temperature

1. Introduction

Plant height, spike number, tiller angle, and morphologies of leaf, stem, and root are important agronomical traits related to plant architecture and yield [1]. Many genes governing morphological traits development in crops have been identified. The IDEAL PLANT ARCHITECTURE (IPA1) from rice encodes a plant-specific transcription factor, regulating plant architecture, growth and development, as well as disease resistance and environmental adaptability [2–6]. The wheat HOMEOBOX DOMAIN-2 (HB-2) encodes a class III homeodomain-leucine zipper transcription factor that increases spikelet production and grain protein content by alterations of leaf and vascular morphology, as well as amino acids [7]. Similarly, a CONSTANS-like protein TaCOL-B5 has been characterized with increased spikelet nodes within a spike, increased tillering and spiking, and the formation of elongated yet slender grains [8].

The von Willebrand factor A (vWA) domain proteins have been identified first in human [9]. vWA containing proteins were found to be related to biotic and abiotic stresses tolerance in Arabidopsis [10,11]. RGLG1, an E3 ubiquitin ligase, contains a vWA domain with two calcium ion binding sites and regulates ABA signaling in Arabidopsis [10]. Arabidopsis vWA domain-containing protein BON1 is temperature sensitive and involved in suppression of immunoreceptor genes and stomatal closure [11]. The vWA domain's allosteric regulation mechanism in Arabidopsis seems to differ from the mechanism in integrin alpha and beta I domains in mammals [11]. Recently, vWA containing tandem kinase protein WTK6-vWA was reported to provide leaf rust resistance in wheat [12]. However, the functions of vWA domain containing proteins in regulating plant architectures, especially in wheat, was not observed.

Bread wheat, renowned for its wide adaptability across diverse regions, is vulnerable to temperature stresses during its late reproductive phases [13]. In cereals, elevated ambient temperatures can accelerate flowering. High ambient temperatures impaired reproductive development and grain yield in barley (Hordeum vulgare L.) [13]. Gene HvVRN1 in barley regulates spikelet number in response to high ambient temperatures [14]. HvPpd-H1 also functions in modulating reproductive development and seed number per spike under high ambient temperatures, exhibiting genetic interaction with HvVRN1 [14]. Recent study [15] in barley have unveiled the function of HvMADS1, a SEPALLATA MADS-box transcription factor, in preserving spike architecture in response to elevated temperatures by regulating cytokinin homeostasis. However, the genes that regulate wheat morphology and architecture in response to temperatures remain largely unexplored.

The availability of reference genome sequences for wheat cultivars, as well as mapping and mutant populations, has improved our ability to identify genes involved in wheat response to temperatures [12,16]. In this study, we applied mutagenesis approach to identify wheat plant architecture phenotypic abnormalities mutants in plant height, leaf shape and spike. Map-based cloning was performed to isolate a key gene that regulated wheat plant architecture and sensitive to temperature.

2. Materials and methods

2.1. Plant materials and growth conditions

Mutants wpa1, wpa-Mut2, wpa-Mut3 and wpa-Mut4 with developmental defects were generated by ethyl methanesulfonate (EMS) mutagenesis of dry seeds of Zhongke 331 (ZK331), a bread wheat-wild emmer wheat introgression line [17]. Seeds of ZK331 (15,000) were treated with 0.6% (w/v) EMS, generating 2030 M1 plants. Mutants wpa-Mut5 and wpa-Mut6 were obtained from an EMS-induced mutation library of cv. Jimai 22 (JM22), and wpaMut7 was identified from EMS-induced mutation library of cv. Nongda 399 (ND399). The mutants with developmental defects in the M2 generation were further validated by progeny (M3) tests. Agronomic trait evaluation of the M3 progenies were conducted at the Gaoyi Experimental Station (Gaoyi, Hebei, China). Each row 2 m in length and 25 cm in width was evenly planted with 21 seeds, and standard field management practices for wheat production were employed. At maturity, plant height, spike length, grain number per plant, and 1000-grain weight were measured, while leaf traits were recorded at the onset of the grain filling stage. Plants used for these measurements were harvested from the central row of each plot, and grain morphometric measurements, including grain width and length, were recorded using vernier calipers. Statistical significance was assessed using two-tailed Student's t-tests at P < 0.05.

For the identification of a WPA1 gene, F2 and F3 mapping populations were derived from self-pollination of F1 hybrids of crosses between wpal and ZK331, Zhoumai 22 (ZM22), and Aikang 58 (AK58). For temperature assessment, young seedlings of both ZK331 and wpal were collected from the field and grown in artificial climate chamber under long-day conditions (12 h light/12 h dark). The chamber maintained a light intensity of 300 µmol m*2 s*1 and a relative humidity of 65%-70%, with controlled temperatures at 16 °C and 25 °C.

2.2. DNA extraction, bulked-segregant analysis, and polymorphic DNA marker development

Genomic DNA extraction was performed using the cetyltrimethyl ammonium bromide (CTAB) method [18]. To validate polymorphic DNA markers, DNA-based bulked segregant analysis (BSA) was performed using plant architecture mutant wpa1 and normal DNA bulks, each comprising twenty F2 plants with distinct phenotypes [19]. For polymorphism analyses, wheat genomic SSRs (the Xgwm, Xwmc, Xbarc, Xcfa, and Xcfd series) were selected. Primer sequences for these genomic SSR and EST markers can be retrieved from GrainGenes (https://wheat.pw.usda.gov). The SSR and STS markers in the WPA1 genomic region were designed using BatchPrimer3 [20] and primer3 v.0.4.0 (https://bioinfo.ut.ee/primer3-0. 4.0/), based on the Chinese Spring genome sequence RefSeqv1.0 at the WheatOmics website [21]. Polymorphic SSR and STS markers between the parental lines and the contrasting DNA bulks were used to genotype the F2 segregating populations (Table S1).

2.3. Independence test and linkage analysis

The goodness of fit of observed to expected segregation frequencies was assessed by chi-squared (x²) test. Linked markers were used to construct the genetic linkage map by genotyping the wpa1 x ZK331 F2 segregating population. The Mapmaker 3.0 software [22], with a logarithm of the odds ratio (LOD) of 3.0, was used to determine the genetic distance between the target gene and its linked markers. Recombination frequencies were converted to centimorgan (cM) map distances using the Kosambi mapping function. Maps were drawn with Mapdraw v.2.1 software [23]. Putative gene annotations were performed using Wheatomics website [21].

2.4. Vector construction and transformation

To generate WPA1 genome editing lines, we designed two single-guide RNAs (sgRNAs, gRNA1 and gRNA2) using CRISPRdirect (https://crispr.dbcls.jp/). These sgRNAs were generated to target the conserved sequences in the exons of all three homoeologs. Then the fragment was ligated into the vector pMETaU6.1 by digestion with BsaI. The resulting construct was digested and cloned into the pLGY-E003 vector, yielding the CRISPR/Cas9 gene-editing construct designated pBUE411:sgRNA. Transformation of this construct into wheat line K35 was performed via Agrobacterium-mediated approach using the EHA105 strain [24].

2.5. WPA1 subcellular localization

For subcellular localization in wheat protoplasts, the CDS of both WPA1 and wpal were inserted into pUbi:GFP to obtain pUbi:WPA1-GFP and pUbi:wpa1-GFP constructs. Wheat protoplasts were isoated as described previously with a few modifications [25]. Leaves of wheat seedlings, grown in darkness for approximately 2 weeks, were excised and immersed in an enzyme solution containing 1.5% cellulose R-10 and 0.3% macerozyme R-10 for vacuum infiltration, followed by a 4.5-h incubation at room temperature. After filtration, centrifugation and several washes with W5 buffer (2 mmol L*¹ MES (pH 5.7), 5 mmol L*¹ KCl, 154 mmol L*¹ NaCl, and 125 mmol L*¹ CaCl2), the protoplasts were resuspended in MMG buffer ([4 mmol L*¹ MES (pH 5.7), 0.4 mol L*¹ D-mannitol, and 15 mmol L*¹ MgCl2). Then, 15 µg of each of the pUbi: GFP, pUbi: WPA1-GFP and pUbi:wpa1-GFP were transfected into protoplasts using 40% PEG-4000 (Sigma-Aldrich). The transformed protoplasts were then incubated for 14 h at 22 °C and treated for another 3 h at 6 °C and 25 °C. The GFP signal was visualized with a confocal microscope (488 nm excitation; emission detected between 491-561 nm) with pUbi: GFP serving as the control.

To investigate subcellular localization in Nicotiana benthamiana, the CDSs of WPA1 and wpal were cloned into Super1300:mScarlet vectors to obtain Super1300:WPA1-mScarlet and Super1300:wpa1- mScarlet constructs. Plasmids were transfected into Agrobacterium strain GV3101 and cultured for 2 d at 30 °C. The resulting cell culture was centrifugated, resuspended in infiltration buffer (10 mmol L*¹ MES-KOH (pH 5.7), 10 mmol L*¹ MgCl2, and 150 µmol L*¹ Acetosyingone) to achieve a final OD600 = 0.5, and infiltrated into the leaves of 3-4-week-old N. benthamiana plants using a needle-less syringe. Following a 48 h incubation at 22 °C the transformed leaves were subjected to temperature treatments at 6 °C and 25 °C for 24 h prior to being observed under a confocal microscope (561 nm, 570-660 nm).

2.6. RNA-seq analyses

RNA was extracted from leaves at the five-leaf stage of both ZK331 and wpal plants grown in a growth chamber at 16 °C and 25 °C. Each sample comprised three biological replicates. Total RNA extraction was conducted using TRIzol reagent (Tiangen, Beijing, China). RNA quality was assessed through gel electrophoresis. RNA purity was confirmed with a NanoPhotometer spectrophotometer (IMPLEN, USA), and RNA integrity was evaluated using the RNA Nano 6000 Assay Kit on the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Libraries were constructed and sequenced, generating 150-nt paired-end reads on the DNBSEQ-T7 (MGI Tech Co., Ltd., Shenzhen, Guangdong, China) and processed using the MGI-2000 (MGI Tech Co., Ltd.). Each library yielded approximately 12 Gb of high-quality reads. The reads were mapped to the wheat reference genome (RefSeq v2.1). Then the gene expression level of each sample was calculated using kallisto v0.46.2 [26,27]. Differential expressed genes (DEGs) were identified based on a criterion of P value < 0.05 and log2 (Fold Change) > 1, as determined by DESeq2 (version 3.11)

2.7. Phylogenetic tree construction and selective sweep detecting

Proteins containing both the vWA domain (PF00092) and the Vwaint domain (PF14624) were selected from representative monocots (Triticum aestivum, Oryza sativa and Zea mays), dicots (Arabidopsis thaliana and Gossypium raimondii), basal angiosperms (Amborella trichopoda), pteridophytes (Ceratopteris thalictroides), bryophytes (Physcomitrella patens and Marchantia polymorpha), and algae (Chlamydomonas reinhardtii and Nostoc punctiforme). We aligned the completed amino acid sequences of WPA1 with proteins from these species to identify those displaying the highest similarity, characterized by the presence of both the vWA domain and the Vwaint domain. These identified proteins, along with representatives from lower animals and fungi, were used for construction of a phylogenetic tree using the neighbor-joining (NJ) method.

We employed the XP-CLR test to identify selective sweeps within the wheat as previously described [29,30]. To assess whether WPA1 underwent selective pressures, we ranked the identified selective sweeps based on their XP-CLR scores, retaining the top 5% regions as indicative of selective sweeps. We assigned WPA1 as a target of selection if its genomic location overlapped with these regions.

3. Results

3.1. Identification of mutant wpa1 with pleiotropic developmental defects in bread wheat

A mutant, designated wheat plant architecture (wpa) mutant wpa1, was identified in the field with pleiotropic developmental defects including reduced plant height, altered leaf shape, defective spike development at the heading stage, and shrunken kernels (Figs. 1A–D, S1). The F1 plants showed similar phenotype to the wild type ZK331 rather than the mutant wpa1 (Fig. 1A–D). To test the inheritance pattern of the mutant wpa1, we also crossed wpa1with bread wheat cultivars AK58 and ZM22 (Fig. S2). Genetic analysis of the resulting progenies showed a 3 normal: 1 mutant segregating ratio in the F2 populations, suggesting that the observed developmental defects in wpa1 are controlled by a single gene, wpa1 (Table S2).

3.2. Map-based cloning of gene WPA1

Among 192 representative SSR markers evenly distributed on 21 wheat chromosomes screened for polymorphisms among the parental lines AK58, ZM22 and mutant wpal, Xwmc506 on chromosome arm 7DS was found to have a linkage with the WPA1 locus in the F2 populations. Additional SSR and STS markers developed based on the Chinese Spring reference genome sequence were further used to genotype 317 and 390 F2:3 families from the wpal x AK58 and wpal × ZM22 crosses, respectively. Six recombinants were identified and the closest STS markers M3 and M7 placed WPA1 within a 0.28 CM genetic interval (Fig. 1E). Further narrowing of the WPA1 locus was achieved by genotyping 403 and 1950 mutant-like F2 plants from the wpa1x AK58 and wpa1x ZM22 populations, respectively. The WPA1 locus was narrowed to a 0.03 CM genetic interval between markers M4 and M5 (Fig. 1E), corresponding to a 21.89 kb region on chromosome 7DS according to Chinese Spring RefSeq v1.0. Within this interval, only one high-confidence gene, TraesCS7D01G010200 (Fig. 1F), encoding a protein with von Willebrand factor type A (vWA) domain and a functional metal ion-dependent adhesion site (MIDAS), was predicted. Amplification of full-length cDNA revealed identical sequences across ZK331, AK58 and ZM22, which comprise a single exon with a CDS of 1575 bp and encode a protein of 525 amino acids while wpal carries a G1103A mutation, resulting R368K change in the encoded protein (Fig. 1G, H). Three more mutants (wpa-Mut2, wpa-Mut3, and wpa-Mut4) with similar developmental defects as mutant wpal were identified from the same screening. Two mutants (wpa-Mut5 and wpa-Mut6) from Jimai 22 and one from Nongda 399 (wpa-Mut7), showed similar developmental defects. All six mutants carried a mutation at the WPA1 locus (Fig. S3), suggesting TraesCS7D01G010200 as the most likely candidate gene for WPA1. Two homoeologs WPA1-7A on chromosome arm 7AS and WPA1-4A on chromosome arm 4AL sharing high nucleotide identity with WPA1 were identified in ZK331 (Fig. 54).

3.3. Functional validation of WPA1 via CRISPR/Cas9 assays

To further validate the function of WPA1, we conducted genome editing of WPA1 in the wheat line K35 using the CRISPR/ Cas9 system. K35 contains the same WPA1 allele on 7DS as ZK331 and two homoeologs WPA17AK35 on 7AS and WPA14AK35 on 4AL with 92.56% and 96.56% amino acid identities to WPA1, respectively (Fig. S5). Two specific guide RNAs (gRNA1 and gRNA2) targeting the 50 and 30 terminuses of the WPA1, WPA1-7AK35 and WPA1-4AK35 exons were introduced into K35 using Agrobacterium-mediated transformation and yielded 5 wpa1-like genome-edited lines (WPA1-Ed#1 to WPA1-Ed#5) (Fig. 2A, B, S6A, B), in which the gRNA targeted genomic regions were all edited, but still heterozygous (Fig. S6C). DNA sequence comparison of the gRNA targeted genomic regions revealed mutations at the WPA1 locus, including 3-bp codon deletions in WPA1-Ed#1, WPA1-Ed#2, WPA1-Ed#3, and WPA1-Ed#5 mutants, along with a 270-bp codon deletion in WPA1-Ed #4 mutant (Fig. S6C). Genome-edited mutants WPA1-Ed#1 to WPA1-Ed#5 exhibited stronger developmental defects compared to mutant wpa1. Five genome-edited lines displayed two types of phenotypes, WPA1-Ed#4 and WPA1-Ed#5 displayed weak developmental defects, whereas WPA1-Ed#1 to WPA1-Ed#3 displayed strong developmental defects, possibly because sequence variation occurs at both Nterminal and Cterminus in WPA1Ed#1 to WPA1-Ed#3. These findings suggest that WPA1 is indeed the causal gene, and an altered WPA1 protein may disrupt the developmental process's sequence.

3.4. Temperature dependent developmental defects of wpa1

As wpal did not show developmental defects until its late reproductive phases, we hypothesized that these defects are triggered in wpal by ambient temperatures (Fig. 57). We therefore examined the phenotype of wpal under controlled temperatures and photoperiod. Fully vernalized plants of ZK331 and wpal were transplanted from field into pots and grown in growth chamber at constant 16 °C and 25 °C with 12 h dark/12 h light conditions. Under 25 °C conditions, we observed differences between ZK331 and wpal in plant height, leaf shape, spike length, grain number per spike, and grain size (Fig. 3). Under 16 °C conditions, mutant wpal plants showed no differences in plant architecture or reproductive development from ZK331 for any trait (Fig. 3). This suggests that 25 °C temperatures promote the developmental defects in the mutant wpa1.

RNA-seq was performed to further understand how wpal influences wheat development at different temperatures. Consistent with the observed developmental anomalies in wpal under 25 °C temperatures, principal component analyses (PCA) revealed a distinct separation in global transcriptional patterns between ZK331 and the mutant wpal at 25 °C compared to 16 °C. Principal component 1 accounted for 59.31% of the variance at high ambient temperature (Fig. 4A). At different temperatures (25 °C or 16 °C), volcano plots of transcriptome data showed that DEGs between ZK331 and wpal at 25 °C (2045 DEGs) were more than that at 16 °C (345 DEGs) (Fig. 4B). This suggests that wpal triggers alterations in transcriptome profile under 25 °C. To elucidate the role of WPA1 in the temperature regulation of wheat development, a Venn diagram was constructed with DEGs from both ZK331 and the mutant wpal across different temperatures. The results uncovered 700 DEGs responsive to temperatures and regulated by both ZK331 and wpal (Fig. 4C). Gene Ontology (GO) enrichment of these DEGs revealed enrichment in the photosynthesis pathway, indicating that genes from this group are primarily influenced by temperatures. To further understand WPA1's role in temperature-regulated development, we conducted another GO enrichment analysis with DEGs affected by wpal in response to temperatures. The results revealed enrichment in pathways associated with light responsiveness, reproductive growth, and flowering (Fig. 4D), suggesting that WPA1 may modulate these developmental processes by influencing gene expression.

To further probe how elevated ambient temperatures lead to the developmental defects in the mutant wpal, the subcellular localization of WPA1 in N. benthamiana and wheat protoplast was determined using transient assays. Fluorescent signals of WPA1-GFP driven by the Ubi promoter and WPA1-mScarlet driven by the Super promoter were observed in cytoplasm (Fig. 58). The mutant version wpal exhibited a similar localization pattern as WPA1 (Fig. 58), suggesting that the wpal (R368K) point mutation does not alter its subcellular localization. To test whether WPA1 localization altered in response to environmental temperatures, we treated the transformed wheat protoplasts at 25 °C and 6 °C for 3 h and N. benthamiana at 25 °C and 6 °C for 24 h. The results showed no subcellular localization difference for WPA1 and wpal in response to environmental temperatures (Fig. S8), suggesting that WPA1 subcellular localization may not be responsible for the temperature regulation of wpal development. Instead, WPA1 and WPA1-7A alleles may regulate the wheat architecture by increasing expression levels at 25 °C temperature (Fig. 4E). These results suggest that WPA1 may be involved in these developmental processes by triggering substantial changes in transcriptome profile due to different temperatures experienced by plants in the field.

3.5. Phylogenetic analysis of WPA1 homologs

To understand the evolutionary origin of WPA1, WPA1 homologs were identified across species. Proteins containing the vWA and Vwaint domains are widely present in most plant species, as well as certain fungi and animals. However, during the course of evolution, these WPA1 homologous proteins have undergone diversification into various forms with or without the RING structural domain in higher plants. Proteins lacking RING domain, such as WPA1, appear to be present only in the monocotyledon (Fig. 5), indicating an uncovered function of WPA1 in regulating development plasticity in response to environments. The sequence variations of WPA1 in a set of 795 resequenced worldwide wheat accessions from different geographic regions were then compared [29,30]. Using the cross-population composite likelihood ratio (XPCLR), no selection signatures were found in the WPA1 locus during wheat domestication (Fig. S9). These results suggest that WPA1 may be highly conserved in wheat adaptation to distinct local climates.

4. Discussion

Global warming has impacted various plant species, influencing their distribution, phenology and biodiversity [31]. Environmental temperatures play a crucial role in regulating the developmental plasticity of plants, including important crops [31]. Improving crop resilience to elevating temperature is becoming increasingly important. In this study, we identified a novel vWA domain containing protein in bread wheat, which is associated with developmental defects in the mutants in field condition. These developmental defects can be influenced by different temperatures. The finding that WPA1 is involved in temperaturemediated regulation of wheat development and architecture suggests it might as a target gene for engineering wheat in the situation of climate change.

Recent advances in genetic analysis of wheat populations have provided insights into the selection signatures present in wheat genomes. Sequence polymorphisms comparison of the WPA1 homeologs between wheat landraces and modern cultivars revealed that the chromosomal regions containing WPA1, WPA17A and WPA1-4A exhibited XP-CLR scores below the top 5%, which is a common threshold for identifying domesticated sweeps (Fig. S9). This result suggests that none of the WPA1 homeologs were under intense human selection pressure during wheat improvement. Despite the absence of strong selection signals, WPA1 remains a viable target for genetic manipulation to create new alleles for designing temperature resilient wheat cultivars.

The vWA domain mediates physical interactions of proteins. We found that WPA1 is involved in different biological processes. However, it remains unexplored whether this vWA domain mediated protein interaction is temperature dependent. In Arabidopsis, BONZAI 1/COPINE 1 (BON1/CPN1) gene, which encodes vWA domain, regulates immunity responses, and loss-of-function mutations in this gene lead to aberrant immune regulation with a temperature-sensitive lesion-mimic phenotype. Transgenic plants expressing the BON1/CPN1 vWA domain displayed a lesionmimic phenotype reminiscent of bon1/cpn1 mutants, suggesting that the exogenous BON1/CPN1 vWA domain fragment may disrupt the normal function of the endogenous BON1/CPN1 protein by interfering with its protein interactions [32]. Recent research [33] has further elucidated that the autoimmunity of bon mutants in Arabidopsis is caused by the activation of TNL-type R proteins, while growth defects in maize and Arabidopsis are largely attributed to the deficient BR signaling. The rice LGD1 gene, which also encodes a vWA domain-containing protein, pleiotropically regulates rice vegetative growth and development, suggesting multiplex regulatory function of vWA domain [34]. vWA domaincontaining proteins undergo conformational changes when binding divalent cations, such as Ca2+, Mg2+, or Mn2+ [9,35]. Most vWA domains carry a key metal ion-dependent adhesion site (MIDAS) [36], which is pivotal for their protein interactions [9,32]. This finding suggests that vWA domain-containing proteins, including BON1/CPN1, function in multiple signaling pathways.

The temperature-dependent regulatory function of WPA1 in wheat plant architecture development need further investigation.

In summary, we cloned a temperature-dependent gene WPA1 that encodes a vWA domain protein and regulates wheat plant architecture. WPA1 is specific to monocotyledonous plants among higher plants, revealing a potentially novel pathway for temperature regulation of development in these species. WPA1 may be a target for design of wheat cultivars resilient to climate change.

CRediT authorship contribution statement

Yongxing Chen: Conceptualization, Resources, Investigation, Data curation, Writing – original draft. Huixin Xiao: Formal analysis, Visualization, Writing – original draft, Writing – review & editing. Yuange Wang: Data curation, Investigation, Writing – original draft. Wenling Li: Investigation. Lingchuan Li: Data curation, Formal analysis. Lingli Dong: Formal analysis. Xuebo Zhao: Methodology. Miaomiao Li: Investigation. Ping Lu: Investigation. Huaizhi Zhang: Investigation. Guanghao Guo: Investigation, Formal analysis. Keyu Zhu: Investigation. Beibei Li: Investigation. Lei Dong: Investigation. Peng Chen: Investigation. Shuming Wu: Formal analysis. Yunbo Jiang: Formal analysis. Fei Lu: Methodology. Chengguo Yuan: Investigation, Resources. Zhiyong Liu: Project administration, Writing – review & editing, Funding acquisition. Yusheng Zhao: Project administration, Writing – review & editing, Funding acquisition. Qiuhong Wu: Project administration, Investigation, Writing – review & editing, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Many thanks to Genying Li of Shandong Academy of Agriculture Sciences, Jinan, Shandong, China, for developing the transgenic wheat. This research was financially supported by the Key Research and Development Program of Zhejiang (2024SSYS0099), the National Key Research and Development Program of China (2022YFD1200203), and Key Research and Development Program of Hebei province (22326305D).

Appendix A. Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2024.05.008.

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ARTICLE INFO

Article history:

Received 5 May 2024

Revised 21 May 2024

Accepted 22 May 2024

Available online 6 June 2024

Footnote

* Corresponding authors.

E-mail addresses: [email protected] (Z. Liu), [email protected] (Y. Zhao), [email protected] (Q. Wu).

1 These authors contributed equally to this work.

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