Investigating the mechanisms of isochorismate synthase: An approach to improve salicylic acid synthesis and increase resistance to Fusarium head blight in wheat

Abstract

Salicylic acid (SA), a vital endogenous hormone, plays a crucial role in plant growth and the response to abiotic and biotic stress. Isochorismate synthase (ICS) and phenylalanine ammonia lyase (PAL) are critical rate-limiting enzymes for SA synthesis. Fusarium head blight (FHB) seriously threatens the safety of wheat production, but increasing the content of SA can enhance FHB resistance. However, the pathway of SA synthesis and regulation in wheat remains unknown. In this study, three wheat ICS (TaICSA, TaICSB, and TaICSD) were identified, and their functions were validated in vitro for isomerizing chorismate to isochorismate. The mutation of one or two homoeoalleles of TaICSA, TaICSB, and TaICSD in the wheat variety 'Cadenza' reduced SA levels under ultraviolet treatment and Fusarium graminearum infection, further enhancing sensitivity to FHB. Overexpression of TaICSA can significantly enhance SA levels and resistance to FHB. To further study SA synthesis pathways in wheat and avoid interference with pathogenicity related genes, the leaves of wild-type Cadenza and different TaICS mutant lines were subjected to ultraviolet treatment for transcriptomic analysis. The results showed that 37 PALs might be involved in endogenous SA synthesis, and 82 WRKY and MYB family transcription factors may regulate the expression of ICS and PAL. These results were further confirmed by RT-PCR. In conclusion, this study expands our knowledge of SA biosynthesis and identifies TaICSA, as well as several additional candidate genes that encode transcription factors for regulating endogenous SA levels, as part of an efficient strategy for enhancing FHB resistance in wheat.

Headnote

ABSTRACT

Salicylic acid (SA), a vital endogenous hormone, plays a crucial role in plant growth and the response to abiotic and biotic stress. Isochorismate synthase (ICS) and phenylalanine ammonia lyase (PAL) are critical rate-limiting enzymes for SA synthesis. Fusarium head blight (FHB) seriously threatens the safety of wheat production, but increasing the content of SA can enhance FHB resistance. However, the pathway of SA synthesis and regulation in wheat remains unknown. In this study, three wheat ICS (TaICSA, TaICSB, and TaICSD) were identified, and their functions were validated in vitro for isomerizing chorismate to isochorismate. The mutation of one or two homoeoalleles of TaICSA, TaICSB, and TaICSD in the wheat variety 'Cadenza' reduced SA levels under ultraviolet treatment and Fusarium graminearum infection, further enhancing sensitivity to FHB. Overexpression of TaICSA can significantly enhance SA levels and resistance to FHB. To further study SA synthesis pathways in wheat and avoid interference with pathogenicity related genes, the leaves of wild-type Cadenza and different TaICS mutant lines were subjected to ultraviolet treatment for transcriptomic analysis. The results showed that 37 PALs might be involved in endogenous SA synthesis, and 82 WRKY and MYB family transcription factors may regulate the expression of ICS and PAL. These results were further confirmed by RT-PCR. In conclusion, this study expands our knowledge of SA biosynthesis and identifies TaICSA, as well as several additional candidate genes that encode transcription factors for regulating endogenous SA levels, as part of an efficient strategy for enhancing FHB resistance in wheat.

Keywords:

Salicylic acid

Chorismate

Isochorismate

RNA-seq

Phenylalamine ammonia lyase

1. Introduction

The phytohormone salicylic acid (SA, 2-hydroxybenzoic acid) is a small phenolic compound that is widely involved in plant growth and the response to abiotic and biotic stress [1,2]. Since the first report of SA functioning as an endogenous signal for plant disease resistance [3], the association between SA and plant disease resistance has received close attention. Many studies have shown that SA accumulation can induce pathogenesis-related gene expression and establish systemic acquired resistance (SAR), a long-lasting, broad-based resistance to subsequent pathogen infection [2,4,5].

The isochorismate synthase (ICS) and phenylalanine ammonia lyase (PAL) pathways are the main synthetic pathways of SA, and the ICS pathway in particular plays a crucial role in SA synthesis and plant resistance to pathogen infection. As a critical enzyme in the ICS pathway, ICS catalyzes the conversion of chorismate to isochorismate in chloroplasts [2]. ICS expression and SA accumulation can be stimulated by subjecting the leaves to ultraviolet (UV) treatment and can be successfully used to explore the mechanism of SA synthesis and regulation in Arabidopsis thaliana [6–8]. In Arabidopsis thaliana, the deletion of AtICS1 and AtICS2 results in a 90% reduction in SA accumulation after UV treatment [9,10]. In Nicotiana benthamiana, silencing of NbICS leads to significantly decreased SA accumulation under UV treatment [11]. The deletion of AtICS1 leads to increased susceptibility to virulent pathogens, decreased resistance to avirulent pathogens, reduced defense gene expression, and failure to develop SAR [2]. Deletion of HvICS in barley results in reduced resistance to Fusarium, while deletion of phenylalanine ammonia-lyase (PAL) has no significant effect on disease resistance [12]. Therefore, ICS genes play an important role in plant immunity.

Many transcription factors have been identified as specifically binding ICS or promoters to regulate SA synthesis [13]. For example, AtWRKY28 and AtWRKY46 bind to the "TGAC" core element in the promoter of AtICS1 to induce high AtICS1 expression and promote SA synthesis [14]. Wang et al. [15] found that transcription factor TCP8 of the TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family can specifically bind to the promoter of AtICS1, promote AtICS1 expression, and interact with TCP9 to inhibit ICS expression, thereby maintaining SA balance in plants [15]. The interaction of Ethylene-insensitive 3 (EIN3) and NAC domain-containing protein 19 (ANAC019) can directly inhibit AtICS1 expression [16], and Non-expressor of PR gene 1 (NPR1) inhibits PR1 expression and negatively regulates AtICS1 expression [17]. Thus, ICS expression is regulated by a variety of transcription factors that regulate SA accumulation in plants.

Fusarium head blight (FHB), which is caused by species in the genus Fusarium seriously threatens the safety of wheat production [18,19] After infection with FHB, mycotoxins, such as nivalenol (NIV), deoxynivalenol (DON), 3-acetyl deoxynivalenol (3-ADON), and 15-acetyl deoxynivalenol (15-ADON), are produced in wheat seeds. These mycotoxins can lead to several symptoms in humans and animals, such as anorexia, diarrhea, vomiting, and gastrointestinal bleeding [20]. Due to the impact of global warming and changes in cropping systems, FHB outbreaks have caused significant global economic losses in wheat, and their severity and frequency appear to be increasing [21]. SA is an important endogenous hormone that helps plants resist infection by Fusarium graminearum, the main pathogen causing FHB. Three main explanations for SA-mediated FHB resistance. 1) SA mediates systemic acquired resistance and promotes the accumulation of pathogenesis-related (PR) proteins to confer FHB resistance [12]; 2) SA triggers membrane lipid nanodomain reorganization, thereby regulating plasmodesmata closure to impede pathogenic spreading [44]; 3) SA directly inhibits the conidial germination and hyphal growth of F. graminearum [22,23]. Due to the complexity of the wheat genome and the functional diversity of homologous genes, as well as the synthetic and regulatory pathways of SA, which are still unclear in wheat, there is a lack of candidate genes for improving the SA content in wheat spikelets.

To investigate the synthetic and regulatory pathways of SA in wheat, we cloned ICS from the wheat genome and identified its enzyme activity in Escherichia coli. Early termination mutants of ICS were constructed to study the role of ICS in wheat growth and FHB resistance. Potential PAL pathways and regulators of ICS expression were explored through transcriptome analysis. We aimed to increase the endogenous SA content and strengthen FHB resistance in wheat by regulating candidate genes involved in SA synthesis.

2. Materials and methods

2.1. Sequence analysis

Wheat gene and protein sequences were downloaded from the Ensembl database (http://plants.ensembl.org/index.html). DNAman9.0 software (Lynnon Biosoft, San Ramon, CA, USA) was used for aligning protein sequences. The SRAMP online tools (http:// www.cuilab.cn/sramp) were used for m6A methylation site prediction. The SMART online tool was used for the specific domain - analysis of protein sequences (https://smart.embl-heidelberg.de/).

2.2. Functional complementation of the PchB strain of E. coli

According to the methods of previous studies [9,24], Pchb (NCBI number: CP124674) was synthesized by Tsingke Biotechnology (Beijing, China), as shown in Fig. S1, and further constructed into PET-32(+) through double digestion with 5' Sacl and 3' Xhol (New England Biolabs, USA) (PET-32(+)-PchB). The cDNA sequences of TalCSA (primer pair: 32aPchB-TaICSA-F + 32aPchB-T aICSA-R), TaICSB (primer pair: 32aPchB-TaICSA-F + 32aPchB-TalCS A-R), and TalCSD (primer pair: 32aPchB-TaICSA-F + 32aPchB-TaIC SA-R) were cloned from the 'Cadenza' variety and used to construct PET-32(+)-PchB through signal digestion with Notl (New England Biolabs, Ipswich, MA, USA). All of the primer sequences used are listed in Table S2. These plasmids were used to transform the E. coli strain (BL21 DE3 PlysS, #D1015M, Beyotime Biotechnology). The transformed cells were grown in Luria-Bertani (LB) medium (tryptone 10 g L*¹, yeast extract 5 g L*¹, and NaCl 10 g L*¹) supplemented with 50 µg mL*¹ ampicillin at 37 °C. Synthesis of the ICS protein was induced at an OD600 of 0.8 by introducing isopropyl B-D-thiogalactopyranoside (IPTG) until it reached a final concentration of 0.1 mmol L*¹. The IPTG-induced culture was grown for 12 h at 37 °C before the cells were collected via centrifugation, washed twice with 10 mmol L*¹ MgSO4, and resuspended in 50 µL of 10 mmol L*¹ MgSO4. Finally, the diluted cells were inoculated with a toothpick and streaked onto mCAS medium (Kulaibo Technology Co., LTD., # pm0820-1L, Beijing, China). The culture was incubated at 37 °C for 24 h, and the color changes around the marked bacteria solution were observed. Fractions containing proteins were assessed using SDS-PAGE.

2.3. Plant materials and growing conditions

All the EMS mutants and transgenic wheat used in this work were in the Triticum aestivum (tetraploid, AABBDD genome) 'Cadenza' ecotype background. All plants were grown in a climate chamber with 16 h of daylight at 25 °C and 8 h of darkness at 18 °C, with 55% humidity (Xunon Instruments, Beijing, China). At least two generations of transgenic and EMS mutants were observed with a consistent phenotype.

2.4. Subcellular localization

The cDNA sequences of TaICSA (primer pair: 163-TaICSA-F + 1 63-TaICSA-R), TaICSB (primer pair: 163-TaICSA-F + 163-TaICSA-R) , and TaICSD (primer pair: 163-TaICSA-F + 163-TaICSA-R) were cloned from 'Cadenza' and used to construct 163-UBI-hGFP5 (kindly provided by the Chinese Academy of Sciences) through double digestion with 50 HindIII and 30 BamHI (New England Biolabs, USA). Then 'Cadenza' protoplasts were extracted and transformed as described by Zong et al. [25]. The localization of the TaICSA, TaICSB, and TaICSD proteins in wheat protoplasts was observed using a Leica microscope (Leica Microsystems Trading Co., Shanghai, China).

2.5. Disruption and overexpression of TaICS

Early termination mutants (Cadenza0236, *TaICSA; Cadenza0917, *TaICSB; and Cadenza0635, *TalCSD) were obtained from the EMS mutant library of 'Cadenza' (middle susceptible to FHB in Sichuan) using TalCSA, TalCSB, and TalCSD. The *TalCSAB mutant was obtained in the F2 generation by crossing *TalCSA with *TaICSB, and then by further crossing with *TaICSD to create the *TalCSABD heterozygous mutant. Finally, backcrosses were performed twice with wild-type 'Cadenza' (WT). Following specific primers (Table S2), the homozygous mutants of WT, *TaICSA, *TaICSB, *TaICSD, *TaICSAB, *TaICSAD, *TaICSBD, and *TaICSABD were selected.

The cDNA sequences of TaICSA (primer pair: UBI-TaICSA-F + U BI-TaICSA-R), TaICSB (primer pair: UBI-TaICSA-F + UBI-TaICSA-R), and TaICSD (primer pair: UBI-TaICSA-F + UBI-TaICSA-R) were cloned from 'Cadenza' and linked to the pCAMBIA1300-BAR construct (kindly provided by Yan-peng Wang, Chinese Academy of Sciences) through double digestion with 50 BamHI and 30 SmaI (New England Biolabs, USA). All vectors were transferred into 'Cadenza' with reference to the method of Zong et al. [25]. The T1 transgenic lines obtained were verified by the primer pair Ubi1899F + Bar496R (Table S2). Phenotypic analyses were completed in the T2 generation, and the results were confirmed in the T3 generation.

2.6. RNA sequencing and data processing

The leaves of all homozygous mutants were treated with 254 nm UV for 30 min at Zadoks stage 13 (Zadoks cereal development scale). The third leaf was used to isolate total RNA using a Plant RNAprep Pure Kit (TIANGEN Biotech, Beijing, China), according to the manufacturer's instructions. RNA sequencing was completed at E-GENE (Shenzhen E-GENE Technology, Shenzhen, Guangdong, China). The expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) was used to standardize the expression level of each gene based on gene length and sequencing depth [26]. Normalization and differential expression analysis were performed using DESeq2 [27]. Genes were considered significantly differentially expressed if they had a log2 (fold change) > 1 or < 1 and a P < 0.01. The annotations for the mRNA transcript sequences were derived from the EnsemblPlants database (http://ensemblgenomes.org/). TopGO was used for GO enrichment analysis [28]. ClusterProfiler 4.0 was used for KEGG pathway analysis (FDR < 0.05) [29].

2.7. Quantitative RT-PCR and qPCR validation

Total RNA was extracted from 100 mg (fresh weight) of plant tissue using the E.Z.N.A. Total RNA Kit I (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer's instructions. The RNA was reverse transcribed using the PrimeScript RT Reagent Kit with Genomic DNA Eraser (Takara, Dalian, Liaoning, China) according to the manufacturer's protocol. The genes selected for RT-PCR and qPCR analysis met two of three criteria: 1) being expressed at a sufficiently high level to be detectable by RT-qPCR and qPCR analysis; 2) having a sufficiently long region with a unique sequence, which allowed for the design of genome-specific primers; or 3) showing a significant difference in expression between the WT and ICS mutants. The primers used are described in Table S2. The 2***CT method was used for the analysis of relative gene expression data [30]. TaGAPDH (TraesCS7B02G213300) was used as a reference for RT-PCR. TaAox (TraesCS2D02G335300), TaGAPDH, and Tahn-RNP-Q (TraesCS2D02G388400) were used as references for qPCR [22].

2.8. Measurement of SA and JA

Two florets from each fully developed spikelet in a whole spike at the mid-anthesis stage were inoculated with 1 x 103 conidia, and the humidity was increased to 85%. The inoculated wheat plants were treated according to the description provided above. At 0, 48, and 96 h, the inoculated spikes were harvested and ground to a fine powder in liquid nitrogen. The Plant RNAprep Pure Kit (TIANGEN Biotech) was used to isolate RNA from 0.2 g of plant material, following the manufacturer's instructions. Analyses were completed with three biological replicates per treatment. The SA content in wheat ears was determined according to a study by Fragnière et al. [6]. The chromatographic results were analyzed using an Agilent (Agilent Technologies, Santa clara, CA, USA) C18 Eclipse (5 µm, 2.1 x 150 mm) column and OpenLab CDS (Agilent Technologies) software. The JA content was determined using a monoclonal antibody-based ELISA (#RXJ1401587PL, Ruixin Biological Technology, Quanzhou, Fujian, China), following the manufacturer's instructions.

2.9. FHB resistance and fungal biomass assay

Fusarium graminearum (PH-1, main pathogen of FHB) conidia were collected according to Capellini and Peterson (1965). Two florets from each fully developed spikelet in a whole spike at the midanthesis stage were inoculated with 1 x 103 conidia. After inoculation, all plants were placed in a climate chamber with 16 h of day-time at 25 °C and 8 h of nighttime at 18 °C, wrapped in moist plastic wrap, and incubated for 48 h at 25 °C. The number of infected spikelets was statistically analyzed 10 d after inoculation. Fungal biomass was estimated using the expressed levels of FgGAPDH and determined according to the method in a previous study [23].

2.10. Statistical analysis

Student's t-test (implemented in GraphPad Prism 9.0 software, San Diego, CA, USA) was used to test the significance of differences among the average values of SA content, JA content, relative expression levels of genes, and disease level. TBtools was used for heatmap and Venn diagram analysis [31].

3. Results

3.1. TaICSA, TaICSB, and TaICSD are predicted as isochorismate synthases

Three homologous genes (TraesCS5A02G193800, TraesCS5B0 2G189100, and TraesCS5D02G196200) were predicted to encode ICS in the wheat genome database, named TaICSA, TaICSB, and TaICSD, respectively. The open reading frames (ORFs) of these three homologous genes contained 15 exons and 14 introns, and their predicted protein sizes were 63.04 (TaICSA), 62.40 (TaICSB), and 62.59 kDa (TaICSD). The coding sequences (CDSs) of TaICSA, TaICSB, and TaICSD were further confirmed by Sanger sequencing in 'Cadenza'. Further analysis of these three homologous protein variations in the wheat genome revealed a conserved sequence in the hexaploid wheat reference accessions (Fig. S2A). Domain analysis results showed that the three homologous proteins have a conserved chorismate binding enzyme (Pfam00425) domain, and protein sequence alignment revealed different sequences at DNAbinding transcription factor activity (SM00380) regions (Fig. S2B). Thus, these differences in protein sequences may lead to differences in efficiency or function between TaICSA, TaICSB, and TaICSD.

3.2. TaICSA, TaICSB, and TaICSD isomerize chorismate into isochorismate in chloroplasts

TaICSA, TaICSB, and TaICSD were predicted to be located in the chloroplast of wheat by means of the web tool Cell-PLoc 2.0 (https://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/). For confirming this prediction, three plasmids were constructed with the CDS of TaICSA, TaICSB, or TaICSD fused with green fluorescent protein (GFP) and driven by a ubiquitin (UBI) promoter. The three fused proteins were expressed using the wheat protoplast system.

The confocal microscopy observation showed that UBI:TaICSAGFP, UBI:TaICSB-GFP, and UBI:TaICSD-GFP were detected only in the chloroplast, whereas UBI:GFP was distributed throughout the entire cell (Fig. 1A). This result supports the bioinformatic prediction that TaICSA, TaICSB, and TaICSD are chloroplast-located isochorismate synthases.

To determine the function of ICS encoded by TaICSA, TaICSB, and TaICSD, we conducted a functional assay system based on the Fenton reaction in Escherichia coli [9,24,32] (Fig. 1B). The CDSs of TaICSA, TaICSB, and TaICSD were constructed into the recombinant pET-32a(+)-PchB vector (Fig. 1C). Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the protein sizes of TaICSA, TaICSB, and TaICSD were approximately 60 kDa, as expected (Fig. 1D). An orange halo was produced by transforming TaICSA, TaICSB, and TaICSD in a modified chrome azurol sulphonate agar (mCAS) medium (Fig. 1E). These results indicate that all three alleles, namely, TaICSA, TaICSB, and TaICSD, are functional alleles involved in the isomerization of chorismate into isochorismate.

3.3. Loss function of ICS reduces SA accumulation under UV stress

To further investigate the effects of ICS on wheat growth and FHB resistance, we screened the nonsense mutations of TalCSA, TaICSB, and TaICSD from TILLING populations. The mutations were confirmed by Sanger sequencing, which showed that *TaICSA, *TaICSB, and *TalCSD had amino acid changes, leading to stop codons (Fig. 2A). The cDNA sequences of *TaICSA, *TaICSB, and *TalCSD were individually inserted into the recombinant pET- 32a-PchB vector for ferrophilic detection (Fig. 2B). None of the transgenic cells produced an orange halo (Fig. 1E), suggesting that nonsense mutations in TalCSA, TaICSB, and TalCSD resulted in the loss of ICS enzyme activity.

To generate mutant plants containing multiple mutations of the TalCS gene, crosses were performed between *TaICSA, *TaICSB, and *TaICSD, resulting in eight combinations of mutations, namely WT, *TaICSA, *TaICSB, *TaICSD, *TaICSAB, *TaICSAD, *TaICSBD, and *TICSABD, which were used for further experiments. Phenotypic analysis showed that the homozygous plants of *TaICSBD and *TICSABD could not survive at Zadoks stage 29 (Fig. 2B), possibly due to insufficient SA [9]. Therefore, WT, *TaICSA, *TaICSB, *TaICSD, *TaICSAB, and *TaICSAD were used for further analysis.

At the three-leaf stage, the ICS mutants showed no significant difference in SA accumulation compared to the WT (Fig. 2C). However, it could be induced by UV treatment, and the simultaneous deletion of TaICSA and TaICSD significantly reduced SA accumulation (Fig. 2C). The deletion of TaICSB alone significantly reduced SA accumulation in wheat, indicating that TaICSB plays a crucial role in SA accumulation under UV stress.

We further quantified the expression levels of TaICSA, TaICSB, and TaICSD using quantitative (q)PCR in the WT under UV treatment. As expected, TaICSA, TaICSB, and TaICSD showed significant expression, with TaICSB exhibiting higher levels than TaICSA and TaICSD. The expression level of TaICSA was significantly upregulated, while TaICSD was significantly downregulated by UV treatment (Fig. 2D). The above results also confirm that TaICSA, TaICSB, and TaICSD exhibit differences in efficiency.

3.4. ICS deletion reduces SA accumulation and increases FHB susceptibility

The FHB resistance assay showed that resistance was significantly reduced in *TalCSA, *TaICSB, *TaICSD, *TaICSAB, and *TaICSAD (Fig. 3A). Analysis of the SA content showed no significant difference in SA accumulation between WT and ICS mutants in the spikelets at 0 h after inoculation with PH-1. However, 48 h after inoculation with PH-1, *TaICSA, *TaICSB, *TaICSAB, and *TalCSAD, but not *TalCSD, showed a significant decrease in SA accumulation (Fig. 3B). We further examined the expression levels of TalCSA, TalCSB, and TalCSD after F. graminearum infection in the WT, and we found that only TalCSA was significantly induced as a result of infection, while TalCSD was dramatically repressed and TalCSB did not change significantly (Fig. 3C). These results indicate that ICS deletion improves wheat FHB susceptibility and that TalCSA is significantly induced by F. graminearum infection, making it a potential candidate gene for improving FHB resistance in wheat.

3.5. Overexpression of TaICSA increases resistance to FHB

Given the significant upregulation of TaICSA under UV treatment and F. graminearum infection (Figs. 2D, 3C), we overexpressed TaICSA in 'Cadenza' (Fig. 4A). The three stable transgenic lines with the highest TaICSA (OE-38, OE-41, and OE-48) expression levels were selected for further analysis (Fig. 4B, C). As expected, the spikelets of OE-41 and OE-48 exhibited a significant increase in SA levels (Fig. 4D), as well as significantly enhanced resistance to FHB compared to the WT (Fig. 4E, F, G).

Compared with OE-41 and OE-48, OE-38 exhibited lower expression of TaICSA and SA accumulation, which may weaken the prevention of hyphal spreading in the early stage of F. graminearum infection [22–44]. This further resulted in no significant difference in biomass after 2 d of infection and a significant increase in spikelets after 10 d of F. graminearum infection (Fig. 4E, F, G). Therefore, adequate TaICSA overexpression can increase the accumulation of endogenous SA in wheat and enhance resistance to FHB.

3.6. Screening of genes regulating ICS expression and endogenous SA synthesis

We observed no significant difference in ICS mutants without UV treatment or F. graminearum infection compared to the WT (Figs. 2C, 3B). These results suggest that unidentified synthetic or regulatory pathways may compensate for the reduction in SA accumulation resulting from ICS deletion. To further investigate any unknown genes that may regulate or participate in SA synthesis and to avoid interference with pathogenicity related genes, RNA sequencing (RNA-seq) data were obtained from WT plants and ICS mutants under UV treatment using tri-leaf wheat leaves. Transcriptome analysis identified 100,266 differentially expressed genes (DEGs) between the WT plants and the ICS mutants (Table S3). When comparing UV-treated WT and ICS mutants with the non-treated WT, the number of DEGs in the ICS mutants was higher than that in the WT, especially for *TaICSA (Fig. 5A). In all DEGs, we observed specific DEGs in *TaICSA (1231 genes), *TaICSB (213 genes), *TalCSD (352 genes), *TaICSAB (583 genes), and *TalCSAD (351 genes) compared to the WT under UV treatment (Fig. 5B).

We further analyzed specific DEGs in ICS mutants using Gene Ontology (GO) enrichment analysis. The results predicted that 154 transcription factors participate in SA synthesis and regulation. These included GO:0009751 (response to SA), GO:1901149 (SA binding), GO:0080142 (regulation of SA biosynthetic process), GO:0052640 (SA glucosyltransferase glucoside-forming activity), GO:0009863 (SA mediated signaling pathway), and GO:2000031 (regulation of SA mediated signaling pathway). Through the functional annotation of these genes, it was found that 51.92% (82) belonged to the WRKY and MYB transcription factor families (Fig. 5C). Details about these genes are listed in Table S4. WRKY and MYB transcription factors may be involved in regulating the differential expression of TaICSA, TaICSB, and TaICSD.

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that 37 DEGs were involved in SA synthesis and significantly differentially expressed in ICS mutants and the WT (Table S5). After UV irradiation, the expression levels of the candidate PALs were induced to varying degrees (Fig. 5D). However, most genes were inconsistently expressed in different ICS mutants. *TalCSA and *TaICSAB were grouped together in a heat-map, while *TaICSB, *TaICSD, and *TalCSAD formed a different cluster (Fig. 5D). We also identified six genes predicted to encode chorismate mutase (CM) within the PAL pathway, and observed significant differences in their expression between the WT and ICS mutants following UV treatment (Table S5). The above results suggest that the PAL pathway may compensate for the obstruction of SA accumulation caused by the loss of ICS enzyme activity.

3.7. Validation of genes regulating ICS expression and SA synthesis

To validate the transcriptome sequencing results, we performed reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qPCR) on a serious of candidate genes, including three genes predicted to encode PAL enzymes (TaPAL1, TaPAL2, and TaPAL3), two genes predicted to be MYB transcription factors (TaMYB1 and TaMYB3), and three genes predicted to be WRKY transcription factors (TaWRKY50, TaWRKY56, and TaWRKY57). The RT-PCR results showed that these genes were significantly differentially expressed in the ICS mutants (Fig. 6A). As expected, further qPCR analysis revealed a significant increase in the expression levels of TaPAL1 in *TaICSB, *TaICSD, *TaICSAB, and *TaICSAD, with significant differential expression (Fig. 6B). The expression levels of TaMYB1 and TaWRKY57 significantly increased in the WT and ICS mutants following UV treatment. Additionally, significant differential expression of these transcription factors was observed in the ICS mutants (Fig. 6B). These results confirm our transcriptome data and suggest that PAL-related genes can compensate for the obstruction of SA accumulation caused by the loss of ICS enzyme activities. Additionally, WRKY and MYB transcription factors play an important regulatory role in SA accumulation.

To further investigate the effects of TalCSA overexpression on the above transcription factors, we performed qPCR analysis to detect the expression levels of MYB and WRKY transcription factors in the WT, *TaICSA, and OE-48 samples. The expression of TaMYB1 and TaMYB3 was significantly induced in *TalCSA, while TaWRKY55 and TaWRKY57 were significantly induced in OE-48 (Fig. 6C). After 48 h of inoculation with F. graminearum, the expression levels of TaMYB1 and TaWRKY55 were significantly inhibited in *TaICSA, TaMYB3 was significantly inhibited in OE-48, and TaWRKY57 was significantly induced in *TaICSA (Fig. 6D). Thus, TaMYB1, TaMYB3, TaWRKY55, and TaWRKY57 may be involved in regulating TalCSA expression in wheat and in the process of SA synthesis.

To determine if the SA synthesis pathways induced by UV and F. graminearum infection are similar, all DEGs of TaMYB and TaWRKY induced by UV were further checked in a previously published transcriptome data in response to F. graminearum infection [45]. The results showed that 75 out of 82 TaMYB and TaWRKY were differentially expressed under F. graminearum infection (Table S4), as well as TaMYB1, TaMYB3, and TaWRKY57 were upregulated in response to UV treatment. Therefore, these results also indicate that transcriptome data obtained from UV treatment can be utilized to identify candidate genes associated with wheat FHB resistance.

JA accumulation was evaluated in the WT, DTaICSA, and OE-48 after inoculation with F. graminearum. The JA content in DTaICSA and OE-48 was significantly higher than that in the WT at 0 h. However, it decreased significantly 48 and 96 h after inoculation with F. graminearum (Fig. 6E). These results indicate that the absence or increase in SA can induce or antagonize JA synthesis after F. graminearum infection.

4. Discussion

SA is a crucial plant hormone that plays a significant role in plant growth and the response to abiotic and biotic stress [1,33]. The ICS and PAL pathways are primarily recognized for SA synthesis [2,34]. In the ICS pathway, chorismate is initially converted into isochorismate by ICS in the chloroplasts. Then, the Multidrug and Toxic Compound Extrusion (EDS5) protein transports it to the cytoplasm [35]. The avrPphB SUSCEPTIBLE3 (PBS3) enzyme has recently been shown to catalyze the conjugation of IC and glutamate to produce isochorismate-9-glutamate, which is further converted into SA by ENHANCEDPSEUDOMONASSUSCEPTIBILITY1 (EPS1) [36]. In the PAL pathway, CM is responsible for catalyzing the conversion of chorismate to prephenate. Unknown steps then result in the production of phenylalanine (Phe) [2]. The PAL enzyme converts Phe into trans-cinnamic acid (tCA) [37], and Abnormal Inflorescence Meristems (AIM1) can catalyze the conversion of tCA into benzoic acid (BA). Then, benzoic acid2hydroxylase (BA2H) catalyzes the conversion of BA into SA [38].

Based on the aforementioned studies and our transcription data, we constructed the ICS pathway and the PAL pathway of SA synthesis in wheat (Fig. 7). Three homologous genes (TaICSA, TaICSB, and TaICSD) encode ICS, which isomerizes chorismate to isochorismate in wheat chloroplasts and participates in SA synthesis (Fig. 1). Six CMs and 54 PALs showed significant differential expression between the WT and ICS mutants under UV treatment (Fig. 5). The remaining enzymes have not yet been annotated. These results will serve as a foundation for enhancing wheat SA accumulation and for improving wheat FHB resistance. Importantly, the ICS mutants created in this study will serve as valuable resources for further investigating the genes associated with SA synthesis in wheat.

In this study, the expression levels of TaICSA, TaICSB, and TaICSD differed significantly. TaICSA and TaICSB were significantly upregulated under UV treatment, while the expression levels of TaICSD were significantly downregulated (Fig. 2D). F. graminearum infection induced the expression of TaICSA (Fig. 3C). According to previous studies, there are three reasons to account for this phenomenon. First, the ICS or its promoter can specifically bind to regulate the differential expression of TaICSA, TaICSB, and TaICSD. For instance, the TCP8 and TCP9 transcription factors can bind to AtICS1 and inhibit its expression in A. thaliana, and OsWRKY6 directly regulates the expression of OsICS1 by binding directly to the OsICS1 promoter in rice [39]. Sequence analysis of the TaICSA, TaICSB, and TaICSD proteins showed significant differences in the region associated with DNA-binding transcription factor activity (SM00380) (Fig. S2B). Second, methylation sites in the sequence of TaICSA, TaICSB, and TaICSD may result in variations in ICS protease activity, leading to differences in gene expression [40]. Finally, ICS proteins must bind to multiple peptides and perform enzymatic activity [8]. The differences in the sequences of the TaICSA, TaICSB, and TaICSD proteins may lead to the binding of different peptides (Fig. S2B). The investigation of these reasons will be the focus of our future research. Among wheat relatives, TaICSA, TaICSB, and TaICSD serve as important references for further genetic improvement of SA content in wheat.

Among all ICS mutants, DTaICSBD and DTaICSABD plants showed leaf dieback at the tri-leaf stage (Fig. 2B). The expression levels of TaICSA, TaICSB and TaICSD in the Ensembl database at various growth stages of wheat showed that the expression levels of TaICSB and TaICSD were significantly higher than those of TaICSA during different growth and development stages (Fig. S3). In contrast, the expression of TaICSA significantly increased in wheat in response to UV treatment and F. graminearum infection (Fig. 2D, 3C). These results suggest that TaICSB and TaICSD are associated with wheat growth and development, while TaICSA may be linked to abiotic and biological stress.

The control of FHB primarily depends on the environmental conditions during the growing season, the characteristics of the pathogenic fungi, and the wheat cultivars. The most important measures involve reducing yield losses and preventing DON toxin accumulation before and during the middle and late stages of wheat flowering. These measures include chemical control, cultivating resistant varieties, biological control, and improving tillage and irrigation conditions. Resistance breeding is considered the most economical and effective method for controlling FHB. Although more than 250 quantitative trait loci related to FHB resistance have been identified on 21 wheat chromosomes, only a few genetic resources are available for breeding [41]. The endogenous hormone SA in wheat can directly inhibit the spore germination and mycelial growth of F. graminearum. Hao et al. [12] showed that FHB resistance in barley can be enhanced by HvICS overexpression. Zhao et al. [42] found that TaICSB, TaNPR1, and TaNPR3 positively regulated FHB resistance in wheat, which is consistent with the results presented in Fig. 3B. However, transgenic plants overexpressing TaICSB or TaICSD did not survive (data not shown). The reason for this is that SA overaccumulation inhibits auxin accumulation, further inhibiting plant growth and development [43]. TaICSA overexpression did not affect wheat growth, but instead increased the SA content and enhanced FHB resistance (Fig. 4D, E). Therefore, TaICSA is an important potential target gene for enhancing SA accumulation and increasing FHB resistance in wheat.

CRediT authorship contribution statement

Ya-Zhou Zhang: Conceptualization, Methodology, Writing – original draft, Writing – review & editing, Funding acquisition. Jie Man: Investigation, Data curation, Writing – original draft. Dan Xu: Investigation, Data curation, Writing – original draft. Lan Wen: . Yinghui Li: Writing – review & editing. Mei Deng: Resources, Formal analysis. Qian-Tao Jiang: Writing – review & editing. Qiang Xu: Writing – review & editing. Guo-Yue Chen: Writing – review & editing. Yu-Ming Wei: Supervision.

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

We thank Professors Jorge Dubcovsky (University of California, Davis, CA, USA) and Francine Paraiso (University of California, Davis, CA, USA) for kindly providing EMS mutants of ICS. We also thank Professor Yan-peng Wang (Institute of Genetics and Development, Chinese Academy of Sciences) for providing wheat protoplast extraction and transgenic services. We are also grateful for the assistance of Wenjun Zhang (University of California, Davis, CA, USA) in editing the language of this manuscript. This work was supported by the National Natural Science Foundation of China (3210170116) and the Science and Technology Department of Sichuan Province (2022YFSY0035).

Appendix A. Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2024.05.012.

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ARTICLE INFO

Article history:

Received 21 February 2024

Revised 16 April 2024

Accepted 23 May 2024

Available online 13 June 2024

Footnote

* Corresponding authors.

E-mail addresses: [email protected] (Y.-Z. Zhang), [email protected]. cn (Y.-M. Wei).

References

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