Identification of Whirly transcription factors in Triticeae species and functional analysis of TaWHY1-7D in response to osmotic stress

Osmotic stress poses a threat to the production and quality of crops. Whirly transcription factors have been investigated to enhance stress tolerance. In this study, a total of 18 Whirly genes were identified from six Triticeae species, which were classified into Whirly1 and Whirly2. The exon–intron structure, conserved motif, chromosomal location, collinearity, and regulatory network of Whirly genes were also analyzed. Real-time PCR results indicated that TaWHY1 genes exhibited higher expression levels in leaf sheaths and leaves during the seedling stage, while TaWHY2 genes were predominantly expressed in roots. Under PEG stress, the expression levels of TaWHY1-7A, TaWHY2-6A, TaWHY2-6B, and TaWHY2-6D were increased, TaWHY1-7D was reduced, and TaWHY1-4A had no significant change. All TaWHY genes were significantly up-regulated in response to NaCl stress treatment. In addition, TaWHY1-7A and TaWHY1-7D mainly enhanced the tolerance to oxidative stress in yeast cells. TaWHY2s mainly improved NaCl stress tolerance and were sensitive to oxidative stress in yeast cells. All TaWHYs slightly improved the yeast tolerance to d-sorbitol stress. The heterologous expression of TaWHY1-7D greatly improved drought and salt tolerance in transgenic Arabidopsis. In conclusion, these results provide the foundation for further functional study of Whirly genes aimed at improving osmotic stress tolerance in wheat.


Introduction
Wheat (Triticum aestivum L.) is one of the most important staple crops worldwide and a major source of calories for the expanding world population.As a sessile organism, wheat has to suffer from a variety of adverse conditions during the growth and development stages, such as drought and salinization, which contribute to a great reduction in the overall wheat yield and quality (Gupta et al., 2020).Therefore, mining stress-resistant genes and developing improved varieties are the most important strategies to improve wheat yield and quality.
Whirly (WHY) proteins are plant-specific transcription factors binding to single-stranded DNA (ssDNA) to modulate growth and defense responses and located in the chloroplasts, mitochondria, and nucleus (Desveaux et al., 2005;Krupinska et al., 2022;Taylor et al., 2022).Whirly domain consists of four structural topologies, which are characterized by two antiparallel four-stranded b-sheets stabilized by a C-terminal helix-loop-helix motif (Desveaux et al., 2005;Cappadocia et al., 2013;Taylor et al., 2022).Due to the structural similarity with "whirligig," Whirly transcription factors are named Whirly (Desveaux et al., 2005).The conserved "KGKAAL" motif in the Whirly domains exists extensively in higher plants, which participate in binding to ssDNA and hexamerization of the tetramers forming hollow sphere structures of 12 nm in diameter (Desveaux et al., 2002;Cappadocia et al., 2012).Additionally, Whirly proteins contain a conserved cysteine residue, which might play a vital role in the formation of disulfide bridges between two Whirly proteins (Foyer et al., 2014).
Whirly genes have been identified in various plant species, such as Arabidopsis, strawberry, tomato, cassava, and barley (Desveaux et al., 2005;Janack et al., 2016;Yan et al., 2020;Hu and Shu, 2021), however, a comprehensive genome-wide analysis of Whirly genes in Triticeae species has not been investigated.In this study, a genomewide analysis of Whirly genes was performed in Triticeae species including Triticum aestivum, Triticum urartu, Triticum dicoccoides, Aegilops tauschii, Hordeum vulgare, and Secale cereale to characterize their sequences, gene structures, evolutionary relationships, expression patterns, and stress tolerance under osmotic stress.These results will provide a valuable foundation for further functional investigations of Whirly genes in response to osmotic stress.

Plant material and growth conditions
Bread wheat cv.Chinese Spring preserved in our laboratory was used in this study, and the sterilized bread wheat seeds were soaked with ddH 2 O in dark and 4°C condition overnight, then cultured on filter paper wetted with ddH 2 O in a culture room at 25/18°C with 16-h light/8-h dark for 1 week.Next, 7-day-old bread wheat seedlings with uniform leaf size and root length were selected for subsequent experiments.For drought and salt stress treatments, 7day-old bread wheat seedlings were cultured under 20% PEG6000 (w/v) and 300 mM NaCl treatments, respectively.In each treatment, the root, leaf sheath, and leaf tissues were collected at 0 h, 1 h, and 6 h, then frozen in liquid nitrogen and stored at −80°C for further investigation.

Multiple sequence alignment and phylogenetic tree construction
Multiple sequence alignment of Whirly amino acid sequences was performed with ClustalW using the default options in MEGA 11 (Tamura et al., 2021) and visualized by ESPript 3.0 (Gouet et al., 2003).The phylogenetic tree was constructed by using the neighbor-joining (NJ) method with 1,000 bootstrap replicates in MEGA 11 software (Tamura et al., 2021) and visualized by Evolview service (Subramanian et al., 2019).
Gene structure, conserved motif, domain, and 3D structure analyses The exon-intron structures of Whirly genes were analyzed based on TGT (Triticeae-Gene Tribe) (Chen et al., 2020b).The conserved motifs and domains of Whirly family proteins were annotated using the MEME program (Bailey et al., 2009) and SMART website (Letunic et al., 2021) and visualized by TBtools (Chen et al., 2020a).The Swiss-Model program was used to predict the three-dimensional (3D) structure of Whirly proteins (Waterhouse et al., 2018).

Chromosome localization, gene duplication, and micro-collinearity analysis
The chromosome localization, micro-collinearity, and paralogous/orthologous gene pairs of Whirly genes were identified by using Triticeae-Gene Tribe (TGT) (Chen et al., 2020b).The gene duplication events were determined by Multiple Collinear Scanning Toolkits (MCScanX) (Wang et al., 2012).TBtools was used to calculate the nonsynonymous rate (K a ), synonymous rate (K s ), and the nonsynonymous and synonymous substitution ratio (K a /K s ) values of the paralogous gene pair with the Nei-Gojobori (NG) method (Chen et al., 2020a).

Regulatory network analysis
The upstream transcription factors and downstream target genes of TaWHYs were predicted by using the wheat integrative gene regulatory network (wGRN) (Chen et al., 2023).Proteinprotein interactions (PPIs) were analyzed using the STRING database (Von Mering et al., 2003) and WheatOmics 1.0 (Ma et al., 2021).

Gene expression analysis by RNA-seq data
To investigate the gene expression patterns in bread wheat under drought stress, bread wheat cv.Chinese Spring was planted in a growth chamber under a photoperiod of 16 h/8 h (light/dark).For drought stress, the seedlings were subjected to water-deficit condition during the seedling stage.The leaf tissues were harvested after 0 days, 2 days, 6 days, and 10 days of treatment, and the total RNA of all the collected samples was extracted.A Nanodrop2000 spectrophotometer was used to determine the quantity and quality of the RNA.A total of 12 bread wheat samples (three biological replicates were conducted for each treatment) were sequenced at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China), and paired-end sequencing was performed with an Illumina Novaseq 6000.The transcriptome data have been submitted to NCBI (BioProject ID: PRJNA1003680).

RNA extraction and real-time PCR
Real-time PCR was performed to detect the expression pattern of Whirly genes according to a previous study (Liu et al., 2022).The total RNA was isolated using RNApure Plant Kit (CWBIO), and the first-strand cDNA was synthesized from 1 mg of total RNA using Prescript III RT ProMix (CISTRO).The real-time PCR was performed using gene-specific primers (Supplementary Table S1) with 2× Ultra SYBR Green qPCR Mix (CISTRO), and the TaActin gene was selected as a reference control.The real-time PCR cycling parameters were 95°C for 30 s, followed by 45 cycles at 95°C for 5 s and 60°C for 30 s, with a melting curve analysis.All reactions were performed on three technical and biological replicates.The relative expression levels of target genes were calculated using the 2 −△△CT method (Livak and Schmittgen, 2001).

Drought and salt tolerance assay in Arabidopsis
The coding sequences of TaWHY1-7D were cloned into the pCAMBIA3301-GFP vector, then transformed into Agrobacterium tumefaciens EHA105, and generated 35S:TaWHY1-7D transgenic Arabidopsis lines via the floral dip method.The primers are shown in Supplementary Table S1.The transgenic Arabidopsis lines were selected via spraying 0.5‰ Basta solution.For drought and salt tolerance assays, WT and 35S:TaWHY1-7D transgenic Arabidopsis were treated with drought (water-deficit) and 500 Mm NaCl conditions.
To elucidate the evolutionary relationship of Whirly genes, a phylogenetic tree was constructed using these 29 Whirly proteins (Figure 1A).According to the results, Whirly genes were classified into two categories, named group 1 (Whirly1) and group 2 (Whirly2).Bread wheat T. aestivum (AABBDD, hexaploid) has undergone two rounds of natural hybridization events (Levy and Feldman, 2022).Thus, the number of gene family members in T. aestivum (AABBDD) is approximately 1.5-and 3-fold than that in T. dicoccoides (AABB, tetraploid) and other diploid Triticeae species, respectively.Consistently, three Whirly1 or Whirly2 genes   1A; Supplementary Table S2).

Gene structure and conserved motif analysis
To investigate the functional divergence of Whirly genes, the exon-intron structures, conserved motifs, and 3D structures of Whirly genes were analyzed in Triticeae species (Figure 1; Supplementary Figure S1).The results revealed that Whirly1 and Whirly2 genes contained six and eight exons in the Triticeae species, respectively.The conserved motif analysis showed that all Whirly proteins contained the Whirly transcription factor domain (PF08536), which consisted of motifs 1, 2, 3, and 7.These also confirmed the reliability of the identified Whirly gene family members.Motif 3 contained the conserved "KGKAAL" sequence, which participated in binding to ssDNA (Supplementary Figure S1) (Desveaux et al., 2002;Cappadocia et al., 2012).Almost all Whirly proteins contained motif 4, except for TdWHY1-4A and TdWHY1-7A, which lacked a portion of the amino acid sequences of motif 4 (Supplementary Figure S1).Motifs 8, 9, and 10 were present in group 1 members, while they were absent in group 2 members.Motif 5 was unique to group 2 members.In addition, all TaWHY proteins contained two anti-parallel four-stranded b-sheets that extend like blades from an a-helical core (Figure 1C), which were consistent with its "whirligig" structure (Desveaux et al., 2005).
To further investigate the evolutionary process of TaWHYs, gene duplication, and micro-collinearity analyses of the Whirly genes were performed (Figure 3; Supplementary Table S4).A total of six paralogous gene pairs of TaWHYs (TaWHY1-4A/TaWHY1-7A/TaWHY1-7D, and TaWHY2-6A/TaWHY2-6B/TaWHY2-6D) were identified in bread wheat genome and expanded by wholegenome duplication (WGD) or segmental duplication events (Figure 2B; Supplementary Table S4).The K a /K s values of paralogous gene pairs were all less than 1, indicating that TaWHY genes underwent purifying selection to avoid functional divergence (Supplementary Table S4).Micro-collinearity analysis contributes to the investigation of the inheritance and variation of specific genes in local regions and detecting the origin of specific genes during the hybridization and polyploidization process (Chen et al., 2020b).To explore the origin of Whirly genes in Triticeae species, TaWHY1-4A, TaWHY2-6A, and TaWHY1-7A were used as query genes to analyze the micro-collinearity by TGT (Figure 3).The homologous genes of TaWHY2-6A were detected in the collinearity regions of T. urartu, Ae. tauschii, subgenomes A and

Expression patterns analysis of TaWHYs
To insight into the biological function of TaWHY genes, the transcriptome data and real-time PCR were used to determine the expression patterns of six TaWHY genes in different tissues (leaves, leaf sheaths, and roots during bread wheat seedling stage) and in response to osmotic (drought and salt) stress (Figures 4, 5).The analysis of the transcriptome data revealed that the TaWHY1 genes (TaWHY1-4A, TaWHY1-7A, and TaWHY1-7D) exhibited the highest expression levels in leaves, and the TaWHY2 genes (TaWHY2-6A, TaWHY2-6B, and TaWHY2-6D) showed the Micro-collinearity analysis of the Whirly gene in Triticeae species.TaWHY1-4A (A), TaWHY1-7A (B), and TaWHY2-6A (C) were used as the query gene, respectively.highest expression levels in roots (Figure 4A).Consistently, the realtime PCR results showed that TaWHY1 genes (TaWHY1-4A, TaWHY1-7A, and TaWHY1-7D) were highest expressed in leaf sheaths, followed by leaves, and roots during the bread wheat seedling stage.TaWHY2 genes (TaWHY2-6A, TaWHY2-6B, and TaWHY2-6D) exhibited the highest expression level in roots, followed by leaf sheaths, and finally in leaves (Figure 4B).
After drought stress treatment, RNA-seq analysis revealed that the TaWHY1 genes exhibited the highest expression levels after 6 days of drought treatment, and the expression of TaWHY2 genes increased with the progression of drought stress duration (Figure 5A).The real-time PCR results demonstrated the expression of TaWHY1-7A was up-regulated under PEG stress, peaking at 1 h with 1.6-fold compared with the control, TaWHY1- Expression pattern analysis of TaWHYs in different tissues.(A) The expression levels of TaWHY genes in root and shoot were determined through RNA-seq analysis.Fragments per kilobase of exon per million mapped fragments (FPKM) values were used to measure the expression levels of genes.(B) The expression levels of TaWHY genes in the root, leaf sheath, and leaf during the bread wheat seedling stage were determined by realtime PCR.The expression level of the bread wheat actin gene was used as the reference control to standardize the RNA samples for each reaction.Data represent the mean ± SD of three replicates.Liu et al. 10.3389/fpls.2023.1297228Frontiers in Plant Science frontiersin.org7D was down-regulated, and TaWHY1-4A was not significantly changed.The expression of TaWHY2-6A, TaWHY2-6B, and TaWHY2-6D (group 2) was gradually up-regulated and reached the highest expression level at 6 h under PEG stress with approximately 2.9-, 2.3-, and 1.6-fold compared with the control, respectively (Figure 5B).After NaCl treatment, the expression levels of TaWHY genes were significantly up-regulated (Figures 5C, D).The real-time PCR results revealed that the expression levels of TaWHY1-4A, TaWHY2-6A, TaWHY2-6B, TaWHY2-6D, TaWHY1-7A, and TaWHY1-7D were all increased, peaking at 1 h, 1 h, 6 h, 6 h, 1 h, and 6 h with approximately 2.6-, 2.7-, 7.4-, 2.9-, 3.2-, and 12.9-fold compared with the control, respectively (Figure 5D).Therefore, we speculated that TaWHYs might play an important role under osmotic stress.

Upstream transcription factors, downstream target genes, and interacting proteins analysis of TaWHYs
Transcription factors can interact with different cis-elements in the promoter region of target genes, exerting diverse functions in plant growth, development, and stress response (Strader et al., 2022).To determine the functions of TaWHY genes, upstream transcription Expression patterns of TaWHY genes in response to osmotic stress.(A) RNA-seq analysis of the expression profiles of TaWHY genes in response to drought stress for 0 days, 2 days, 6 days, and 10 days, respectively.Fragments per kilobase of exon per million mapped fragments (FPKM) values were used to measure the expression levels of genes.(B) The expression profiles of TaWHY genes in bread wheat seedling leaves at 0 h, 1 h, and 6 h after PEG stress treatment.(C) RNA-seq analysis of the expression profiles of TaWHY genes in response to 0 mM, 100 mM, 200 mM, and 300 mM NaCl treatment.FPKM values were used to measure the expression levels of genes.(D) The expression profiles of TaWHY genes in bread wheat seedling leaves at 0 h, 1 h, and 6 h after NaCl stress treatment.The expression level of the bread wheat actin gene was used as the reference control to standardize the RNA samples for each reaction.Data represent the mean ± SD of three replicates.The asterisk indicates significant differences compared with 0 h (control, as 1) based on Student's t-test (*p < 0.05; **p < 0.01).
These results suggested the regulatory mechanism of TaWHY genes to avoid or defend against osmotic stress.

TaWHYs improve the tolerance to osmotic and oxidative stresses in yeast cells
To further investigate the function of TaWHY genes under osmotic (D-sorbitol and NaCl) and oxidative (H 2 O 2 ) stresses, TaWHY2-6A, TaWHY2-6B, TaWHY2-6D, TaWHY1-7A, and TaWHY1-7D were cloned into the pGADT7 vector, and then transformed into the yeast cells BY4741 or stress-sensitive yeast mutant BY4741 (Dhog1) to confirm the ability to improve stress resistance in yeast cells (Figure 8).The results suggested that the growth of the BY4741 or Dhog1 yeast cells carrying these TaWHY genes was not obviously different compared with the control (pGADT7 empty vector) under normal growth conditions.After D-sorbitol treatment, Dhog1 yeast cells overexpressing TaWHYs slightly enhanced their tolerance to D-sorbitol stress in comparison to the negative control.The Dhog1 yeast overexpressing TaWHY2- The upstream transcription factor (A) and downstream target gene (B) analyses of TaWHY genes.
6A, TaWHY2-6B, and TaWHY2-6D obviously improved the resistance to NaCl stress, but the colonies of Dhog1 with TaWHY1-7A and TaWHY1-7D were slightly increased compared with the negative control under NaCl stress.
Adverse environmental conditions induce ROS production; ROS accumulation can cause oxidative damage to membranes, proteins, and RNA and DNA molecules and even lead to the oxidative destruction of the cell in a process termed oxidative stress; thereby, ROS scavenging is essential for plants to avoid or defend against adverse stress (Choudhury et al., 2017).To determine whether TaWHYs enhanced stress tolerance by scavenging ROS in yeast cells, Dhog1 yeast cells carrying pGADT7-TaWHYs or pGADT7 were grown on YPD medium containing 4.0 mM H 2 O 2 , suggesting TaWHY1-7A and TaWHY1-7D strongly enhanced the oxidative stress tolerance in yeast, but the colonies of Dhog1 overexpressing TaWHY2-6A, TaWHY2-6B, and TaWHY2-6D were reduced compared with control.These results indicated that the TaWHY1 and TaWHY2 genes performed diverse functions.TaWHY1 mainly enhanced the tolerance to oxidative stresses; TaWHY2 mainly improved NaCl stress tolerance and was sensitive to oxygen stress; and TaWHY1 and TaWHY2 genes slightly improved the tolerance to D- sorbitol stress.

TaWHY1-7D confers drought and salt tolerance in Arabidopsis
In order to further confirm the potential role of TaWHY1-7D in response to drought and salt stresses, we generated 35S:TaWHY1-7D transgenic Arabidopsis lines.Three independent transgenic lines (OE4, OE8, and OE10) and wild-type (WT) were chosen for the functional analysis of TaWHY1-7D in response to drought and salt stresses (Figure 9; Supplementary Figure S6).The results showed that there were no obvious phenotypic differences between transgenic and WT plants under normal conditions.After an 8-day drought treatment, the wild-type (WT) plants exhibited wilting and subsequent yellowing.In contrast, the transgenic Arabidopsis overexpressing TaWHY1-7D Protein-protein interaction (PPI) network analysis of TaWHY proteins.
remained predominantly green.After NaCl treatment for 8 days, both WT and transgenic Arabidopsis lines exhibited growth inhibition compared with CK.The growth inhibition was more severe in WT plants compared to transgenic Arabidopsis.Thus, the heterologous expression of TaWHY1-7D greatly improved drought and salt tolerance in transgenic Arabidopsis.
Recently, Whirly genes were reported to improve osmotic stress resistance in plants, such as MeWHYs, which could interact with MeCIPK23 to activate ABA biosynthesis and regulate drought resistance in cassava (Yan et al., 2020).In this study, TaWHY1-7A and three TaWHY2 genes were up-regulated under PEG stress, TaWHY1-7D was down-regulated, and TaWHY1-4A was not significantly changed (Figure 5), suggesting that functional differentiation of Whirly genes occurred.All TaWHYs were upregulated under NaCl stress (Figure 5) and improved the resistance of NaCl stress in yeast, respectively (Figure 8).The heterologous expression of TaWHY1-7D greatly improved drought and salt tolerance in transgenic Arabidopsis (Figure 9).In addition, Whirly genes have been reported to regulate ROS homeostasis (Lin et al., 2019), and our results also showed that TaWHY1-7A and TaWHY1-7D strongly enhanced the oxidative stress tolerance in yeast cells (Figure 8).ROS scavenging also might be an important reason for the improvement of stress resistance in TaWHY1 genes.However, the growth of Dhog1 overexpressing TaWHY2-6A, TaWHY2-6B, and TaWHY2-6D was inhibited under oxidative stress; these were consistent with a previous study that found that The phenotype of the 35S: TaWHY1-7D transgenic Arabidopsis under drought and NaCl stress.Three independent 35S:TaWHY1-7D transgenic Arabidopsis lines (OE4, OE8, and OE10) and wild type (WT) were chosen for functional analysis of TaWHY1-7D under normal conditions (CK), drought (water-deficit), and salt (NaCl) stress treatments.
overexpression of AtWHY2 caused the accumulation of ROS in the plant (Cai et al., 2015).The ROS accumulation might cause cellular stress, thus activating the alternative pathway to reduce ROS levels and eliminate the stress (Cai et al., 2015).GO enrichment analysis also showed that TaWHY1-7D and TaWHY2-6D regulated downstream target genes to respond to H 2 O 2 and oxidative stress (Supplementary Figure S4).Based on the above research, we speculate that the Whirly genes may play a vital role in plant resistance to osmotic stress.These results provide useful information for further functional studies of Whirly genes and lay a foundation to improve wheat yield and quality via molecular breeding under osmotic stress.
FIGURE 2 Chromosomal localizations (A) and syntenic relationships (B) among TaWHY genes in T. aestivum.(B) Red lines in the highlight indicate the syntenic TaWHY gene pairs.

FIGURE 9
FIGURE 9 found in T. aestivum, while T. dicoccoides and other diploid Triticeae included two and one Whirly1 or Whirly2 gene, respectively (Figure were