Genome-wide identification, characterization and expression pattern analysis of TIFY family members in Artemisia argyi

Abstract

Background

Plant-specific TIFY proteins play crucial roles in regulating plant growth, development, and various stress responses. However, there is no information available about this family in Artemisia argyi, a well-known traditional medicinal plant with great economic value.

Results

A total of 34 AaTIFY genes were identified, including 4 TIFY, 22 JAZ, 5 PPD, and 3 ZML genes. Structural, motif scanning, and phylogenetic relationships analysis of these genes revealed that members within the same group or subgroup exhibit similar exon-intron structures and conserved motif compositions. The TIFY genes were unevenly distributed across the 15 chromosomes. Tandem duplication events and segmental duplication events have been identified in the TIFY family in A. argyi. These events have played a crucial role in the gene multiplication and compression of different subfamilies within the TIFY family. Promoter analysis revealed that most AaTIFY genes contain multiple cis-elements associated with stress response, phytohormone signal transduction, and plant growth and development. Expression analysis of roots and leaves using RNA-seq data revealed that certain AaTIFY genes showed tissue-specific expression patterns, and some AaTIFY genes, such as AaTIFY19/29, were found to be involved in regulating salt and saline-alkali stresses. In addition, RT-qPCR analysis showed that TIFY genes, especially AaTIFY19/23/27/29, respond to a variety of hormonal treatments, such as MeJA, ABA, SA, and IAA. This suggested that TIFY genes in A. argyi regulate plant growth and respond to different stresses by following different hormone signaling pathways.

Conclusion

Taken together, our study conducted a comprehensive identification and analysis of the TIFY gene family in A. argyi. These findings suggested that TIFY might play an important role in plant development and stress responses, which laid a valuable foundation for further understanding the function of TIFY genes in multiple stress responses and phytohormone crosstalk in A. argyi.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-024-10856-4.

Background

The TIFY gene family, characterized by a highly conserved motif (TIF[F/Y]XG), is a plant-specific gene family that encodes transcription factors. The first TIFY family gene was identified in Arabidopsis thaliana and named as ZIM (Zinc-finger protein expressed in inflorescence meristem) [1]. Based on the differences in its domain structural characteristics, the TIFY family has been divided into four subfamilies, namely the TIFY, ZML (ZIM and ZIM-LIKE), JAZ (JASMONATE ZIM-Domain), and PPD (PEAPOD) [2]. Among them, the TIFY subfamily proteins only have the TIFY domain [3]; In addition to the TIFY domain, the ZML subfamily proteins also contain a GATA zinc finger and a CCT domain [4]. The JAZ subfamily contains a C-terminal Jas domain (SLX2FX2KRX2RX5PY), and these Jas sequences have similarity to that of the CCT domain’s N-terminus, and some members can interact with the MYC2 proteins to inhibit the jasmonate acid (JA) signaling pathway [1]. For the PPD subfamily, there is also an N-terminal PPD domain and a modified Jas domain with a defective proline-tyrosine in the C-terminus [5]. The structural differences among the four subfamilies of the TIFY family lay an important foundation for the functional differentiation and functional diversity of the TIFY genes.

The TIFY gene family has been studied for over 20 years and has been functionally validated to play multiple regulatory roles in cell signal transduction. These roles include regulation of plant growth and development, plant stress response, and phytohormone treatments [2]. For instance, AtTIFY1 is the first identified TIFY gene in plants, and it can promote the elongation of the petioles and hypocotyls [1]. SlJAZ2 plays an important role as a regulator of the transition from vegetative growth to reproductive growth in tomato [6]. AtTIFY4a and AtTIFY4b are involved in the regulation of lamina size and curvature [5]. Extensive studies have shown that many TIFY genes are widely involved in abiotic stress. For example, AtTIFY10a and AtTIFY10b, GmTIFY10e and GmTIFY10g, TdTIFY11a, and OsJAZ8 are involved in salt stress [7–10]; Additionally, ZmTIFY16 responds to drought and salt tolerance in Arabidopsis and Zea mays [11]; while CaTIFY7 and CaTIFY10b are involved in cold stress in Capsicum annuum [12]. In recent years, molecular studies on medicinal plants have shown that JA can induce the synthesis and accumulation of secondary metabolites. Especially, the JAZ subfamily, as a key negative regulator of the JA signaling pathway, plays pivotal roles in regulating the synthesis of secondary metabolites in medicinal plants. For example, in Arabidopsis thaliana, JAZ binds to MYB/bHLH/WD40 complexes, leading to the inhibition of flavonoid biosynthesis [13]. In Salvia miltiorrhiza, JA has been reported to stimulate the production of salvianolic acids and tanshinones by releasing SmMYC2a from SmJAZ1 and SmJAZ8 [14, 15]; Additionally, SmJAZ3 and SmJAZ9 play a role in regulating of tanshinone biosynthesis and function as suppressive transcriptional regulators in the JA signaling pathway [16]. In tea plants, CsJAZ1 and its alternative splicing are negatively associated with flavan-3-ols biosynthesis [17]. Therefore, because the TIFY family plays a role in regulating the synthesis of secondary metabolites, and different medicinal plants contain various secondary metabolites, an increasing amount of research has been conducted on the study of the TIFY family in medicinal plants.

Artemisia argyi Lévl. et Van. is a well-known traditional medicinal plant, and is widely distributed in Asian countries, especially throughout in China. The dried leaf of A. argyi is a well-known traditional Chinese medicine called ‘‘Aiye” or “Chinese mugwort’’. It is widely used for moxibustion heat therapy to treat of hemostasis, epistaxis, hemorrhage, inflammation, abdominal cold pain, uterine cold infertility, itching, and menstruation-related symptoms in China and many other Asian countries [18]. Modern pharmacological analysis has revealed that A. argyi contains a diverse range of bioactive compounds, including volatile oil, terpenoids, flavonoids, eudesmane, and organic acids. These bioactive compounds exhibit remarkable pharmacological effects, such as a broad-spectrum antihistamine, antibacterial, and antiviral properties [19, 20]. In addition, A. argyi is also widely used as forage for animals, food, in cultural presentation, for cosmetics, and as daily necessities in various geographical regions [21]. The distribution of A. argyi indicates a wide range of adaptability, as it can withstand a variety of unfavorable conditions. Based on the demand for A. argyi, it is of great significance to study its growth, development, and stress regulation. Based on previous studies showing that TIFY proteins play multiple regulatory roles in plant growth and development, stress response, and phytohormone regulation [5–12]. How the TIFY gene regulates the growth and development of A. argyi, as well as its response to adversity, is therefore a scientific question worth investigating.

In the present study, genome-wide identification and investigation of the TIFY family genes conducted using from whole-genome genome data of A. argyi [22]. Phylogenetic relationships, gene structure, conserved motifs and domains, Gene Ontology (GO) annotation, and research on promoter cis-elements were conducted to gain insight into their potential functions. Furthermore, the expression patterns of TIFY genes in different tissues, and their response to salt or saline-alkali stresses, were analyzed using transcriptome data. The expression patterns in response to different hormones were investigated using quantitative real-time RT-PCR (qRT-PCR). Our study will provide preliminary contributions to clarifying the functions of the TIFY family and will offer valuable information for further studies on TIFY genes in A. argyi.

Results

Identification of TIFY family genes in the A. argyi genome

To identify all putative TIFY genes in A. argyi, we conducted HMMER and BLASTP searches, and verified the presence of the TIFY domain (PF06200) using Pfam. A total of 34 TIFY gene members were identified in the A. argyi genome (Table 1). The TIFY gene members in A. argyi were relatively less than Brassica, Glycine max, Z. mays, Triticum aestivum, and Gossypium hirsutum, but higher than Vitis vinifera, A. thaliana, Oryza sativa, Nicotiana tabacum, etc. (Figure S1). Genes were named from AaTIFY1 to AaTIFY34 based on their positions on the chromosomes. The coding sequences (CDS) and amino acid lengths of these identified TIFY sequences varied significantly. The lengths of the CDS varied from 399 bp (AaTIFY11) to 1140 bp (AaTIFY15). Similarly, the amino acid sequence lengths of AaTIFYs ranged from 132 aa (AaTIFY11) to 379 aa (AaTIFY15). The predicted molecular weight of AaTIFYs also varied greatly, ranging from 15,181.13 Da (AaTIFY11) to 39,519.55 Da (AaTIFY15). The expected theoretical isoelectric points range from 5.16 (AaTIFY23) to 9.54 (AaTIFY8). Twenty-five out of 34 AaTIFYs had values higher than 7.0, indicating that most of the A. argyi TIFYs were alkaline proteins. Protein instability coefficients of AaTIFYs proteins ranged from 34.28 (AaTIFY16) to 78.93 (AaTIFY12), and most of them are unstable, except AaTIFY2, AaTIFY10, AaTIFY16, and AaTIFY32, with instability indices lower than 40. The aliphatic index varies from 54.04 (AaTIFY9 and AaTIFY27) to 83.19 (AaTIFY6). The grand average of hydropathicity varies from -0.903 (AaTIFY12) to -0.351 (AaTIFY17), indicating that all these proteins are hydrophilic (Table 1). In addition, all AaTIFY proteins were predicted to be located in the nucleus using Plant-mPLoc Online Software, indicating that they are likely to function as transcription factors and play important roles in the nucleus (Table 1).

Table 1.

Physical and chemical characters of TIFY proteins in A. argyi

Gene name	Gene ID	Amino acids number (aa)	Molecular weight (Da)	Isoelectric point (pI)	Instability index	Aliphatic index	Grand average of hydropathicity	Subcellular localization
AaTIFY1	Aarg01G011340.1	273	30774.04	7.71	64.1	75.71	-0.626	Nucleus
AaTIFY2	Aarg02G015240.1	358	39102.93	5.71	34.72	63.18	-0.896	Nucleus
AaTIFY3	Aarg02G021200.1	219	24243.37	6.32	42.03	68.22	-0.521	Nucleus
AaTIFY4	Aarg02G031700.1	154	17025.13	5.44	42.18	66.04	-0.422	Nucleus
AaTIFY5	Aarg03G000100.1	252	27783.32	8.82	41.37	75.91	-0.658	Nucleus
AaTIFY6	Aarg03G029130.1	191	21339.34	9.01	47.3	83.19	-0.414	Nucleus
AaTIFY7	Aarg03G029140.1	191	21354.4	9.46	44.76	75.08	-0.49	Nucleus
AaTIFY8	Aarg03G029150.1	191	21304.26	9.54	45.68	78.12	-0.483	Nucleus
AaTIFY9	Aarg04G028560.1	188	20414.92	9.15	45.85	54.04	-0.669	Nucleus
AaTIFY10	Aarg04G028570.1	162	17555.47	9	37.8	54.94	-0.809	Nucleus
AaTIFY11	Aarg05G003550.1	132	15181.13	9.21	72.29	68.71	-0.864	Nucleus
AaTIFY12	Aarg05G003560.1	138	15958.01	8.98	78.93	72.75	-0.903	Nucleus
AaTIFY13	Aarg05G010380.1	284	31297.4	5.98	46.94	74.86	-0.449	Nucleus
AaTIFY14	Aarg06G012000.1	360	38086.32	8.29	45.62	65.56	-0.517	Nucleus
AaTIFY15	Aarg08G011150.1	379	39519.55	8.89	44.25	55.09	-0.623	Nucleus
AaTIFY16	Aarg08G018090.1	266	29261.42	5.99	34.28	56.43	-0.698	Nucleus
AaTIFY17	Aarg09G008010.1	318	33487.76	9.1	46.56	63.81	-0.351	Nucleus
AaTIFY18	Aarg09G026580.1	256	27820.53	9.04	47.36	59.14	-0.368	Nucleus
AaTIFY19	Aarg09G031130.1	185	20141.9	9.52	67.19	78.05	-0.471	Nucleus
AaTIFY20	Aarg10G012540.1	298	33062.17	8.83	65.72	62.89	-0.859	Nucleus
AaTIFY21	Aarg10G013160.1	298	33053.03	8.64	67.06	61.24	-0.886	Nucleus
AaTIFY22	Aarg10G054640.1	219	24301.4	5.97	42.76	68.22	-0.535	Nucleus
AaTIFY23	Aarg10G062020.1	154	16922.91	5.16	41.11	65.39	-0.457	Nucleus
AaTIFY24	Aarg11G000310.1	249	27368.9	8.69	40.69	75.66	-0.637	Nucleus
AaTIFY25	Aarg11G032570.1	191	21187.08	9.23	40.72	78.64	-0.432	Nucleus
AaTIFY26	Aarg11G032580.1	191	21398.42	9.34	50.56	72.51	-0.542	Nucleus
AaTIFY27	Aarg12G027070.1	188	20414.92	9.15	45.85	54.04	-0.669	Nucleus
AaTIFY28	Aarg13G015630.1	285	31322.19	5.98	48.99	69.47	-0.529	Nucleus
AaTIFY29	Aarg13G018300.1	185	20447.42	9.39	59.77	73.24	-0.464	Nucleus
AaTIFY30	Aarg14G013310.1	347	36615.63	9.01	49.57	67.2	-0.525	Nucleus
AaTIFY31	Aarg16G010670.1	378	39378.46	8.89	44.16	56.27	-0.603	Nucleus
AaTIFY32	Aarg16G018370.1	264	29189.4	6.34	38.28	57.23	-0.689	Nucleus
AaTIFY33	Aarg17G008320.1	318	33544.82	9.1	46.49	63.81	-0.361	Nucleus
AaTIFY34	Aarg17G025320.1	256	27792.48	9.04	47.69	58.4	-0.378	Nucleus

Analysis of the structures, phosphorylation sites, and signal peptides of AaTIFY proteins

The secondary structures of AaTIFY proteins were analyzed using SOPMA. The results showed that the secondary structure of AaTIFY proteins was composed of α-helix, extension, β-angle, and random coil (Table S1). Among them, the random coil was the most common secondary structure in A. argyi TIFY amino acid sequences, accounting for a range from 49.35% (AaTIFY4) to 72.78% (AaTIFY14), followed by alpha helix from 6.86 to 31.17%, extended strand from 5.7 to 16.36%, and β-angle from 2.46 to 9.09% (Table S1). For example, the secondary structure of the AaTIFY14 protein consists of 72.78% random coil, 8.06% alpha helix, 14.44% extended strand, and 4.72% β-angle (Table S1). Furthermore, to obtain more structural insights, the 3D structures of AaTIFY proteins were also modeled. All these 3D structures had an almost similar structural composition with secondary structures (Fig. 1).

Fig. 1.

Predicted 3D structures of AaTIFY proteins in A. argyi

For most cellular proteins, post-translational modifications play an important role in regulating membrane proteins, thereby influencing cell physiological functions [23]. Among them, phosphorylation is an important post-translational modification process in organisms. It can effectively regulate the normal physiological activities of cells and plays a crucial role in controlling the development of cancer cells [24]. In this study, phosphorylation sites of AaTIFY proteins were analyzed by using NetPhos3.1. The results showed that 34 members of AaTIFYs had multiple phosphorylation sites ranging from 16 to 49 (Table S1). Among them, AaTIFY15 had the most phosphorylation sites (49), followed by AaTIFY31 (48) and AaTIFY28 (46). Furthermore, for different types of phosphorylation sites, serine phosphorylation sites were the most distributed, ranging from 10 to 40, followed by threonine phosphorylation sites ranging from 2 to 19, and tyrosine phosphorylation sites only ranging from 0 to 6. Among them, AaTIFY4/6/7/8/9/10/15/23/26/27/29/31 proteins do not contain tyrosine phosphorylation sites. In addition, the predicted signal peptides of AaTIFY proteins were analyzed using SignalP-5.0. The results indicated that the signal peptide values of AaTIFY proteins were all below 0.5, demonstrating the absence of signal peptides in AaTIFY proteins (Table S1). This suggests that TIFY proteins in A. argyi are not secreted proteins.

Chromosomal distribution of AaTIFY genes

Based on the chromosome-scale genome assembly of A. argyi [22]. The results of chromosomal distribution analyzes showed that the 34 AaTIFY genes in A. argyi were irregularly distributed on 15 of the 17 chromosomes of A. argyi, with the numbers of TIFY members varying from 1 to 4 (Fig. 2). Among them, Chr.03 and Chr.10 had the most genes distributed with four AaTIFY members, followed by Chr.02, Chr.05, Chr.09, and Chr.11 with three AaTIFY members. Chr.04, Chr.08, Chr.13, Chr.16, and Chr.17 each have two AaTIFY members, while Chr.01, Chr.06, Chr.12, and Chr.14 have only one AaTIFY member each.

Fig. 2.

Chromosome distribution mapping of TIFY family in A. argyi

Phylogenetic tree, domains, conserved motifs, and gene structure of TIFY family in A. argyi

To determine the homology relationships of the identified TIFY family in A. argyi, the alignment of the 34 TIFY protein sequences of A. argyi was used to conduct phylogenetic analysis (Fig. 3). The topology of the resulting phylogenetic tree indicated that 34 AaTIFY proteins can be divided into three groups: Group I, II, and III. Among them, Group I include AaTIFY1/13/20/21/28; Group II contains 16 members and can be divided into II-A and II-B subgroups. Both subgroups can be further divided into two categories: II-A-1 (AaTIFY31/15/30/14), II-A-2 (AaTIFY4/23/29), II-B-1 (AaTIFY3/5/22/24), and II-B-2 (AaTIFY6/7/8/25/26), respectively. Group III contains 13 members and can be divided into III-A (AaTIFY11/12/19) and III-B (AaTIFY2/9/10/16/17/18/27/32/33/34). III-B can be further divided into two categories: III-B-1 (AaTIFY9/10/17/18/27/33/34) and III-B-2 (AaTIFY2/16/32). Moreover, the evolutionary tree showed that most TIFY members have sister pairs of genes. There was a total of 10 pairs of sister pair genes, including AaTIFY17/33, AaTIFY18/34, AaTIFY9/27, AaTIFY11/12, AaTIFY4/23, AaTIFY14/30, AaTIFY15/31, AaTIFY13/28, AaTIFY5/24, and AaTIFY3/22.

Fig. 3.

Phylogenetic tree and gene structure of AaTIFY gene family. A The neighbor-joining phylogenetic tree of TIFY family; B Distribution of the conserved motifs in AaTIFY proteins. Ten conserved motifs are marked with different colored boxes. C The distribution of conserved domains in AaTIFY proteins. D Exon/intron organization of AaTIFY genes. Green boxes represent exons and black lines represent introns. The upstream/downstream regions (UTR) of AaTIFY genes are indicated by yellow boxes. the numbers 0–2 represents Phase Num, which is the phase information of the intron-exon junctions

Using the MEME suite, ten conserved motifs were identified and visualized in AaTIFY proteins (Fig. 3B). The number of preserved motifs varies from 2 to 6 among different AaTIFY members. All members of the AaTIFY proteins share motif 2, which corresponds to the TIFY conserved domain (Fig. 3B, C). Motif 1 is present in all AaTIFY members except AaTIFY14/15/30/31. Motif 3 is present in 15 members of AaTIFY2/3/4/5/6/7/8/16/22/23/24/25/26/29/32 proteins. Motif 4 is present in all AaTIFY members except AaTIFY4/11/23 proteins. Motif 5 is present in AaTIFY1/3/13/20/21/22/28 proteins. Motif 6 is present in AaTIFY1/5/6/7/8/13/14/15/20/21/24/25/26/28/30/31 proteins. Motif 7 is present in AaTIFY17/18/33/34 proteins. Motif 8 is present in AaTIFY14/15/30/31 proteins. Motif 9 is present in AaTIFY3/4/6/7/8/22/23/25/26/29 proteins. Motif 10 is present in AaTIFY9/10/17/18/27/33/34 proteins.

Furthermore, the conserved domains in AaTIFY proteins were evaluated using the Conserved Domains in NCBI tools (Fig. 3C). Four potential conserved domains were identified: the TIFY domain, the Jas motif domain (also known as CCT_2), the CCT domain, and the ZnF_GATA domain. Among them, all AaTIFY proteins contain the TIFY domain, indicating that all 32 members belong to the TIFY family. The CCT domain and the ZnF_GATA domain are only found in AaTIFY2/16/30, indicating that AaTIFY2/16/30 belongs to the ZML subfamily. AaTIFY14/15/30/31 proteins only contain the TIFY domain. These members may have undergone structural loss during evolution. The remaining 29 members all contain two conserved domains, including the TIFY domain and the CCT_2 domain (Fig. 3C). In addition, by combining the evolutionary tree of AaTIFY proteins, it was observed that the proteins in the same clade exhibited similar domains and conserved motifs (Fig. 3).

Furthermore, the exon-intron structure of all 34 TIFY genes in A. argyi was analyzed to enhance the understanding of the structural diversity of these genes. As shown in Fig. 3D, the number of introns ranged from 1 to 7 in the 34 AaTIFY members. Among them, AaTIFY13/17/20/21/28/33 contains the largest number of 7 introns; followed by AaTIFY1/2/16/18/32/34 with 6 introns; AaTIFY9/14/15/27/30/31 with 5 introns; AaTIFY10 with 4 introns; AaTIFY4/19/23/29 with 3 introns; AaTIFY11/12 with 2 introns; and the remaining 9 members belong to the JAZ subfamily containing only 1 intron. The combination of phylogenetic tree and gene structure analysis showed that the members in the same branch mostly have the same structure, indicating that the members in the same branch may have the same or similar functions.

Analysis of cis-elements in the promoter regions of AaTIFY genes

The 2000 bp upstream promoter regions were scanned to identify cis-elements in order to gain insight into the potential function of the TIFY gene family (Fig. 4, Table S2). The results showed that AaTIFY genes contain a variety of cis-elements, including light responsiveness (412), abscisic acid responsiveness (155), MeJA-responsiveness (126), anaerobic induction (103), low-temperature responsiveness (32), auxin responsiveness (30), defense and stress responsiveness (23), salicylic acid responsiveness (22), gibberellin-responsiveness (17), and others. Among them, 30 members contain abscisic acid responsiveness cis-elements, excluding AaTIFY14 and AaTIFY30. The numbers of abscisic acid responsiveness cis-elements in AaTIFY33, AaTIFY13, AaTIFY8, and AaTIFY26 were 11, 10, 9, and 9, respectively. Regarding the MeJA-responsiveness elements, they are present in 24 AaTIFY genes, with notable occurrences in AaTIFY16 (30), AaTIFY14 (10), AaTIFY30 (10), and AaTIFY34 (8). For the other plant hormone response cis-elements, there are 13, 11, and 18 AaTIFY genes containing salicylic acid, gibberellin-responsive, and auxin responsiveness cis-elements, respectively. Some members contain multiple hormone-responsive elements. For example, AaTIFY16/17/27/29/32 contain four types of hormone response elements, including auxin, gibberellin, salicylic acid, and MeJA responsiveness elements. Thirteen AaTIFY genes contain three types of hormone response cis-elements, and ten AaTIFY genes contain two types of hormone response cis-elements. The existence of these cis-regulatory elements indicates a potential function of the TIFY genes in crosstalk regulation by multiple hormones.

Fig. 4.

Cis-elements analysis in the promoters of TIFY genes in A. argyi. The 2,000 bp promoter sequences of AaTIFY genes were used to analyze cis-elements

For the environmental response element, all members contain multiple cis-acting elements involved in light responsiveness. 31 members contain cis-acting regulatory elements essential for anaerobic induction, except for the AaTIFY19 gene. Additionally, 14 genes (AaTIFY3/6/8/11/12/14/15/17/19/22/23/25/30/31) contain defense and stress responsiveness elements; 17 genes (AaTIFY1/4/5/10/11/12/13/14/16/18/23/24/28/29/30/32/34) contain low-temperature responsiveness elements; and 8 genes (AaTIFY5/11/15/17/24/29/32/33) contain drought-inducibility responsiveness elements. Additionally, some genes contain tissue-specific cis-elements or other functional regulations. For example, AaTIFY6/8/13/15/24/25/32 contain cis-regulatory elements involved in endosperm expression; AaTIFY5/7/24 are involved in the regulation of flavonoid biosynthetic genes; AaTIFY1/2/7/14/15/16/21/26/28/30/32 have cis-regulatory elements related to meristem expression; AaTIFY3/8/10/25 have cis-acting elements involved in cell cycle regulation; and AaTIFY27 has a cis-acting regulatory element involved in seed-specific regulation (Fig. 4).

Sequence similarity, and collinear prediction of AaTIFY proteins

We visualized the sequence similarity of AaTIFY proteins in A. argyi using Circoletto [25]. As shown in the Fig. 5A, there are 4 pairs of 8 members with red ribbons indicating they have the highest similarly in homology proteins, including AaTIFY14/AaTIFY30, AaTIFY15/AaTIFY31, AaTIFY17/AaTIFY33, and AaTIFY20/AaTIFY21. 8 pairs of 15 members with orange ribbons indicating they also have 75% < orange ≤ 99% similarity. 26 members with green ribbons showed 50% < orange ≤ 75% similarly. These results indicate that there were collinearity events among the TIFY proteins of A. argyi.

Fig. 5.

The chromosomal distribution and collinear relationships prediction of TIFY genes in A. argyi. A Chromosomal distribution and collinear prediction of AaTIFY genes in A. argyi. Gray lines indicate all synteny blocks in A. argyi genomes, black lines indicate duplicated TIFY gene pairs. B Visualising sequence similarity of AaTIFY genes in A. argyi. Different colored ribbons represent different levels of similarity, blue ≤ 50%, green ≤ 75%, orange ≤ 99%, red > 99%. C Collinear prediction of AaTIFY family genes between A. thaliana and A. argyi. Gray lines indicate all synteny blocks between A. thaliana and A. argyi; red lines highlight the syntenic TIFY gene pairs

There are two tandem duplication events and segmental duplication events in the TIFY gene family in A. argyi, including AaTIFY5, AaTIFY6, and AaTIFY7 on Chr.3; and AaTIFY23 and AaTIFY24 with tandem duplication on Chr.11 (Fig. 5A, B). In addition to the tandem duplication event, 19 AaTIFY members showed 45 collinear events that were also identified with the MCScanx method in the AaTIFY family (Fig. 5B, Table S3). In addition, to explore the potential evolutionary clues of the TIFY gene family in A. argyi, a comparative syntenic graph of A. argyi associated with A. thaliana was constructed (Fig. 5C, Table S4). The results showed that a total of 21 AaTIFY genes and 10 AtTIFY genes had a collinearity relationship, and 25 collinear gene pairs were identified between the AaTIFY gene family and the AtTIFY gene family. The results showed an expansion of TIFY gene families in A. argyi compared to A. thaliana.

Phylogenetic analysis of TIFY proteins from four species

To gain insight into the evolutionary relationship of TIFY proteins in A. argyi, a phylogenetic tree was constructed based on the multiple sequence alignment of 77 TIFY protein sequences. This set included 34 TIFYs in A. argyi, 18 in A. thaliana, and 25 in Populus, which were used to build the neighbor-joining phylogenetic tree (Fig. 6). According to the constructed phylogenetic relationship, the 77 TIFYs were classified into 9 groups, named Group 1 to Group 9. The members of the AaTIFY family were mainly distributed in Groups 3, 6, 7, and 9. Among these, Group 9 had the highest distribution number (7), and Group 4 did not have any AaTIFY members. According to the structural characteristics of TIFY, it can be divided into four categories, namely JAZ, ZML, TIFY, and PPD. Group 7 belongs to the TIFY subfamily and contains four members, including AaTIFY14/15/30/31. Group 6 belongs to the PPD subfamily and contains 6 members, including AaTIFY1/13/20/21/28. Group 5 belongs to the ZIM subfamily and contains three members, including AaTIFY2/16/32. There are 8 members in Populus and 3 members in A. thaliana. The remaining six groups, including Group 1/2/3/4/8/9, belong to the JAZ subfamily and contain 23 members. Additionally, this phylogenetic tree also indicates that TIFY proteins from the same group tend to cluster together, similar to the phylogenetic tree constructed by AaTIFY proteins in A. argyi (Figs. 3 and 6).

Fig. 6.

Phylogenetic relationships of TIFY family proteins of A. argyi, A. thaliana, and Populus. The phylogenetic tree was created by MEGA 7.0 using the ClustalW and NJ method with 1000 bootstrap replicates. The black five-pointed star represents the Populus TIFYs, the black dot represents Arabidopsis TIFYs, and the black triangle represents A. argyi TIFYs

Protein-protein interaction network of AaTIFY proteins in A. argyi

In order to further understand the potential functions of 34 AaTIFY proteins and the interactions among its family members, the STRING software was utilized to construct an interaction network based on the orthologs of A. thaliana in the STRING database (Fig. 7). The results showed that 13 out of 34 AaTIFY proteins had potential interactions with 64 edges among family members, and 18 AaTIFY proteins had potential interactions with 102 edges when additional proteins were added, including MYC1, MYC3, WRKY57, COI1, and K20J1.8 proteins (Fig. 7). Among them, the AaTIFY10 protein had the most interaction partners, interacting with 15 proteins. This was followed by AaTIFY11, AaTIFY19, and AaTIFY27, which had 13, 10, and 9 predicted interaction partners, respectively. For the node 2 analysis, AaTIFY34, MYC2, and MYC3 proteins can interact with 11 proteins. AaTIFY32 and COI1 can interact with 10 proteins. Followed by AaTIFY29 and AaTIFY31, which can interact with 8 proteins. These findings suggest that MYC2, MYC3, and COI1 proteins play an important role in regulating the function of the TIFY gene family. Above all, the protein interaction analysis provides an important basis for further verification of the function of AaTIFY proteins.

Fig. 7.

Protein–protein interaction network of AaTIFY proteins in A. argyi based on their orthologs in Artemisia annua. Line thickness indicates the strength of data support

Expression profiling of AaTIFY genes in different tissues of A. argyi

To comprehend the spatial pattern expression and the potential function of AaTIFY genes in A. argyi, we investigated the transcript levels of AaTIFY genes in leaf and root tissues of A. argyi based on the RNA-seq data available from our previous study (NCBI accession: PRJNA1054765). The expression heatmap showed a tissue pattern where 25 out of 34 members had relatively low expression levels in leaves and increased expression in root tissues (Fig. 8, Table S5). Among them, four members, including AaTIFY6, AaTIFY7, AaTIFY10, and AaTIFY26, showed an average FPKM of 0 in leaves but were expressed in roots. Sixteen members showed significantly higher expression in roots compared to leaves. Nine out of 32 members had relatively higher expression levels in leaves than in roots, and AaTIFY18/23/34 showed significant expression. These results indicate that the expression of AaTIFYs has tissue preference and specificity, and they may play important roles in the development of different tissues.

Fig. 8.

Expression profiles of AaTIFY genes in leaves and roots of A. argyi. The heatmap of FPKM datas were normalized by Z-score; The bar chart shows the log₂(Root/Leaf) values of root vs. leaf; Each set had three replicates

Expression profiling of AaTIFY genes in response to salt and saline-alkali treatments

To analyze the expression profile of AaTIFY genes, RNA-seq data (NCBI accession: PRJNA1054765) from A. argyi leaves and roots subjected to salt and saline-alkali treatments were analyzed using FPKM values (Fig. 9, Table S6). The expression patterns of AaTIFY8/19/25/29 genes in leaves were significantly increased under salt treatment (Fig. 9A), and AaTIFY19/25/26 genes in roots were also significantly increased (Fig. 9B). Under saline-alkali treatment, the expression patterns of four AaTIFY genes in leaves and roots were significantly altered. AaTIFY4/23 genes were down-regulated, while AaTIFY19/29 genes were up-regulated in leaves (Fig. 9C). In roots, AaTIFY3/22/29 genes were significantly decreased (Fig. 9D). The results indicate that salt and saline-alkali treatments have varying effects on the expression levels of AaTIFY genes. Most AaTIFY genes exhibit similar regulatory profiles in response to salt and saline-alkali stress. It is important to note that this analysis is objective and does not include any subjective evaluations. AaTIFY19/29 members may play a crucial role in regulating plant growth under salt or saline-alkali stress.

Fig. 9.

Expression profiles of AaTIFY genes in response to salt and saline-alkali treatments based on RNA-seq data. A salt treatment in leaves, B salt treatment in roots, C saline-alkali treatment in leaves, D saline-alkali treatment in roots. The FPKM data were normalized by Z-score; The bar chart shows the log₂(Treatment/CK) values; Each set had three replicates

Expression pattern of AaTIFY genes in response to different hormone treatments

To examine the expression pattern of the AaTIFY genes in response to different hormones, 11 genes with FPKM > 1 and no sister pair genes were selected for examination of their expression under MeJA, SA, ABA, and IAA treatments. The results of RT-qPCR show the expression of these 11 AaTIFY genes in response to hormones of MeJA, IAA, SA, and ABA hormones to varying degrees (Fig. 10). Under MeJA treatment, the transcript level changes of 11 AaTIFY genes were investigated using RT-qPCR. Our results showed that almost all 11 AaTIFY genes were induced by MeJA application (Fig. 10A). Among them, the expression patterns of AaTIFY19/23/29 were rapidly induced within 3 h of MeJA treatment, and their expression were increased by more than 1000 folds; AaTIFY17/18/27 could also be rapidly induced to more than 30 folds, these gene exhibited a similar pattern, rapidly increasing within 3 h, followed by a significant decrease in expression level. The other five members including AaTIFY1/15/20/30/32 genes, also exhibited significantly increased expression under MeJA treatment at 3 h and 12 h, and then decreased at 24 h and 48 h. These results suggest that the TIFY family plays an important role in the early response to MeJA treatment.

Fig. 10.

Expression profiles of AaTIFY genes under different hormone treatments determined by RT-qPCR in A. argyi. RT-qPCR data were normalized using Actin as the reference gene and were displayed relative to 0 h. Results are expressed as mean ± standard deviation (SD). Error bars represent the standard error of the mean of three independent replicates. Columns with different letters are significantly different (P < 0.05) by ANOVA test. A MeJA treatment, B SA treatment, C ABA treatment, D IAA treatment

Under SA treatment, the expression of AaTIFY18/19/23 showed a pattern of initially increasing and then decreasing. They reached their peak expression at 3 h and exhibited decreased expression at 24 and 48 h. AaTIFY15/27/29/30/32 reach their peak at 12 h; The expression patterns of AaTIFY1/17/20 genes showed a decrease (Fig. 10B). The AaTIFY19/23/27/29 genes were found to be up-regulated, with their expression levels increasing by 14.12, 12.86, 4.98, and 2.86 folds, respectively. While these four genes also exhibited significantly decreased expression at other treatment time points. The above results indicate that various members of the TIFY family exhibit significant differences in their response to SA treatment, suggesting that different members may have distinct functions in response to SA treatment.

Under ABA treatment, the expression levels of AaTIFY genes were altered to various degrees (Fig. 10C). In general, the expression of AaTIFY genes was up-regulated under ABA treatment. In particular, AaTIFY29 increased by 27.19 folds at 12 h, AaTIFY23 by 19.88 folds at 3 h, AaTIFY19 by 9.46 folds at 48 h, followed by AaTIFY27, AaTIFY32 and AaTIFY18 by 8.01, 3.55 and 3.17 folds, respectively. In addition, the remaining 5 members also showed increases to varying degrees.

Under IAA treatment, the expression of AaTIFY genes was generally up-regulated to varying degrees (Fig. 10D). Among them, AaTIFY29 increased to 51.50 folds at 12 h, AaTIFY19 increased to 25.02-fold at 3 h, AaTIFY23 increased to 13.78 folds at 3 h, and AaTIFY27 increased to 10.06 folds at 12 h. These results suggest that members of AaTIFY29/19/23/27 may play an important role in the response to IAA treatment. In addition, the remaining 8 members also showed low fold up-regulation by IAA treatment. These results suggest that TIFY also plays an important role in regulating plant growth and development.

Most importantly, through comprehensive analysis of these expression patterns, we found that AaTIFY19/23/27/29 members were extremely significant under multiple hormone stresses, suggesting that these four genes, which may act as core members, play an important role in responding to hormones to regulate growth and development of A. argyi.

Discussion

With the improvement of people’s living standards, individuals are increasingly focusing on leading a healthy lifestyle, leading to a growing demand for medicinal plants. A. argyi, which has a variety of functional uses and is one of the important medicinal materials for regulating human health, has attracted more and more attention from researchers. Among these studies, research on its growth and development, stress response, and regulation of active ingredients is increasing. TIFY is a plant-specific transcription factor that has been identified in many plants, such as Arabidopsis [4], maize [26], and Camellia Sinensis [27]. Numerous previous studies have shown that the TIFY family plays a critical role in plant growth and development, stress responses, as well as in the regulation of secondary metabolite synthesis. Therefore, the study of the TIFY family in A. argyi is of great significance for elucidating the growth and development, stress responses, and secondary metabolite synthesis of A. argyi.

The characterization of the TIFY family in A. argyi

In the present study, the TIFY genes were systematically identified and analyzed, revealing at least 34 AaTIFY genes in the genome of A. argyi. The number of AaTIFY members in A. argyi was much higher than in Vitis vinifera (19), A. thaliana (19), Oryza sativa (20), Nicotiana tabacum (33), and relatively less than in Brassica rapa (36), Glycine max (38), Z. mays (47), Triticum aestivum (49), and Gossypium hirsutum (50) (Figure S1). The analysis of physicochemical properties reveals a high variation among the different members. The variation range of the amino acid sequence of AaTIFYs was 132 aa to 379 aa, with molecular weights ranging from 15181.13 Da to 39519.55 Da, and the expected theoretical isoelectric point ranging from 5.16 to 9.54. In terms of gene structure, the range of variation in AaTIFY gene introns ranged from 1 to 7. The variation range of the conserved motif was 3 to 6. According to the phylogenetic tree, we noticed that most of the AaTIFYs in the same clade have a similar pattern of exon-intron organization and conserved motifs, although the length of introns varied for some members. This phenomenon suggests that the intron organization of homologous genes tends to be conserved during evolution. These results were similar to those in kiwifruit [28]. Above all, these differences also lay the foundation for the TIFY family classification and reveal that TIFY family members may have functional differentiation.

Previous studies have shown that a gene family generally undergoes either tandem duplication or large-scale segmental duplication to maintain a high number of members during its evolution [29]. Gene duplication and loss were the primary evolutionary driving forces for the expansion or contraction of genomes. Duplicated genes could result in gene redundancy [30]. Previous studies have found significant differences in gene duplications within the TIFY family across species. For example, Populus trichocarpa, cassava, and Brassica rapa have 10, 18, and 18 duplicated TIFY genes, respectively [31–33]. In this study, 34 TIFY genes were classified in 4 subfamilies which were TIFY, JAZ, PPD, the ZML with 4, 22, 5 and 3 members, respectively. Compared with other species, such as in cassava (16 JAZ, 3 PPD, 7 ZML, and 2 TIFY) [32], Brassica rapa (21 JAZ, 7 TIFY, 6 ZML, and 2 PPD) [33], and walnut (2 TIFY, 12 JAZ, 2 ZML, and 2 PPD) [34], indicates that the phenomenon of gene multiplication and compression exists in different subfamilies. This includes the expansion of gene members in the JAZ and PPD subfamilies, while the TIFY and ZML subfamilies may experience gene member contraction in A. argyi. Furthermore, both tandem duplications (two tandems) and segmental duplications (44 pairs) were detected in the A. argyi TIFY gene family, suggesting that both types of duplication events may be the key mechanisms for the formation of the TIFY family in A. argyi. These results support the concept that duplication events play a significant role in the development and expansion of the TIFY gene family in plants. These duplication events may have originated from segmental or large-scale duplication events [35]. These phenomena were similar to those in the kiwifruit, which also has two tandem duplications and high segmental duplications [28]. In addition, the genome data of A. argyi is only half assembled into haplotype group A, containing pseudochromosomes 1 ~ 17. In our study, the analysis of the TIFY family was based on the genome data of haplotype group A, which may reduce or halve the number of TIFY family members [22]. Therefore, the number of AaTIFY members should be more than 34 or twice 34. According to genome studies in A. argyi, the assembled genome data reveals at least three rounds of whole-genome duplication (WGD) events, including a recent WGD event in the A. argyi genome, and a recent burst of transposable elements. These may contribute to its large genome size, indicating that the expansion or contraction of members in different TIFY subfamilies may have been caused by WGD events that occurred during A. argyi evolution [22].

Based on the phylogenetic tree of A. thaliana and A. argyi, all TIFYs can be divided into 9 groups. Group 1 consisted of AtJAZ7/8/10 and AaTIFY11/12/19. Studies on this group of genes from Arabidopsis suggested that they may play a role in stress and leaf senescence mediated by jasmonate signalling [36, 37]. Group 2 consisted of AtJAZ1/2 and AaTIFY4/23/29, which have been suggested to play a role in auxin response, abiotic stress and development of root growth [38, 39]. Group 3 consisted of only 9 AaTIFYs, group 3 is relatively close to group 2 and may have similar functions in concert with group 2. Group 5 consisted of AtZML1/2 and AaTIFY2/16/32, which belong to the ZML subfamily and may be involved in hypocotyl and petiole elongation [40]. Group 6 contains AtPPD1/2 and AaTIFY1/13/20/21/28, which belong to the PPD subfamily and regulate the development of lamina size and curvature [41]. Group 7 includes AtTIFY8 and AaTIFY14/15/30/31, may function as a repressor of leaf senescence [42]. Group 8/9 includes AtJAZ3/4/9 and AaTIFY9/10/18/27/17/33/34, studies of this group of genes indicated that they may play a role in plant defence, growth and development [43]. Most importantly, the results of the phylogenetic tree analysis contribute to our understanding of the functions of different AaTIFY genes.

TIFY genes involved in salt or saline-alkali stress in A. argyi

Previous studies have shown that the TIFY family plays an important role in the regulating various stresses, such as salt, drought, and cold stress. In apple, overexpression of MdJAZ2 increased the tolerance to salt and drought stresses during seedling development in Arabidopsis [44]. In Populus trichocarpa, PtrJAZ1/3/4/5/7/8/9/10/12 showed increased expression levels under heat, drought, and cold stresses, indicating that the PtrTIFY family plays important roles in responding to these abiotic stresses [31]. In rice, overexpression of OsTIFY11a/OsJAZ9 significantly enhanced tolerance to salt and dehydration stress [45]. Promoter cis-elements, which regulate gene transcription and expression, are often utilized to predict the potential function of genes [46]. In this study, the presence of multiple cis-regulatory elements in the promoter region of TIFY genes demonstrates that these genes may be activated in response to a variety of stressors. For example, all members contain multiple cis-acting elements involved in light responsiveness, indicating that the expression of AaTIFY genes was regulated by light, which in turn regulates the growth and development of A. argyi; 14 AaTIFY genes contain defense and stress responsiveness elements; 16 AaTIFY genes contain low-temperature responsiveness elements; and 8 AaTIFY genes contain drought-inducibility responsiveness elements. These cis-element analyzes lay an important foundation for further functional verification of the TIFY family. Furthermore, combined with transcriptome data, we found that some AaTIFY genes, such as AaTIFY3/4/19/22/23/25 genes, respond to salt stress or saline-alkali stress. They also contain defense and stress responsiveness cis-elements. It is speculated that these genes may respond to salt or saline-alkali stress through the regulation of these cis-elements.

TIFY genes respond to crosstalk between different phytohormones

Previous studies have shown that TIFY plays an important role in responding to phytohormone regulation, especially for jasmonic acid (JA) and abscisic acid (ABA). In our study, almost all AaTIFY genes, except AaTIFY6/11/12, possessed at least one phytohormone-related promoter cis-element, indicating that AaTIFY genes respond to multiple exogenous phytohormone treatments. Among them, the JAZ subfamily acts as a key protein in the plant-specific JA signaling pathway, and the importance of the JAZ protein in mediating JA-regulated responses is well described in many species. In this study, our results showed that almost all 11 selected AaTIFY genes’ expressions were induced by MeJA treatment to various degrees, which is consistent with a previous study on TIFY family genes in cassava [32]. Among them, AaTIFY17/18/27 belong to the JAZ subfamily and were rapidly induced by more than 30-fold. These results are consistent with the rapid triggering of JAZ gene expression by JA treatment, which may be responsible for moderating the JA response.

There is no doubt that ABA and its signaling pathway play important roles in regulating various stress responses [47]. In grapes, six out of the 11 abiotic stress-responsive TIFY genes can be regulated by ABA [48]. In our study, AaTIFY18/19/23/27/29/32 were significantly stimulated by exogenous ABA treatment, and their expression levels increased by 3.1–27.2 folds, suggesting that the function of the AaTIFY family in response to various abiotic stresses may be regulated at an ABA-dependent transcriptional level. These results were similar to the function of TIFYs in cassava by Zheng et al. [32]. Furthermore, Abscisic acid (ABA) and Jasmonic acid (JA) have been reported to act synergistically during drought stress signaling. For instance, OsJAZ1 was induced by ABA and drought stress. Overexpression of OsJAZ1 led to a drought-sensitive phenotype under ABA treatment [49]. These results also indicate the direction for studying the synergistic regulation of ABA and JA by TIFY genes in A. argyi in response to stress.

IAA is a very important phytohormone that plays vital roles during the growth and development of plants [50]. In our study, the expression of most AaTIFY genes was also regulated to varying degrees by IAA treatment, particularly for AaTIFY19/23/27/29, indicating that these genes play a crucial role in the growth and development of A. argyi. Previous studies have revealed that IAA can interact with JA, and their mainly embodied in regulating the root meristem activity and stem cell maintenance through antagonistic effects in plants [51, 52]. Therefore, we assumed that AaTIFY19/23/27/29 directly participate in the JA-IAA crosstalk, and they might also be also involved in regulating of primary root growth in A. argyi.

Salicylic acid (SA) plays a crucial role in the signal transduction pathway of abiotic responses [53]. In Populus trichocarpa, most PtrJAZ subfamily members are induced by salicylic acid (SA) treatment [31]. In our study, for the SA treatment, different members of AaTIFY also show varying levels of regulatory patterns, particularly AaTIFY23, AaTIFY29, AaTIFY19, and AaTIFY27. In addition, the TIFY gene has been reported to mediate salicylic acid (SA) and stress responses. SA can alleviate the adverse effects of salt stress [54] and heat stress [55]. In the expression profiling study of AaTIFY genes in response to salt and saline-alkali treatments, AaTIFY23, AaTIFY29, and AaTIFY19 also showed significant responses, suggesting that these three genes may play important roles in the cross-talk between salicylic acid (SA) and salt or saline-alkali stress.

Above all, AaTIFY genes were induced by multiple treatments such as JA, SA, ABA, and IAA, as well as by abiotic stresses including salt and saline-alkali stress. Previous studies have shown that phytohormone crosstalk is universally present in plants, constituting a very complex signaling regulatory network that plays a critical role in the regulation of growth and development in plants [56]. In our study, we postulate that TIFY genes in A. argyi respond to various stresses through different hormone signaling pathways, such as JA-ABA stress crosstalk, JA-IAA crosstalk, or SA-stress crosstalk, regulating the growth and development of A. argyi. For future research, the functional of AaTIFY genes in the regulation of growth and development, adversity stress and hormone crosstalk regulation in A. argyi plants can be further investigated. In addition, based on previous studies, JAZ genes, act as a crucial interface in the jasmonate signalling cascade, can be involved in the regulation of biosynthesis of plant metabolic components [13–17]. In-depth research on the JAZ genes of A. argyi to regulate the synthesis of active components of A. argyi, which in turn can improve the quality of A. argyi and lay the foundation for the molecular breeding of A. argyi.

Conclusions

In this study, the A. argyi TIFY family genes were comprehensively and systematically characterized. A total of 34 AaTIFY genes were identified and their physical and chemical characters, chromosomal distribution, conserved domain, different types of structures, promoter cis-elements, gene collinear, protein-protein interaction, and phylogenetic tree were analyzed. The results showed that 34 AaTIFY genes could be classified into four main groups of JAZ, ZML, TIFY and PPD, and members within the same group had similar motif compositions and gene structures. The expression profiling of the AaTIFY genes in different tissues, response to salt, saline-alkali, and different hormone treatments were analyzed. The extensive bioinformatics and expression analysis of the AaTIFY genes contributes to our understanding of the functions of these genes in multiple stress responses and phytohormone crosstalk.

Materials and methods

Plant material, growth conditions and hormone treatments

The plant materials used in our study were obtained from the medicinal botanical garden of Henan University of Chinese Medicine, Zhengzhou, Henan Province, China. The root buds of A. argyi were used as explants for disinfected, and sterile tissue culture seedlings of A. argyi were obtained. Aseptic tissue culture seedlings of A. argyi plants which grown in tissue culture bottle with ½ Murashige and Skoog (½MS) culture medium with 0.7% agar and 3% sucrose. Seedlings were grown in a sterile culture room under a 16 h light/8 h dark photoperiod (120 µmol m^− 2 s^− 1) at 24 ~ 26℃. After 1-month of growth, A. argyi seedlings were treated with different hormones (SA, MeJA, IAA, and ABA) as described by Lian, et al. [57]. All samples collected were immediately frozen in liquid nitrogen and stored at -80℃ for later analysis.

Identification and annotation of the TIFY genes in A. argyi

The A. argyi genome annotation (PRJCA010808) was downloaded from the National Genomics Data Center (NGDC, https://ngdc.cncb.ac.cn) [22]. HMMER and Basic Local Alignment Search Tool (BLASTP) were used to search for A. argyi TIFY proteins, using the published Arabidopsis TIFY protein sequences as the initial protein query. The Hidden Markov Model of Pfam3 tool was used to investigate the TIFY domain (PF06200). Information on DNA sequences, cDNA sequences, protein sequences and feature information of TIFYs were obtained from the GFF file available on the A. argyi genome project website [22]. The physiochemical parameters of each gene were calculated using ExPASy (https://web.expasy.org/protparam/) [58]. The subcellular localization prediction of TIFY proteins was estimated using the Plant-mPLoc online tool (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) [59]; the secondary structure of TIFY proteins was analyzed using the SOPMA online tool (https://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) [60]; the 3D structures of AaTIFY proteins were analyzed using SWISS-MODEL (https://swissmodel.expasy.org/) [61]; the protein phosphorylation site prediction of TIFY proteins was analyzed using NetPhos3.1 online tool (http://www.cbs.dtu.dk/services/NetPhos) [62]; the protein signal peptide prediction of TIFY proteins was analyzed using SignalP-5.0 (http://www.cbs.dtu.dk/services/SignalP/) [63].

Chromosomal positions, duplication, collinearity analysis and protein-protein interaction prediction

Based on the chromosome-scale genome assembly of A. argyi by Hongyu Chen et al., the genome was assembled into 17 pseudochromosomes, and the chromosomal positions of each TIFY gene member were confirmed using the A. argyi genome annotation dataset [22]. Multiple collinear scanning toolkits (MCScanX) were used to analyze gene duplication events using default parameters [64]. To reveal the syntenic relationship of orthologous TIFY genes between A. argyi and Arabidopsis, the syntenic analysis maps were constructed using the Dual Systeny Plotter software (https://github.com/CJ-Chen/TBtools) [65]. Furthermore, the visualization of sequence similarity of AaTIFY genes in A. argyi was analyzed using the Circoletto online tool (https://bat.infspire.org/circoletto/) [25]. In addition, the protein-protein interaction network of AaTIFY proteins was analyzed using STRING online software (https://cn.string-db.org/), and an interaction network of AaTIFY proteins in A. argyi was constructed based on the orthologues of Arabidopsis in STRING database [66].

Phylogenetic analysis, gene structural, and conserved motif of TIFY in A. argyi

A total of 34 TIFY protein sequences from A. argyi were aligned using ClustalW with the default parameters, and then a phylogenetic unrooted tree was constructed using MEGA6.0 software by using the Neighbor-joining (NJ) algorithm, and the support of the tree topology was assessed by bootstrap analysis with 1000 replicates. For the phylogenetic analysis of TIFY protein in different species, 92 TIFY protein sequences from A. argyi (32), Arabidopsis thaliana (18) [4], poplar (25) were used to construct the neighbor-joining phylogenetic tree. Finally, the constructed phylogenetic tree was annotated and visualized using the online tool of Evolviewv2 (https://www.evolgenius.info/evolview-v2/) [67].

To assess the exon-intron organization of the TIFY genes, GFF files of the A. argyi genome were obtained from the National Genomics Data Center, and the gene structure of the AaTIFY genes was analyzed using TBtools. For the MEME of conserved motif analysis, the MEME website (https://meme-suite.org/meme/) was used to predict the conserved motif of TIFY proteins with the number of conserved motifs was set to 10. The conserved domain of TIFY proteins were analyzed using the Batch Web CD-Search Tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). An integrated graphic of the phylogenetic tree, conserved motif, and conserved domain was mapped using the TBtools [65].

Cis-regulatory element analysis in the promoter regions of TIFY genes in A. argyi

To identify the cis-elements in the promoter region of each TIFY gene in A. argyi, the 2000 bp upstream sequences of the TIFY-encoding DNA sequences were extracted from the the available A. argyi genome data using the TBtools program, and possible cis-acting elements were analyzed using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/index.html). A total of 22 cis-elements were selected by removing cis-elements of functional ambiguity as well as core promoter elements. The cis-element distributions were visualized using TBtools [65].

Transcriptome analysis based on RNA-seq data

To reveal the expression profile of TIFY genes in response to salt and saline-alkali stress in A. argyi, one-month-old sterile seedlings were transplanted to the greenhouse under 70% relative humidity, and under 16:8 h light: dark conditions at 24 ~ 26℃, after 2-month growth, seedlings were treated with or without 200 mmol/L salt (NaCl : Na₂SO₄ = 1 : 1, pH = 7.0) and saline-alkali stress (NaCl : Na₂SO₄ : NaHCO₃: Na₂CO₃ = 1 : 1 : 1 : 1, pH = 9.0) for 3 days, then the leaves between three and six nodes and roots were harvested for RNA-seq. Then, we performed the 2 × 150 bp paired-end sequencing on an Illumina Novaseq™ 6000 at the (LC Sceiences, USA) following the vendor’s recommended protocol. Firstly, Cutadapt [68] and perl scripts in house were used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases. Then sequence quality was verified using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), including the Q20, Q30 and GC-content of the clean data. StringTie and ballgown were used to estimate the expression levels of all transcripts and perform expression abundance for mRNAs by calculating FPKM (fragment per kilobase of transcript permillion mapped reads) value. The RNA-seq data were submitted to NCBI with SRA accession number PRJNA1054765. Heatmaps were generated using OmicStudio tools at https://www.omicstudio.cn/tool [69] based on the mean FPKM values of three replicates of TIFY genes.

RNA extraction and RT-qPCR analysis

Total RNA of A. argyi was extracted using the EASYspin Plus Plant RNA Kit (AidLab, Beijing, China). According to the manufacturer’s instructions, 1 µg of qualified RNA samples were prepared for cDNA strand synthesis using the EasyScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit (Transgen, Beijing, China). RT-qPCR experimental steps were performed according to a previously published protocol [70]. Actin was used as the internal reference gene. Each assay was detected by 3 biological replicates and 3 technical replicates. Relative expression level was calculated using the 2^−△△Ct method [71]. All primers in this paper were listed in Table S7.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, CL and SC; Formal analysis, CL, BZ , JJL and HY; Funding acquisition, CL and SC; Investigation, JJL, HY and JL; Resources, FZ and JXL; Supervision, CL and JY; Validation, XL, RM, and JXL; Writing – original draft, CL and BZ; Writing – review & editing, CL, JXL and SC. All authors read and approved the final manuscript.

Funding

This research was funded by the Key Scientific Research Project of Higher Education Institutions in Henan Province (22A360012); Chinese herbal medicine industry technology system of Henan Province (Yucaike [2024]8); Key project at central government level: The ability establishment of sustainable use for valuable Chinese medicine resources (2060302).

Data availability

All data generated or analyzed during this study are included in this article and supplementary materials. The genome sequences of A. argyi was downloaded from National Genomics Data Center (https://ngdc.cncb.ac.cn with BioProject accession number PRJCA010808), and the RNA-seq data were downloaded from NCBI with SRA accession number PRJNA1054765). The TIFY protein sequences of Arabidopsis thaliana and Populus trichocarpa were downloaded from TAIR (https://www.arabidopsis.org/) and Phytozome 13 (https://phytozome-next.jgi.doe.gov/info/Ptrichocarpa_v4_1) databases, respectively.

Declarations

Ethics approval and consent to participate

No specifc permit is required for the samples in this study. We comply with relevant institutional, national, and international guidelines and legislation for plant studies.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.