Identification of MYC genes in four Cucurbitaceae species and the response to temperature stress

doi:10.21203/rs.3.rs-4203459/v1

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Research Article

Identification of MYC genes in four Cucurbitaceae species and the response to temperature stress

https://doi.org/10.21203/rs.3.rs-4203459/v1

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published 16 Sep, 2024

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Background

Myelocytomatosis (MYC) transcription factors are crucial mediators of plants responding to environmental stresses through binding DNA regulatory regions. However, little systematic characterization of MYC genes is available in Cucurbitaceae species.

Results

In this study, we identified 10, 8, 12, and 10 MYC genes separately in Cucumis sativus, Cucumis melo, Citrullus lanatus, and Benincasa hispida. Characterization analysis revealed that all of the MYC proteins contain a highly conserved H4-V5-E6-E8-R9-R11-R12 sequence, which is essential for the binding DNA regulatory regions. The evolutionary analysis enabled us to categorize the predicted 40 MYC proteins from seven species into five distinct groups, which was also discovered that the expansion of the MYC genes occurred before the divergence of monocots and dicots. The upstream promoter region of the MYC genes contain a variety of developmental, stress, and hormone-responsive regulatory elements. The expression of cucumber MYC genes varies significantly across organs, with particularly high expression of CsaV3_3G001710 observed across all organs. Transcriptomic analysis reveals that certain cucumber MYC genes undergo specific upregulation or downregulation in response to both biotic and abiotic stressors. Particularly under temperature stress, cucumber genes CsaV3_3G007980 and CsaV3_3G001710 showed significant upregulation. Interestingly, the homologous genes of these two in C. lanatus exhibited a similar expression pattern to C. sativus, while in B. hispida, they displayed a significant downregulation, which is quite the opposite. These findings indicated that these two genes indeed responded to temperature stress with different expression patterns, highlighting the divergent functions of homologous genes across different species.

Conclusions

This study analyzed the size and composition of the MYC gene family in four Cucurbitaceae species, and investigated stress-responsive expression profiles, especially under temperature stress. All the results showed that MYC play important roles in development and stress-responsive, laying a theoretical foundation for further investigating its response mechanisms.

Myelocytomatosis oncogene (MYC), an important class of transcription factors (TFs) belong to basic helix-loop-helix (bHLH) TF family, contains two conserved functional domains, namely bHLH_MYC_N domain in the N-terminal region and bHLH region in the C-terminal [1–3]. As a sub-genetic family in the bHLH family member, MYC play an important role in plant growth and development, secondary metabolism and signal transduction [4].

The first MYC gene was cloned form Arabidopsis thaliana, named AtMYC1, functional studies showed that it played a certain role in plant seed development [5]. Successively, more MYC genes were found in Arabidopsis. It was found that MYC2 could regulate leaf aging by antagonizing with bHLH Ⅲd subfamily transcription factors [6], and could also interact with JAZ7 to inhibit leaf aging under dark conditions [7]. In Arabidopsis thaliana, AtMYC2 could cooperate with AtMYC3 and AtMYC4 in regulating leaf development [8], chlorophyll degradation [9], seed production and seed storage protein accumulation [10, 11]. Previous studies also found that AtMYC2 could inhibit the growth of leaf veins by inhibiting the synthesis of auxin in plant leaves [12]. Others, the Arabidopsis Aborted MicrosporeS (AMS) gene, encoded a MYC class transcription factor, plays a crucial role in tapetum cell development and pollen wall formation [13]. In addition, MYC genes in other plants have been identified to be involved in plant growth and development. In apple (Malus pumila Mill.), at the fruit ripening stage, MdMYC2 can affect ethylene biosynthesis and promote fruit ripening by promoting the expression of MdACS1 and MdACO1 [14]. In rice (Oryza sativa), overexpression of OsMYC2 could interact with OsJAZ1 and activate the downstream gene OsMADS1, and then regulate the development of spikelet [15]. The MYC genes also have important effect on the accumulation of plant secondary metabolites. Such as, overexpressed the AtMYC3 and AtMYC4 resulted in the excessive accumulation of anthocyanins in Arabidopsis. Likewise, wheat (Triticum aestivum) MYC1 could also regulate anthocyanin synthesis in pericarp [16]. The CrMYC2 could control the jasmonate-responsive expression of the ORCA genes that regulated alkaloid biosynthesis in Catharanthus roseus. In few, TcJAMYC could bind to E-box and activate the gene promoter related to paclitaxel pathway, which can effectively improve the yield of paclitaxel in suspension cell culture system [17]. In Artemisia annua, AaMYC2 could bind to AaJAZ1-4 and activate the expression of artemisinin biosynthetic enzymes CYP71AV1 and DBR2, which positively regulates artemisinin biosynthesis [18].

Although a large number of studies on MYC genes in various species have been conducted, studies on MYC genes in Cucurbitaceae crops is still weak. Cucurbitaceae is one of the most important edible plant families in the world, among which cucumbers, melons, watermelons and wax gourds are widely grown worldwide, coming into being the great economic benefits. In this study, we aim to identify MYC genes in four Cucurbitaceae crops and compare the evolution and variations of MYC genes among species through bioinformatics analysis. We will also analyze the expression patterns of MYC genes under biotic and abiotic stresses, focusing on identifying MYC genes involved in temperature stress response, for providing theoretical support for stress-resistant breeding in Cucurbitaceae crops.

Identification and bioinformatics analysis of the MYC gene family in Cucurbitaceae crops

Download the HMM model file (PF14215.7 and PF00010) for the MYC gene family from the Pfam database (http://pfam.xfam.org/), and download the protein sequence file of Cucumis sativus L., Cucumis melo L., Citrullus lanatus, Benincasa hispida from the Cucurbitaceae Genomic Database (http://cucurbitgenomics.org; [19–22] ). Use the Hidden Markov Model (HMM) plugin in the HMMER v3 software package to predict candidate MYC gene family members in the Cucumis sativus L., Cucumis melo L., Citrullus lanatus, Benincasa hispida genome [23], and extract the sequence information of high-quality candidate proteins using Perl scripts (E < 1 × 10^− 5). Then, identify the candidate MYC gene family genes using the BLASTP program and the NCBI-Conserved Domain Data (CDD) (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi; [24]).

MYC gene family characterization and phylogenetic tree construction in Cucurbitaceae crops

Utilizing the online tool ExPASy (https://web.expasy.org/protparam/) [25], we analyzed the amino acid count, molecular weight, isoelectric point, instability coefficient, aliphatic index, and average hydrophobicity of the members of the MYC gene family. We used the online website CELLO (http://cello.life.nctu.edu.tw/) for subcellular localization prediction. We employed the online software MEME (http://meme-suite.org/) to analyze the conserved motifs of MYC family proteins, with the parameters set as follows: 10 motifs, optimal motif width range from 6 to 200. The multiple sequence alignment was performed using the ClustalX 2.0 and visualized by Jalview [26]. Phylogenetic analysis was performed using MEGA7 [27] with the neighbor-joining (NJ) method, and the parameters were set as the Poisson model, pairwise deletion, and 1000 bootstrap replications [28].

Chromosomal distribution and collinearity analysis of MYC genes

Based on the physical location information in the genome database, we used the mapchart to map the MYC gene family members onto Cucurbitaceae crops chromosomes, respectively [29]. The distribution of the Cucurbitaceae crops SRS gene family on the chromosomes were visualized using TBtools software [30]. Gene duplication events were analyzed using the multiple collinearity scan tool (MCScanX) [31]. A collinearity analysis plot was generated using the Dual Systeny Plotter software (https://github.com/CJ-Chen/TBtools) [32].

Gene Expression Analysis

Transcriptome sequencing data related to cucumbers were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/). The SRA to Fastq plugin in TBtools was used to convert the downloaded SRA data into Fastq format. Data quality was assessed using the FastQC plugin [33], and adapter sequences and low-quality sequences were removed with the Trimmomatics plugin [34], resulting in clean data. The filtered transcriptome data was aligned to the cucumber ChineseLong_V3 genome using the STAR plugin, generating SAM files [35]. Gene expression levels were analyzed using the StringTie Quantify plugin [36]. Finally, differential gene expression analysis was conducted using the DESeq2 plugin [37].

Tissue-specific expression analysis of Cucumber MYC genes

Using transcriptome sequencing data from the NCBI database (PRJNA80169) [38], we analyzed the tissue-specific expression of the cucumber MYC gene family in various tissues and organs, including cucumber leaves, stems, female flowers, male flowers, unfertilized ovaries, fertilized ovaries, ovaries, roots, tendrils, and the base of tendrils. We utilized the TBtools software to create an expression heatmap, depicting the specific expression patterns of the cucumber MYC gene family in different cucumber tissues and organs.

Stress-responsive expression analysis of Cucumber MYC genes

Using transcriptome sequencing data from the NCBI database, including PRJNA634519 [39], PRJNA438923 [40], PRJNA477930 [41], PRJNA321023 [42], and PRJNA419665 [43], we analyzed the specific expression patterns of the cucumber MYC gene family in response to various stress conditions, such as high temperature, low temperature, high salinity, silicon, powdery mildew, downy mildew, and southern root-knot nematodes. We used TBtools software to create expression heatmaps, illustrating the gene expression responses of the cucumber MYC gene family undering abiotic and biotic stress conditions.

Temperature stress treatment, RNA extraction and qRT-PCR

The cultivars Jinyan-4(Cucumis sativus), Harukei-3 (Cucumis melo), B227 (Benincasa hispida), and 8424 (Citrullus lanatus) provided by Hebei Engineering Research Center for Seedling Breeding of Solanaceae and Fruit Vegetables of Hebei University of Engineering were used to explore the genes expression under temperature stress. The seedlings of four cultivars were treated at 42°C, and the leaves of the seedlings were taken at 0, 3, 6 and 12 h after treatment for qRT-PCR. At the same time, the seedlings of four cultivars were treated at 10°C, and the leaves of the seedlings were taken at 0, 3, 6 and 12 h after treatment for qRT-PCR. All samples have three biological replicates, and frozen in liquid nitrogen and then immediately stored at -80℃.

Total RNA was extracted using the RNAprep Pure Plant Kit (DP432, Tiangen Bio-tech, Beijing, China) according to the manufacturer's instructions. The cDNA was synthesized from the total RNA using the PrimeScript RT Kit (Takara). Specific primers for each gene were designed through primer 6 (Table 1). Subsequently, the synthesized cDNA underwent qRT-PCR on an Opticon thermocycler (CFX96 Connect Real-Time System; Bio-Rad, Hercules, CA) using SYBR Green PCR master mix (Vazyme, Nanjing, China) according to the manufacturer's instructions. The 2-ΔΔCT method was employed to calculate the relative expression of the MYC genes [44].

Table 1

List of primers used in quantitative RT-PCR. All primers are designed by Primer 6.
Gene	Sense Primer	Anti-sense Primer
CsaV3_3G007980	TAGTCAGTGGAGTCAGAGAT	CTACACGGTTAATCACAGAAG
CsaV3_3G001710	GGATGCGATGATAAGGATTC	TCTTCACTGTTGCTTGTTG
Bhi01M000362	GCGGTTCTATGCTCTACG	TTGAGGTGAGGCTGGATT
Cla97C05G080890	CTAGTTAATGGAGTCAGAGATG	CACGGTTAATCACAGAAGG
MELO3C006016	CGGTGAGGAATGATGAGAA	AATGAGACAGTTGCCAGAA
MELO3C013851	CGAGACGAGTTCTTGGATT	ACGGTGGTGGTAATTGAAT
Bhi11M000137	CGACTCAGACCACTCAGA	GATTCAATGGCTCTTCTCTTC
Cla97C10G186220	GGACTCAGACCACTCAGA	GATTCAATGGCTCTTCTCTTC
Bhi10G001911(Actin)	ATGTTCACAACCACTGCCGA	GTCGAGCGCAACATAAGCAA
MELO3C008032(Actin)	CATGTTCACCACCACTGCCGA	TGGCTGGAATAGAACTTCTGGGC
ClActin	CCATGTATGTTGCCATCCAG	GGATAGCATGGGGTAGAGCA
CuActin	GGAGAAGATCTGGCATCACA	CTCCAATCCAGACACTGTACT

Identification and physicochemical property analysis of MYC genes

A total of 40 MYC genes were identified in the genomes of four Cucurbitaceae crops. 10 in C. sativus, 8 in C. melo, 12 in C. lanatus, and 10 in B. hispida. All the MYC genes were unequally distributed on each chromosome. For example, in C. sativus L., there are 6 MYC genes on chromosome 3, while there are none on chromosome 1, 2, 4, and 5. In C. melo L., except for chromosomes 2, 3, 5, 7, 8, 9, 10, and 12 where there is no distribution, the other chromosomes each have 1, 2, or 3 MYC genes (Fig. 1). Sequence analysis revealed that the amino acid lengths encoded by CsMYC genes varied from 431aa (CsMYC8) to 694aa (CsMYC5), by CmMYC genes varied from 433aa (CmMYC8) to 745aa (CmMYC1), by ClMYC genes varied from 423aa (ClMYC3) to 969aa (ClMYC1), and by BhMYC genes varied from 501aa (BhMYC4) to 968aa (BhMYC3). In Cucurbitaceae crops, except for the CsMYC1, CmMYC5, ClMYC10 and BhMYC1 were acidic proteins, the others are alkaline proteins. The most proteins are unstable (instability index greater than 40), while the CsMYC5, CmMYC1, ClMYC12, BhMYC7, and BhMYC9. The average hydrophilicity values of all are less than 0, indicating that all the proteins are hydrophobic. Subcellular localization prediction results revealed that the most MYC proteins are localized in the cell nucleus, followed by chloroplasts. (Table 2).

Table 2

Protein information of MYC gene family members in Cucurbitaceae crops.
Species	Gene ID	Name	Number of amino acids (aa)	Molecular weight (D)	pI	Instability index	Aliphatic index	Average of hydropathicity	Prediction of subcellular location
Cucumis sativus L.	CsaV3_3G000850.1	CsMYC1	447	49398.41	8.65	43.61	82.37	-0.416	Nucleus
	CsaV3_3G001710.1	CsMYC2	642	69911.19	6.21	50.44	64.14	-0.578	Nucleus
	CsaV3_3G007980.1	CsMYC3	649	71943.89	5.83	49.00	77.66	-0.490	Nucleus
	CsaV3_3G022420.1	CsMYC4	501	55301.14	5.70	44.81	78.60	-0.445	Nucleus
	CsaV3_3G034600.1	CsMYC5	694	78486.44	5.24	38.28	84.84	-0.370	Chloroplast\Nucleus
	CsaV3_3G049150.1	CsMYC6	688	75666.20	5.11	58.15	69.71	-0.617	Nucleus
	CsaV3_6G000530.1	CsMYC7	644	72769.50	5.51	43.25	78.57	-0.500	Nucleus
	CsaV3_6G008940.1	CsMYC8	431	48394.52	5.42	47.63	76.43	-0.429	Nucleus
	CsaV3_6G037080.1	CsMYC9	650	71869.01	5.91	48.30	83.31	-0.347	Nucleus
	CsaV3_7G027460.1	CsMYC10	691	76190.07	5.66	41.31	76.58	-0.372	Nucleus
Cucumis melo L.	MELO3C015748.2.1	CmMYC1	745	82003.24	5.70	37.16	81.25	-0.269	Nucleus
	MELO3C003412.2.1	CmMYC2	723	79597.55	5.32	56.07	68.62	-0.628	Nucleus
	MELO3C024041.2.1	CmMYC3	501	55262.12	6.16	48.09	78.06	-0.486	Nucleus
	MELO3C006016.2.1	CmMYC4	586	65067.91	5.55	47.76	80.00	-0.535	Nucleus
	MELO3C013772.2.1	CmMYC5	442	48984.93	7.68	48.03	81.52	-0.439	Nucleus
	MELO3C013851.2.1	CmMYC6	662	72281.88	6.03	49.58	64.24	-0.570	Nucleus
	MELO3C021212.2.1	CmMYC7	656	74227.22	5.61	42.86	79.07	-0.473	Nucleus
	MELO3C022250.2.1	CmMYC8	433	48684.93	5.59	47.11	76.07	-0.442	Nucleus
Citrullus lanatus	Cla97C03G062520.1	ClMYC1	969	105839.97	6.15	47.45	78.86	-0.415	Nucleus
	Cla97C05G080890.1	ClMYC2	618	68589.08	5.85	46.81	76.99	-0.547	Nucleus
	Cla97C06G112130.1	ClMYC3	423	47569.45	5.14	52.02	71.70	-0.513	Nucleus
	Cla97C06G112140.1	ClMYC4	427	47692.74	5.09	52.44	52.44	-0.424	Nucleus
	Cla97C06G113160.1	ClMYC5	645	72922.74	5.78	46.22	79.64	-0.469	Nucleus
	Cla97C07G128490.1	ClMYC6	637	70287.21	6.36	45.59	81.93	-0.368	Nucleus
	Cla97C07G129080.1	ClMYC7	501	55179.91	5.96	49.98	76.47	-0.504	Nucleus
	Cla97C09G170270.1	ClMYC8	690	76046.16	5.87	40.82	80.36	-0.345	Nucleus
	Cla97C09G174730.1	ClMYC9	680	74878.38	5.24	53.48	70.68	-0.610	Nucleus
	Cla97C10G185380.1	ClMYC10	463	51021.92	8.77	53.53	79.74	-0.471	Nucleus
	Cla97C10G186220.1	ClMYC11	694	76681.99	6.35	49.20	64.81	-0.573	Nucleus
	Cla97C10G204640.1	ClMYC12	695	78085.01	4.97	38.55	83.87	-0.326	Chloroplast\Nucleus
Benincasa hispida	BhiUN179M27	BhMYC1	453	49798.89	8.19	47.72	83.43	-0.371	Nucleus
	Bhi01M000362	BhMYC2	617	68250.67	5.77	44.99	78.36	-0.520	Nucleus
	Bhi02M001191	BhMYC3	968	105559.53	6.12	44.22	79.96	-0.421	Nucleus
	Bhi05M000179	BhMYC4	501	55182.03	6.02	45.65	79.20	-0.464	Nucleus
	Bhi05M000251	BhMYC5	647	71471.31	5.91	43.64	80.06	-0.370	Nucleus
	Bhi05M000336	BhMYC6	682	75237.76	5.22	48.52	71.17	-0.632	Nucleus
	Bhi09M000958	BhMYC7	604	66789.67	6.01	39.23	80.83	-0.350	Nucleus
	Bhi11M000137	BhMYC8	660	72104.63	5.99	47.48	65.35	-0.570	Nucleus
	Bhi11M001900	BhMYC9	697	78638.85	5.07	39.16	84.61	-0.314	Chloroplast\Nucleus
	Bhi12M001654	BhMYC10	643	72375.09	5.60	47.35	80.34	-0.451	Nucleus

Structure and phylogenetic analysis of Cucurbitaceae crops MYC gene

Motif prediction analysis on the protein sequences of the MYC family members showed that when the number of motifs is limited to 10, no MYC family member contains all the motifs (Fig. 2). And the motif 1, 2, 5, 7, and 8 were relatively conservative, common to all MYC genes. Gene structure analysis revealed that the number of exons in MYC genes ranges from 1 to 15 (Fig. 2). The gene structures of most MYC genes within the same lineage are similar, further indicating the conservation of protein motifs and gene structures within the same evolutionary branch of MYCs.

According to the alignment of bHLH domains in MYC proteins, there was a basic amino acid region (Basic) composed of approximately 12 amino acids. This region contains a highly conserved H4-V5-E6-E8-R9-R11-R12 sequence, which is essential for the binding of bHLH to target genes. This region also includes two helical structures, comprising approximately 37 amino acids. Notably, the 22th and 38th leucine (Leu) amino acids in the HLH domain are highly conserved, indicating their necessity for dimer formation (Fig. 3).

To clarify the evolutionary relationships among the MYC gene family in Cucurbitaceae crops, Zea mays, Brachypodium distachyon, and Oryza sativa, we constructed a phylogenetic tree. As the result showed that All MYC proteins could be divided into five subgroups, labeled as I to Ⅴ. In each subgroup, there are both monocotyledonous and dicotyledonous plants, indicating that the MYC genes were relatively conserved during the evolutionary processes of both monocots and dicots. Group Ⅳ is the largest subgroup, consisting of 4, 4, 6, 3, 2, 1, and 1 MYC proteins from C. sativus L., C. melo L., C. lanatus, B. hispida, Z.mays, B. distachyon, and O. sativa, respectively. while the group I was the smallest, only with 4 MYC genes (Fig. 4). In group Ⅱ, there were 7 MYC genes, while only 1 belongs to monocotyledonous plants.

Collinearity analysis of MYC gene among Cucurbitaceae crops

To infer the evolution of MYC genes, synteny analysis was carried out among the four Cucurbitaceae species (Fig. 5; Table 3). A total of 36 MYC genes (9 in C. sativus L., 7 in C. melo L., 11 in C. lanatus, and 9 in B. hispida) were located within synteny blocks of the four Cucurbitaceae genomes. We found five orthologous gene pairs which exist among all four species. The result showed that these MYC genes were conserved during the evolution of all the four Cucurbitaceae species, suggesting the conserved roles in Cucurbitaceae species. Furthermore, it was also observed that some MYC genes were lost in some species. For instance, certain MYC genes were found in C. sativus L., C. melo L., and B. hispida, but were lost in B. hispida, such as the CsaV3_3G000850/MELO3C013772.2/Cla97C10G185380 collinear gene pair. The result showed that the specific traits in different Cucurbitaceae species during the evolution and the amplification of genome. In addition, there were two collinear gene pairs only existed in C. lanatus, and B. hispida.

Table 3

The collinear gene pairs existed in four *Cucurbitaceae* genomes.
Number	Cucumber	Melon	Watermelon	Waxgourp
1	CsaV3_3G000850	MELO3C013772.2	Cla97C10G185380	-
2	CsaV3_7G027460	MELO3C015748.2	Cla97C09G170270	Bhi09M000958
3	CsaV3_6G037080	-	Cla97C07G128490	Bhi05M000251
4	CsaV3_6G008940	MELO3C022250.2	Cla97C06G112130	-
5	CsaV3_6G000530	MELO3C021212.2	Cla97C06G113160	Bhi12M001654
6	CsaV3_3G049150 CsaV3_3G001710	MELO3C003412.2 MELO3C013851.2	Cla97C09G174730 Cla97C10G186220	Bhi05M000336 Bhi11M000137
7	CsaV3_3G049150 CsaV3_3G001710	MELO3C003412.2 MELO3C013851.2	Cla97C09G174730 Cla97C10G186220	Bhi05M000336 Bhi11M000137
8	CsaV3_3G007980	MELO3C006016.2	Cla97C05G080890	Bhi01M000362
9	CsaV3_3G034600	-	Cla97C10G204640	Bhi11M001900
10	-	-	Cla97C03G062520	Bhi02M001191
11	-	-	Cla97C07G129080	Bhi05M000179

The regulatory TFs of MYC genes

The 1.5-kb upstream sequences of MYC genes were selected to predicate theirs regulatory TFs. As the results showed that three types of cis-elements related to development, hormone stress, and abiotic stresses were identified (Fig. 6). Among the cis-elements related to development, the number of G-box (CACGTC) elements is the highest, which is a light-responsive element. For example, genes of MELO3C021212.2.1, MELO3C003412.2.1, and Cla97C10G186220.1 contain 9 G-box elements, indicating that they might be regulated by the light environment. For the cis-elements related to hormone stress, the abscisic acid (ABA)-responsive element ABRE (ACGTG) occupies a relatively large proportion, 8 in genes of MELO3C021212.2.1, MELO3C003412.2.1, and Cla97C10G186220.1, 6 in genes of Bhi02M001191, CsaV3_3G001710.1, and Bhi11M000137. Among the cis-elements related to abiotic stresses, anaerobic induction ARE (AAACCA) were detected in a series of members, such as 6 in Cla97C07G128490.1 and CsaV3_3G007980.1, 5 in Cla97C05G080890.1.

Tissue-specific expression analysis of MYC genes in C. sativus

To investigate the expression profiles of the MYC gene family in different tissues, using cucumber as a representative, we conducted the transcriptome analysis based on publicly available cucumber transcriptome sequencing data among various tissues (PRJNA80169). We utilized the cucumber ChineseLong_V3 genome information for this reanalysis, focusing on the expression levels of the cucumber MYC gene family in 10 different tissues or organs, including root, stem, leave, tendril, male flower, female flower, ovary, ovary unfertilized, ovary fertilized and tendrils base. As the result showed that there was significant expression variation of the MYC gene family across different tissues (Fig. 7). Such as, While the CsaV3_3G001710 gene showed a relatively high level of expression across all tissues or organs, three genes (CsaV3_3G000850, CsaV3_6G008940, and CsaV3_6G037080), on the other hand, exhibited relatively low expression levels across all tissues or organs. Some genes exhibited significant tissue-specific expression patterns. For example, the CsaV3_3G049150 gene showed relatively high expression in root and fertilized ovary, while demonstrating low expression in other tissues or organs. Similarly, compared to other tissues or organs, the CsaV3_7G027460 gene displayed higher expression in root. The above results indicate that the cucumber MYC family genes played distinct roles in the development of tissues or organs, contributing to various functions in the growth and development of the plant.

Expression analysis of cucumber MYC genes under different stress conditions.

Based on publicly available transcriptome data from the NCBI SRA database, we conducted a analysis of the expression levels of the cucumber MYC genes under both biotic and abiotic (high temperature, low temperature, salt and silicon stress, powdery mildew, and southern root-knot nematode) stress conditions.

Under high-temperature stress, most MYC genes did not show significant differential expression (Fig. 8). For instance, genes of CsaV3_6G037080, CsaV3_3G000850, and CsaV3_6G008940 exhibited no change in expression levels under high-temperature stress, and their expression levels were relatively low. However, gene CsaV3_3G001710 demonstrated a significant upregulation at 6 hours post high-temperature treatment (6hph), while gene CsaV3_3G007980 showed high expression levels at both 3hph and 6 hph. These results suggest that gene CsaV3_3G007980 is likely involved in the response of cucumber to high-temperature stress.

Under low-temperature stress, the expression levels of four genes (CsaV3_6G037080, CsaV3_3G000850, CsaV3_6G008940, and CsaV3_6G000530) showed no significant change and remained at relatively low levels. Two genes exhibited relatively high expression levels during low-temperature treatment, with CsaV3_3G007980 gene showing a significant upregulation at 6hph. The expression levels of the other genes remained unchanged before and after low-temperature treatment. Additionally, CsaV3_3G049150 showed a significant downregulation in expression both at 6hph and 12hph. Worth noting is that the expression levels of all genes did not undergo significant changes at 3hph. These results suggest that CsaV3_3G007980 and CsaV3_3G049150 genes play a key role in the response of cucumber to prolonged low-temperature stress, and CsaV3_3G007980 was positively regulated, while CsaV3_3G049150 was negatively regulated (Fig. 9).

Under salt and silicon stress, most genes did not show differential expression after NaCl and Silicon treatments. One gene (CsaV3_3G000850) exhibited significant downregulation after NaCl treatment and show significant upregulation after Silicon treatment. However, when treated simultaneously with NaCl and Silicon, it displayed a higher degree of downregulation (Fig. 10). Despite the significant differential expression observed for CsaV3_3G00850 gene after treatment, its expression level remained relatively low under both control and stress.

Similarly, we analyzed the response of the MYC gene to biological stress. After inoculation with powdery mildew for 48 hours, most genes showed no significant difference in expression in both resistant (SSL508-28) and susceptible (D8) material (Fig. 11). However, certain MYC genes exhibited differential expression patterns between SSL508-28 and D8. For instance, CsaV3_3G000850, treated with powdery mildew, demonstrated a significantly higher level of upregulation in D8, compared to a relatively lower fold-change in SSL508-28. Interestingly, post-inoculation, the absolute expression level of CsaV3_3G000850 in D8 was much lower than its expression in SSL508-28. In addition, after inoculation with powdery mildew, CsaV3_3G049150 showed a significantly downregulated expression in D8 and a certain degree of upregulation in SSL508-28.

After inoculation with root-knot nematode (Meloidogyne incognita), the expression levels of most genes in both resistant (IL10-1) and susceptible (CC3) material generally exhibited similar trends, such as CsaV3_3G000850 and CsaV3_3G034600 (Fig. 12). However, there were two genes, CsaV3_6G000530 and CsaV3_6G037080, that showed an upregulation trend in resistant materials and a downregulation trend in susceptible materials. Nevertheless, the degree of differential expression for these genes is relatively low, whether in resistant or susceptible materials.

The responding of eight genes of four Cucurbitaceae crops under temperature stress

These eight genes in four Cucurbitaceae crops (CsaV3_3G007980, CsaV3_3G001710, MELO3C006016, MELO3C013851, Bhi01M000362, Bhi11M000137, Cla97C10G186220, Cla97C05G080890) were selected for analysis in response to temperature stress. As the result showed (Fig. 13), in Cucumis sativus, that genes CsaV3_3G007980 and CsaV3_3G001710 showed upregulated expression under both low and high temperature stress, which is relatively consistent with the transcriptome results, indicating their involvement in cucumber response to temperature stress. This expression pattern also appeared in Cucumis melo and Citrullus lanatus. The homologous genes MELO3C006016 and Cla97C05G080890 of CsaV3_3G007980 showed mainly upregulated expression under high temperature stress. However, under low temperature stress, The homologous gene MELO3C013851 exhibited significant downregulation at 3 and 6 hours of low temperature treatment, and then showed upregulated expression at 12 hours of low temperature treatment. While, in Benincasa hispida, these two homologous genes Bhi01M000362 and Bhi11M000137 show significantly downregulated expression in all time periods of both low and high temperature treatments.

Characteristics of MYC genes in Cucurbitaceae crops

The MYC transcription factor has been reported to participate in various life activities in plants, playing a crucial role in regulating the growth and development of plant organs as well as in modulating tolerance to abiotic stress responses. The MYC protein has a bHLH_MYC_N domain in the N-terminal region, which consists of two subdomains: JID and TAD. The former is essential for interacting with JAZ proteins, while the latter is a putative transcriptional activation domain [45]. In the C-terminal region, the conserved bHLH domain present determines its specificity and affinity for DNA sequence binding, and it can facilitate the formation of various homodimers and heterodimers [46]. The bHLH domain comprises a basic region and a HLH region. The basic region is located at the N-terminus of the domain, containing sites for DNA recognition and binding. In this study, a highly conserved H4-V5-E6-E8-R9-R11-R12 sequence were detected in basic region, in which the highly conserved Leu in the 22th and 38th were necessity for dimer formation (Fig. 3) [47] had proved that the mutations at the two Leu sites significantly affect bHLH dimerization in Arabidopsis. According to the differences in the recognition mode between the basic region and the cis-acting elements, bHLH-type transcription factors can be classified into six main groups (designated A to F) [1]. Most of the MYC genes in Cucurbitaceae crops can specifically bind to the G-box (5’-CACNTG-3’), and belongs to group B. Consistent with previous findings, the C-terminus of the domain includes a conserved helix-loop-helix (HLH) structure that can form homodimers or heterodimers with other proteins.

According to the analysis of gene structure, the MYC genes in group Ⅲ and Ⅳ had fewer introns (less than or equal to 2), with even 50% of all MYC genes lacking introns. In contrast, MYC genes in the other three groups contain a large number of introns. Previous studies have indicated that introns and exons play important roles in the diversity and evolution of gene families through gain/loss and insertion/deletion events [48, 49]. The significant difference in the number of introns among MYC genes suggests that Cucurbitaceae crops have undergone intron loss events during their evolutionary process to adapt to environmental changes. [50] found that having fewer introns in genes enables plants to respond more rapidly to environmental changes. In addition, evolutionary analysis between monocots and dicots revealed that all groups include both monocot and dicot species, indicating the MYC genes were relatively conserved during the evolutionary processes of both monocots and dicots.

Functions of MYC genes and its responding to the temperature stree

A wealth of research has shown that MYC transcription factors play significant roles in the growth and development of plants. For instance, MYC is involved in regulating processes such as plant seed production [51], stamen development [52], hormone regulation [53], and secondary metabolism [54]. In this study, the majority of MYC genes are expressed in roots, leaf, and unfertilized ovary (Fig. 7). Coupled with the identification of numerous cis-elements related to development, and hormone stress, these findings further underscore their functions in growth and development.

Meanwhile, MYC genes play important roles in response to abiotic and biotic stresses. Judging from the results of this study, gene (CsaV3_3G000850) exhibited significant downregulation after NaCl treatment and show significant upregulation after Silicon treatment. These results indicate that silicon treatment induced high expression of CsaV3_3G000850, thereby enhancing salt tolerance in cucumber. Similar results have been reported in Arabidopsis, where overexpression of the AtMYC2 gene significantly increased osmotic stress tolerance [55]. In recent years, there has been a growing focus on the response of the MYC gene to temperature stress. Overexpressing SlICE1, which encodes a MYC-type transcription factor, enhances cold tolerance in tomatoes [56]. In Arabidopsis, MYC67 and MYC70 interact with ICE1, leading to negative regulation of cold tolerance [57]. Overexpression of PtrbHLH, a basic helix-loop-helix transcription factor from Poncirus trifoliata, confers enhanced cold tolerance in pummelo (Citrus grandis) by regulating CAT to modulate the level of H₂O₂ [58]. Under cold conditions, StICE1 of potato enhanced the stability of cell membranes by upregulating the expression of the StLTI6A gene, thereby increasing its tolerance [59]. The MYC-type TF MdbHLH4 negatively regulates apple cold tolerance by inhibiting the expression of MdCBF1/3 and the promoter-binding activity of MdICE1L, as well as by promoting the expression of MdCAX3L-2 and the cold-induced degradation of MdICE1L [60]. In this study, we found two MYC genes (CsaV3_3G007980 and CsaV3_3G001710) in cucumber that show significant differential expression under temperature stress (Figs. 8 and 9). To validate the involvement of these two genes in temperature stress response in other cucurbit species, we extracted their homologous genes for analysis of their reactions under temperature stress. Gene expression analysis revealed differential expression of these two genes across all four species, albeit with varying patterns. Homologous genes of these two genes in Citrullus lanatus exhibit a closer resemblance to those in cucumber, showing a certain degree of upregulation under both low and high temperature stress. In Cucumis melo, homolog of CsaV3_3G001710 demonstrate a downregulation trend under temperature stress. Remarkably different is the case in Benincasa hispida, where homologs of these two genes exhibit significant downregulation under both low and high temperature stress, opposite to what is observed in Cucumis sativus. Comparative functional genomics research indicated that if evolutionarily related species regulatory elements are conserved, then gene expression characteristics within species will correspondingly be conserved [61]. In this study, cis-regulatory element analysis revealed certain differences in both the type and quantity of these elements among the eight homologous genes, which could potentially account for the differential expression of homologous genes across species. All these findings suggest that CsaV3_3G007980 and CsaV3_3G001710, along with their homologs in other Cucurbitaceae crops, are highly responsive to temperature stress. However, the differential expression patterns between species remain unresolved. Further exploration of these genes' response mechanisms to temperature stress will be the focus of future research.

In summary, we had identified 10, 8, 12, and 10 MYC genes respectively in C. sativus, C. melo, C. lanatus, and B. hispida, each playing distinct roles in the developmental processes of plant. Particularly under environmental stress, some genes respond actively to external pressures through upregulation or downregulation of expression. Additionally, we had observed two genes that are more sensitive to temperature stress, labeled as CsaV3_3G007980 and CsaV3_3G001710. However, these two genes exhibit contrasting expression patterns across different species. This implied that there had been some degree of alterations in gene function following species divergence. All those results provide valuable insights for future functional studies of MYC genes and present potential candidate genes for enhancing environmental adaptability of Cucurbitaceae species.

MYC Myelocytomatosis

TF Transcription Factor

AMS Arabidopsis Aborted MicrosporeS

CDD Conserved Domain Data

ABA Abscisic Acid

hph Post High-temperature Treatment

HLH Helix-Loop-Helix

Ethics approval and consent to participate

This study has not directly involved humans, animals or plants.

Consent for publication

Not applicable.

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the manuscript.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the National Natural Science Foundation of China (32302542), which provide support for design of the study; Science Research Project of Hebei Education Department (QN2022062), which provide support for data collection; Science and Technology Research and Developmental Guidance Program of Handan (23313014019) and Construction of Innovative Teams in Modern Agricultural Industry Systems in Hebei Province (HBCT2024140206), which support for the analysis and interpretation of data.

Authors' contributions

W.X. designed, performed the experiments, analyzed data and wrote the paper; L.T. prepared the material and wrote the paper; Z.Y.N., Y.J.Y., L.R.R. and C.H. analyzed data; L.N.Y. revised the paper; W.S.N. and W.L.P. wrote and revised the paper. All authors read and approved the final version of the manuscript.

Acknowledgements

Not applicable.

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Identification of MYC genes in four Cucurbitaceae species and the response to temperature stress

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Journal Publication

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Abstract

Background

Results

Conclusions

Figures

Background

Methods

Chromosomal distribution and collinearity analysis of MYC genes

Gene Expression Analysis

Tissue-specific expression analysis of Cucumber MYC genes

Stress-responsive expression analysis of Cucumber MYC genes

Temperature stress treatment, RNA extraction and qRT-PCR

Results

Identification and physicochemical property analysis of MYC genes

Collinearity analysis of MYC gene among Cucurbitaceae crops

The regulatory TFs of MYC genes

The responding of eight genes of four Cucurbitaceae crops under temperature stress

Discussion

Characteristics of MYC genes in Cucurbitaceae crops

Functions of MYC genes and its responding to the temperature stree

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1