Background: Auxin transcription factor (ARF) is an important transcription factor that transmits auxin signals and is involved in plant growth and development as well as stress response. However, genome-wide identification and responses to abiotic and pathogen stresses of the ARF gene family in Cucurbita pepo L, especially pathogen stresses, have not been reported. Results: Finally, 33 ARF genes (CpARF01 to CpARF33) were identified in C.pepo from the Cucurbitaceae genome database using bioinformatics methods. The putative protein contains 438 to 1071 amino acids, the isoelectric point is 4.99 to 8.54, and the molecular weight is 47759.36 to 117813.27 Da, the instability index ranged from 40.74 to 68.94, and the liposoluble index ranged from 62.56 to 76.18. The 33 genes were mainly localized in the nucleus and cytoplasm, and distributed on 16 chromosomes unevenly. Phylogenetic analysis showed that 33 CpARF proteins were divided into 6 groups. According to the amino acid sequence of CpARF proteins, 10 motifs were identified, and 1,3,6,8,10 motifs were highly conserved in most of the CpARF proteins. At the same time, it was found that genes in the same subfamily have similar gene structures. Cis-elements and protein interaction networks predicted that CpARF may be involved in abiotic factors related to the stress response. QRT-PCR analysis showed that most of the CpARF genes were upregulated under NaCl, PEG, and pathogen treatment compared to the control. Subcellular localization showed that CpARF22 was localized in the nucleus. The transgenic Arabidopsis thaliana lines with the CpARF22 gene enhanced their tolerance to salt and drought stress. Conclusion: In this study, we systematically analyzed the CpARF gene family and its expression patterns under drought, salt, and pathogen stress, which improved our understanding of the ARF protein of zucchini, and laid a solid foundation for functional analysis of the CpARF gene.
Keywords: Zucchini; ARF gene family; Drought and Salt stress; Pathogen stress; Transgenic Arabidopsis thaliana
Supplementary Information The online version contains supplementary material available at https://doi.org/10.1186/s12864-024-09992-8.
Auxin-response factor (ARFs) is an essential family of transcription factors that bind to the Auxin-response elements (AuxRE) and play critical roles in a variety of plant development and physiological processes, as it has been shown to control the processes of cell differentiation, elongation, and division in conjunction with other plant-growth regulators [[
At present, with the development of sequencing technology, a large number of ARF family members have been identified in numerous plants. There are 23 ARF genes in Arabidopsis thaliana, Under osmotic stress, the growth of Arabidopsis thaliana leaves was inhibited by enhancing the ARF-mediated auxin response [[
Zucchini has strong regeneration ability due to its thick root system, its stem can be up to 5 m long, and its branch is strong, its petiole is stout and its growth is strong, therefore, Cucurbitaceae crops generally have a stronger environmental adaptability than other plants [[
The whole genome sequences of zucchini were downloaded from the Cucurbitaceae database (
Expasy (https://web.Expasy.org/) online tool ProtParam was used to calculate physical and chemical properties of CpARF proteins, such as theoretical isoelectric point, instability index, relative molecular weight and hydrophobicity index. The subcellular location of the CpARF protein was analyzed by PSORT (
The GFF annotation files of zucchini was imported into GSDS 2.0 (Gene Structure Display Server 2.0) (
TBtools was used to map CpARF genes on chromosome mapping by GFF annotation file. Moreover, we detected duplicate gene pairs using the plant genome replication database server (
According to the GFF annotation file of zucchini, the Tbtools were used to extract the 2000 bp of DNA sequence upstream of the transcription start site of the CpARF family genes. PlantCARE was used to analyze the cis-acting elements of the CpARF genes promoter region [[
Based on amino acid sequences of CpARF proteins, BLAST2GO (https://
The seeds used for this investigation originated from the college of plant protection, Gansu Agricultural University China. The seeds of zucchini were disinfected in 2% NaClO solution for 7 min to sterilize, washed three times with sterile distilled water, and the seeds were placed on filter paper and covered with petri dishes. The seeds were cultured for 3 days under dark conditions at 25 °C, and sterile water was sprayed every day to keep the filter paper moist. Ten seeds were placed in each petri dish for germination, and six petri dishes were shared. When the embryo grew to 2 cm, it was moved into the pot. Salt and drought stress treatments of zucchini: temperature 25℃, relative humidity 70%, light intensity 130 μmol·m
Total RNA of leaves and roots was extracted from frozen samples using RNA simple Total RNA kit (Wuhan, China). Reverse transcription of RNA into cDNA used the Prime Script RT reagent kit (Wuhan, China). The SYBR Green Pro Taq HS premix qPCR kit (containing Rox) was used for qRT-PCR analysis. To detect the specificity of the primers, the target gene and the reference gene (β-Actin) were compared and verified in Cu Gen DB. The reaction system for qRT-PCR analysis on ABI7500 Real-Time PCR System (Applied Biosystems) was 20 μL, including 10 μL 2 × SYBR Green Pro Taq HS Premix (ROX plus)*
The cloned CpARF22 gene was transferred to the expression vector containing the green fluorescent protein (GFP) by homologous recombination method, and the constructed GV1300: CpARF22-GFP vector plasmid was transformed into Agrobacterium GV1300. Agrobacterium strains containing plasmids were infiltrated into tobacco leaves for 48 h. The GFP fluorescence signal of CpARF22 protein was observed by confocal laser scanning microscopy (LSCM).
The CDS region of the CpARF22 gene was submitted to the website of homologous recombination primer design In-Fusion Cloning: general information (
All the statistical analyses were performed using Microsoft Excel 2010 and SPSS software (IBM, Armonk, NY). The means among various groups were compared by Duncan's multiple range tests. The data were analyzed and are expressed as the means standard deviations (SDs), and P < 0.05 indicated significance difference.
By analyzing the whole genome sequence of zucchini, 33 candidate ARF members (Table 1, Table S1) were identified, named as CpARF01-CpARF33. The number of amino acids of the family members varied greatly, ranging from 438 aa (CpARF19) to 1071aa (CpARF20), and the average number of amino acids was 749. The molecular weight of the protein was 47,759.36 Da (CpARF19) to 117,813.27 Da (CpARF20). The PI values of the encoded proteins ranging from 4.99 to 8.54, and the PI values of 27 proteins were less than 7 (except CpARF04, CpARF06-CpARF09, and CpARF26), which belonged to acidic proteins, suggesting that they may play a role in the acidic subcellular environment. The average coefficient of hydrophilicity ranged from -0.669 (CpARF05) to -0.350 (CpARF26). The instability index ranged from 40.74 (CpARF11) to 68.94 (CpARF03), both of which belonged to unstable protein, while the liposoluble index ranged from 62.56 (CpARF06) to 76.18 (CpARF32). The physical and chemical properties analysis showed that 33 CpARF proteins were unstable hydrophobic proteins, and the hydrophobicity index of all the proteins was less than 0, so 33 proteins were hydrophilic proteins. Prediction of subcellular localization showed that 13 proteins were located in the nucleus, 14 in the cytoplasm, 3 in the chloroplast matrix, 1 in the microbody, 1 in the cell membrane, and 1 in the periphery. 82% of CpARF proteins have the characteristics of transcription factors and play a role in the nucleus and cytoplasm.
Table 1 Basic physicochemical properties of the proteins encoded by the 33 CpARF family genes identified in the Zucchini Genome
Gene accession No Gene Size (aa) Molecular weight Isoelectric point Instability index Aliphatic index GRAVY Subcellular Localization phosphorylation sites Cp4.1LG01g00450.1 699 78,183.79 6.94 56.17 68.71 -0.595 nucleus 151 Cp4.1LG01g17160.1 550 61,055.49 5.32 48.26 73.73 -0.475 endoplasmic reticulum 106 Cp4.1LG01g19620.1 839 93,309.36 5.68 68.94 75.39 -0.466 cytoplasm 150 Cp4.1LG01g24390.1 688 76,157.80 7.29 47.21 72.56 -0.433 nucleus 126 Cp4.1LG01g22980.1 769 83,523.68 5.47 61.35 66.33 -0.669 chloroplast stroma 147 Cp4.1LG02g08050.1 589 63,259.83 7.73 54.61 62.56 -0.642 cytoplasm 161 Cp4.1LG05g07840.1 584 63,467.64 8.42 64.78 66.13 -0.532 cytoplasm 143 Cp4.1LG05g08160.1 838 91,462.81 8.03 55.03 74.14 -0.411 nucleus 150 Cp4.1LG05g08310.1 591 63,958.99 7.48 50.65 67.50 -0.568 cytoplasm 150 Cp4.1LG06g00800.1 880 96,839.30 5.09 54.17 73.55 -0.413 cytoplasm 142 Cp4.1LG07g01180.1 835 92,787.48 6.51 40.74 72.40 -0.491 outside 138 Cp4.1LG07g07700.1 953 105,600.58 6.20 62.16 70.55 -0.547 nucleus 124 Cp4.1LG07g10790.1 880 97,981.09 6.20 62.35 69.26 -0.584 cytoplasm 123 Cp4.1LG08g13310.1 607 67,287.59 5.29 60.25 70.64 -0.473 chloroplast stroma 124 Cp4.1LG09g07430.1 890 98,822.89 5.85 66.11 69.01 -0.511 cytoplasm 151 Cp4.1LG10g06190.1 891 98,898.43 6.18 63.98 75.30 -0.411 cytoplasm 165 Cp4.1LG10g05170.1 711 77,877.45 6.65 59.60 67.61 -0.435 nucleus 153 Cp4.1LG10g04760.1 587 64,771.64 7.00 48.47 69.47 -0.357 nucleus 105 Cp4.1LG11g04200.1 438 47,759.36 4.99 41.30 73.42 -0.372 microbody (peroxisome) 64 Cp4.1LG11g06740.1 1071 117,813.27 5.98 62.42 73.39 -0.494 nucleus 179 Cp4.1LG11g12020.1 847 93,282.59 5.62 57.99 70.06 -0.458 cytoplasm 111 Cp4.1LG13g00970.1 650 72,058.42 5.95 58.87 70.03 -0.570 chloroplast stroma 156 Cp4.1LG13g05520.1 836 93,036.11 5.56 66.38 72.39 -0.509 cytoplasm 132 Cp4.1LG13g09970.1 684 75,624.89 5.88 43.58 69.85 -0.439 nucleus 129 Cp4.1LG14g02660.1 727 81,703.23 6.74 51.59 69.42 -0.567 nucleus 169 Cp4.1LG16g05330.1 680 74,740.74 8.54 48.72 74.56 -0.350 nucleus 142 Cp4.1LG16g05510.1 719 79,772.38 5.84 54.87 67.09 -0.528 cytoplasm 153 Cp4.1LG17g10130.1 680 76,155.03 5.94 54.78 72.93 : -0.516 cytoplasm 144 Cp4.1LG18g09430.1 850 94,359.18 6.05 50.70 69.96 -0.516 nucleus 138 Cp4.1LG19g04050.1 705 78,158.47 6.83 48.27 69.84 -0.424 nucleus 128 Cp4.1LG19g06940.1 723 78,850.37 7.28 61.64 65.27 -0.413 nucleus 134 Cp4.1LG19g05880.1 840 93,309.15 5.86 65.38 76.18 -0.422 cytoplasm 171 Cp4.1LG20g05550.1 883 97,693.10 5.92 65.77 73.34 -0.396 cytoplasm 148
In order to study the genetic relationship between the ARF family members of zucchini and the ARF family such as Arabidopsis thaliana and pumpkin, the phylogenetic trees of 101 protein sequences of three species were constructed (Fig. 1, Table S2). The results showed that 101 members were divided into seven evolutionary branches, and the CpARFs genes of zucchini were distributed in six branches, indicating that the CpARFs family proteins were diverse. It was found that one pair of paralogous CpARF gene pairs (CpARF14 and CpARF02); 23 pairs of orthologous gene pairs were found between pumpkin and zucchini(CpARF08/ CmARF03, CpARF24/ CmARF26, CpARF04/ CmARF11, CpARF30/ CmARF18, CpARF11/ CmARF22, CpARF25/ CmARF29, CpARF01/ CmARF08, CpARF22/ CmARF28, CpARF07/ CmARF23, CpARF28/ CmARF19, CpARF05/ CmARF12, CpARF27/ CmARF31, CpARF31/ CmARF17, CpARF17/ CmARF06, CpARF15/ CmARF30, CpARF32/ CmARF16, CpARF23/ CmARF27, CpARF03/ CmARF10, CpARF10/ CmARF20, CpARF06/ CmARF01, CpARF12/ CmARF04, CpARF20/ CmARF14 and CpARF21/ CmARF15), it suggests that there is a close relationship between pumpkin and zucchini.
Graph: Fig. 1Phylogenetic analysis of CpARF protein families in Arabidopsis thaliana , pumpkin and zucchini. Pumpkin, Arabidopsis thaliana and zucchini are represented by the black pentagram, purple pentagram, and circle, respectively. The same species is represented by the same symbol with the same color. The numbers outside the branch represent the guided values based on 1000 repetitions
Gene structure analysis showed that 33 CpARF genes contained exons and introns, and the number of exons ranged from 3 to 16. Among them, the number of exons of 25 CpARF genes were greater than 10. The number of exons of CpARF05, CpARF08, CpARF19, CpARF24, and CpARF26 genes ranged from 2 to 4, and the number of exons of the remaining 3 genes ranged from 5 to 6. In addition, we found that most genes have terminal non-coding sequences, of which 22 genes have 5 ' and 3 ' terminal non-coding sequences, CpARF08, CpARF11, CpARF16, CpARF19, CpARF21, CpARF26, CpARF31 have 3 ' terminal non-coding sequences, CpARF12, CpARF14 have 5 ' terminal non-coding sequences, CpARF23, CpARF30 do not have terminal non-coding sequences, 94% of the genes have terminal non-coding sequences. At the same time, it was found that the genes in different subfamilies have distinct gene structures, such as CpARF11 and CpARF16, the length difference was large, suggesting that the genes had mutated during evolution (Fig. 2). These results indicated that the structural differences of CpARFs genes may affect their functions. Therefore, it is necessary in order to further analyze the functions of these genes.
Graph: Fig. 2Phylogenetic relationship and exon–intron structure analysis of ARF family in zucchini. Exon–intron structure analysis was performed using the online tool GSDS. The length of exons and introns of each CpARF gene shows that the value includes CDS, UTR, and introns. The yellow value is the CDS coding region, and the blue value is the UTR coding region
Through Motif analysis of 33 CpARF proteins (Fig. 3), it was found that Motif 1 exists in all CpARFs(expect CpARF19, CpARF22), Motif 2 exists in 20 CpARFs (CpARF02, CpARF03, CpARF05, CpARF10, CpARF12-CpARF15, CpARF19, CpARF21, CpARF23, CpARF27, CpARF32), and Motif 3 exists in all CpARFs. Motif 4 exists in all CpARFs (expect CpARF11, CpARF17, CpARF19, CpARF31), Motif 5 exists in all CpARFs (except CpARF02, CpARF05, CpARF14), Motif 6 exists in all CpARFs (except CpARF12), Motif 7 exists in all CpARFs (CpARF02, CpARF11, CpARF17, CpARF19), Motif 8 exists in all CpARFs. Motif 9 exists in all CpARFs (except CpARF02, CpARF11, CpARF19, and CpARF21), and motif 10 exists in all CpARFs (except CpARF03 and CpARF18). At the same time, we can note that the proteins in the same subfamily have similar basic motifs. The 33 CpARF proteins have conserved motifs between 5–10, of which 14 CpARF proteins have 10 conserved motifs, 10 CpARF proteins have 9 conserved motifs, 5 CpARF proteins have 8 conserved motifs (CpARF05, CpARF12, CpARF14, CpARF21, CpARF31), and 2 CpARF proteins have 7 conserved motifs (CpARF11, CpARF17). CpARF02 protein has 6 conserved motifs, and CpARF19 protein has 5 conserved motifs. It can be observed that motifs 1,3,6,8 and 10 are particularly conserved in most CpARF proteins. This difference in conserved motifs may also serve as one of the reasons for the functional differences in CpARF genes.
Graph: Fig. 3Phylogenetic relationship and conserved motif analysis of ARF family in zucchini analysis of Chromosome Location and Gene Duplication
The results showed that 33 CpARF genes were unevenly distributed on all 16 chromosomes of zucchini. There were 5 CpARF genes on chromosome 1, 3 CpARF genes on chromosomes 5,7,10,11,13, and 19 respectively, 2 CpARF genes on chromosome 16, and 1 CpARF gene on chromosomes 2,6,8,9,14,17,18, and 20 respectively (Fig. 4).
Graph: Fig. 4The chromosome location map of the ARF gene in zucchini was drawn by the MapDraw program, and the name of the gene was marked on the right side of the chromosome
The evolutionary selection of zucchini CpARF genes was tested by calculating non-synonymous (Ka) and synonymous (Ks) substitutions (Table 2). A total of 20 pairs of duplicated gene pairs were discovered in the CpARF genes, and all of them were fragment duplications. In general, the ratio of Ka/Ks > 1 indicates positive selection, Ka/Ks < 1 indicates underwent purifying selection pressure, and Ka/Ks = 1 indicates neutral selection. The results showed that the Ka/Ks ratios of 20 pairs of CpARF genes were < 1 (Table 3), indicating that these genes underwent purifying selection pressure during evolution. At the same time, we found that the time of duplication of these genes varied greatly, and the repeated years ranged from 6.785 (CpARF07/CpARF22) and 119.870 MYA (CpARF26/CpARF30).
Table 2 The Ka/Ks ratios and predicted duplication dates for duplicated CpARF Genes in Zucchini
Duplicated CpARF gene1 Duplicated CpARF gene2 Ka Ks Ka/Ks Date (MYA) = Ks/2λ Selective pressure Duplicate type 0.265 1.362 0.195 45.387 Purifying selection Segmental 0.041 0.322 0.127 10.718 Purifying selection Segmental 0.112 0.560 0.200 18.665 Purifying selection Segmental 0.058 0.355 0.162 11.830 Purifying selection Segmental 0.068 0.204 0.332 6.785 Purifying selection Segmental 0.039 0.444 0.087 14.811 Purifying selection Segmental 0.058 0.266 0.217 8.850 Purifying selection Segmental 0.219 0.318 0.689 10.601 Purifying selection Segmental 0.074 0.533 0.139 17.767 Purifying selection Segmental 0.080 0.379 0.210 12.624 Purifying selection Segmental 0.149 1.737 0.086 57.898 Purifying selection Segmental 0.143 1.455 0.098 48.516 Purifying selection Segmental 0.053 0.345 0.153 11.491 Purifying selection Segmental 0.071 0.378 0.189 12.612 Purifying selection Segmental 0.287 2.188 0.131 72.933 Purifying selection Segmental 0.053 0.371 0.143 12.366 Purifying selection Segmental 0.262 1.593 0.165 53.100 Purifying selection Segmental 0.307 2.349 0.131 78.312 Purifying selection Segmental 0.327 1.832 0.179 61.051 Purifying selection Segmental 0.263 3.596 0.073 119.870 Purifying selection Segmental
Table 3 Secondary Structure of CpARF proteins
Gene Alpha helix Extended strand Random coil 169 (24.18%) 145 (20.74%) 385 (55.08%) 126 (22.91%) 109 (19.82%) 315 (57.27%) 192 (22.88%) 165 (19.67%) 482 (57.45%) 133 (19.33%) 151 (21.95%) 404 (58.72%) 192 (24.62%) 130 (16.67%) 458 (58.72%) 175 (18.72%) 203 (21.71%) 557 (59.57%) 135 (20.71%) 149 (22.85%) 368 (56.44%) 147 (20.14%) 182 (24.93%) 401 (54.93%) 143 (18.26%) 171 (21.84%) 469 (59.90%) 177 (20.11%) 164 (18.64%) 539 (61.25%) 242 (28.98%) 152 (18.20%) 441 (52.81%) 254 (26.65%) 155 (16.26%) 544 (57.08%) 247 (28.07%) 133 (15.11%) 500 (56.82%) 99 (16.31%) 127 (20.92%) 381 (62.77%) 195 (21.91%) 164 (18.43%) 531 (59.66%) 219 (24.58%) 157 (17.62%) 515 (57.80%) 120 (16.88%) 140 (19.69%) 451 (63.43%) 95 (16.18%) 128 (21.81%) 364 (62.01%) 86 (19.63%) 86 (19.63%) 266 (60.73%) 278 (25.96%) 164 (15.31%) 629 (58.73%) 217 (25.62%) 147 (17.36%) 483 (57.02%) 131 (20.15%) 145 (22.31%) 374 (57.54%) 177 (21.17%) 158 (18.90%) 501 (59.93%) 132 (19.30%) 145 (21.20%) 407 (59.50%) 177 (24.35% 167 (22.97%) 383 (52.68%) 129 (18.97%) 177 (26.03%) 374 (55.00%) 137 (19.05%) 149 (20.72%) 433 (60.22%) 191 (28.09%) 124 (18.24%) 365 (53.68%) 177 (20.82%) 178 (20.94%) 495 (58.24%) 103 (14.61%) 159 (22.55%) 443 (62.84%) 92 (12.72%) 134 (18.53%) 497 (68.74%) 214 (25.48%) 148 (17.62%) 478 (56.90%) 191 (21.63%) 170 (19.25%) 522 (59.12%)
The secondary structure of the ARF proteins of zucchini showed that the proteins encoded by CpARF genes of zucchini contained α-helix, extended chain, and random coil. The overall structural similarity was very different and the complexity was general. The distribution of random coils in ARF proteins is the most as shown in Table 3. The proportion of CpARF10, CpARF14, CpARF17-19, CpARF27, CpARF30, and CpARF31 in random coils is greater than 60%, and the remaining 25 are between 50 and 60%. The distribution of α-helix and extended strand in ARF proteins is second. In the extended chain, CpARF01, CpARF04, CpARF06-CpARF09, CpARF14, CpARF18, CpARF22, CpARF24-27, CpARF29, and CpARF30 accounted for more than 20%, and the remaining 18 CpARF proteins were between 15 and 20%. In the α-helix, CpARF01-CpARF03, CpARF05, CpARF07, CpARF08, CpARF10-13, CpARF15, CpARF16, CpARF20-23, CpARF25, CpARF28, CpARF29, CpARF32, and CpARF33 accounted for more than 20%, and the remaining 12 CpARF proteins are between 12% -20%. The prediction shows that most of them are composed of random coils, which is in line with the secondary structure prediction results.
In order to predict the interaction between CpARF proteins, String software was utilized to construct the ARF protein interaction network of zucchini based on the homologous sequence of Arabidopsis thaliana (Fig. 5). Depending to the prediction results, 12 CpARF proteins appeared in the known interaction network of Arabidopsis thaliana ARF proteins, indicating that there is a complex relationship between the two. We observed that the protein sequence of ARF9 was closely related to five CpARF protein sequences (CpARF01, CpARF07, CpARF22, CpARF25, CpARF28), ARF8 protein and two CpARF protein (CpARF03, CpARF23) are homologous, ARF16 protein and four CpARF protein sequences (CpARF04, CpARF18, CpARF24, CpARF30) are homologous, ARF2 protein and two CpARF protein sequences (CpARF05, CpARF29) are homologous, ETT and two CpARF proteins (CpARF17, CpARF31) were highly similar, these results suggested that these homologue zucchini genes may have similar functions. At the same time, Arabidopsis thaliana ARF interacts with multiple proteins with known functions and unknown functions, indicating that it has a complex regulatory mechanism, so similar CpARF proteins may also have analogous functions. ARF6 and ARF8 regulate the maturation of male and female floral organs in Arabidopsis thaliana, and the removal of these proteins in ARF6 ARF8 double mutants or plants over-expressing miR167 can lead to infertility [[
Graph: Fig. 5The prediction of the interaction network of CpARF proteins based on the interactions of their orthologs in Arabidopsis thaliana
Cis-acting elements are transcription factor-specific binding sites and thus play an important role in regulating genes responsible for the growth, differentiation, and development of organisms, including plants (Fig. 6, Table S3). According to the functional annotation, we obtained 34 cis-acting elements and found that 29.4% (
Graph: Fig. 6The cis-regulatory element in the promoter region of CpARF genes of zucchini. The colors and numbers on the grid indicate the number of different cis-regulatory elements in the CpARF genes
We analyzed the function of these genes based on KEGG database and found that 13 ARF genes (CpARF01, CpARF02, CpARF06, CpARF07, CpARF10, CpARF13, CpARF14, CpARF17, CpARF21, CpARF22, CpARF25, CpARF28 and CpARF31) were involved in plant hormone signal transduction pathway. In addition, GO analysis showed that ARF family genes were involved in 18 terms, involving biological process (15 terms), molecular function (2 terms) and cellular component (1 term). We found that 33 genes were involved in cellular process, 31 genes (expect CpARF19 and CpARF22) were involved in metabolic process, regulation of biological process, response to stimulus, biological regulation (Fig. 7).
Graph: Fig. 7Function annotation Analysis of KEGG and GO. A: KEGG Function annotation; B: GO Function annotation
Auxin response factors (ARFs) play a major role in the differential expression of auxin in response to various abiotic stresses. In addition, there were significant differences in the expression levels of zucchini genes under salt stress (Fig. 8, Table S4, Table S5). Here, we treated zucchini seedlings with 200 mmol / L NaCl for 0 h, 3 h,6 h, 9 h, 12 h, and 24 h, and then assessed the expression of 33 CpARF genes using qRT-PCR. All the CpARF genes (except CpARF24 and CpARF27) showed a rapid response to salt stress. Among them, CpARF04, CpARF23, CpARF25, and CpARF26 reached the most meaningful level of NaCl treatment after 3 h. After 6 h of salt treatment, only CpARF02 had the most significant expression level. CpARF18, CpARF20, CpARF29, and CpARF31 reached the maximum at 9 h, and some genes had the most significant expression at 12 h (CpARF05, CpARF08, CpARF09, CpARF11, CpARF12, CpARF14). The expression values of CpARF10, CpARF21, and CpARF28 were the highest under 24 h salt treatment. Among them, the expression trends of six CpARF genes (CpARF03, CpARF10, CpARF17, CpARF19, CpARF22, CpARF28) were first up-regulated and then down-regulated and then up-regulated. The expression trends of two genes (CpARF07, CpARF16) were continuously up-regulated, and the expression of CpARF21 was the most complex, it was up-regulated first, then down-regulated, then up-regulated, then down-regulated and finally up-regulated, the expression trends of the remaining 19 genes were first up-regulated, then down-regulated, then up-regulated and then down-regulated.
Graph: Fig. 8The expression profiles of auxin response factor (ARF) genes in zucchini in response to NaCl treatment. The relative expression levels of 33 ARF genes in leaves were measured at 0, 3, 6,9,12,24 h of treatment. The relative mRNA levels of the group at 0 h were used for reference. The relative expression was calculated using the method of 2 −△△Ct. Values are means ± SD (n = 3). * represent significance at p < 0.05 compared with the references
The same method was utilized to treat zucchini seedlings with 20% PEG (Fig. 9, Table S4, Table S5). It was found that the most significant genes were expressed at 3 h (CpARF07, CpARF11, CpARF12, CpARF15, CpARF16, CpARF18, CpARF20, CpARF23, CpARF25, CpARF26, CpARF28, CpARF30, CpARF31, CpARF32), CpARF05, CpARF14, and CpARF17 were the most significant at 6 h, and CpARF02, CpARF10, CpARF21, and CpARF33 were the highest at 9 h. The expression level of CpARF03, CpARF08, and CpARF09 was the most significant at 12 h, and the expression level of CpARF22 was the highest at 24 h. Only CpARF24 was down-regulated under drought stress. CpARF06 was almost not expressed before and after treatment, while the expression trend of CpARF01 and CpARF22 were first down-regulated and then up-regulated. Moreover, the expression trends of 9genes (CpARF02, CpARF12, CpARF14, CpARF16, CpARF21, CpARF23, CpARF27, CpARF29CpARF31) were first up-regulated and then down-regulated, and the expression trends of 12 genes (CpARF05, CpARF11, CpARF15, CpARF17-CpARF20, CpARF26, CpARF28, CpARF30, CpARF32) were first up-regulated, then down-regulated and then up-regulated. Eight genes (CpARF03, CpARF04, CpARF07-CpARF09, CpARF13, CpARF25, CpARF33) were up-regulated first, then down-regulated, then up-regulated, and then down-regulated. The expression tendency of CpARF10 was up-regulated first, then down-regulated, then up-regulated, then down-regulated, and finally up-regulated. Of concern, most of these CpARF genes were up-regulated under different treatments, indicating that they responded positively to drought stress.
Graph: Fig. 9The expression profiles of auxin response factor (ARF) genes in zucchini in response to PEG treatment. The relative expression levels of 33 ARF genes in leaves were measured at 0, 3, 6,9,12,24 h of treatment. The relative expression levels of the group at 0 h were used for reference. The relative expression level was calculated using the method of 2 −△△Ct. Values are means ± SD (n = 3). * represents significance at p < 0.05 compared with the reference
In order to analyze the expression patterns of ARF genes in zucchini after infection by pathogenic bacteria (Fig. 10, Table S4, Table S6), the zucchini growing for about 15 days was inoculated with F. oxysporum isolated in our laboratory, and the leaves were taken at different time points (0,12,24 and 48 h) for qRT-PCR analysis. The results showed that the expression of 25 ARF genes in the leaves of zucchini increased significantly at 12 h in the initial stage of inoculation, and decreased to varying degrees at 24 h or 48 h after inoculation. Four ARF genes (CpARF01, CpARF09, CpARF26, CpARF33) had the most significant expression at 24 h, and four ARF genes (CpARF16, CpARF18, CpARF23, CpARF25) had the highest expression at 48 h. The expression pattern of 18 genes (CpARF01-CpARF13, CpARF21, CpARF26, CpARF29, CpARF30, CpARF33) was up-regulated first and then down-regulated. The expression pattern of 14 genes (CpARF14-CpARF20, CpARF22-CpARF25, CpARF27, CpARF28, CpARF31) was up-regulated first, then down-regulated and then up-regulated. The expression pattern of CpARF32 was down-regulated first, then up-regulated, and then down-regulated.
Graph: Fig. 10The auxin response factor (ARF) gene in the leaves of the zucchini responded to pathogen expression profiles. The relative expression levels of 33 ARF genes in the leaves of zucchini were measured at 0,12,24,48 h after treatment. The relative expression was calculated using the method of 2 −△△Ct. Values are means ± SD (n = 3). * represents significance at p < 0.05 compared with the references
In the root, the expression level of the CpARF05 gene decreased after inoculation with F.oxysporum, and other genes increased significantly (Fig. 11, Table S4, Table S6). The expression level of six genes (CpARF12, CpARF20, CpARF25, CpARF28, CpARF29, CpARF30) was the most significant at 12 h, and the expression of five genes (CpARF04, CpARF07, CpARF08, CpARF16, CpARF26) was the highest at 24 h, and the expression of the remaining 21 genes was the most significant at 48 h. Among them, the expression pattern of 14 genes (CpARF01, CpARF06, CpARF09-CpARF11, CpARF13, CpARF15, CpARF17, CpARF21, CpARF24, CpARF27, CpARF31-CpARF33) was continuously up-regulated. Nine genes (CpARF04, CpARF07, CpARF08, CpARF16, CpARF20, CpARF25, CpARF26, CpARF29, CpARF30) were up-regulated first and then down-regulated, and four genes (CpARF05, CpARF14, CpARF18, CpARF23) were down-regulated first and then up-regulated. Six genes (CpARF02, CpARF03, CpARF12, CpARF19, CpARF22, CpARF28) were originally up-regulated, then down-regulated, and then up-regulated. The above results showed that the ARF family genes of zucchini had a rapid response to the infection of F.oxysporum, and the expression patterns of various CpARFs were different, which indicated that different ARF members of zucchini played different roles in the stress regulation pathway of pathogens.
Graph: Fig. 11The auxin response factor (ARF) gene in the roots of the zucchini responded to pathogen expression profiles. The relative expression levels of 33 ARF genes in the root of zucchini were measured at 0,12,24,48 h after treatment. The relative expression was calculated using the method of 2 −△△Ct. Values are means ± SD (n = 3). * represents significance at p < 0.05 compared with the references
The chimeric CpARF22 gene DNA in Agrobacterium tumefaciens was immersed in tobacco leaves, and the fluorescence number in the cells was observed by a laser scanning confocal microscope. As shown in Fig. 12, the CpARF22-GFP fusion protein is expressed in the nucleus.
Graph: Fig. 12Subcellular localization. Localization of CpARF22 in tobacco leaves, CpARF22-GFP represents the gene and GFP represents the control is an empty vector. GFP stands for green fluorescence field, DAPI stands for DAPI field (nuclear staining), DIC stands for indicating field, and Merge stands for superposition field. The images were taken by LEICA DMi8, Japan fluorescence microscopy
In order to further study the function of CpARF22 gene, we successfully cultivated transgenic Arabidopsis thaliana plants over-expressing CpARF22 through Agrobacterium tumefaciens-mediated transformation and obtained a homozygous T
Graph: Fig. 13Effects of salt and drought stress on root length of wild-type (WT) and Arabidopsis thaliana overexpressing CpARF22 (OE-1, OE-2, and OE-3)
Auxin response factors (ARFs) are a class of transcription factors widely involved in plant growth and development and stress resistance regulation, so they become a key gene family for understanding plant biology. At present, there is not any detailed analysis of the ARF genes in zucchini. With the continuous exploration of the nutritional value of zucchini, its economic value is also constantly improving. In order to better elucidate the function of ARF in affecting specific auxin responses in zucchini, we carried out a systematic and comprehensive analysis of the ARF family genes in zucchini.
In this study, we identified 33 CpARF genes by analyzing the whole genome of zucchini, the number of genes was close to that of maize [[
Cis-acting elements, as key molecular switches, are involved in regulating the transcriptional regulation of gene activity in various biological processes. They regulate the precise initiation and transcription efficiency of gene transcription by binding to transcription factors [[
At present, many studies have shown that ARF genes have potential regulatory effects under various abiotic stresses [[
In this study, systematically analyzed the related information, phylogeny, gene structure, conserved motifs, chromosome localization, gene replication, cis-acting elements, and protein interactions of the ARF gene family in zucchini. In addition, we analyzed the expression patterns of zucchini under two different stress treatments and zucchini fusarium wilt infection. The cis-element found in the CpARF gene promoter was considered to be associated with plant hormones abiotic stress and pathogenic stress. QRT-PCR analysis showed that under salt stress and drought stress, the expression of CpARF08 gene was down-regulated most obviously at different time periods. Under pathogen stress, the expression of CpARF21 gene in leaves was down-regulated most obviously at different time periods, and the expression of CpARF28 gene in roots was down-regulated most obviously at different time periods. The transgenic Arabidopsis thaliana lines with the CpARF22 gene enhanced their tolerance to salt stress and drought stress.
We would like to thank the editor and reviewers for critically evaluating the manuscript and providing constructive comments for its improvement.
This work was conceived by Ming-jun Zhang and Ying-yu Xue. Ming-jun Zhang, Shuang Xu, Xuan-ru Jin, Xing-chu Man led the bioinformatic analyses. Ming-jun Zhang wrote the first version of the manuscript while Ming-jun Zhang, Ying-yu Xue, Shuang Xu, Xuan-ru Jin, Xing-chu Man contributed to the improvement of the final version. All authors approved the final manuscript.
This project was supported by the National Natural Science Foundation of China (31760577, 32360759), Star of Innovation project for excellent graduate students in Gansu province(2023CXZX-693).
The reference genome assembly used for data analysis was obtained from the National Center for Biotechnology Information (NCBI). The datasets analyzed during this study are included in this published article and its supplementary information files.
This study does not include human or animal subjects.
Not applicable.
The authors declare no competing interests.
Graph: Additional file 1: Table S1. The 33 ARF gene-coding protein sequence information in this study.
Graph: Additional file 2: Table S2. Amino acid sequence information of 101 ARF genes.
Graph: Additional file 3: Table S3. Cis-acting elements in the promoter region of CpARF genes.
Graph: Additional file 4. Table S4. The primer is designed for qRT-PCR.
Graph: Additional file 5: Table S5. Relative expression of 33 genes by qRT-PCR in different stress.
Graph: Additional file 6: Table S6. Relative expression of 13 genes by qRT-PCR in Pathogen stress.
• qRT-PCR
- Quantitative RT-PCR
- CpARF
- Cucurbita pepo Auxin Response Factor
• UTR
- Untranslated region
• GFP
- Green fluorescent protein
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
By Ming-jun Zhang; Ying-yu Xue; Shuang Xu; Xuan-ru Jin and Xing-chu Man
Reported by Author; Author; Author; Author; Author