Background

As signal molecules, jasmonics, including jasmonic acid (JA) and its cyclopentanous derivatives, play an important role in plant growth [1], development [2, 3], and response to biotic and abiotic stresses [3, 4]. According to previous reports, the core members of the JA-signaling pathway, including SCFCOI1 (SCF, SKP1 + Cdc53/cullin+Rbx1 + F-box; COI1, coronatine insensitive 1) E3 ubiquitin ligase, jasmonate ZIM-domain (JAZ) repressor proteins, and myelocytomatosis2 (MYC2) transcription factor, have been defined as the COI1/JAZs/MYC2 module [5, 6].

In recent years, the mechanistic details of the JA signal transduction pathway and its regulatory network have gradually been revealed [7,8,9]. Research on jasmonics mainly focuses on their metabolism and signal transduction [9], their interaction with other hormones [10,11,12], and the responses of JA signals to pathogenic bacteria or pests [10, 13, 14]. JA content is low in plants under normal growth conditions, and JAZ inhibits the expression of JA-response genes through direct interaction with transcription factors such as MYC2. JA-mediated responses are therefore repressed by JAZ proteins [6, 15]. In response to stresses, such as that caused by insect feeding or pathogen infection, JA is accumulated rapidly, and jasmonoyl-l-isoleucine (JA-Ile) is formed under the action of jasmonic acid-amido synthetase (JAR1) [6, 16]. The increase of JA-Ile levels promotes the interaction between the JAZ repressor protein and the F-box protein encoded by COI1, making JAZs ubiquitinate and degrade via the 26S proteasome pathway, releasing DNA-binding transcription factors (such as MYC2) and inducing the expression of JA response genes [6, 15]. In addition, the deletion of the COI1 locus or its functionally deficient mutations can lead to the elimination or weakness of plant responses to JA [13, 17]. These results illustrate the importance of COI1 in JA signal transduction.

It has been reported that COI1 is not only involved in plant developmental processes, such as leaf senescence [18, 19], seed maturation [13], flowering [20], male fertility [21], anthocyanin formation [22], and root growth [22, 23], but also plays a part during various physiological processes in the plant defense response to insect attack and pathogen infection [13, 24, 25]. COI1 belongs to a multigene family. There are one F-box domain and 16 leucine-rich repeats (LRR) in COI1 protein [26, 27]. The F-box protein participates in the formation of the SCFCOI1 E3 ubiquitin ligase complex involved in the ubiquitin-dependent proteolytic pathway [28], and regulates the expression of JA-responsive genes [28, 29]. As a JA receptor, COI1 protein is maintained at a protein level essential for proper biological functions during plant development and defense, which is strictly regulated by the dynamic balance of SCFCOI1-mediated stabilization and 26S proteasome pathway-mediated degradation [30, 31]. To date, 35 COI1-dependent JA-regulated proteins have been identified in Arabidopsis thaliana [19]. Among them, rubisco activase (RCA), which is correlated with JA-induced leaf senescence, can be down-regulated by JA in a COI1-dependent manner [19]. In addition, Arabidopsis coi1 mutants were observed to be male-sterile [17], apical dominance defective (coi1–37) [32], susceptible to pests and bacterial pathogens [17], insensitive to JA [17], and lacking the expression of JA-induced proteins [33]. Arabidopsis coi1–1 mutant plants were infertile and showed a stay-green phenotype under dark-induced senescence conditions, but those phenotypes could be rescued in mutants overexpressing 35S:OsCOI1a or 35S:OsCOI1b due to the fact that the JA signaling insensitivity of coi1–1 mutants was complemented. This finding suggests that coi1 plays a key role in leaf senescence and fertility [18, 34]. In a study by Huang et al. [22], amino acid changes in COI1 could significantly attenuate its function, not only in regulating JA-inhibited root growth and JA-induced anthocyanin accumulation, but also in JA-mediated plant response to inoculation with the pathogen Pst DC3000. However, different mutations in the COI1 gene have distinct effects on COI1 function in regulating male fertility. GhCOI1 silencing in Gladiolus hybridus impaired inducible defense and increased susceptibility to the necrotrophic pathogenic fungus Alternaria brassicicola [24]. The above findings have provided evidence of the importance of the COI1 gene during the processes of plant growth and development, as well as defense responses. However, there have been no reports on the COI1 gene in sugarcane (Sacharum spp.). Hence, a systematic investigation of the characteristics of the COI1 multigene family in sugarcane should provide an efficient basis for the study and manipulation of the JA signaling pathway.

Sugarcane is an important sugar and biofuel crop in the world [35, 36]. However, various stresses, such as pathogens, low temperatures, and drought, seriously restrict the healthy development of the sugarcane industry [37]. Due to the complex genetic background and long growth period of sugarcane, genetic engineering has great advantages in the cultivation of resistant sugarcane varieties compared to traditional cross breeding [38]. Therefore, the discovery of resistance candidate genes is of great significance. In the present study, first, 21 SsCOI1, three ShCOI1, and seven SbCOI1 genes were identified from the genomes of the sugarcane-related wild species Saccharum spontaneum [36], the sugarcane-related cultivated species Saccharum spp. hybrid cultivar R570 [38], and the sugarcane proximal species Sorghum bicolor [39], respectively. Second, the protein physicochemical properties, chromosome location, evolutionary relationship, protein motif, gene structure, cis-acting elements, tissue-specific expression, and expression profiles of the COI1 gene family under methyl jasmonate (MeJA), cold, drought, and Sporisorium scitamineum stresses were analyzed [40,41,42]. Third, the full-length sequences of three ShCOI1 genes (ShCOI1–4, ShCOI1–5, and ShCOI1–6) were isolated from Saccharum spp. hybrid cultivar ROC22 using a homologous cloning method. In addition, the real-time quantitative PCR (RT-qPCR) technique was used to analyze the gene expression patterns of ShCOI1–4, ShCOI1–5, and ShCOI1–6 under cold, drought, salicylic acid (SA), abscisic acid (ABA), and S. scitamineum stresses [42,43,44]. This study aims to uncover and identify the COI1 gene family in sugarcane, understand their sequence characteristics and gene expression patterns, and thus provide candidate gene resources for sugarcane resistance molecular breeding.

Results

Identification, classification, and phylogenetic analysis of COI1 gene family

A total of 156 COI1 proteins were obtained from 19 sequenced plant species among five lineages, including 33 COI1s in five eudicots (eight in Medicago truncatula; seven in A. thaliana, Capsella rubella, and Vitis vinifera; and four in Fragaria vesca), 87 COI1s in 10 monocots (21 in S. spontaneum; 13 in Zea mays; eight in Brachypodium distachyon and Triticum aestivum; seven in Oryza sativa, Panicum hallii, Setaria italica, and S. bicolor; six in Ananas comosus; and three in R570), four COI1s in one basal angiosperm (Amborella trichopoda), 28 COI1s in two mosses (15 in Sphagnum fallax and 13 in Physcomitrella patens), and four COI1s in one lycophyte (Selaginella moellendorffii). However, no COI1 was identified in five algae plants that belonged to Rhodophyta (Chondrus crispus, Cyanidioschyzon merolae, and Galdieria sulphuraria) and Chlorophyta (Coccomyxa subellipsoidea C169 and Micromonas pusilla CCMP1545) (Fig. 1 and Supplemental Table S1).

Fig. 1
figure 1

The number of COI1 homologue genes and the evolutionary relationship of 24 species. These 24 species used in this study belonged to seven lineages (Monocots, Eudicots, Basal angiosperms, Lycophytes, Mosses, Chlorphyta, and Rhodophyta) that are derived from terrestrial plants and algae

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On the basis of the topology of phylogenetic trees and the conserved amino acid sites of F-box and JAZ-binding sites on COI1 proteins (Supplemental Fig. S1 and Supplemental Table S2) [45], 156 COI1 proteins were classified into four groups (group A, group B, group C, and group D) (Fig. 2). Among them, COI1 genes from the same lineage, such as mosses, monocots, and eudicots, tended to be clustered to the same branch in group A, group B, and group D, and only COI1 proteins from mosses were clustered in group C. In detail, group A contained nine SsCOI1s (SsCOI1–4a, −4b, −4c, −4e, −5, −6a, −6b, −6c, and −6d) and two SbCOI1s (SbCOI1–5 and SbCOI1–6). Group B included five SsCOI1s (SsCOI1–1a, −1b, −3a, −3b, and −3c), two SbCOI1s (SbCOI1–2 and SbCOI1–4), and ShCOI1–2. There were seven SsCOI1s (SsCOI1–2a, −2b, −7a, −7b, −8a, −8b, and −8c), three SbCOI1s (SbCOI1–1, SbCOI1–3, and SbCOI1–7), and two ShCOI1s (ShCOI1–1 and ShCOI1–3) in group D.

Fig. 2
figure 2

Phylogenetic analysis and classification of 156 COI1 proteins in 19 plant species. The phylogenetic tree was constructed using the maximum likelihood method (JTT + G model, complete deletion, and 1000 bootstrap replicates) using MEGA 6.60 based on the full-length sequences of COI1 proteins. Four colored arcs indicated four groups of COI1 proteins. The different colored shapes corresponded to various species were shown in labels. All the accession numbers of COI1 proteins were listed in Supplemental Table S1

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Characteristics of the COI1 gene family in S. bicolor, R570, and S. spontaneum

As shown in Supplemental Table S3, the number of amino acids of 31 COI1s (including 21 SsCOI1s, seven SbCOI1s, and three ShCOI1s) was 434–665, and their corresponding molecular weights (MWs) ranged from 47.94 to 73.14 kDa. The predicted isoelectric point (pI) values of 31 COI1s varied from 5.25 to 8.40. The results of the instability index suggested that 30 of 31 COI1s were unstable proteins (instability index > 40), and the grand average of hydropathicity (GRAVY) showed that 22 of 31 COI1s were hydrophilic proteins (GRAVY < 0). There were no signal peptides or transmembrane structures in these COI1 proteins, suggesting that they were all non-secreted proteins (Supplemental Table S3). Moreover, among 31 COI1 proteins, nine were predicted to be located in the cytoplasm and nucleus, four in the cytoplasm, and 18 in the nucleus (Supplemental Table S3). For the secondary structures of these proteins encoded by the SsCOI1s, ShCOI1s, and SbCOI1s, alpha helixes (45.41–53.46%) and random coils (28.80–37.89%) were the main components, the extended chain (10.90–14.60%) was secondary, and beta turns (3.20–5.84%) accounted for the smallest proportion (Supplemental Table S4).

Conserved motifs and gene structures of the COI1 gene family

Homology analysis showed that the amino acid sequence similarity among 31 COI1 proteins ranged from 27.00 to 100.00% (Supplemental Table S5). A phylogenetic tree (Fig. 3) demonstrated that the protein sequences of 21 SsCOI1s, seven SbCOI1s, and three ShCOI1s were divided into three groups (groups A, B, and D), which was consistent with the above classification in Fig. 2. The number of conserved motifs in 31 COI1 proteins varied from eight to 12, and motifs 1–7 and motif 10 were included in all of these COI1 protein sequences (Fig. 3). Motif 3, motif 2, and motif 1 represented an F-box_5 domain (pfam18791), a typical LRR sequence domain, and a transport inhibitor response 1 protein domain (cl40087), respectively (Supplemental Table S6). However, several motifs were specific in subgroup members. For example, motif 9 was present in all members of group B and group D, but only in three members of group A. Group D members had two motif 8 and one motif 4, except for SsCOI1–2b. In contrast, motifs in the members of group A were relatively irregular. Likewise, among 11 members of group A, four had two motif 4, five had two motif 7, three had motif 5, and three had two motif 8. These results indicate that motif 1, motif 3, motif 6, and motif 10 are relatively conserved in the evolution of the COI1 gene family. The number of introns contained in the COI1 gene family of S. bicolor, S. spontaneum and R570 ranged from two to six (Fig. 3). The numbers of group B had two introns. In group D, except for SsCOI1–2b, SsCOI1–2a, and SsCOI1–8b, all the other nine members had two introns. However, the gene structures of group A members were irregular, with intron numbers ranging from two to six (Fig. 3).

Fig. 3
figure 3

Protein motif and gene structure of SbCOI1, ShCOI1, and SsCOI1 genes. SbCOI1, ShCOI1, and SsCOI1 represented the COI1 gene in Sorghum bicolor, Saccharum spp. hybrid cultivar R570, and S. spontaneum, respectively. The clustering tree on the left side of the figure was constructed using the maximum likelihood method (JTT + G model, complete deletion, and 1000 bootstrap replicates) using MEGA 6.60, and three evolutionary groups (group A, group B, and group D) were shown in different colors. Motifs were identified using Multiple Em for Motif Elicitation. Motif 1–10 was represented by different colored squares. Details of the individual motifs were shown in Supplementary Table S6. The yellow box, blue box, and black line in the gene structures represented exons, untranslated regions (UTR), and introns, respectively. The sizes of motifs, exons, and introns can be estimated using the scale below

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Chromosomal location, duplication events, and synteny analysis of COI1 gene family

Chromosome mapping showed that 21 SsCOI1 genes were unevenly distributed on 18 of 32 S. spontaneum chromosomes (Supplemental Fig. S2 and Supplemental Table S1). Among them, chromosomes Ss1A, Ss4B and Ss5C had two SsCOI1 genes, and each of the remaining 15 chromosomes had one SsCOI1 gene. Three ShCOI1 genes were evenly distributed on chromosomes Sh01, Sh04, and Sh09 among 10 R570 chromosomes (Supplemental Fig. S2). However, SbCOI1–1 and SbCOI1–2 were distributed on Sb01, and the other five SbCOI1 genes were evenly distributed on chromosomes Sb03, Sb05, Sb05, Sb06, and Sb09 of S. bicolor (Supplemental Fig. S2).

To explore explored the expansion mechanisms, the gene types of COI1 in S. spontaneum, R570 and S. bicolor, including singleton, dispersed, proximal, tandem, and whole-genome duplication (WGD)/segmental duplications, were analyzed (Fig. 4a and Supplemental Table S7). In 21 SsCOI1 genes, 14 WGD/segmental (66.67%), three dispersed (14.29%), two proximal repeat genes (9.52%), one tandem (4.76%) duplication, and one singleton gene (4.76%) were found. Interestingly, all three ShCOI1 genes in R570 had dispersed duplications. Among seven SbCOI1 genes, five were detected as dispersed genes (71.43%), and two were WGD/segmental duplications (28.57%) (Fig. 4a and Supplemental Table S7). Therefore, it can be speculated that the SsCOI1 gene family mainly expanded through WGD or segmental duplication events, and the dispersed duplications appear to be the main expansion mechanisms for the SbCOI1 gene family and ShCOI1 gene family.

Fig. 4
figure 4

Expansion mechanisms and synteny analysis of COI1 gene family. a Expansion mechanisms of COI1 gene family in Sorghum bicolor, Saccharum spp. hybrid cultivar R570, and S. spontaneum. The numbers in the pie chart represented the effects of WGD or segmental (whole-genome duplication/segmental duplication), Dispersed (dispersed duplication), Proximal (proximal duplication), Tandem (tandem duplication), and Singleton (single copy). b Synteny analysis of COI1 genes in S. spontaneum and R570. c Synteny analysis of COI1 genes in S. bicolor, R570, and S. spontaneum. All replicated genes in the genome were represented by gray lines in figures b and c; the red lines in figure b represented the replicated SsCOI1 genes; the red lines in figure c represented the homologous COI1 gene pairs in S. spontaneum and R570; the green lines represented the homologous COI1 gene pairs in S. bicolor and R570; and the blue lines represented the homologous COI1 gene pairs in S. spontaneum and S. bicolor. “Ss”, “Sh”, and “Sb” represented the name of each chromosome in S. bicolor, R570, and S. spontaneum, respectively. The detailed information was shown in Supplemental Table S7 and Table S8

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The gene collinearity among S. spontaneum, R570, and S. bicolor was analyzed to investigate the evolutionary mechanism of the COI1 gene family (Fig. 4b, c and Supplemental Table S8). In S. spontaneum, 12 pairs (14 genes) of collinearity relationships of 21 SsCOI1s were observed (Fig. 4b and Supplemental Table S8). Furthermore, all of these 14 genes had WGD/segmental duplications, including 11 pairs (13 SsCOI1 genes) of homoeologous genes that were distributed in different chromosomes. In R570, there was no collinear relationship among three ShCOI1 genes in R570 (Fig. 4b and Supplemental Table S8). As shown in Fig. 4c, there were 12 orthologous pairs between S. spontaneum and S. bicolor, four between S. spontaneum and R570, and four between S. bicolor and R570. The nonsynonymous (Ka)/synonymous (Ks) ratios of all duplicated COI1 genes in S. bicolor, R570, and S. spontaneum were < 1, indicating that the COI1 gene family might have experienced strong purifying selective pressure during evolution (Supplemental Table S8).

Cis-acting elements in the promoter regions of the COI1 gene family

The cis-acting regulatory elements in the promoters of COI1 genes were predicted to assist the gene function elaboration. There were many core elements in the promoter sequences of 31 COI1s, which were involved in stress responsiveness, hormone responsiveness, light responsiveness, and growth and development (Fig. 5 and Supplemental Table S9). The light, ABA and MeJA response elements were the most numerous in the COI1 gene promoter regions, followed by anaerobic induction, drought-inducibility, and low-temperature responsiveness. Among them, light response elements were observed in all COI1 promoter regions. In 31 COI1 promoter regions, 30 COI1s contained MeJA response elements (CGTCA-motif and TGACG-motif), except for SbCOI1–5, 26 (83.87%) contained abscisic acid response elements (ABRE), and 24 (77.42%) contained anaerobic induction elements (ARE), while 22 (70.97%) contained drought-inducibility (MBS) and low-temperature response elements (LTR). In addition, the numbers of COI1 promoter regions that contained auxin (IAA), gibberellin (GA), and SA response elements, were 16, 18, and 12, respectively. The existence of these functional elements indicates that COI1 genes may participate in the induction of multiple stress responses and thus play a role in sugarcane defense against various environmental stresses.

Fig. 5
figure 5

Promoter cis-regulatory element analysis of SbCOI1, ShCOI1, and SsCOI1 gene family. SsCOI1, ShCOI1, and SbCOI1 represented COI1 genes from Saccharum spontaneum, Saccharum spp. hybrid cultivar R570, and Sorghum bicolor. Different colors boxes in cis-regulatory elements correspond to different elements. The clustering tree on the left side of the figure was constructed using the maximum likelihood method (JTT + G model, complete deletion, and 1000 bootstrap replicates), and different color on the phylogenetic tree represented different groups of COI1. The detailed information about promoter cis-regulatory elements of SbCOI1, ShCOI1, and SsCOI1 genes was shown in Supplemental Table S9

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Tissue expression profiles of COI1 genes in sugarcane

The results of transcriptome sequencing (RNA-seq) showed that 21 SsCOI1 genes were constitutively expressed in the root, bud, leaf, stem pith, and epidermal tissues of sugarcane cultivar ROC22 (Saccharum spp. hybrid), but with various expression levels (Fig. 6). Among them, SsCOI1–6d had low expression in all tissues, and the expression levels of SsCOI1–1a, −1b, −3a, −3b, and −3c (clustered into group B) were the highest in the bud and the lowest in the leaf. SsCOI1–2a and SsCOI1–2b had the lowest expression levels in the bud and the highest in the stem pith. The expression levels of SsCOI1–6a, −6b, −6c, −7a, −7b, −8a, −8b, and −8c were the highest in the stem pith and the lowest in the root. The highest expression levels of SsCOI1–4a, −4b, −4c, and −5 were observed in bud tissues, followed by those in the epidermis, stem pith, root, and leaf. There were different expression patterns between SsCOI1–4e and its duplicated gene, SsCOI1–4c. It was noteworthy that the SsCOI1 genes that clustered to group D showed a higher expression level compared with the other group in general. These results suggest that SsCOI1 genes may play a role in sugarcane growth and development, but with different function modes.

Fig. 6
figure 6

Expression pattern of SsCOI1 genes in different sugarcane tissues of mature ROC22. Epidermis, Stem pith, Root, Leaf, and Bud represented different tissues of mature ROC22. The fragments per kilobase of transcript per million mapped (FPKM) shown in the box represented the SsCOI1s expression levels. The color bar represented the normalized values (log2 FPKM). The clustering tree on the left side of the figure was constructed using the maximum likelihood method (JTT + G model, complete deletion, and 1000 bootstrap replicates) by MEGA 6.60, and different colors and letters (A, B, and D) on the phylogenetic tree represented three groups of SsCOI1s

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Expression profiles of COI1 genes under cold and drought treatments

Due to the fact that the drought-inducibility (MBS) and low-temperature response elements (LTR) were observed in most of the promoter sequences of SsCOI1s, their expression profiles under drought and cold stresses were analyzed to further investigate the function of SsCOI1 genes. As shown in Fig. 7, SsCOI1 responded to both drought and cold stresses, but with different expression patterns.

Fig. 7
figure 7

Expression profiles of SsCOI1s under drought and cold stresses. The trimmed mean of M-values (TMM) shown in the box represented the expression levels of SsCOI1s. The color bar represented the normalized values (log2 TMM). The clustering tree on the left side of the figure was constructed using the maximum likelihood method (JTT + G model, complete deletion, and 1000 bootstrap replicates) by MEGA 6.60, and different colors and letters (A, B, and D) on the phylogenetic tree represented three groups of SsCOI1s. Drought_0 d, Drought_2 d, Drought_6 d, Drought_10 d, Recovery_10 d, Cold_0 h, Cold_0.5 h, Cold_1 h, Cold_6 h represented the time that sugarcane cultivated under drought or cold treatments

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Under drought stress, SsCOI1–1a, −1b, −3a, −3b, −3c, −8a, −8b, and −8c had the highest transcription levels at 10 d and the lowest expression levels after water recovery. The expression levels of SsCOI1–2a and SsCOI1–2b were down-regulated at 6 d, but up-regulated at 10 d and after water recovery. Compared with the control, the expression levels of SsCOI1–4a, −4b, −4c, −4e, −5, −7a, and −7b were increased and reached a single peak at 2 d, but decreased to the lowest after 10 d water recovery. The transcription levels of SsCOI1–6a, −6b, −6c, and −6d were increased and had the highest levels at 10 d after drought treatment, but all of them were decreased after water recovery. Under cold stress, the expression levels of all SsCOI1s were elevated. For instance, SsCOI1–4b and SsCOI1–4e showed the highest expression levels at 0.5 h and the lowest expression levels at 1 h. The expression levels of SsCOI1–1a, −1b, −4c, −5, −8a, −8b, and −8c were all enhanced to a peak at 0.5 h and then decreased. The transcription levels of SsCOI1–2b, −3a, −3c, −4a, −6b, −6c, and −6d were continuously up-regulated from 0 to 6 h after cold treatment, while SsCOI1–2a, −3b, and −6a decreased first and then increased and showed the highest levels at 6 h. In addition, SsCOI1–7a and SsCOI1–7b were up-regulated at 0.5 h and remained stable at 1 h after cold treatment.

Expression profiles of COI1 genes in response to sugarcane smut pathogen infection

To study the function of SsCOI1s in response to smut pathogen infection, the gene expression patterns during the interaction between two different sugarcane genotypes and S. scitamineum were analyzed. As shown in Fig. 8, in sugarcane smut-resistant Saccharum spp. hybrid cultivar YC05–179, SsCOI1–3a had no significant expression change while SsCOI1–1a and SsCOI1–1b were down-regulated. SsCOI1–4a and SsCOI1–6b were up-regulated and reached the highest expression levels at 1 day post-inoculation (dpi), while SsCOI1–2a, −2b, −3b, −3c, −4b, −4c, and −4e were up-regulated and reached a single peak at 2 dpi. The transcription levels of SsCOI1–5, −6b, −6c, and −6d were increased to a peak at 5 dpi. In addition, the expression levels of SsCOI1–7a, −7b, −8a, −8b, and −8c were decreased at 1–2 dpi and then increased at 5 dpi. In sugarcane smut-susceptible Saccharum spp. hybrid cultivar ROC22, the transcription levels of SsCOI1–1a, −1b, −2a, −2b, −3a, −3c, −4a, −4b, and −4e were inhibited after smut pathogen inoculation. The expression levels of SsCOI1–3b, −4c, and −5 were up-regulated at 1 dpi, and then down-regulated from 2 dpi to 5 dpi. The expression levels of SsCOI1–6a, −6b, −6c, −6d, −8a, −8b, and −8c were decreased and reached the lowest point at 2 dpi, and then increased at 5 dpi. The expression levels of SsCOI1–7a and SsCOI1–7b remained stable from 1 dpi to 2 dpi, and then were up-regulated at 5 dpi.

Fig. 8
figure 8

Expression patterns of SsCOI1s in the interaction between different sugarcane genotypes and Sporisorium scitamineum. The fragments per kilobase of transcript per million mapped (FPKM) shown in the box represented the SsCOI1s expression levels. The color bar represented the normalized values (log2 FPKM). The clustering tree on the left side of the figure was constructed using the maximum likelihood method (JTT + G model, complete deletion, and 1000 bootstrap replicates) by MEGA 6.60, and different colors and letters (A, B, and D) on the phylogenetic tree represented three groups of SsCOI1s. YC05–179_ 0 d/ 1 d/2 d/5 d and ROC22_0 d/1 d/2 d/5 d represented the sugarcane smut-resistant cultivar YC05–179 and smut-susceptible cultivar ROC22 under S. scitamineum treatment for 0 d, 1 d, 2 d and 5 d, respectively

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These results indicated that all 21 members of the SsCOI1 gene family could be induced during the interaction between sugarcane and smut pathogen, and these allele genes showed similar expression patterns.

Expression profiles of COI1 genes under MeJA treatment via RT-qPCR

The COI1 gene is the core member of the JA signaling pathway. To understand the expression pattern of the COI1 gene in response to JA, the expression levels of seven SsCOI1 haplotype genes under MeJA stress were evaluated via RT-qPCR (Fig. 9). After MeJA treatment, the expression levels of SsCOI1–4 and SsCOI1–6 (the members of group A) remained unchanged from 0 h to 24 h. The expression levels of the SsCOI1–1 gene (a member of group B) decreased significantly at 3 h, but increased significantly at 12 h and remained at a high level at 24 h, at levels 1.62- and 1.92-fold that of the control, respectively. The expression levels of SsCOI1–3 (a member of group B) were increased remarkably at 3 h and 12 h, at levels 1.54- and 1.40- fold higher than the control, and returned to the control level at 24 h. The transcription levels of SsCOI1–2 and SsCOI1–7 (members of group D) were up-regulated at 3 h and then stayed at a relatively stable level. For the SsCOI1–8 gene (a member of group D), its expression levels increased with a single peak at 12 h, which was 1.67-fold higher than the control. The above results show that various SsCOI1 genes might play different roles in response to MeJA.

Fig. 9
figure 9

Expression profiles of SsCOI1s under MeJA treatment via RT-qPCR. MeJA, 100 μM jasmonic acid. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene to normalize the expression data. The 2−ΔΔCT method was applied to obtain the relative expression levels. All data was shown as mean ± standard error (n = 3). Different letters representing significant differences were assessed by Duncan test (*p < 0.05)

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Cloning and sequence analysis of three COI1 genes in the sugarcane cultivar ROC22

To further understand the functions of COI1 genes in a sugarcane cultivar, three candidate COI1 genes, SsCOI1–4b (clustered into subgroup A), SsCOI1–1b (clustered into group B), and SsCOI1–3b (clustered into group B), were cloned from ROC22 and termed as ShCOI1–4, ShCOI1–5, and ShCOI1–6, respectively. The full lengths of the cDNA of the ShCOI1–4, ShCOI1–5, and ShCOI1–6 genes were 1953 bp, 2152 bp, and 2322 bp, respectively, with 592, 600, and 661 encoding amino acids, respectively. The amino acid sequence similarities of ShCOI1–4, ShCOI1–5, and ShCOI1–6 to SsCOI1–4b, SsCOI1–1b, and SsCOI1–3b, respectively, were 99.50, 99.70, and 99.2%, respectively (Supplemental Table S5). All three ShCOI1 proteins contained relatively conservative F-box domains, Transp_inhibit domains, and AMN1 domains (leucine-rich repeat protein) (Supplemental Fig. S1). Furthermore, compared with ZmCOI1a (GRMZM2G125411), ZmCOI1b (GRMZM2G151536), ZmCOI1c (GRMZM2G353209), and ZmCOI2 (GRMZM2G079112), three ShCOI1 proteins possessed only 4–5 of 16 key amino acid residues (asterisks and sites 1–4 in Supplemental Fig. S1) that are supposed to be the binding sites of JA-Ile or JAZ proteins, suggesting that there may be functional differentiation among these three ShCOI1s and ZmCOIs.

Gene expression patterns of ShCOI1–4, ShCOI1–5, and ShCOI1–6 in response to different abiotic stresses via RT-qPCR

According to previous reports, JA and SA are mostly related to plant resistance to pathogen infection [11, 46], and ABA is mostly associated with abiotic stress [47, 48]. In this study, RT-qPCR was used to analyze the expression levels of ShCOI1–4, ShCOI1–5, and ShCOI1–6 in ROC22 under SA, ABA, cold (4 °C), and drought (polyethylene glycol, PEG) treatments (Fig. 10). Under ABA stress, the transcripts of ShCOI1–4 were up-regulated by 2.86- and 2.99-fold at 3 h and 6 h, respectively, and returned to the control levels at 12 h. After SA treatment, the expression levels of ShCOI1–4 increased with a single peak at 12 h that was 1.56-fold higher than the control. Under cold and drought stresses, compared to the control, the transcripts of ShCOI1–4 were decreased. Under SA treatment, the transcripts of the ShCOI1–5 gene increased with a single peak at 12 h that was 2.17-fold higher than the control. For ABA treatment, the expression levels of ShCOI1–5 remained unchanged at 0–6 h and were reduced at 12 h. In addition, the transcription levels of ShCOI1–5 were increased remarkably at 0.5 h, 3 h, and 6 h to levels 2.77-, 1.66-, and 1.66-fold higher than the control under drought stress, respectively, but the transcription levels remained unchanged under cold stress. The expression levels of the ShCOI1–6 gene were stable under cold stress, but increased under SA, ABA, and drought stresses. After SA treatment, the expression level of ShCOI1–6 was increased remarkably at 3 h to a level 1.86-fold higher than the control. Under ABA treatment, the expression levels of ShCOI1–6 were increased at 3–12 h, and peaked at 12 h. The expression level of ShCOI1–6 was significantly increased at 2.14-fold higher than the control at 24 h after drought treatment.

Fig. 10
figure 10

Expression patterns of ShCOI1–4, ShCOI1–5, and ShCOI1–6 in response to different abiotic stresses via RT-qPCR. ABA, 100 μM abscisic acid; SA, 5 mM salicylic acid; Cold, 4 °C low temperature; PEG, 25% polyethylene glycol 8000. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. The 2−ΔΔCT method was applied to obtain the relative expression levels. All data was shown as mean ± standard error (n = 3). Different letters representing significant differences was assessed by Duncan test (*p < 0.05)

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Based on these findings, it is speculated that ShCOI1–4, ShCOI1–5, and ShCOI1–6 actively respond to biotic and abiotic stresses in plants via different signal pathways.

Gene expression patterns of ShCOI1–4, ShCOI1–5, and ShCOI1–6 in response to smut pathogen infection via RT-qPCR

In the sugarcane-smut pathogen biosystem, the expression patterns of ShCOI1–4, ShCOI1–5, and ShCOI1–6 in six Saccharum spp. hybrid cultivars (including smut-resistant cultivars YZ03–258, LC05–136, and YT96–86, and smut-susceptible cultivars GT02–467, FN40, and YZ03–103) were evaluated using RT-qPCR (Fig. 11). Compared with the control, the expression levels of ShCOI1–5 were all significantly increased in three smut-resistant sugarcane cultivars and two smut-susceptible cultivars (GT02–467 and YZ03–103), but were significantly decreased in FN40 at 7 dpi. Except for the increased expression levels of ShCOI1–4 in LC05–136 at 7 dpi and in GT02–467 at 3 dpi, the expression levels of the ShCOI1–4 and ShCOI1–6 genes were both significantly increased in YZ03–258 and YT96–86 and decreased or remained stable in the other four sugarcane cultivars. In summary, ShCOI1–4, ShCOI1–5, and ShCOI1–6 can be induced by smut pathogen attack, but their expression patterns vary during the interaction between different sugarcane cultivars and the smut pathogen.

Fig. 11
figure 11

Expression patterns of ShCOI1–4, ShCOI1–5, and ShCOI1–6 in the interaction between sugarcane and Sporisorium scitamineum. YZ03–258, LC05–136, and YT96–86 were smut-resistant sugarcane cultivars (R); GT02–467, FN40, and YZ03–103 were smut-susceptible sugarcane cultivars (S). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. The 2−ΔΔCT method was applied to obtain the relative expression levels. All data was shown as mean ± standard error (n = 3). Different letters representing significant differences were assessed by Duncan test (*p < 0.05)

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Discussion

According to previous reports, jasmonic signal molecules can induce plants to activate resistance-related genes and systematically accumulate defense-related proteins to resist biotic and abiotic stresses [7, 8, 49]. As the essential member in the JA signaling pathway, COI1 has received increasing attention in recent years. It has been reported that the expression levels of AsCOI1 in Aquilaria sinensis can be significantly induced by MeJA, mechanical wounding, and heat [26]. The transcription levels of HbCOI1 in latex can be induced by JA and tapping [50]. In O. sativa, the mutation of OsCOI1b can delay leaf senescence, down-regulate several senescence-associated genes (including homologs of A. thaliana ETHYLENE INSENSITIVE 3 and ORESARA 1), and result in significant decreases in spikelet fertility and grain filling [18]. When OsCOI1 was inhibited by RNA interference (RNAi), O. sativa plants showed a decrease in resistance to Cnaphalocrocis medinalis and activity declines of trypsin protease inhibitor (TrypPI), polyphenol oxidase (PPO), and peroxidase (POD) [25].

Currently, as a polygenic family, the COI1 gene has been identified and reported in many plants, such as A. thaliana [31], O. sativa [18], Artemisia annua [51], T. aestivum [27], and Hevea brasiliensis [50]. In this study, a total of 156 COI1 proteins were identified from 19 land plant genomes, including 21 SsCOI1s from S. spontaneum, three ShCOI1s from R570, and seven SbCOI1s from S. bicolor, but no COI1 was obtained from five algae plants. It is speculated that COI1 may only exist in terrestrial plants because the COI1 gene family originated after the divergence of the algae and the ancestor of terrestrial plants. Of four groups, only group D in the phylogenetic tree was present in all terrestrial plants, indicating that a common ancestor was shared among the COI1 gene family from terrestrial plants after the divergence from algae (Fig. 2). In the present study, the numbers of COI1 observed in five eudicots were eight in M. truncatula (2n = 4x = 32, autotetraploid) [52]; seven in A. thaliana (2n = 2x = 10, diploid) [53], C. rubella (2n = 2x = 16, diploid) [54], and V. vinifera (2n = 2x = 38, diploid) [55]; and four in F. vesca (2n = 2x = 14, diploid) [56] (Fig. 1 and Supplemental Table S1). This result is in conflict with previous reports that the diploid dicots have a single copy of the COI1 gene, while the polyploid or paleopolyploid dicots may possess a number of COI1 orthologues in their genomes [21]. In addition, 31 COI1 proteins (three ShCOI1s, seven SbCOI1s, and 21 SsCOI1s) consisted of 434–665 amino acids with MWs ranging from 47.94 to 73.14 kDa, and most of them were predicted as unstable hydrophilic non-secreted proteins (Supplemental Table S3). These sequence characteristics of ShCOI1s, SbCOI1s, and SsCOI1s are common with those in other plants, such as the TaCOI gene family [27, 57], AsCOI1 [26], and HbCOI1 [50]. Furthermore, ShCOI1, SbCOI1, and SsCOI1 proteins were predicted to be located in the cytoplasm or nucleus (Supplemental Table S3), which was consistent with the subcellular localization of the AsCOI1 [26], SmCOI1 [58], and TaCOI proteins [57]. However, more empirical evidence needed to be provided.

As it shown in phylogenetic tree (Fig. 2), the COI1 genes from the same lineage, such as mosses, monocots, and eudicots, tended to be clustered to the same clade in group A, group B, and group D, and only COI1 proteins from mosses were clustered in group C. It is speculated that lineage-specific expansion and divergence events have occurred. Interestingly, COI1 proteins from the same species were clustered into different clades. For example, seven SbCOI1 and 21 SsCOI1 proteins were clustered into three groups (A, B, and D) and three ShCOI1s from R570 were clustered into two groups (B and D), revealing that the COI1 gene family exhibited differences in evolution among species.

According to the phylogenetic tree, protein motifs, and gene structure analysis, those COI1 proteins in the same group showed a similar motif composition and exon/intron structure, but varied among different groups (Fig. 3). For example, most members of COI1 clustered into group B and group D had two introns, while there were more intron numbers in group A. Furthermore, the number, length, and positions of introns exhibited diversity. Thus, we speculated that the intron loss or gain events occurred during the process of structural evolution of the COI1 gene family [59, 60]. Motif analysis results revealed the diversity of protein-conserved motifs among the COI1 gene family, such as the different numbers of motifs 4, 5, 7, 8, and 9 among various COI1 proteins that clustered into different clades. Therefore, the classification and evolution of COI1 genes might be related to their structural divergence and diversification.

As reported, gene duplication events can provide a primary source of material for the origin of evolutionary novelties, including new gene functions and expression patterns [61]. Chromosomal location, gene type, and gene collinearity analysis are often used to investigate the expansion and evolutionary mechanism of gene families [61, 62]. In this study, 21 SsCOI1 genes were unevenly distributed among 18 of 32 chromosomes of S. spontaneum, and not every COI1 had homoeologous genes on the homologous chromosomes A, B, C, and D, suggesting that some homologous COI1 genes may have been lost during the polyploidization of the genome [63]. There was a wide homologous relationship among S. spontaneum, R570, and S. bicolor, and the Ka/Ks ratios of all duplicated COI1 genes were < 1, indicating that the COI1 gene family might have experienced strong purifying selective pressure during evolution (Fig. 4b, c, and Supplemental Table S8). In addition, it was observed that the COI1 gene family was expanded by various genome duplication events in S. spontaneum, S. bicolor, and R570 (Fig. 4a and Supplemental Table S7). The different expansion mechanisms demonstrated that the driving forces in the evolution of each COI1 gene or the COI1 gene family among different species were diverse, and there may have been functional differentiation among various COI1 gene family members.

As a key component in the regulation of gene expression, the analysis of cis-acting regulatory elements in gene promoters can assist to elaborate the regulation and function of individual genes and their interaction with other genes [64, 65]. In the present study, a large number of promoter core elements were identified in the promoter sequences of ShCOI1s, SbCOI1s, and SsCOI1s that were involved in stress responsiveness (such as drought, low-temperature, and wound stress), hormone responsiveness (like SA, ABA, and MeJA), light responsiveness, and growth and development (Fig. 5 and Supplemental Table S9). This finding was similar to that observed in the promoter regions of HbCOI1 [50] and TaCOI genes [57]. The existence of these functional elements indicates that COI1 genes may play a role in sugarcane development and defense against various environmental stresses via participating in different regulatory mechanisms.

Gene expression patterns are usually related to their function [66]. As reported, the expression levels of COI1 genes in different plants are spatiotemporal [26, 50, 57, 58]. For instance, in H. brasiliensis, HbCOI1 has high transcription levels in laticifers, but low levels in bark and leaf tissues [50]. In Solanum melongena, the transcription levels of SmCOI1 are significantly down-regulated in anther indehiscence, which is related to the normal development of anthers [58]. In A. sinensis, the AsCOI1 gene was highly expressed in roots and stems, the two major organs of agarwood formation [26]. In addition, the members of the TaCOI gene family are expressed differently in various tissues, with the higher expression levels in stem, leaf, petal, pistil, stamen, and glume tissues than in roots [57]. Analogously, COI1 genes were constitutively expressed in sugarcane cultivar ROC22, but their expression patterns were diverse in different tissues (Fig. 6). For example, the expression levels of the group B genes (SsCOI1–1a, −1b, −3a, −3b, and −3c) were the highest in the bud and the lowest in the leaf. Compared with the other group genes, SsCOI1 genes, which clustered into group D, showed abundant transcripts and had the highest expression levels in stem pith. These results indicate that COI1 genes exhibit a tissue-specific pattern, and the same expression pattern suggests a similar function in the growth and development process.

RNA-seq data revealed that the COI1 gene family played a positive role in sugarcane response to drought and cold stresses with different expression patterns (Fig. 7). Interestingly, this finding is consistent with the prediction that the COI1 promoter sequence contains a large number of drought and low temperature response elements. To validate the function diversities of sugarcane COI1 genes, the expression levels of SsCOI1s under MeJA treatment were assessed by RT-qPCR (Fig. 9). The results show that various SsCOI1 genes may play different roles in response to MeJA. To further elucidate the functions of the COI1 gene family in a sugarcane cultivar, three ShCOI1 genes homologous with SsCOI1–4b, SsCOI1–1b, and SsCOI1–3b were cloned from ROC22. RT-qPCR results revealed that the transcription of ShCOI1–4 was decreased under cold (4 °C) and drought (PEG) stresses, while it was up-regulated significantly after SA and ABA treatments. The expression levels of ShCOI1–5 were up-regulated remarkably under SA and drought stresses, and down-regulated under ABA stress, and remained unchanged under cold stress. The transcriptions of the ShCOI1–6 gene were increased under SA, ABA, and drought stresses, but remained unchanged under cold stress (Fig. 10). These results were consistent with previous reports that COI1 was involved in the response of plants to various abiotic stresses and exhibited functional divergence [21, 57]. Likewise, in the TaCOI gene family, TaCOI2 (TaCOI2-A and TaCOI2-B) and TaCOI6 (TaCOI6-A, TaCOI6-B, and TaCOI6-D) were clustered into group A, while TaCOI5 (TaCOI5-A, TaCOI5-B, and TaCOI5-D) was clustered into group D [57]. The expression levels of TaCOI2 were up-regulated under the ABA, GA, and low-temperature treatments, but down-regulated under the IAA and MeJA treatments [57]. The transcripts of TaCOI6 could be induced by ABA and MeJA, but were suppressed by IAA and PEG [57]. The transcription levels of TaCOI5 were increased under the GA, low temperature, and PEG treatments, while they were decreased after the ABA, IAA, MeJA, and salinity treatments [57]. In Z. mays, the expression levels of four ZmCOIs (clustered into group A) responded to plant hormones were detected, and the results showed that ZmCOI1a and ZmCOI1b were strongly induced by JA and ABA, while ZmCOI1c and ZmCOI2 were less-expressed in maize tissues and slightly induced by JA and ABA, but there was no significant induction for ZmCOI1a, ZmCOI1b, ZmCOI1c and ZmCOI2 by 1-Aminocyclopropane-1-carboxylic acid, GA, 1-Naphthylacetic acid, and SA [21]. In addition, the restoration of male fertility in Arabidopsis mutant coi1–1 could result in plants that overexpressed ZmCOI1a, ZmCOI1b, or ZmCOI1c, but not ZmCOI2, indicating the successful complementation of coi1–1 sterility by ZmCOI1a, ZmCOI1b, and ZmCOI1c and the functional divergence of ZmCOIs [21]. It should be stressed that COI1 genes may exhibit inconsistent expression patterns under certain environmental stresses even if they are clustered into the same group.

It has been reported that JA and SA are mostly related to plant resistance to pathogen infection [11, 46]. Therefore, the COI1 gene family may be involved in sugarcane response to pathogen infection. Transcriptome analysis showed that SsCOI1 genes could be induced during the interaction between sugarcane and the smut pathogen, and the alleles showed similar expression patterns. Among them, the expression levels of SsCOI1–4b and SsCOI1–3b were up-regulated in smut-resistant cultivar YC05–179, but down-regulated in smut-susceptible cultivar ROC22, while SsCOI1–1b was down-regulated in both YC05–179 and ROC22. Furthermore, the expression patterns of ShCOI1–4, ShCOI1–5, and ShCOI1–6, the homologous genes of SsCOI1–4b, SsCOI1–1b, and SsCOI1–3b, respectively, were different in the interaction between the six different sugarcane cultivars and the smut pathogen (Fig. 11). Similarly, TaCOI1 took part in the early defense of compatible and incompatible wheat responses to Blumeria graminis (Bgt), and the response time was earlier in the resistant cultivars than in the susceptible ones [27]. Using virus-induced gene silencing, the expression of TaCOI1 decreased significantly, and the rate of successful penetration by Bgt was higher than that of the control. This indicates that TaCOI1 may play a key role in wheat-Bgt interactions [27]. In A. thaliana, two mutant alleles of coil conferred hypersusceptibility to the necrotrophic pathogen Sclerotinia sclerotiorum than wild-type or heterozygous plants [67]. Furthermore, overexpressed ZmCOIs in the Arabidopsis coi1–1 mutant plants can cause the restoration of resistance to the leaf pathogen Botrytis cinerea and the soil-borne pathogen Pythium aristosporum [21]. Taking the above findings into consideration, we conclude that COI1 genes have multiple functions and participate in sugarcane defense against various environmental stresses via different regulatory mechanisms.

Conclusion

A total of 156 COI1s, including 21 SsCOI1s, seven SbCOI1s, and three ShCOI1s, were identified from 19 species and could be clustered into four groups. The analysis of cis-acting elements, tissue-specific expression, and expression profiles under various stresses suggests that COI1 genes participate in growth, development, and response to various stresses in sugarcane. Furthermore, three COI1 genes, ShCOI1–4, ShCOI1–5, and ShCOI1–6, were obtained by homologous cloning in the sugarcane cultivar ROC22 and could be induced by the stresses of drought, cold, ABA, SA, and S. scitamineum with divergent expression profiles. The results illustrate the fact that sugarcane COI1 genes may actively respond to biotic and abiotic stresses via different regulatory mechanisms. The present study laid a foundation for the functional identification of sugarcane COI1 genes and provided a theoretical basis for molecular breeding of sugarcane resistance.

Materials and methods

Plant materials

Eight Saccharum spp. hybrid cultivars (including YT96–86, LC05–136, YZ03–258, ROC22, GT02–467, YZ03–103, FN40, and YC05–179) and smut whip were obtained from the Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs (Fuzhou, China).

The root, stem pith, leaf + 1, bud, and epidermis tissues of nine consistent 10-month-old ROC22 plants (the prevalent sugarcane cultivar in mainland China) were collected [42]. Four-month-old hydroponic ROC22 tissue-cultured plantlets were sprayed with 100 mM ABA, 5 mM SA (containing 0.01% Tween-20, v/v), 100 μM MeJA (containing 0.1% ethanol and 0.05% Tween-20, v/v), and 25% PEG 8000 at 28 °C with 16 h light and 8 h darkness [42, 68]. The leaves under SA and MeJA treatments were harvested at 0, 3, 12, and 24 h, the leaves under ABA treatment were collected at 0, 3, 6, and 12 h, and the leaves under PEG stress were harvested at 0, 0.5, 3, 6, and 24 h [42, 68]. For cold stress, the whole ROC22 plantlets were kept at a low temperature of 4 °C with 16 h light and 8 h darkness for 0, 6, 12, 24, and 48 h [42, 68].

The stems of eight 10-month-old sugarcane cultivars, including smut-resistant cultivars YC05–179, YZ03–258, LC05–136, and YT96–86; and smut-susceptible cultivars GT02–467, ROC22, FN40, and YZ03–103, were cut into two-bud sets, immersed for 1 day in flowing water, and cultivated under a light–dark regime (16 h of light and 8 h of darkness) at 32 °C until the germinating seedlings with a bud height of about 2 cm [44, 69]. Then, the bud was inoculated with 5 × 106 spores·mL− 1 S. scitamineum (0.01% Tween-20, v/v), and the control group was inoculated with sterile water (0.01% Tween-20, v/v) [43, 44]. All the materials were cultivated at 28 °C with a photoperiod of 16 h light and 8 h darkness [43, 44]. Five buds of YZ03–258, LC05–136, YT96–86, GT02–467, FN40, and YZ03–103 at 0, 1, 3, and 7 dpi were harvested and immediately frozen in liquid nitrogen for gene expression analysis [44, 69]. Five buds of YC05–179 and ROC22 were collected at 0, 1, 2, and 5 d after S. scitamineum inoculation for RNA-seq [70].

Each treatment included three biological replicates. All samples were immediately frozen in liquid nitrogen and stored at − 80 °C.

RNA extraction and first-strand cDNA synthesis

Total RNA was extracted from the collected samples using TRIzol™ (Invitrogen, Carlsbad, USA). RNA (1.0 μg) was reverse transcribed to the first-strand cDNA using a Prime-Script™ RT Reagent Kit (TaKaRa, Dalian, China) for RT-qPCR analysis. The cDNA used as cloning templates was synthesized from the RNA of ROC22 buds using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China).

Identification of the COI1 gene family

To identify the COI1 gene family, the genomic data of a total of 24 species were collected (Supplemental Table S10). The genomic data of 19 plants (A. thaliana, A. trichopoda, A. comosus, B. distachyon, C. rubella, F. vesca, M. truncatula, O. sativa, P. hallii, P. patens, R570, S. italica, S. bicolor, S. spontaneum, S. fallax, S. moellendorffii, T. aestivum, V. vinifera, Z. mays, C. subellipsoidea C169, and M. pusilla CCMP1545) were downloaded from Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html). The genomic data of C. crispus, C. merolae, and G. sulphuraria were downloaded from Ensembl (http://plants.ensembl.org/index.html). The genomic data of S. spontaneum was downloaded from the link of http://www.life.illinois.edu/ming/ downloads/Spontaneum_genome/ [36]. The monoploid reference genome of R570 was obtained from the Sugarcane Genome Hub (http://sugarcane-genome.cirad.fr/) [38]. Two Hidden Markov Model (HMM) profiles (PF18511.1 and PF18791.1), which were predicted by Pfam (http://pfam.xfam.org/search#tabview=tab1), were download from HMMER (https://www.ebi.ac.uk/Tools/hmmer) and used for the HMMER search [71]. Hmmsearch (HMMER package version 3.1b2) was used to search candidate COI1s from the genomic data of 24 species [71]. All obtained sequences were input into the Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/cdd) to search the protein domain [72]. The COI1 gene family members were confirmed after removing incomplete sequences. Allele genes were designated as the same name followed by the letters “a,” “b,” “c,” and “d”, and duplicated genes were designated as the same name followed by the letter “e” in S. spontaneum. The COI1 genes in T. aestivum and Z. mays were named according the research of Bai et al. [57] and An et al. [21], respectively.

Sequence characteristics of the COI1 gene family

All the identified COI1 genes in S. bicolor, R570, and S. spontaneum were submitted to ExPASy (http://web.expasy.org/protparam/) to analyze their amino acid numbers, MW, theoretical pI, instability index, and GRAVY. All full-length proteins were submitted to SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) for secondary structure analysis. The predictions of signal peptides, transmembrane structures, and subcellular localizations were conducted by SignalP-5.0 (http://www.cbs.dtu.dk/services/SignalP/), TMHMM (http://www.cbs.dtu.dk/services/TMHMM/), and Plant-mPloc (http://www.csbio.sjtu.edu.cn/bioinf/euk-multi-2/), respectively. In addition, the percent identity matrixes between COI1 proteins in S. bicolor, R570, S. spontaneum, and sugarcane hybrid cultivar ROC22 were calculated using DNAMAN.

Multiple sequence alignment and phylogenetic analysis

Multiple sequence alignment (MSA) of COI1 proteins was conducted by ClustalW in MEGA 6.60 with default parameters. Three phylogenetic trees in this study, including one phylogenetic tree of the 156 COI1 proteins from 19 plant species, one phylogenetic tree of 21 SsCOI1, seven SbCOI1, and three ShCOI1 proteins, and one phylogenetic tree of 21 SsCOI1s, were constructed using the maximum likelihood method (JTT + G model, complete deletion, and 1000 bootstrap replicates) based on the above alignments [73]. Evolview (https://evolgenius.info/evolview-v2/#mytrees/SHOWCASES/showcase%2002) [74] was used to display and edit the phylogenetic tree.

Motif and gene structure analysis of SbCOI1s, ShCOI1s, and SsCOI1s

Amino acid sequences of SbCOI1s, ShCOI1s, and SsCOI1s were submitted to the Multiple Em for Motif Elicitation online program (http://meme-suite.org/tools/meme) to identify the conserved motifs [75]. The parameters were as follows: maximum motif number, 10; maximum motif width, 50; minimum motif width, 6; and distribution of motif occurrences with zero or one per sequence. Diagrams of exon-intron structures were drawn using the Gene Structure Display Server 2.0 (http://gsds.gao-lab.org/). TBtools (Toolbox for Biologists) v1.09832 and Adobe Illustrator CS6 were used to display and edit the phylogenetic tree, conserved motifs, and gene structures [76].

Chromosomal locations and collinearity analysis of SbCOI1s, ShCOI1s, and SsCOI1s

The physical locations of COI1s on the chromosomes of S. bicolor, R570, and S. spontaneum were analyzed using MapGene2Chrom (MG2C) software (http://mg2c.iask. in/mg2c_v2.1/). Multiple Collinearity Scan toolkit (MCScanX) and TBtools were used with the default parameters to analyze the synteny block and gene duplication pattern [76, 77]. The values of Ka/Ks between orthologous gene pairs were calculated by TBtools to study the selection pressure acting on the evolution of the COI1 gene family [76].

Cis-acting regulatory element analysis in the promoter regions of COI1 genes

A 2000 bp sequence upstream of the start site of gene translation of SbCOI1s, ShCOI1s, and SsCOI1s was retrieved from genomic data as the promoter sequence, and its cis-regulatory elements were predicted using the PlantCARE online program (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [78]. The results of the prediction were visualized using TBtools [76].

Expression profiles of SsCOI1s in sugarcane based on RNA-seq

The RNA of the roots, stem piths, leaves, buds, and epidermis in ROC22 and the buds of YC05–179 and ROC22 inoculated with S. scitamineum for 0, 1, 2, and 5 d [70] were sequenced and assembled by the Biomarker Technologies Company limited (Beijing, China). The original data were obtained by Illumina technology, and after passing quality control, the data were analyzed using the S. spontaneum genome as the reference annotation library. The fragments per kilobase of transcript per million mapped (FPKM) was used as an indicator to measure the expression levels of transcripts or genes. For drought and cold treatments, the original data (PRJNA590595 and PRJNA636260) were downloaded from the Sequence Read Archive database (https://www.ncbi.nlm.nih.gov/sra/). The leaves of Saccharum hybrid cultivar Co 8021 under drought treatment were harvested at 0, 2, 6, and 10 d, and water-recovered at 10 d. The leaves of S. spontaneum under cold treatment were collected at 0, 0.5, 1, and 6 h. The sequence quality of these data was improved by Fastp [79]. The Hisat2 program was used to map sequence data to the S. spontaneum genome [80]. The count read and normalization of the data were conducted by the featurCounts in the Subread package and the trimmed mean of M-values (TMM) [81, 82]. The gene IDs of these transcriptomes followed the format of the original gene ID of S. spontaneum, which was relevant to the related search of their homologous genes. The expression levels of SsCOI1s in different sugarcane tissues and in response to drought, cold, and S. scitamineum stresses were mined from these RNA-seq data. The heat map showing the log2 (FPKM or TMM) expression profiles was generated by TBtools [76].

Cloning and sequences analysis of candidate S. spontaneum SsCOI1 genes in sugarcane cultivar

According to the sequences of SsCOI1s, the specific primers for ShCOI1–4, ShCOI1–5, and ShCOI1–6 (Supplemental Table S11) which were clustered into two different groups of COI1 gene family and with different expression patterns in response to MeJA were designed using Primer premier 5.0 software. The cDNA of ROC22 bud was used as a template for gene cloning. The transcription-polymerase chain reaction (RT-PCR) system contained 1.0 μL cDNA template, 1.0 μL each of the forward and reverse primers (10 μM), 12.5 μL 2× Phanta Max buffer, 0.5 μL dNTPs (2.5 mM), and 0.5 μL Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China), and 8.5 μL ddH2O. The PCR reaction conditions were as follows: 95 °C for 3 min; 35 cycles of 95 °C for 15 s, 56 °C for 15 s, and 72 °C for 2 min 30 s; and 72 °C for 5 min. PCR products were gel-purified, cloned into pMD19-T vector (TaKaRa, Dalian, China), and sequenced [42]. Amino acid sequence alignment among ShCOI1–4, ShCOI1–5, ShCOI1–6, and ZmCOIs (ZmCOI1a, GRMZM2G125411; ZmCOI1b, GRMZM2G151536; ZmCOI1c, GRMZM2G353209; ZmCOI2, GRMZM2G079112) was performed by NTI software [21, 31, 45].

RT-qPCR analysis

Seven primer pairs of non-allelic SsCOI1 genes were designed by Beacon Designer 8.0 (Supplemental Table S11). Due to the high amino acid sequences similarity (Supplemental Table S5), the primer pairs of SsCOI1–4, SsCOI1–1, SsCOI1–3 used in RT-qPCR analysis were the same as those of ShCOI1–4, ShCOI1–5, and ShCOI1–6, respectively (Supplemental Table S5 and Supplemental Table S11). The expression levels of the seven SsCOI1 genes under MeJA stress and those of ShCOI1–4, ShCOI1–5, and ShCOI1–6 in the six sugarcane cultivars (YZ03–258, LC05–136, YT96–86, GT02–467, FN40, and YZ03–103) infected by smut pathogen and under hormones and abiotic stresses (ABA, SA, cold, and PEG) were detected using RT-qPCR. The RT-qPCR was performed on Applied biosystems Q3 (ThermoFisher, Waltham, USA) system using the SYBR-green dye method with the conditions of 50 °C for 2 min; 95 °C for 10 min; 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The total volume of the RT-qPCR reaction system was 20 μL, which included 10 μL of the 2 × ChamQ Universal SYBR qPCR Master Mix, 0.4 μL of the primer (10 μM), 1.0 μL of the template (10 × cDNA diluted liquid), and 8.2 μL ddH2O. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Supplemental Table S11) was used as a reference gene [83]. Each sample was assessed using three replicates. Expression levels of COI1 genes were calculated using the 2−∆∆CT algorithm [84]. Significant differences (*p < 0.05) and standard error (SE) were determined by the Duncan’s new multiple range test by using Data Processing System v9.50 software, and the histogram was graphed by Origin 9.0.