Genome-wide identification of ABCC gene family and their expression analysis in pigment deposition of fiber in brown cotton (Gossypium hirsutum)

Abstract

ABC (ATP-binding cassette) transporters are a class of superfamily transmembrane proteins that are commonly observed in natural organisms. The ABCC (ATP-binding cassette C subfamily) protein belongs to a subfamily of the ABC protein family and is a multidrug resistance-associated transporter that localizes to the tonoplast and plays a significant role in pathogenic microbial responses, heavy metal regulation, secondary metabolite transport, and plant growth. Recent studies have shown that the ABCC protein is also involved in the transport of anthocyanins/proanthocyanidins (PAs). To clarify the types and numbers of ABCC genes involved in PA transport in Gossypium hirsutum, the phylogenetic evolution, physical location, and structure of ABCC genes were classified by bioinformatic methods in the upland cotton genome, and the expression levels of these genes were analyzed at different developmental stages of the cotton fiber. The results showed that 42 ABCC genes were initially identified in the whole genome of upland cotton; they were designated GhABCC1-42. The gene structure and phylogenetic analysis showed that the closely related ABCC genes were structurally identical. The analysis of chromosomal localization demonstrated that there were no ABCC genes on the chromosomes of AD/At2, AD/At5, AD/At6, AD/At10, AD/At12, AD/At13, AD/Dt2, AD/Dt6, AD/Dt10, and AD/Dt13. Outside the genes, there were ABCC genes on other chromosomes, and gene clusters appeared on the two chromosomes AD/At11 and AD/Dt8. Phylogenetic tree analysis showed that some ABCC proteins in G. hirsutum were clustered with those of Arabidopsis thaliana, Vitis vinifera and Zea mays, which are known to function in anthocyanin/PA transport. The protein structure prediction indicated that the GhABCC protein structure is similar to the AtABCC protein in A. thaliana, and most of these proteins have a transmembrane domain. At the same time, a quantitative RT-PCR analysis of 42 ABCC genes at different developmental stages of brown cotton fiber showed that the relative expression levels of GhABCC24, GhABCC27, GhABCC28, GhABCC29 and GhABCC33 were consistent with the trend of PA accumulation, which may play a role in PA transport. These results provide a theoretical basis for further analysis of the function of the cotton ABCC genes and their role in the transport of PA.

Introduction

Cotton (Gossypium spp.) is an economically important crop that is cultivated worldwide. Cotton fiber is a necessity for daily life and is an important raw material for the textile industry. Colored cotton is an eco-friendly textile raw material that does not need to be dyed, bleached or otherwise treated to obtain a certain color in the process of making textiles. The common varieties of colored cotton are brown cotton and green cotton, and brown cotton is the primary cultivated variety [1]. However, colored cotton has a number of disadvantages, such as low yield, short fiber length, low color and genetic instability of the pigment [2], which limits its application and marketing. Therefore, it is urgently important to elucidate the molecular mechanism governing colored cotton fiber pigmentation formation. At present, there are in-depth studies on the metabolic pathways of brown cotton fiber pigments. The key functional genes and regulatory factors in the proanthocyanidin (PA) biosynthesis pathway have been well studied [3], but the mechanism underlying transport and oxidative polymerization has not been elucidated to date.

Studies have found that PAs in plant cells are stored in the large central vacuole; therefore, the polymerization of PAs may occur in the vacuole [4]. Some researchers have shown that some key enzymes in the PA metabolism pathway, such as ANR, ANS, and DFR, are located in the cytoplasm [5], and hypothesized that these three enzymes may perform anthocyanin and flavan-3-ol biosynthesis in a certain area of the cytoplasm [6, 7]. Studies have shown that in Arabidopsis thaliana seed coat cells, epicatechin and cyanidin are initially synthesized in the cytoplasm, are transported to the vacuole by transporters, and finally aggregate to form multimers in the vacuole [8]. Previous studies have shown that during the transport of epicatechin and catechin to the vacuole, four types of proteins are involved in the transport process: ABCC protein, MATE protein (TT12), GST protein (TT19) and P-ATPase protein (TT13) [9]. A study of A. thaliana seed coat PAs found that TT12 is involved in the transport of epicatechin glycosides and anthocyanin glycosides, while TT19 is involved in the transport of anthocyanin glycosides and epicatechin, and the proton pump encoded by TT13 provides a concentration gradient transport epicatechin and catechin [10–12]. Studies have shown that the ABCC protein may be involved in the transport of flavonoids in some plants and can cotransport PA precursor substances with the GST protein [9].

ABC (ATP-binding cassette) is a family of ancient and large transmembrane proteins that are commonly observed in natural organisms. Most ABC transporters have activities in vivo and relying on the energy generated by ATP hydrolysis to achieve transmembrane transport of substrates inside and outside the cell, which include amino acids, liposomes, polysaccharides, peptides, heavy metal chelates, alkaloids and drugs [13, 14]. Multidrug resistance (MDR) is the first ABC transporter identified in eukaryotes and is involved in the process of excretion of intracellular drugs to prevent excessive accumulation of drugs in cells [15, 16]. Fifty-six and 53 ABC transporters have been identified in Drosophila and Bombyx mori, respectively, in which the Bmwh3 protein of B. mori and the white and brown ABC proteins of Drosophila have been shown to have pigment transport functions [17–21]. The corn bronze-2 (bz2) mutant lacks a glutathione S-transferase encoded by the bz2 gene, resulting in the inability of anthocyanins to accumulate in vacuoles. Because glutathione S-transferase has a very important effect on the activity of MRP transporter binding substrates, it is speculated that in the maize bz-2 mutant, the MRP type ABC transporter is likely to participate in the anthocyanin transport process [22]. Studies have found that sodium orthovanadate, an inhibitor of ABC transporter, can significantly reduce the secretion of flavonoids. It is speculated that ABC transporter may be related to the secretion of flavonoids from soybean roots [23].

Plant ABC transporters were first discovered during plant detoxification, and subsequently, a large number of ABC transporters were identified in plants. To date, the functions of ABC transporters have exceeded the scope of the detoxification mechanism. Some studies have confirmed that ABC transporters play important roles in plant pathogenic microbial responses, regulation of heavy metals, and transport of secondary metabolites [24]. With the sequencing and implementation of the plant genome, the ABC transporter has been fully identified and studied in plants. At present, the number of ABC transporters identified in plants is considerably higher than those in animals or microorganisms; for instance, there are 123 ABC transporters in Oryza sativa, 127 ABC transporters in A. thaliana, and 89 ABC transporters in leguminous plants [25–27]. A large number of ABC transporters in plants may be involved in complex metabolic activities.

The ABCC protein (ATP-binding cassette C subfamily) belongs to a subfamily of the ABC protein family and is a multidrug resistance-associated transporter. Most ABCC transporters have a transmembrane domain consisting of 3 to 5 transmembrane helices and are involved in many physiological processes, such as intracellular detoxification, transport of chlorophyll metabolites, and regulation of ion channels [28]. In addition, plant ABCC transporters play an important role in the process of storing glycosides and pigment metabolites in vacuoles. Previous studies have shown that ABCG10 regulates the expression level of isoflavones in sputum [29]; MRP3 is transformed into the leaves by constructing an interference vector and changes the color of the leaves in maize [30]; and VvABCC1 is involved in anthocyanins in grape skins [31].

Cotton is the most important fiber crop worldwide and exhibits a wide range of varieties, among which upland cotton (Gossypium hirsutum) is the most widely cultivated. With the completion of whole-genome sequencing of upland cotton [32], the genome-wide database can be used for systematic screening, identification and comparative genomics research and provides a rich resource for research on the biological functions of ABCC gene family members. At present, the ABCC genes of A. thaliana, Vitis vinifera, Zea mays and other species have been identified, and there are many studies on the function of the model plant ABCC gene family. In this study, the ABCC gene family of upland cotton was identified by bioinformatics. The number, sequence characteristics and evolutionary relationship of ABCC genes in upland cotton are analyzed at the genetic level. The function of these genes is predicted by qRT-PCR and homology comparison, and the role of the genes in the transport and accumulation of pigment in brown cotton fiber is further discussed to provide a theoretical basis for breeding pigment-stable varieties of brown cotton.

Results

Identification of the ABCC gene family in upland cotton

Using bioinformatic methods, ABCC family members were screened from upland cotton. At the same time, the basic information of all ABCC genes was searched using the EsPAsy online website, and the physical and chemical properties of protein length, molecular weight and isoelectric point were obtained and analyzed. The results showed that 42 ABCC genes were obtained in upland cotton (Table 1). The ABCC protein sequences appeared significant differences, and their amino acid lengths ranged from 435 aa (GhABCC39) to 1624 aa (GhABCC24). The molecular weights ranged from 48.52 kD (GhABCC39) to 182.95 kD (GhABCC24). The isoelectric points ranged from 5.4 (GhABCC39) to 8.96 (GhABCC40). It can be observed from the basic characteristics of these ABCC proteins that the gene family varies considerably regarding gene lengths and protein properties, indicating that the members of the gene family have different characteristics and potentially play different biological roles.

Table 1. Characteristics of GhABCC genes identified in G. hirsutum.

Gene name	Gene ID	Chromosomal localization	Cds Position	Numbers of Amino acid	Molecular weight (Da)	pI	Numbers of exon
GhABCC1	CotAD_02682	Dt_chr9	41311889–41317949	1428	159320.98	6.92	11
GhABCC2	CotAD_75551	sca	1711–6407	1231	137429.86	6.38	11
GhABCC3	CotAD_27409	Dt_chr3	39527379–39532708	1465	162797.03	6.43	11
GhABCC4	CotAD_21899	Dt_chr11	59886597–59892332	1465	163846.02	7.67	12
GhABCC5	CotAD_72273	At_chr11	62166046–62171020	1298	144934.19	7.44	11
GhABCC6	CotAD_21900	Dt_chr11	59965930–59971598	1431	160252.41	7.90	11
GhABCC7	CotAD_48319	Dt_chr1	3925636–3935180	1540	170682.03	7.47	11
GhABCC8	CotAD_62215	At_chr1	6160978–6165643	1250	138003.72	5.94	10
GhABCC9	CotAD_15441	sca	576384–581146	1286	142314.02	6.20	10
GhABCC10	CotAD_15407	sca	144115–149898	1623	180455.27	6.44	10
GhABCC11	CotAD_22397	sca	870353–878207	1565	undefined		12
GhABCC12	CotAD_15406	sca	120149–125156	1399	155348.33	6.36	9
GhABCC13	CotAD_15404	sca	93416–98079	1212	134306.59	6.16	11
GhABCC14	CotAD_26386	At_chr7	11009721–11015682	1504	169401.33	8.12	11
GhABCC15	CotAD_16330	Dt_chr8	38634127–38639663	1540	173246.96	7.32	10
GhABCC16	CotAD_22657	Dt_chr4	22246611–22251942	1508	169327.55	6.34	9
GhABCC17	CotAD_71461	Dt_chr7	9958770–9964794	1524	171597.89	8.11	11
GhABCC18	CotAD_21687	Dt_chr5	25328345–25333684	1487	167184.25	8.41	10
GhABCC19	CotAD_39801	sca	131301–136337	1415	158915.88	8.00	9
GhABCC20	CotAD_57277	At_chr3	15120096–15122856	827	92300.62	7.47	3
GhABCC21	CotAD_13839	Dt_chr12	3046390–3054079	1435	161389.18	6.81	13
GhABCC22	CotAD_57278	At_chr3	15127049–15129617	655	72410.90	5.82	8
GhABCC23	CotAD_72276	At_chr11	62142290–62145829	858	94684.55	6.07	12
GhABCC24	CotAD_14237	Dt_chr8	3735646–3752579	1624	182946.63	7.51	27
GhABCC25	CotAD_46251	sca	447046–453542	907	101297.70	5.69	9
GhABCC26	CotAD_04577	At_chr4	36364268–36368203	1012	113332.26	6.63	9
GhABCC27	CotAD_23475	At_chr8	40396551–40404026	1160	129215.35	6.07	15
GhABCC28	CotAD_14239	Dt_chr8	3694851–3704893	1127	125785.73	8.55	16
GhABCC29	CotAD_14243	Dt_chr8	3607141–3614778	1086	122059.63	7.24	14
GhABCC30	CotAD_21686	Dt_chr5	25335414–25338543	787	88197.96	6.21	9
GhABCC31	CotAD_23481	At_chr8	40244764–40271890	1313	147940.48	8.40	21
GhABCC32	CotAD_14236	Dt_chr8	3763762–3771149	1086	121250.91	5.69	14
GhABCC33	CotAD_57900	At_chr8	36145902–36153374	1031	115657.03	6.07	13
GhABCC34	CotAD_62212	At_chr1	6136757–6138947	658	72785.47	7.55	3
GhABCC35	CotAD_44535	At_chr9	45977082–45988622	999	110655.69	5.60	23
GhABCC36	CotAD_72272	At_chr11	62224591–62226533	451	50419.82	8.64	7
GhABCC37	CotAD_01493	Dt_chr9	60823161–60843906	1072	119006.68	6.04	26
GhABCC38	CotAD_43419	sca	4322–8619	606	67994.50	6.20	11
GhABCC39	CotAD_74385	At_chr1	61527124–61529014	435	48521.00	5.40	7
GhABCC40	CotAD_49953	At_chr11	61728158–61729963	533	59826.99	8.96	3
GhABCC41	CotAD_23476	At_chr8	40366247–40379559	1078	121925.83	8.71	25
GhABCC42	CotAD_62211	At_chr1	6115177–6125803	676	74854.98	5.57	5

Phylogenetic analysis of the ABCC gene family

To better clarify the genetic relationship between monocotyledonous and dicotyledonous species, a phylogenetic tree was constructed according to the ABCC sequences from Z. mays (19), G. hirsutum (42), A. thaliana (94) and V. vinifera (23) (Fig 1). According to the classification method described by Sun et al. [33], the GhABCC gene family was divided into four subfamilies: I, II, III, and IV. The results indicated that more ABCC genes in cotton and A. thaliana are clustered together, and the evolutionary relationship between them is closely related. The AtABCC1 and AtABCC2 genes in A. thaliana have been demonstrated to be involved in the transport of PAs [34, 35]; therefore, we hypothesize that members of subfamily III in G. hirsutum may be involved in the transport of anthocyanins in vacuoles.

Fig 1. Phylogenetic tree of ABCC proteins from G. hirsutum, A. thaliana, V. vinifera and Z. mays.

The tree was generated with MEGA 7.0 software (1000 bootstrap replicates) using the neighbor-joining method, Different colors indicate different subfamilies of ABCC.

Characteristics of GhABCC gene structures

In this study, to better understand the evolutionary relationship of GhABCC genes, a phylogenetic tree was constructed with the ABCC proteins from G. hirsutum (Fig 2). As shown in Fig 2, there are 15 homologous pairs in the GhABCC gene, of which 11 pairs of bootstrap values are 100, indicating that the 11 pairs of GhABCC genes are closely related. The gene sequences of members of the same subfamily are very similar, indicating the possibility of their similarity of functions, reflecting the conservation of the GhABCC gene during evolution.

Fig 2. Phyletic evolution and gene structure of the ABCC gene family in G. hirsutum.

The exon-intron structure map of the upland cotton ABCC family was obtained using the GSDS online tool. Cotton ABCC family phylogenetic tree (left) and gene structure (right). Yellow boxes, blue boxes and black lines represent exons, 5’ or 3’ untranslated region (UTR) and introns, respectively.

Distribution of conserved motifs of GhABCCs

Twenty conserved sequences of ABCC protein in upland cotton were identified by the online software MEME. Also, it can be observed that all GhABCCs contain motifs 1, 2, 3 and 7 (Fig 3), and the conserved regions of different subfamilies are different, indicating that these motifs may have some specific functions (Fig 4). The similarity and difference of gene structure and conserved motifs reflect the relative conservation of the GhABCC gene family in the lengthy evolutionary process and the diversity generated for adapting to the environment.

Fig 3. Conserved motif compositions of ABCC genes from upland cotton.

Twenty putative conserved motifs were elucidated using MEME with complete protein sequences. All motifs have been labeled by different colors.

Fig 4. Distribution of conserved motifs of ABCC proteins in G. hirsutum.

The x-axis indicates the conserved sequences of the domain. The height of each letter indicates the conservation of each residue across all proteins. The y-axis is a scale of the relative entropy, which reflects the conservation rate of each amino acid.

Chromosome localization of GhABCC genes

To further study the effect of gene evolution on the GhABCC gene family, chromosome localization of ABCC genes was analyzed using Mapinspect software in upland cotton. The results showed that ABCC genes were primarily distributed on At1, Dt1, At3, Dt3, At4, Dt4, Dt5, At7, Dt7, At8, Dt8, At9, Dt9, At11, Dt11 and Dt12 chromosomes in upland cotton, and most of them were in the middle and lower parts of chromosomes; only a small number were in the upper part (Fig 5). Forty-two ABCC genes were randomly distributed, among which 5 ABCC genes were distributed on the Dt8 chromosome and were the most abundant. It is generally believed that a 200-kb nucleotide group with more than three genes is considered a gene cluster. There is a gene cluster on chromosome DT8 in upland cotton, which may encode structural genes that catalyze different steps of the same metabolic pathway. Tandem gene replication is a process in which DNA molecules replicate one or more adjacent copies, which achieve the evolution of gene families through high-frequency gene production and death. According to the definition of gene tandem replication, GhABCC14, 16, 26; GhABCC14, 15, 17; GhABCC4, 5; GhABCC6, 40; GhABCC24, 41; GhABCC27, 32; and GhABCC29, 31 have gene tandem replication.

Fig 5. Chromosomal localization of ABCC genes in G. hirsutum.

The chromosome numbers are indicated at the top of each bar, while the size of a chromosome is indicated by its relative length. The unit on the left scale is Mb, and the short line indicates the approximate position of the GhABCC gene on the corresponding chromosome. Segmental duplication gene pairs are connected with color lines.

Protein secondary structure prediction and subcellular localization analysis of GhABCCs

Using the online analysis tool SOPMA to make predictions, it was determined that the secondary structure of ABCC protein is mainly alpha helix and random coils, and the proportion of extended strand and beta turn is relatively small (Table 2). Using Cell-PLoc 2.0 software, it was found that ABCC proteins were mainly located on the cell membrane, and only GhABCC24, 27, 28, 29, 31, 32, 33 were located on the vacuole membrane (Table 2). It can be seen from the evolutionary tree that most of these 7 genes are in subfamily III (Fig 1); therefore, it is hypothesized that genes in subfamily III may play important roles in the transport of anthocyanins/PAs.

Table 2. Protein secondary structure prediction and subcellular localization analysis.

Gene name	Alpha helix %	Extended strand %	Beta turn %	Random coil %	Subcellular localization
GhABCC1	53.43	17.10	5.99	23.48	Cell membrane, Cytoplasm
GhABCC2	52.23	15.84	5.85	26.08	Cell membrane, Cytoplasm
GhABCC3	50.03	15.70	5.32	28.94	Cell membrane, Cytoplasm
GhABCC4	53.92	14.61	4.85	26.62	Cell membrane
GhABCC5	52.62	15.33	5.32	26.73	Cell membrane
GhABCC6	53.67	15.30	5.03	26.00	Cell membrane
GhABCC7	50.65	14.61	5.45	29.29	Cell membrane
GhABCC8	50.64	16.88	6.40	26.08	Cell membrane, Cytoplasm
GhABCC9	50.08	15.94	6.38	27.60	Cell membrane, Cytoplasm
GhABCC10	49.60	17.62	7.15	25.63	Cell membrane, Cytoplasm
GhABCC11	50.10	14.95	6.01	28.95	Cell membrane
GhABCC12	50.32	15.80	6.22	27.66	Cell membrane, Cytoplasm
GhABCC13	50.00	16.01	6.60	27.39	Cell membrane, Cytoplasm
GhABCC14	50.73	15.69	5.39	28.19	Cell membrane
GhABCC15	49.94	16.36	5.00	28.70	Cell membrane
GhABCC16	50.99	15.25	5.31	28.45	Cell membrane
GhABCC17	51.38	15.35	5.25	28.02	Cell membrane
GhABCC18	53.67	15.33	5.51	25.49	Cell membrane
GhABCC19	50.39	15.41	5.02	29.19	Cell membrane, Cytoplasm
GhABCC20	54.41	11.37	3.63	30.59	Cell membrane
GhABCC21	52.33	15.61	5.64	26.41	Cell membrane, Cytoplasm
GhABCC22	51.60	16.49	6.11	25.80	Cell membrane
GhABCC23	43.94	19.58	6.76	29.72	Cell membrane
GhABCC24	57.20	13.36	4.00	25.43	Vacuole
GhABCC25	45.20	19.85	7.06	27.89	Cell membrane
GhABCC26	48.42	19.27	5.53	26.78	Cell membrane, Cytoplasm
GhABCC27	54.74	15.09	4.74	25.43	Vacuole
GhABCC28	52.71	16.33	5.68	25.29	Vacuole
GhABCC29	55.06	15.29	4.97	24.68	Vacuole
GhABCC30	49.30	19.06	6.35	25.29	Cell membrane
GhABCC31	51.94	15.84	4.87	27.34	Vacuole
GhABCC32	55.25	13.26	5.71	25.78	Vacuole
GhABCC33	53.83	16.68	5.43	24.05	Vacuole
GhABCC34	54.56	12.61	4.26	28.57	Cell membrane, Cytoplasm
GhABCC35	51.35	15.42	4.90	28.33	Cell membrane
GhABCC36	48.34	21.73	6.65	23.28	Cell membrane
GhABCC37	51.96	16.04	5.32	26.68	Cell membrane
GhABCC38	47.85	18.15	8.09	25.91	Cell membrane
GhABCC39	52.41	19.31	6.21	22.07	Cell membrane
GhABCC40	56.10	17.26	4.13	22.51	Cell membrane
GhABCC41	54.17	14.38	3.53	27.92	Cell membrane
GhABCC42	43.20	18.20	4.73	33.88	Cell membrane, Cytoplasm

Accumulation of PAs and expression analysis of ABCC genes in brown cotton

The different developmental stages of fiber in white cotton and brown cotton were observed. Before the boll stage, there is no difference in appearance between brown cotton and white cotton; during the period of boll opening, the color of brown cotton fiber gradually darkens, and white cotton is still white (Fig 6). It can be seen that environmental conditions may promote the accumulation of PAs in the brown cotton fiber and lead to the deposition of pigments, thereby darkening the color. Therefore, the contents of PAs at different development stages of brown cotton fiber were measured. The contents of PAs increased gradually with the development of fiber, reached the highest level at 12 DPA, and thereafter decreased gradually (Fig 7). The main reason for this phenomenon may be related to the expression level of related genes in the procyanidin biosynthesis pathway.

Fig 6. The phenotypes of white fiber and brown fiber at different stages of pigment deposition.

The photographs are taken from the budding stage to the end of the boll opening stage during different growth and development periods of cotton.

Fig 7. PA content at different development stages of brown cotton fibers.

Abscissa indicates different days post anthesis of cotton fifibers, and ordinate indicates PA content, the error bars indicate SE.

To analyze the expression levels of GhABCC genes related to PA accumulation during the development stages of brown cotton fiber, fluorescence quantitative primers were designed according to the GhABCC gene sequences (S1 Table), and quantitative RT-PCR was performed using RNA from fibers at 6 DPA, 12 DPA, 18 DPA, 24 DPA and 30 DPA. Since the content of PA in brown cotton fiber changes significantly during these five periods [36], these periods were choosed to determine the relative expression of ABCC genes. The results showed that the relative expression levels of most GhABCCs were inconsistent with the accumulation of PAs in brown cotton fibers, while the relative expression levels of GhABCC24, GhABCC27, GhABCC28, GhABCC29 and GhABCC33 were consistent with the trend of PA accumulation (Fig 8). These GhABCC genes belong to subfamily III, and it was also shown that the GhABCC genes related to PA transport are primarily located in subfamily III according to the analyses of subcellular localization. Therefore, it is hypothesized that one or several genes of GhABCC24, GhABCC27, GhABCC28, GhABCC29 and GhABCC33 may be involved in the accumulation of PAs. In addition, these genes were compared with A. thaliana ABCC1 through DNAMAN software. Among these genes, GhABCC27 and AtABCC1 had the highest homology, reaching 56.19%, and the evolutionary relationship between GhABCC27 and AtABCC1 was the closest in the composite phylogenetic tree (Fig 1). This result suggests that GhABCC27 may have the same function as AtABCC1, which is involved in the transport of PAs.

Fig 8. Expression patterns of ABCC genes in G. hirsutum.

Relative expression levels of the ABCC genes at different development stages of brown cotton fiber. The relative expression level was calculated using the 2^−ΔΔCt method. Different colors represent expression level; 0 and 12 indicates the expression level, different colors represent expression level; red indicates high expression, and blue indicates low expression.

Analysis of cis-acting elements of GhABCC27 promoter

The online promoter element prediction tool PlantCARE was used to analyze the cis-acting elements of GhABCC27. It was found that in addition to the basic core elements of the promoter such as CAAT-box and TATA-box, the promoter also includes a large number of elements involved in the light response (AE-box, ATCT-motif, Box 4, GT1-motif, LAMP-element, TCT-motif and chs-CMA1a), as well as plant abiotic stress inducing elements (ARE and MBS) and gibberellin response elements (GARE-motif and P-box). It has the same cis-acting elements as the GST transporter GhTT19, so it is speculated that GhABCC27 may have the same transport function as GhTT19. In addition, it also includes a cis-acting regulatory element related to endosperm expression (GCN4_motif) and a cold-responsive cis-acting element and other cis-acting elements (Table 3). These results indicate that the expression of GhABCC27 gene in brown cotton may be regulated by external environmental conditions such as light, plant hormones, and adversity stress.

Table 3. Functional prediction of cis acting elements of GhABCC27 promoter.

Element name	Sequence (5′–3′)	Copy number	Function
AE-box	AGAAACTT	1	part of a module for light response
ARE	AAACCA	1	cis-acting regulatory element essential for the anaerobic induction
ATCT-motif	AATCTAATCC	1	part of a conserved DNA module involved in light responsiveness
Box 4	ATTAAT	1	part of a conserved DNA module involved in light responsiveness
GARE-motif	TCTGTTG	1	gibberellin-responsive element
CAAT-box	CCAAT	5	common cis-acting element in promoter and enhancer regions
CGTCA-motif	CGTCA	2	cis-acting regulatory element involved in the MeJA-responsiveness
GCN4_motif	TGAGTCA	1	cis-regulatory element involved in endosperm expression
GT1-motif	GGTTAA	1	light responsive element
LAMP-element	CTTTATCA	2	part of a light responsive element
LTR	CCGAAA	1	cis-acting element involved in low-temperature responsiveness
MBS	CAACTG	1	MYB binding site involved in drought-inducibility
P-box	CCTTTTG	1	gibberellin-responsive element
TATA-box	TATA	7	core promoter element around -30 of transcription start
TCT-motif	TCTTAC	1	part of a light responsive element
TGACG-motif	TGACG	2	cis-acting regulatory element involved in the MeJA-responsiveness
chs-CMA1a	TTACTTAA	2	part of a light responsive element

Discussion

In recent years, there have been many reports on the structure and function of each member of the ABC gene family, but few reports on cotton have been published. Plant ABCC transporter plays an important role in the process of vacuolar storage of glycosides and pigment metabolites, and is generally named MRP in related research reports [37]. Studies have shown that both ZmMrp3 and ZmMrp4 of corn are involved in the accumulation of pigments in vacuoles [30]. Among the 129 ABC genes, 15 ABC genes encoding MRP proteins have been identified [38], but only the structure and function of the MRP gene family has been studied in A. thaliana. The initial discovery of the plant MRP gene was due to the observation that the entry of glutathione compounds into the vacuole was dependent on ATP for energy, rather than the proton potential difference inside and outside the membrane [39]. Both AtMRP1 and AtMRP2 have glutathione-conjugated transport activity, and AtMRP2 is more active than AtMRP1 in Arabidopsis [40]. There is evidence that AtABCC1 functions as an anthocyanin transporter that depends on GSH without the formation of an anthocyanin-GSH conjugate [41].

In this study, the ABCC gene families of Arabidopsis, grape, maize and upland cotton were identified and analyzed at the genomic level. 42 ABCC genes were identified in upland cotton. The high number of family members determines the diversity and specificity of ABCC gene family functions. According to the homology and domains of the conserved sequences of ABCC transporters, GhABCCs are divided into 4 subfamilies, and the members of each subfamily are named systematically. The increase in the number of genes among species is considered to be the way to promote the evolution of species. The main way to increase the number of gene families is gene replication. Gene replication can be divided into intragene replication and intergene replication. The upland cotton genome undergoes several gene duplication events in the process of replication, and the copies of these genes are usually free from selection pressure [42]. This phenomenon not only guarantees the evolution of upland cotton but also enriches the diversity of the upland cotton gene family. Previous studies have shown that epicatechin is synthesized in the cytoplasm, transported by transporters to the vacuole, aggregates and accumulates in the vacuole. Previous studies have found that the AtABCC1 and AtABCC2 genes play an important role in the accumulation of PAs in Arabidopsis seed coats [34, 35]. VvABCC17 has also been shown to be located in the vacuolar membrane and participate in the transport of glycosylated anthocyanins. From the evolutionary tree, we can clearly observe that AtABCC1, AtABCC2 and VvABCC17 [41] and G. hirsutum are clustered in subfamily III. The subcellular location predicts that the genes of subfamily III are primarily located on the vacuole membrane (Table 2). Therefore, we hypothesize that members of subfamily III may also play an important role in the synthesis of brown cotton fiber PAs. Through fluorescence quantitative RT-PCR analysis, it was found that the relative expression levels of GhABCC24, GhABCC27, GhABCC28, GhABCC29 and GhABCC33 were consistent with the trend of PA accumulation in brown cotton fibers (Figs 7 and 8). Therefore, we hypothesize that these genes may be related to the transport of PAs. Sequence alignment analysis of these genes with Arabidopsis ABCC1 through DNAMAN software shows that GhABCC27 and AtABCC1 have the highest homology, and the evolutionary relationship was the closest in the phylogenetic tree (Fig 1). This result suggests that GhABCC27 may have the same function as AtABCC1, which is involved in the transport of PAs.

The online promoter element prediction tool PlantCARE was used to analyze the cis-acting elements of GhABCC27. The results showed that in addition to the basic core elements of the promoter such as CAAT-box and TATA-box, the promoter also includes a large number of elements involved in the light response (AE-box, ATCT-motif, Box 4, GT1-motif, LAMP-element, TCT-motif and chs-CMA1a), as well as plant abiotic stress inducing elements (ARE and MBS) and gibberellin response elements (GARE-motif and P-box) (Table 3). These results indicate that the expression of GhABCC27 gene in brown cotton may be regulated by external environmental conditions such as light, plant hormones, and adversity stress. In addition, because GhABCC27 and GST transporter GhTT19 have the same cis-acting elements, GhABCC27 may have the same function of transporting PAs as GhTT19.

Materials and methods

Experimental material and genome databases

Zongcaixuan 1, which has natural brown fiber, is a kind of upland cotton line bred by our laboratory. This line is planted in the high-tech agricultural park of Anhui Agricultural University (Hefei, PR China) in accordance with normal field management. The genome-wide database for upland cotton is available from the website (http://mascotton.njau.edu.cn) [32]. The A. thaliana genome data are from the database (http://www.arabidopsis.org/).

Identification of ABCC gene in upland cotton genome

A local database of the whole genome sequence of G. hirsutum, V. vinifera, A. thaliana and Z. mays was established using DNATOOLS software. Using the amino acid sequence of A. thaliana AtABCC1 (PF00005.27) as a query, TblastN (E-value = 0.001) sequence alignment was performed on the established local database of amino acid sequences of four species [42, 43], and the ABCC family genes were initially screened. The results were tested in the Pfam [44] database (http://pfam.xfam.org/) and CDD [45] (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to screen the ABCC sequence of the gene signature domain (ABC_membrane, ABC_tran). Use ExPAsy [46] (http://www.expasy.org/) to analyze the amino acid sequence online to determine the isoelectric point (PI) of amino acid, the molecular weight (MW) of the protein, and the instability coefficient.

Construction of the phylogenetic tree of the GhABCC gene family

The ClustalW tool involved in the MEGA7.0 [47] software was utilized to perform multiple sequence alignment according to the amino acid sequence of the upland cotton ABCC gene, and the phylogenetic tree was subsequently constructed by the neighboring method (NJ, Neighbor-Joining). Branch support values indicate nonparametric bootstrap values (in percentages of 1000 replicates). Meanwhile, a total of 178 ABCC protein sequences from A. thaliana, V. vinifera and Z. mays were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/). A comprehensive analysis was performed, and a composite phylogenetic tree of the ABCC proteins was drawn by the above method.

Analyses of chromosomal localization, gene structure and conserved motifs

The position information of each ABCC gene on the chromosome was obtained from the cotton genome database, and the physical position of these genes on the chromosome was mapped using MapInspect (http://mapinspect.software.informer.com) software. The exon-intron structure map of the upland cotton ABCC family was obtained using the GSDS [48] online tool (http://gss.cbi.pku.edu.cn/). According to the obtained protein sequence, the MEME [49] online analysis tool (http://meme.sdsc.edu/) was employed to analyze the motif pattern of the upland cotton ABCC family protein.

Signal peptide detection and subcellular localization prediction

The secondary structures of the ABCC proteins were predicted using the online analysis tool SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html). The Cell-PLoc 2.0 online analysis platform can perform subcellular localization of proteins of eukaryotes, humans, plants, gram-positive bacteria, gram-negative bacteria and viruses. The subcellular localizations of these proteins were predicted by the Cell-PLoc 2.0 [50] algorithm (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/).

Gene expression analysis of GhABCC

The primers for fluorescent quantitative RT-PCR were designed based on the selected ABCC subfamily gene sequences in upland cotton (S1 Table). The RNA of the fibers of different developmental stages of brown cotton was extracted, and the RNA was reverse transcribed into cDNA and subjected to qRT-PCR analysis. The qRT-PCR volume was 20 μL, including 10 μL of SYBR premix Ex Taq enzyme, 2 μL of cDNA, and 0.8 μL of upstream and downstream primers. The reaction procedure was as follows: 50°C for 2 min; 40 cycles of 95°C for 30 s, 95°C for 5 s, and 60°C for 20 s followed by 72°C for 10 min. The UBQ7 gene was used as an internal reference [51]. Each sample was subjected to three biological replicates, and the relative expression levels were calculated using the 2 ^−ΔΔCt method [52].

Determination of PA content

According to methods from Ikegami [53], the soluble and insoluble PAs of brown cotton fifibers at different developmental stages (6 DPA, 12 DPA, 18 DPA, 24 DPA, and 30 DPA) were extracted, and the content of PAs was determined by spectrophotometry according to the standard curve of catechins, which were used as controls [54]. For each experiment, three biological replicates were executed.

Analysis of promoter cis-acting elements

The sequence of about 2000 bp before the ATG upstream of the GhABCC27 gene is obtained from the upland cotton genome, which is the promoter sequence. Use PlantCARE [55] (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) software to conduct bioinformatics analysis of GhABCC27 promoter and predict its main cis-acting elements.

Conclusions

In this study, the ABCC gene family was analyzed by bioinformatics in different species, and 23, 19, 94 and 42 ABCC genes were identified in V. vinifera, Z. mays, A. thaliana and G. hirsutum, respectively. These genes were analyzed to determine their phylogenetic evolution, chromosome localization, gene structure, orthologous genes and gene expression patterns. ABCC genes could be divided into 4 major subfamilies in upland cotton; members of the same subfamily had the same or similar gene structure, and there was a large gap in the gene structure of members of the different subfamily. In the phylogenetic tree, through homology comparison with A. thaliana and prediction of subcellular location, it was preliminarily determined that the genes related to PA transport were located in subfamily III. By comparing the expression of ABCC subfamily genes and PA content at different developmental stages in brown cotton fiber, five candidate genes related to PA transport were screened out. Through bioinformatic analyses, the role of ABCC family genes in upland cotton in the process of pigment transport was initially explored, which may help to establish a theoretical foundation for further research studying the function of ABCC genes and analyzing the molecular mechanism of PA transport across membranes in brown cotton.

Supporting information

S1 Table. Sequences of primers used for RT-PCR in this study.

(DOCX)

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This research was supported by the National Natural Science Foundation of China (No.31672497) and Key Research and Development Plan of Anhui province (No. 202004e11020002). The funding bodies played no role in the design of the experiment and data collection, analysis, or preparation of the manuscript.