Comparative Analysis of Structural Characteristics and Expression of the CPS Gene in Williams Banana Dwarf Mutant and Its Wild-type Parent

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Jiaqi LinCollege of Agriculture, Guangxi University, Nanning 530005, China

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Weiyan XuanCollege of Agriculture, Guangxi University, Nanning 530005, China

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Yanpei LiCollege of Agriculture, Guangxi University, Nanning 530005, China

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Shixiang XiaoCollege of Agriculture, Guangxi University, Nanning 530005, China

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Dou FengCollege of Agriculture, Guangxi University, Nanning 530005, China

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Banana (Musa sp.) is one of the world’s most important crops, and a source of extreme economic importance in many countries around the world. However, the height of banana plant poses a significant challenge in both harvesting fruit and their tolerance to extreme weather. Gibberellin (GA) is one of the important endogenous hormones affecting plant height. Copalyl diphosphate synthase (CPS) is the first key enzyme in the GA biosynthesis pathway. In this paper, two full-length coding sequences of CPS genes were cloned from ‘William B6’ dwarf mutant banana and its wild-type parent (Musa AAA group), named CPS-A and CPS-G, respectively. The full-length complementary DNA (cDNA) sequences of CPS-G and CPS-A were both 2163 base pairs (bp), and encoded 720 amino acid residues. There were eight differences between the two speculative amino acid sequences in the alignment analysis. The molecular weights of CPS-G and CPS-A were 82,359.00 and 82,412.15 Da, respectively, and their isoelectric points were 6.17 and 6.03, respectively; there were no signal peptides and transmembrane structures. The banana CPS was mainly located in the cytoplasm by subcellular localization prediction. The results of reverse quantitative real-time polymerase chain reaction showed that CPS gene expression levels in the leaves and false stems of dwarf banana were lower than those of wild banana except for the developmental stage of the 10th leaf. Its expression level in the dwarf banana stem was significantly lower than that of the wild type at the 15th, 20th, and 25th-leaf age, respectively. The results showed that the dwarfism of the ‘Williams B6’ dwarf mutant might be related to the mutation of the CPS sequence and the difference of expression level. This study laid a foundation for further research on functional verification and the genetic regulation mechanism of the CPS gene.

Abstract

Banana (Musa sp.) is one of the world’s most important crops, and a source of extreme economic importance in many countries around the world. However, the height of banana plant poses a significant challenge in both harvesting fruit and their tolerance to extreme weather. Gibberellin (GA) is one of the important endogenous hormones affecting plant height. Copalyl diphosphate synthase (CPS) is the first key enzyme in the GA biosynthesis pathway. In this paper, two full-length coding sequences of CPS genes were cloned from ‘William B6’ dwarf mutant banana and its wild-type parent (Musa AAA group), named CPS-A and CPS-G, respectively. The full-length complementary DNA (cDNA) sequences of CPS-G and CPS-A were both 2163 base pairs (bp), and encoded 720 amino acid residues. There were eight differences between the two speculative amino acid sequences in the alignment analysis. The molecular weights of CPS-G and CPS-A were 82,359.00 and 82,412.15 Da, respectively, and their isoelectric points were 6.17 and 6.03, respectively; there were no signal peptides and transmembrane structures. The banana CPS was mainly located in the cytoplasm by subcellular localization prediction. The results of reverse quantitative real-time polymerase chain reaction showed that CPS gene expression levels in the leaves and false stems of dwarf banana were lower than those of wild banana except for the developmental stage of the 10th leaf. Its expression level in the dwarf banana stem was significantly lower than that of the wild type at the 15th, 20th, and 25th-leaf age, respectively. The results showed that the dwarfism of the ‘Williams B6’ dwarf mutant might be related to the mutation of the CPS sequence and the difference of expression level. This study laid a foundation for further research on functional verification and the genetic regulation mechanism of the CPS gene.

Banana (Musa sp.) is a tall, perennial monocotyledon plant. It is the second-most productive fruit in the world. Because its fruit nutritional value is large, it is deeply loved by people. Most of the main banana-producing areas are in the tropical and subtropical monsoon climate areas in the world (Perrier et al., 2011). At present, the banana cultivars popularized and planted in large areas are all cultivars with tall stems, large leaves, and large canopies and have high yields. However, due to the poor wind resistance ability of banana with tall stems, they are often damaged by typhoons, resulting in stem breaking and lodging, which seriously affects the growth and development of banana, resulting in greatly reduced production and heavy losses in the banana industry (Chen et al., 2016; Shu et al., 2016). Dwarf mutant plants often appear in the banana population, and dwarf banana lines have the advantages of shorter plant heights, smaller crown widths, convenient management, and tolerance of dense planting, but they are difficult to popularize and apply in production because of short fruit and irregular comb fingers. Cultivated banana is triploid and sterile, so it is difficult to improve and breed high-quality and high-yield cultivars with suitable plant heights through crossbreeding (Čížková et al., 2015). However, the plant height traits of banana can be improved by biotechnology and transgenic technology. First, the molecular mechanism and key regulatory genes of banana stem dwarfing must be understood to improve the plant height traits of banana cultivars by genetic engineering and molecular breeding. Therefore, it is of great significance to study the mechanism of the banana dwarfing mutation and explore its key regulatory genes.

A large number of studies have shown that plant height is regulated by multiple endogenous hormones. Most of the dwarfing variation is mainly related to the content changes of gibberellin (GA) and brassinolide (BR), and a few are related to auxin (IAA) (Wei et al., 2012). It has been reported that GA is the most important plant hormone for regulating plant height during morphogenesis. GA regulates plant height mainly through GA synthesis and signal transduction (Wei et al., 2012; Yamaguchi, 2008; Zentella et al., 2007). In higher plants, the biosynthesis pathway of GA is regulated by multiple enzymes (Dayan et al., 2012). These enzymes include copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurene acid oxidase (KAO), GA20 oxidase (GA20ox), GA3 oxidase (GA3ox), and GA2 oxidase (GA2ox), in which CPS, KS, KO, and KAO play a role in the early stage of GA biosynthesis (Schomburg et al., 2003; Yamaguchi, 2008). These enzymes only participate in one-step reactions and are encoded by a single gene. In Arabidopsis thaliana, the single genes encoding CPS, KS, and KO are GA1, GA2, and GA3, respectively. Mutations at these loci can lead to severe dwarfing and even sterility in plants. GA20ox, GA3ox, and GA2ox play a regulatory role in the late stage of GA synthesis. They are encoded by multigene families and participate in multistep reactions. Mutations in these loci can lead to semidwarf phenotype in plants (Sakamoto et al., 2004). CPS is the first key enzyme in the GA biosynthesis pathway, which is located in the plant precursor (Silverstone et al., 1997; Smith et al., 1998). In the first stage of GA synthesis, geranylgeranyl pyrophosphate (GGPP) will be transformed into ent-copalyl pyrophosphate (CPP), which will form a precursor of GA under the catalysis of KS. This is the key reaction that determines the synthesis of GGPP to GA (Xu et al., 2004). CPS plays an important role in the regulation of plant growth and development, and the activity of CPS is closely related to the expression level of the CPS gene. If the CPS gene is completely mutated, the synthesis pathway of GA will be blocked, and the plant will not be able to form any GA, which is manifested by the inability of seeds to germinate, dwarf plants, and male sterility (Koornneef and Veen, 1980; Sun and Kamiya, 1994). At present, the dwarfing phenotypes caused by CPS gene mutations include A. thaliana GA1 (Ogawa et al., 2003), Oryza sativa OSCPS1 (Sakamoto et al., 2004), Pisum sativum LS (Ait-Ali et al., 1997), and Zea mays AN1 (Bensen et al., 1995), among others. The nine Gal mutants found in A. thaliana all had dwarf phenotypes due to varying degrees of CPS gene mutation, among which gal-3 was extremely dwarfing because of the loss of the 5-kb base in CPS (Hedden and Phillips, 2000; Sun et al., 1992). In O. sativa, the OsCPS1 gene encodes the first enzyme of the GA biosynthesis pathway. Two alleles of OsCPS1, OSCPS1–1 and OSCPS1–2, both showed extreme dwarfism, and the plants could not blossom and bear fruit (Sakamoto et al., 2004). P. sativum ls-1 mutants also had a mutation at the CPS site. In Z. mays An1 mutants, due to the insertion of a mu transposon into the CPS gene, the plants were semidwarf, with internode shortening and other phenotypes, and the normal phenotype could be restored by exogenous endoroot kaurene treatment (Bensen et al., 1995). In ‘Williams’ banana 8818 (Musa AAA group), it was found that GA content in banana dwarf mutant 8818–1 was significantly lower than that in wild type. Further studies showed that there were differences in the expression levels of MaGA20ox4, MaGA20ox5, and MaGA20ox7 of the MaGA20ox gene family as well as MaGA2ox7, MaGA2ox12, and MaGA2ox14 of the MaGA2ox gene family in dwarf mutants and their parents (Chen et al., 2016). However, the molecular mechanism of the banana dwarfing mutation has not been deeply understood and revealed. It is not clear whether the dwarfing mutation of banana is related to the CPS gene. In this study, the ‘Williams B6’ dwarf mutant and its wild-type parents were used as research materials, the full-length coding sequence of CPS was cloned by reverse transcription-polymerase chain reaction (RT-PCR) techniques, and the expression patterns of the CPS gene at different stages and in different organs of ‘Williams B6’ dwarf mutant banana were analyzed and studied. This will lay a foundation for exploring whether the CPS gene is the key gene affecting ‘Williams B6’ dwarf mutant banana.

Materials and Methods

Test materials.

In this study, the banana cultivars were William B6 and its dwarf mutant. We obtained a natural mutant bud from a plant of banana cultivar Williams B6 (Musa AAA group) planted in the field. The plant height of mutant bud offspring was only ≈1.7 m high, ≈0.8 to 1 m shorter than the wild-type parental plant. Meanwhile, its leaf length and leaf width were shorter than that of the wild-type parent, and the fruit finger was shorter.

Test methods.

From Feb. 2020 to Jan. 2021, the experiment was carried out in the Key Laboratory of Cultivation and Breeding of Agricultural and Forestry Crops at the Agricultural College of Guangxi University, Nanning, Guangxi Zhuang Autonomous Region, China. In the early and midterm growth stages of the ‘Williams B6’ dwarf mutant and its wild-type parent, samples were taken of leaves and false stems at the developmental stage of the 10th, 15th, 20th, and 25th leaf, respectively. Fruit finger samples of the first, second, third, fifth, seventh, and eighth fruit combs were collected during the flowering stage, frozen in liquid nitrogen, and stored at −80 °C.

Cloning of the CPS gene.

The total RNA were extracted from the tender leaves of the dwarf mutant and its wild-type parent according to the instructions of the RNA extraction kit (Tiangen Biotech Co., Beijing, China), and the integrity of RNA was determined by gel electrophoresis. The concentration and purity of RNA were detected by a ultraviolet visible photometer (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA). The RNA with an optical density value at 260 nm (OD260) and optical density value at 280 nm (OD280) ratio of ≈2:1 was selected and transformed into cDNA. The cDNA of the dwarf mutant and its parent leaves were used as a template. The specific primers P1 5'-ATGGTGGGTGAAGTCAGAGCG-3' and P2 5'-CTAAGCCACAGGCTTGAAG-3' were designed according to the ORF of the CPS encoding gene of Musa acuminata spp. malaccensis published in the National Center for Biotechnology Information (NCBI) (accession no. XM_018830081.1). The CPS gene of banana was amplified by PCR using a high-fidelity Taq enzyme (Sangon Biotech Co., Shanghai, China). The PCR product was detected by 1% agarose gel electrophoresis. The target fragment was recovered and purified using the SanPrep column DNA gel recovery kit (Sangon Biotech Co.) connected to the PUCI-Blunt Zero vector (Sangon Biotech Co.) and transferred to Escherichia coli DH5 α competent cells. Then the transformed cells were cultured and screened by colony PCR. The positive clone colonies were sent to Shanghai Biological Engineering Co. (Shanghai, China) for sequencing.

Bioinformatics analysis of CPS gene.

The physicochemical properties, phosphoric acid sites, secondary structures, subcellular localization, signal peptides, and functional domains of CPS proteins were analyzed through a series of online software. NCBI (Bethesda, MD) BlastP was used to search the homologous sequence of CPS amino acid sequence in dwarf banana. DNAMAN software (version 5.0; Lynnon Biosoft, San Ramon, CA) was used to analyze the homologous amino acid sequence, and MEGA X6.0 software (Kumar et al., 2018) was used to construct the phylogenetic tree by the neighbor-joining method.

Expression of CPS gene in different tissues of dwarf banana and its wild-type.

Based on the CPS full-length cDNA sequences of dwarf and wild-type bananas, a specific fragment of 144 bp was selected as a template. The specific primers PCPSQ1 5'-TAACGGTGACTCGTCCTAA-3' and PCPSQ2 5'-CTACTGCCTTGTCCTTGC-3' for quantitative RT-PCR (qRT-PCR) were designed. CPS expression was analyzed using banana actin (GenBank accession no. AB022041) as the reference gene and using cDNA templates from the young leaves, false stems, and fruit of dwarf and wild-type bananas at different developmental stages. All qRT-PCR analysis results were repeated three times, and the relative expression levels of CPS genes were calculated by using the 2−ρρCT method (Livak and Schmittgen, 2001).

Statistical analysis.

The data results of qRT-PCR were analyzed by an analysis of variance with statistical software (IBM SPSS Statistics version 20.0; IBM Corp., Armonk, NY). Different letters indicate significant differences (P < 0.05).

Results and Analysis

Structural analysis on the full-length cDNA sequence of CPS gene in the dwarf mutant and its wild-type parent.

Two amplified bands of CPS sequences were obtained from the dwarf mutant and its wild-type parent (Fig. 1), and were named CPS-A and CPS-G, respectively. Sequencing results showed that the full-length ORF of the two CPS genes was 2163 bp, encoding 720 amino acids. The amino acid sequences alignment result of CPS-G and CPS-A is shown in Fig. 2. There were eight differences between the amino acid sequences of CPS-G and CPS-A at 278, 347, 369, 446, 504, 507, 642, and 652 (Fig. 2). The nucleotide sequence alignment result of CPS-G and CPS-A with MaCPS1 (XP-009414733.1), MaCPS2 (XP-009414734.1), and MaCPS3 (XP-0094174635.1) are shown in Fig. 3. According to the alignment results and the RT-PCR primer positions, the CPS-A and CPS-G most likely come from the same gene locus as MaCPS3.

Fig. 1.
Fig. 1.

Gel electrophoresis plot of polymerase chain reaction (PCR)-amplified product of the ‘William B6’ dwarf mutant and wild-type parental banana copalyl diphosphate synthase (CPS) gene: M = DL 5000 DNA marker; A = PCR amplification product with complementary DNA (cDNA) of ‘William B6’ dwarf mutant plants as template; B = PCR amplification product with cDNA of wild-type parental banana as template.

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05185-22

Fig. 2.
Fig. 2.

Amino acid sequence alignment analysis of the ‘William B6’ dwarf mutant and wild-type parental banana copalyl diphosphate synthase (CPS) protein: CPS-A = ‘William B6’ dwarf mutant banana CPS protein; CPS-G = wild-type parental banana CPS protein.

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05185-22

Fig. 3.
Fig. 3.

Alignment of complementary DNA (cDNA) sequences of the copalyl diphosphate synthase (CPS) gene of the ‘William B6’ dwarf mutant and wild-type parental banana and banana genomes: CPS-A = ‘William B6’ dwarf mutant CPS gene; CPS-G = wild-type parental CPS gene; MaCPS1, MaCPS2, and MaCPS3 = CPS genes from the banana genome of banana species hub (Musa acuminata). Red boxes indicate the primer sequence parts.

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05185-22

Analysis of the physicochemical properties of the proteins encoded by the CPS genes in dwarf mutants and wild-type banana.

The analysis results showed that the molecular formulas of CPS-G and CPS-A were C3672 H5699 N1029 O1061 S35 and C3676 H5694 N1024 O1060 S38, respectively, the molecular weights were 82,359.00 and 82,412.15 Da, and the theoretical isoelectric points were 6.17 and 6.03, respectively. The total numbers of negatively charged residues were 96 and 97, the total numbers of positively charged residues were 87 and 86, the fat index values were 86.71 and 86.03, and the average hydrophilicity (GRAVY) values were –0.244 and –0.231, respectively. The instability indices (II) were 151.5 and 151.84, respectively, indicating that it is an unstable protein. The phosphorylation sites analysis of CPS-G and CPS-A showed that there were three phosphorylation sites of serine, threonine, and tyrosine, including serine 42 and 40, threonine 7 and 15, as well as tyrosine 11. Function domain analysis of CPS-A and CPS-G proteins revealed that both have three domains, including the isoprenoid-biosyn-C1 superfamily, ent-copalyl diphosphate synthase, and terpene synthase domain (Fig. 4).

Fig. 4.
Fig. 4.

The function domain of the ‘William B6’ dwarf mutant and wild-type parental banana copalyl diphosphate synthase (CPS) protein. (A) Functional domains of the ‘William B6’ dwarf mutant CPS protein. (B) Functional domains of the wild-type parental banana CPS protein.

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05185-22

Prediction of CPS protein secondary structure in the dwarf mutant and its wild-type parent.

The secondary structure of the CPS protein was predicted by Sopma (Geourjon and Deleage, 1995). The results showed that the secondary structures of CPS-A and CPS-G proteins were composed of α-helix, extended strand, β-turn, and random coil. Among these, the α-helix accounted for 63.06%, the extended strand for 4.31% and 4.86%, the β-turn for 4.17% and 4.31%, and the random coil for 28.47% and 27.78%, respectively.

Phylogenetic tree analysis of CPS protein in the dwarf mutant and its wild-type parent.

Sequence alignment analysis of CPS-A and CPS-G proteins with CPS proteins from other plants showed that CPS-A protein sequences were 66.39%, 66.94%, and 64.59%, similar to those of Phoenix dactylifera (XP_026657336.1), Elaeis guineensis (XP_010918399.1), and Cocos nucifera (AIC82453.1), respectively. Phylogenetic tree was constructed by combining CSP-A and CPS-G with banana CPS protein family and other plant CPS proteins. It was found that CPS-A and CPS-G were most closely related to M. acuminata MaCPS3, and clustered into the same branch with Ananas comosus AcCPS, E. guineensis EgCPS, C. nucifera CnCPS, and P. dactylifera PdCPS (Fig. 5).

Fig. 5.
Fig. 5.

Phylogenetic tree analysis of dwarf mutant and wild-type parental banana copalyl diphosphate synthase (CPS) and their homologous proteins. Neighbor-joining method of MEGA 6.0 (Kumar et al., 2018) was used to construct the phylogenetic tree with a bootstrap value of 1000 [Musa acuminata (MaCPS1, MaCPS2, MaCPS3), Nelumbo nucifera (NnCPS), Elaeis guineensis (EgCPS), Phoenix dactylifera (PdCPS), Cocos nucifera (CnCPS), Ananas comosus (AcCPS), Asparagus officinalis (AoCPS), Gossypium arboreum (GaCPS), Hevea brasiliensis (HbCPS), Populus trichocarpa (PtCPS), Populus alba (PaCPS), Vitis riparia (VrCPS)].

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05185-22

Comparative analysis of CPS gene expression in the dwarf mutant and its wild-type parent.

The test results showed that CPS was expressed in different tissues and varied in leaves, stems, and fruits of dwarf and wild-type parents (Fig. 6). There was no significant difference in the CPS gene expression levels in the leaves between the dwarf and its wild-type parents, except for 25th leaf, at the same time expression level of the CPS gene in the false stem between the dwarf mutants and its wild-type parent were significantly higher than that in the leaves. In the dwarf mutant false stem, the expression level of the CPS gene reached the highest at the 10-leaf age, whereas in the wild-type banana false stem, the expression level of the CPS gene increased with the increase of leaf age, and the expression level of CPS reached the highest at the 25-leaf age. At the same time, except at the 10-leaf age, the expression level of the CPS gene in dwarf mutant false stems was significantly lower than that in wild-type plants. Among them, at 20-leaf age, the expression level of the CPS gene in dwarf mutant false stems was 1.91 times lower than that in wild-type plants. At 25-leaf age, the expression level of CPS gene in dwarf mutant false stem was 2.13 times lower than that in wild-type plants.

Fig. 6.
Fig. 6.

Expression levels of the copalyl diphosphate synthase (CPS) gene in leaves and pseudostems during different periods of the ‘William B6’ dwarf mutant and their wild-type parents. Data are means of three replicates. Different lowercase letters represent a significant difference at the level of P < 0.05 using least significant difference statistical analysis. Mean labeled by the same letter are not significantly different.

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05185-22

In our study, the dwarf mutant bananas were found to have shorter fruits than those of wild-type bananas. To further explore the expression of the CPS gene in different fruit fingers during banana fruit development, the first, second, third, fifth, seventh, and eighth fruit fingers of dwarf and wild-type plants were collected for quantitative fluorescence detection. The results, as shown in Fig. 7, were that the CPS gene was expressed in different fruit fingers of dwarf and wild-type plants, and the expression level was different. The expression level of the CPS gene in the fruit fingers of dwarf plants was higher than that of wild-type bananas, and the difference was significant.

Fig. 7.
Fig. 7.

Expression levels of the copalyl diphosphate synthase (CPS) gene in the first, second, third, fifth, seventh, and eighth fruit fingers of the ‘William B6’ dwarf mutant and wild-type parents. Data are means of three replicates. Different lowercase letters represent a significant difference at the level of P < 0.05 using least significant difference statistical analysis. Means labeled by the same letter are not significantly different.

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05185-22

Discussion and Conclusions

The CPS gene is one of the early key enzymes in the GA synthesis pathway in plants. It is located at the forefront of the synthesis pathway and catalyzes the synthesis of CPP from GGPP. At present, the obtained research results showed that there are differences in the copy number of CPS in plant genomes, and there is one copy of gene locus in most genomes, whereas there are three copies of gene loci CPS1, CPS2, and CPS3 in banana genome A that has been sequenced. In this study, a CPS gene sequence of 2163 bp was cloned from ‘Williams B6’ banana and its dwarf mutant. From the results of sequencing and sequence alignment and the positions of upstream and downstream primers used in RT-PCR, the cloned CPS in this study is likely to be CPS3, rather than CPS1 and CPS2, in the genome of banana A that has been sequenced. Therefore, it is speculated that there is only one CPS3 locus, lacking the CPS1 and CPS2 locus, in both the experimental materials of this experiment.

The protein domains of dwarf mutant CPS-A and wild-type banana CPS-G were analyzed. It was found that they all contain three domains, including isoprenoid-biosyn-C1 superfamily, ent-copalyl diphosphate synthase, and terpene synthase domain, which has a similar domain to the CPS proteins of plants such as Triticum aestivum L. TaCPS1 protein (Guo et al., 2016) and A. thaliana AtCPS (Zhao et al., 2022). At the same time, through phylogenetic tree analysis, it was found that the dwarf banana and wild-type banana CPS had the closest relationship with the three CPS proteins in the banana A genome, indicating that the two CPS genes cloned in this experiment are indeed banana CPS genes.

Complete mutation of CPS can seriously affect the synthesis of GA in plants, thus affecting the growth and development of plants (Sun and Kamiya, 1994). CPS-related dwarf mutants have been found in a variety of plants, such as Medicago truncatula MNP1 (Guo et al., 2020), P. sativum LS (Ait-Ali et al., 1997), A. thaliana Gal-3, O. sativa S2–47 (Li et al., 2013), and Z. mays An1 (Zhang et al., 2020). These mutants are related to the regulation of CPS. In O. sativa extreme dwarf mutant S2–47, the loss of a single base in the coding region of OsCPS1 gene led to frameshift mutation, which reduced the synthesis of active GA and resulted in severe dwarfing and failure of flowering and fruiting (Li et al., 2013); P. sativum LS-1 mutant is caused by the mutation of a base guanine (G) to adenine (A) at the CPS site, resulting in impaired messenger RNA splicing, reduced plant endogenous GA1 content, and dwarfing traits (Ait-Ali et al., 1997). Because of a mutation at a site in the structural domain of AtCPS protein, the synthesis of GA in A. thaliana dwarf mutant Gal-168 was significantly reduced, and the plants showed shorter plant height, shorter roots and hypocotyls, and late flowering. Exogenous GA3 spraying could partially restore its phenotype (Zhao et al., 2022). In this study, it was found that the CPS gene had eight differences in amino acid sequence alignment between dwarf mutants and wild-type bananas, and all eight loci were in the isoprene synthase superfamily domain, among which three loci were in the subterpene synthase family domain. Therefore, it is speculated that the mutation of ‘Williams B6’ dwarf mutant may be caused by the mutation of multiple sites of CPS sequence, leading to the change of the function of its encoding product CPS, and such structural difference may lead to the partial blocking of GA synthesis pathway. Meanwhile, the expression of the CPS determines the activity of the CPS enzymes. Zhang (2020) showed that the CPS1 gene plays a major negative regulation role in GA anabolism. In dwarfed O. sativa lines with Dof34 gene transfer, the decreased expression of CPS1 gene led to a decrease in GA anabolism level, resulting in a semidwarfing phenotype in O. sativa. This study found that the expression levels of CPS in both dwarf mutant leaves and false stems were significantly lower than in wild type, indicating that CPS expression may also affect GA synthesis in plants. Therefore, it is speculated that the expression regulation of key enzyme genes of GA biosynthesis pathway leads to the emergence of a banana dwarfing phenotype.

The results of this study suggest that CPS gene mutation and expression level change play an important role in the regulation of banana stem dwarfing variation, which provides a certain reference for further research on the function of this gene. However, it is necessary to further analyze and verify the function of CPS to determine whether the mutation and reduced expression level of CPS are the main causes of banana stem dwarfing variation. For example, using gene editing technology for site-directed editing, making the CPS of the wild-type parent mutated at the site corresponding to its mutant, whether it can cause similar phenotypic mutations.

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  • Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. 2018 MEGA X: Molecular evolutionary genetics analysis across computing platforms Mol. Biol. Evol. 35 6 1547 1549 https://doi.org/10.1093/molbev/msy096

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  • Li, C., Hou, L., Yin, L., Zhao, J. & Li, X. 2013 GA responsiveness and gene mapping of rice extreme dwarf mutant s2-47 Zuo Wu Xue Bao 39 10 1766 1774 https://doi.org/10.3724/SP.J.1006.2013.01766

    • Search Google Scholar
    • Export Citation
  • Livak, K.J. & Schmittgen, T.D. 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method Methods 25 4 402 408 https://doi.org/10.1006/meth.2001.1262

    • Search Google Scholar
    • Export Citation
  • Ogawa, M., Hanada, A., Yamauchi, Y., Kuwahara, A., Kamiya, Y. & Yamaguchi, S. 2003 Gibberellin biosynthesis and response during Arabidopsis seed germination Plant Cell 15 7 1591 1604 https://doi.org/10.1105/tpc.011650

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  • Perrier, X., Langhe, D.E., Donohue, M., Lentfer, C., Vrydaghs, L., Bakry, F., Carreel, F., Hippolyte, I., Horry, J.P., Jenny, C., Lebot, V., Risterucci, A.M., Tomekpe, K., Doutrelepont, H., Ball, T., Manwaring, J., Maret, P. & Denham, T. 2011 Multidisciplinary perspectives on banana (Musa spp.) domestication Proc. Natl. Acad. Sci. USA 108 28 11311 11318 https://doi.org/10.1073/pnas.1102001108

    • Search Google Scholar
    • Export Citation
  • Sakamoto, T., Miura, K., Itoh, H., Tatsumi, T., Ueguchi-Tanaka, M., Ishiyama, K., Kobayashi, M., Agrawal, G.K., Takeda, S., Abe, K., Miyao, A., Hirochika, H., Kitano, H., Ashikari, M. & Matsuoka, M. 2004 An overview of gibberellin metabolism enzyme genes and their related mutants in rice Plant Physiol. 134 4 1642 1653 https://doi.org/10.1104/pp.103.033696

    • Search Google Scholar
    • Export Citation
  • Schomburg, F.M., Bizzell, C.M., Lee, D.J., Zeevaart, J.A. & Amasino, R.M. 2003 Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants Plant Cell 15 1 151 163 https://doi.org/10.1105/tpc.005975

    • Search Google Scholar
    • Export Citation
  • Shu, H., Sun, W., Wang, Z., Silver, M., Yin, M., Han, Q. & Zhou, Z. 2016 The possible analysis for breeding banana varieties with high resistance to typhoon. molecular plant breeding Mol. Plant Breed. 12 3511 3515 https://doi.org/10.13271/j.mpb.014.003511

    • Search Google Scholar
    • Export Citation
  • Silverstone, A.L., Chang, C., Krol, E. & Sun, T.P. 1997 Developmental regulation of the gibberellin biosynthetic gene GA1 in Arabidopsis thaliana Plant J. 12 1 9 19 https://doi.org/10.1046/j.1365-313X.1997.12010009.x

    • Search Google Scholar
    • Export Citation
  • Smith, M.W., Yamaguchi, S., Ait-Ali, T. & Kamiya, Y. 1998 The first step of gibberellin biosynthesis in pumpkin is catalyzed by at least two copalyl diphosphate synthases encoded by differentially regulated genes Plant Physiol. 118 4 1411 1419 https://doi.org/10.1104/pp.118.4.1411

    • Search Google Scholar
    • Export Citation
  • Sun, T., Goodman, H.M. & Ausubel, F.M. 1992 Cloning the Arabidopsis GA1 locus by genomic subtraction Plant Cell 4 2 119 128 https://doi.org/10.1105/tpc.4.2.119

    • Search Google Scholar
    • Export Citation
  • Sun, T.P. & Kamiya, Y. 1994 The Arabidopsis GA1 locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis Plant Cell 6 1509 1518 https://doi.org/10.1105/tpc.6.10.1509

    • Search Google Scholar
    • Export Citation
  • Wei, L., Cheng, J., Li, L. & Wu, J. 2012 Regulation of plant height by GAs biosynthesis and signal transduction Chin. J. Biotechnol. 28 2 144 153 https://doi.org/10.13345/j.cjb.2012.02.002

    • Search Google Scholar
    • Export Citation
  • Xu, M., Hillwig, M.L., Prisic, S., Coates, R.M. & Peters, R.J. 2004 Functional identification of rice syn-copalyl diphosphate synthase and its role in initiating biosynthesis of diterpenoid phytoalexin/allelopathic natural products Plant J. 39 3 309 318 https://doi.org/10.1111/j.1365-313X.2004.02137.x

    • Search Google Scholar
    • Export Citation
  • Yamaguchi, S. 2008 Gibberellin metabolism and its regulation Ann. Rev. Plant Biol. 59 225 251 https://doi.org/10.1146/annurev.arplant.59.032607.092804

    • Search Google Scholar
    • Export Citation
  • Zentella, R., Zhang, Z.L., Park, M., Thomas, S.G., Endo, A., Murase, K., Fleet, C.M., Jikumaru, Y., Nambara, E., Kamiya, Y. & Sun, T.P. 2007 Global analysis of DELLA direct targets in early gibberellin signaling in Arabidopsis Plant Cell 19 10 3037 3057 https://doi.org/10.1105/tpc.107.054999

    • Search Google Scholar
    • Export Citation
  • Zhang, J., Zhang, Y., Xing, J., Yu, H., Zhang, R., Chen, Y., Zhang, D., Yin, P., Tian, X., Wang, Q., Duan, L., Zhang, M., Peters, R.J. & Li, Z. 2020 Introducing selective agrochemical manipulation of gibberellin metabolism into a cereal crop Nat. Plants 6 67 72 https://doi.org/10.1038/s41477-019-0582-x

    • Search Google Scholar
    • Export Citation
  • Zhang, S.Q. 2020 Genetic transformation of rice and screening and identification of its dwarf line Hebei Univ. Sci. Technol. DOI: 10.27107/d.cnki.ghbku.2020.000113

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    • Export Citation
  • Zhao, S., Kong, Y., Xin, P., Chu, J., Wan, Y., Ling, H. & Liu, Y. 2022 AtCPS V326M significantly affect the biosynthesis of gibberellins (in Chinese, abstract in English) Hereditas. 44 3 245 252 https://doi.org/10.16288/j.yczz.21-405

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Contributor Notes

J.L., W.X., Y.L., S.X., and D.F. contributed equally to this work.

D.F. is the corresponding author. E-mail: fengdou@gxu.edu.cn.

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    Fig. 1.

    Gel electrophoresis plot of polymerase chain reaction (PCR)-amplified product of the ‘William B6’ dwarf mutant and wild-type parental banana copalyl diphosphate synthase (CPS) gene: M = DL 5000 DNA marker; A = PCR amplification product with complementary DNA (cDNA) of ‘William B6’ dwarf mutant plants as template; B = PCR amplification product with cDNA of wild-type parental banana as template.

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    Fig. 2.

    Amino acid sequence alignment analysis of the ‘William B6’ dwarf mutant and wild-type parental banana copalyl diphosphate synthase (CPS) protein: CPS-A = ‘William B6’ dwarf mutant banana CPS protein; CPS-G = wild-type parental banana CPS protein.

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    Fig. 3.

    Alignment of complementary DNA (cDNA) sequences of the copalyl diphosphate synthase (CPS) gene of the ‘William B6’ dwarf mutant and wild-type parental banana and banana genomes: CPS-A = ‘William B6’ dwarf mutant CPS gene; CPS-G = wild-type parental CPS gene; MaCPS1, MaCPS2, and MaCPS3 = CPS genes from the banana genome of banana species hub (Musa acuminata). Red boxes indicate the primer sequence parts.

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    Fig. 4.

    The function domain of the ‘William B6’ dwarf mutant and wild-type parental banana copalyl diphosphate synthase (CPS) protein. (A) Functional domains of the ‘William B6’ dwarf mutant CPS protein. (B) Functional domains of the wild-type parental banana CPS protein.

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    Fig. 5.

    Phylogenetic tree analysis of dwarf mutant and wild-type parental banana copalyl diphosphate synthase (CPS) and their homologous proteins. Neighbor-joining method of MEGA 6.0 (Kumar et al., 2018) was used to construct the phylogenetic tree with a bootstrap value of 1000 [Musa acuminata (MaCPS1, MaCPS2, MaCPS3), Nelumbo nucifera (NnCPS), Elaeis guineensis (EgCPS), Phoenix dactylifera (PdCPS), Cocos nucifera (CnCPS), Ananas comosus (AcCPS), Asparagus officinalis (AoCPS), Gossypium arboreum (GaCPS), Hevea brasiliensis (HbCPS), Populus trichocarpa (PtCPS), Populus alba (PaCPS), Vitis riparia (VrCPS)].

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    Fig. 6.

    Expression levels of the copalyl diphosphate synthase (CPS) gene in leaves and pseudostems during different periods of the ‘William B6’ dwarf mutant and their wild-type parents. Data are means of three replicates. Different lowercase letters represent a significant difference at the level of P < 0.05 using least significant difference statistical analysis. Mean labeled by the same letter are not significantly different.

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    Fig. 7.

    Expression levels of the copalyl diphosphate synthase (CPS) gene in the first, second, third, fifth, seventh, and eighth fruit fingers of the ‘William B6’ dwarf mutant and wild-type parents. Data are means of three replicates. Different lowercase letters represent a significant difference at the level of P < 0.05 using least significant difference statistical analysis. Means labeled by the same letter are not significantly different.

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    • Search Google Scholar
    • Export Citation
  • Li, C., Hou, L., Yin, L., Zhao, J. & Li, X. 2013 GA responsiveness and gene mapping of rice extreme dwarf mutant s2-47 Zuo Wu Xue Bao 39 10 1766 1774 https://doi.org/10.3724/SP.J.1006.2013.01766

    • Search Google Scholar
    • Export Citation
  • Livak, K.J. & Schmittgen, T.D. 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method Methods 25 4 402 408 https://doi.org/10.1006/meth.2001.1262

    • Search Google Scholar
    • Export Citation
  • Ogawa, M., Hanada, A., Yamauchi, Y., Kuwahara, A., Kamiya, Y. & Yamaguchi, S. 2003 Gibberellin biosynthesis and response during Arabidopsis seed germination Plant Cell 15 7 1591 1604 https://doi.org/10.1105/tpc.011650

    • Search Google Scholar
    • Export Citation
  • Perrier, X., Langhe, D.E., Donohue, M., Lentfer, C., Vrydaghs, L., Bakry, F., Carreel, F., Hippolyte, I., Horry, J.P., Jenny, C., Lebot, V., Risterucci, A.M., Tomekpe, K., Doutrelepont, H., Ball, T., Manwaring, J., Maret, P. & Denham, T. 2011 Multidisciplinary perspectives on banana (Musa spp.) domestication Proc. Natl. Acad. Sci. USA 108 28 11311 11318 https://doi.org/10.1073/pnas.1102001108

    • Search Google Scholar
    • Export Citation
  • Sakamoto, T., Miura, K., Itoh, H., Tatsumi, T., Ueguchi-Tanaka, M., Ishiyama, K., Kobayashi, M., Agrawal, G.K., Takeda, S., Abe, K., Miyao, A., Hirochika, H., Kitano, H., Ashikari, M. & Matsuoka, M. 2004 An overview of gibberellin metabolism enzyme genes and their related mutants in rice Plant Physiol. 134 4 1642 1653 https://doi.org/10.1104/pp.103.033696

    • Search Google Scholar
    • Export Citation
  • Schomburg, F.M., Bizzell, C.M., Lee, D.J., Zeevaart, J.A. & Amasino, R.M. 2003 Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants Plant Cell 15 1 151 163 https://doi.org/10.1105/tpc.005975

    • Search Google Scholar
    • Export Citation
  • Shu, H., Sun, W., Wang, Z., Silver, M., Yin, M., Han, Q. & Zhou, Z. 2016 The possible analysis for breeding banana varieties with high resistance to typhoon. molecular plant breeding Mol. Plant Breed. 12 3511 3515 https://doi.org/10.13271/j.mpb.014.003511

    • Search Google Scholar
    • Export Citation
  • Silverstone, A.L., Chang, C., Krol, E. & Sun, T.P. 1997 Developmental regulation of the gibberellin biosynthetic gene GA1 in Arabidopsis thaliana Plant J. 12 1 9 19 https://doi.org/10.1046/j.1365-313X.1997.12010009.x

    • Search Google Scholar
    • Export Citation
  • Smith, M.W., Yamaguchi, S., Ait-Ali, T. & Kamiya, Y. 1998 The first step of gibberellin biosynthesis in pumpkin is catalyzed by at least two copalyl diphosphate synthases encoded by differentially regulated genes Plant Physiol. 118 4 1411 1419 https://doi.org/10.1104/pp.118.4.1411

    • Search Google Scholar
    • Export Citation
  • Sun, T., Goodman, H.M. & Ausubel, F.M. 1992 Cloning the Arabidopsis GA1 locus by genomic subtraction Plant Cell 4 2 119 128 https://doi.org/10.1105/tpc.4.2.119

    • Search Google Scholar
    • Export Citation
  • Sun, T.P. & Kamiya, Y. 1994 The Arabidopsis GA1 locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis Plant Cell 6 1509 1518 https://doi.org/10.1105/tpc.6.10.1509

    • Search Google Scholar
    • Export Citation
  • Wei, L., Cheng, J., Li, L. & Wu, J. 2012 Regulation of plant height by GAs biosynthesis and signal transduction Chin. J. Biotechnol. 28 2 144 153 https://doi.org/10.13345/j.cjb.2012.02.002

    • Search Google Scholar
    • Export Citation
  • Xu, M., Hillwig, M.L., Prisic, S., Coates, R.M. & Peters, R.J. 2004 Functional identification of rice syn-copalyl diphosphate synthase and its role in initiating biosynthesis of diterpenoid phytoalexin/allelopathic natural products Plant J. 39 3 309 318 https://doi.org/10.1111/j.1365-313X.2004.02137.x

    • Search Google Scholar
    • Export Citation
  • Yamaguchi, S. 2008 Gibberellin metabolism and its regulation Ann. Rev. Plant Biol. 59 225 251 https://doi.org/10.1146/annurev.arplant.59.032607.092804

    • Search Google Scholar
    • Export Citation
  • Zentella, R., Zhang, Z.L., Park, M., Thomas, S.G., Endo, A., Murase, K., Fleet, C.M., Jikumaru, Y., Nambara, E., Kamiya, Y. & Sun, T.P. 2007 Global analysis of DELLA direct targets in early gibberellin signaling in Arabidopsis Plant Cell 19 10 3037 3057 https://doi.org/10.1105/tpc.107.054999

    • Search Google Scholar
    • Export Citation
  • Zhang, J., Zhang, Y., Xing, J., Yu, H., Zhang, R., Chen, Y., Zhang, D., Yin, P., Tian, X., Wang, Q., Duan, L., Zhang, M., Peters, R.J. & Li, Z. 2020 Introducing selective agrochemical manipulation of gibberellin metabolism into a cereal crop Nat. Plants 6 67 72 https://doi.org/10.1038/s41477-019-0582-x

    • Search Google Scholar
    • Export Citation
  • Zhang, S.Q. 2020 Genetic transformation of rice and screening and identification of its dwarf line Hebei Univ. Sci. Technol. DOI: 10.27107/d.cnki.ghbku.2020.000113

    • Search Google Scholar
    • Export Citation
  • Zhao, S., Kong, Y., Xin, P., Chu, J., Wan, Y., Ling, H. & Liu, Y. 2022 AtCPS V326M significantly affect the biosynthesis of gibberellins (in Chinese, abstract in English) Hereditas. 44 3 245 252 https://doi.org/10.16288/j.yczz.21-405

    • Search Google Scholar
    • Export Citation
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