View of In Silico Characterization of Lycopene Beta Cyclase (LCYB) and Lycopene Epsilon Cyclase (LCYE) Genes from DH-Pahang (Musa acuminata, A Genome) and DH-PKW (Musa balbisiana, B Genome)

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2023, Vol. 13, No. 1, 81 – 92

http://dx.doi.org/10.11594/jtls.13.01.09

How to cite:

Wiprayoga IPP, Meitha K, Dwivany FM (2023) In Silico Characterization of Lycopene Beta Cyclase (LCYB) and Lycopene Research Article

In Silico Characterization of Lycopene Beta Cyclase (LCYB) and Lycopene Epsilon Cyclase (LCYE) Genes from DH-Pahang (Musa acuminata, A Genome) and DH- PKW (Musa balbisiana, B Genome)

I Putu Prakasa Wiprayoga, Karlia Meitha, Fenny M. Dwivany *

School of Life Sciences and Technology, Institut Teknologi Bandung, Bandung 40132, Indonesia

Article history:

Submission February 2022 Revised February 2022 Accepted September 2022

ABSTRACT

Alpha (α) and beta (β)-carotene are among the nutritious contents of bananas, with the unique feature of a high ratio of α-/β-carotene. Research on the gene and protein of the key enzymes determining the ratio of α-/β-carotene in bananas, namely lycopene beta cyclase (LCYB) and lycopene epsilon cyclase (LCYE), is currently not well de- fined. Hence, this study aimed to compare the characters of the LCYB and LCYE genes and their putative proteins from Musa acuminata 'DH-Pahang' and Musa balbisi- ana 'DH-PKW'. The corresponding nucleotide sequences from both species were aligned to detect similarities in the gene structure. Their protein products were char- acterized at the primary and tertiary levels. The phylogenetic tree was constructed based on nucleotide and protein sequences. The result showed that the gene structure between these two species is similar in LCYB in chromosome 9 but different in LCYB in chromosome 7 and LCYE. The presence of cis-acting regulatory elements in response to light dominated the 2000 nucleotide region of the 5'UTR of LCYB and LCYE genes in both species. Based on protein alignment and domain analysis, the NADB_Rossmann superfamily domain was detected in both LCYB and LCYE.

Alignment of the three-dimensional protein structure showed a significant difference between MaLCYB.c07 and MbLCYB.c07 only. The phylogenetic tree based on protein sequences indicated the distant relationship of MaLCYB.c07 and MbLCYB.c07 with other LCYB ingroup OTUs. The results of this study could provide a molecular basis related to the exploration of bananas as a promising functional food to meet the needs of provitamin A.

Keywords: LCYB, LCYE, Musa acuminata, Musa balbisiana, provitamin A

*Corresponding author:

E-mail: [email protected]

Introduction

Vitamin A deficiency (VAD) has been causing serious health problems, especially in poor and de- veloping countries. Data on the prevalence of VAD in the period of 1995-2005 showed a mod- erate-severe problem status (>10%) in preschool children in 122 countries [1]. Moreover, VAD in Asia and Africa also increases mortality rates in children and pregnant women [2, 3].

The discovery of banana cultivars with high provitamin A carotenoids (pVACs) piqued the interest to promote bananas as a functional food in treating VAD cases [4]. Various commonly eaten banana cultivars were derived from Musa acu-

minata (A genome) and Musa balbisiana (B ge- nome). Research in 2009 showed that consump- tion of 7 banana cultivars imported from the Pa- cific Islands, with a pure composition of A genome (AA and AAA) and a combination of A and B genomes (AAB and ABB), successfully im- proved vitamin A intake of vulnerable population in Africa [3]. Hence, the distinctive content of pVACs in bananas with pure A or B genomes and their combination becomes an interesting basis for studying the genes involved in the biosynthesis of carotenoids in each genome.

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Alpha (α) and beta (β)-carotene are important compounds in the pVACs group. In the biosyn- thetic pathway (figure 1), these two compounds are synthesized from the cyclization reaction, an essential pathway that generates carotenoid diver- sity with different cyclic end groups. LCYE and LCYB compete on lycopene substrates to form α- carotene or b-carotene. At the pathway catalyzed only by LCYB, molecules with two beta rings are formed, namely b-carotene. In another branching pathway, a reaction catalyzed simultaneously by LCYB and LCYE resulted in the formation of beta and epsilon ring molecules, namely α -carotene [5]. Thus, the relative activity of these two enzymes will determine the ratio of α/β-carotene [6].

It is also known that the conversion efficiency of β-carotene into vitamin A is higher than α-carotene [7].

D'Hont et al. [8] and Wang et al. [9] have car- ried out whole genome sequencing of M. acu- minata 'DH-Pahang' and M. balbisiana 'DH- PKW', respectively. However, the characteriza- tion of LCYB and LCYE genes and proteins in these two genomes have not been well defined.

Therefore, this study compares the characters of LCYB and LCYE genes and their putative proteins from M. acuminata 'DH-Pahang' and M. balbisi-

ana 'DH-PKW'. The results suggested key regula- tory areas and domains of the enzymes for future improvement of pVACs content in banana.

Material and Methods

Sequence retrieving and gene identification Nucleotide sequences annotated by KEGG (www.genome.jp/kegg/) [10] database were used as the reference for determining LCYB and LCYE genes. Each coding sequence (CDS) was retrieved from KEGG and served as the query for the Basic Local Alignment Search Tool (BLAST) feature on the Banana Genome Hub database (https://banana- genome-hub.southgreen.fr/) [11]. BLASTN was conducted on the whole genome of M. acuminata 'DH-Pahang' (v4.3) for DH-Pahang, and whole ge- nome annotation of M. balbisiana 'DH-PKW' (v1.1) for DH-PKW. The sequences with identity percentage higher than 90% was then identified as LCYB or LCYE gene sequences.

Gene structure prediction

Gene structure prediction was performed with FGENESH+(www.softberry.com/berry.phtml?to- pic=fgenesh&group=help&subgroup=gfind) thro- ugh ab-initio principle [12]. Furthermore, Scipio was used to confirm the exon-intron junctions [13]. Gene structure prediction in the form of the length of nucleotide sequence of genes from the transcription start site (TSS) to the poly-A tail, the number of exons and introns, and the position of each exon and intron, were then visualized with Il- lustrator for Biological Sequences (IBS) software (ibs.biocuckoo.org/) [14]. To find the percentage of identity and similarity between two sequences, a global pairwise sequence alignment was performed using the Emboss Needle in the region of open reading frame (ORF) [15].

Analysis of Cis-Acting Regulatory Element (CAREs)

The presence of cis-acting regulatory elements (CAREs) in the upstream of 2000 nucleotides (5') relatives to the start codon (ATG) of the first exon of each gene was detected using the PlantCARE tool (https://bio.tools/plantcare) [16]. The identified CAREs motifs were then grouped according to their function related to hormonal regulations and abiotic stresses. The frequency of occurrence of CAREs in DH-Pahang and DH-PKW was cal- culated for each gene.

Figure 1. Carotene biosynthesis pathway. GGDP:

geranylgeranyl diphosphate; PSY: phytoene synthase; PDS: phytoene desaturase;

ZDS: z-carotene desaturase; CrtISO: carotenoid isomerase; LCYE: lycopene - cyclase; LCYB: lycopene -cyclase) (Adapted from Badejo [5].

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Analysis of protein motif and domain

The predictive protein sequences obtained from FGENESH+ were analyzed to find motifs and domains [12]. Domains were discovered using the NCBI Conserved Domain Search tool (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [17]. The identified domains were then visualized with Illustrator for Biological Sequences (IBS) software.

The search for motifs was carried out based on common expression principle, with reference to the literature. Initially, protein sequences from four other plants were obtained by performing BLASTP using queries from DH-PKW in the GenBank database (blast.ncbi.nlm.nih.gov/

Blast.cgi) [18]. Next, multiple sequence alignment (MSA) was performed with the muscle algorithm.

Alignment results with the marked motifs were visualized using GeneDoc software [19].

Prediction of Three-Dimensional Protein Struc- ture

The protein sequences were submitted on the i-TASSER webpage (https://zhanglab.dcmb.med.

umich.edu/I-TASSER/) to find the predictive three-dimensional (tertiary) structure of the protein [20]. In this study, we used multiple threading alignment and iterative structural assembly approach. Table 1 summarizes templates for structural modelling. The selected three dimensional structure is the model with the highest C-score. To make comparisons between three-dimensional structures, the root means square deviation (RMSD) value displayed by PyMol (pymol.org/2/) was used, with an RMSD value

<3Å indicating that the two aligned proteins are similar [21].

Table 1. Template for each protein.

Organism Protein Template (PDB)

DH- Pahang

MaLCYB.c09 3ATQ MaLCYB.c07

MaLCYE

7CTQ 3ATQ

DH-PKW

MbLCYB.c09 3ATQ MbLCYB.c07

MbLCYE

3ATQ 3ATQ

Construction of phylogenetic tree

Phylogenetic trees were constructed based on CDS and protein sequences to examine the rela- tionship between LCYB and LCYE in DH-Pahang

and DH-PKW, relative to related genes in selected plants. CDS from other plants were obtained by performing BLASTP using queries from DH-

PKW in the GenBank database

(blast.ncbi.nlm.nih.gov/Blast.cgi) [18]. Ten sequences with the highest max score were selected as operational taxonomic units (OTUs). The CDS of these ten species were then used to retrieve protein sequences to build a second type of phylogenetic tree.

The phylogenetic tree construction was carried out in MEGA X software [22]. Initially, multiple sequence alignment (MSA) was performed with the muscle algorithm, and sequence trimming was performed as necessary. The phylogenetic tree was constructed using the maximum likelihood (ML) algorithm, with a bootstrap of 1000.

Results and Discussion

Determination of LCYB and LCYE genes in DH- Pahang and DH-PKW

A total of six LCYB and LCYE loci were found in DH-Pahang and DH-PKW. In DH-Pahang, two LCYB loci were identified in chromosome 9 (MaL- CYB.c09) and chromosome 7 (MaLCYB.c07), and one LCYE locus in chromosome 1 (MaLCYE). In DH-PKW, two LCYB loci were found, which were also in chromosome 9 (MbLCYB.c09) and 7 (MbLCYB.c07), as well as one LCYE locus in chromosome 1 (MbLCYE).

Structure Prediction of LCYB and LCYE genes In DH-Pahang, the sequence length of MaL- CYB.c09 and MaLCYB.c07 gene were 2514 and 2262 base pairs, respectively. In DH-PKW, the MbLCYB.c09 and MbLCYB.c07 gene had se- quence length of 2639 and 3141 base pairs, respec- tively. Comparison of the LCYB gene structure in DH-Pahang and DH-PKW is displayed in figure 2 (a).

The LCYB genes in chromosome 9, MaL- CYB.c09 and MbLCYB.c09, each consists of one exon. After conducting pairwise sequence alignment, the identity percentage value was 99% be- tween MaLCYB.c09 and MbLCYB.c09. This very high identity percentage indicates conserved nucleotide sequences.

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In DH-Pahang, the MaLCYB.c07 gene was de- tected to have two exons and one intron, whereas in DH-PKW, the MbLCYB.c07 gene had three ex- ons and two introns. Confirmation by Scipio re- vealed that there was no confirmed splicing site between exon-intron junctions in the MaL- CYB.c07 gene. Fu et al. [6] found the presence of a premature stop codon at the end of the first exon

of the MaLCYB.c07 gene, which caused this tran- script produces a nonfunctional protein. Hence, the CDS and protein sequence of the MaLCYB.c07 gene were only derived from the first exon (marked with the green box in figure 3). Pairwise sequence alignment between MaLCYB.c07 and MbLCYB.c07 showed the identity percentage value of 94.8%, indicating highly conserved nucleotide sequences.

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Figure 2. Comparison of exons (green box) and intron (gray line) of LCYB (a) and LCYE (b). Blue triangle is TSS, red rhombus is poly-A tail. Yellow box in MaLCYB.c07 is an unconfirmed exon. There is a scale difference in gene length between figure (a) dan (b).

Figure 3. Further analysis of the MaLCYB.c07 ORF. In the inset, the green box indicates the first exon, the yellow box indicates the second exon.

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For the LCYE gene, one locus was found in DH-Pahang and DH-PKW. The sequence length of MaLCYE and MbLCYE were 9730 and 9381 base pairs, respectively. Comparison of the struc- ture of the MaLCYE and MbLCYE genes is dis- played in Figure 2 (b). Prediction of the LCYE gene between DH-Pahang and DH-PKW showed difference in the number of exons-introns. In the MaLCYE gene, there were eleven exons and ten introns. In the MbLCYE gene, there were twelve exons and eleven introns. The comparison of sim- ilarities between the MaLCYE and MbLCYE genes showed an identity percentage of 80.2%.

Cis-Acting Regulatory Elements (CAREs) of LCYB dan LCYE

Differences in the frequency of occurrence of CAREs were observed on LCYB DH-Pahang and DH-PKW in chromosome 9 and chromosome 7, as shown in Figure 4 (a). Furthermore, specific CAREs motifs are shown in Table 2. The predominance of the CAREs motifs that responsible for the light response was observed in all LCYB of both species. The highest abundance of specific CAREs motifs for light response in LCYB in chro- mosome 9 of both species was Box-4, whereas in LCYB in chromosome 7 was G-box.

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Figure 4. Frequency of CAREs in LCYB (a) and LCYE (b) related to hormonal and stress responses. Chromo- some 9 DH-Pahang and DH-PKW refers to MaLCYB.c09 and MbLCYB.c09, respectively; chromo- some 7 DH-Pahang and DH-PKW refers to MaLCYB.c07 and MbLCYB.c07, respectively. DH-Pahang and DH-PKW refers to MaLCYE and MbLCYE, respectively.

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For the LCYE gene, difference in the fre- quency of occurrence of the CAREs were also observed between DH-Pahang and DH-PKW, as can be seen in Figure 4 (b), and the specific CAREs motifs in Table 2. The appearance of the CAREs motifs responsible for the light response also pre- dominates in the LCYE of both species. The high- est abundance of specific CAREs motif on MaL- CYE for light response was the Box-4, whereas in MbLCYE was the G-box.

The presence of CAREs that respond to abiotic stress indicates the role of environmental factors in the regulation of LCYB and LCYE gene expres- sion. The predominance of light-responding CAREs in LCYB and LCYE of both species indi- cates an important role of light for increasing gene expression in carotenoid metabolism related to the function of carotenoids in photo-inhibition and photo-oxidation prevention [23]. Plants that are subjected to anaerobic stress will produce compounds protecting their tissues from damage caused by abnormalities in oxygen-dependent re- actions. Drought, like other types of stress, can

lead to accumulation of reactive oxygen species (ROS), and controlling of LCYB and LCYE gene expression in drought stress can regulate certain carotenoid levels for protection against these ROS [24].

Auxin, gibberellin, abscisic acid, and MeJa are hormones that play important roles in the expression of various genes in carotenoid metabolism.

Auxins have been shown to interact in signaling pathways involving light in the regulation of carotenoid levels [25]. In the study of Moreno et al.

[26], it was known that LCYB gene expression in Daucus carota induces positive feedback on gib- berellin-associated genes. Abscisic acid is a carotene-derived compound that acts as an important hormone in plant stress responses involving carotenoids [27]. MeJa administration is known to moderately increase LCYB gene expression in Co- riandrum sativum [28].

Domain and motif of LCYB and lcye putative proteins

Table 2. Abundance of specific CAREs motifs in LCYB and LCYE Respond to Motif

Abundance MaLCYB

.c07

MaLCYB .c09

MbLCYB .c07

MbLCYB

.c09 MaLCYE MbLCYE

Auxin TGA-box 1 0 0 0 1 1

TGA-element 1 1 1 2 0 0

Gibberellin P-box 0 0 1 0 1 1

TATC-box 0 1 0 0 0 0

Absicic Acid ABRE 12 1 11 1 3 1

Methyl Jasmonate

CGTCA-motif 2 1 1 0 2 0

TGACG-motif 2 1 1 0 2 0

Light

AE-box 0 0 0 0 1 0

3-AF1 1 0 0 0 0 0

ATCT-motif 0 1 0 0 0 0

Box 4 0 3 0 2 5 0

chs-CMA2a 0 1 0 1 0 1

GA-motif 1 0 1 0 0 0

GATA-motif 2 0 2 0 0 1

GATT-motif 0 1 0 1 0 0

G-box 11 0 10 0 4 3

GT1-motif 0 1 0 0 2 2

I-box 1 0 1 0 0 0

LAMP-element 0 1 0 1 0 0

MRE 1 0 1 0 0 0

Sp1 3 0 3 0 0 0

GTGGC-motif 0 0 0 0 0 1

TCCC-motif 1 1 1 1 0 0

TCT-motif 1 0 1 0 0 2

Anaerobic ARE 2 1 3 2 2 4

GC-motif 1 0 1 0 1 3

Drought MBS 0 1 0 1 0 1

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Pairwise sequence alignment between the putative protein sequences of MaLCYB.c09 and MbLCYB.c09 showed the percentages of identity and similarity of 99.4% and 99.8%, respectively.

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Furthermore, the percentages of identity and similarity between MaLCYB.c07 and MbLCYB.c07

were 19.2% and 21.0%, respectively, indicating the sequences are not conserved. The indication of Figure 5. Domain protein structure of LCYB (a) and LCYE (b). Domain is shown with blue box.

Figure 6. MSA for finding motifs in LCYB. Black-highlighted amino acids indicate highly conserved sequence.

The lighter the highlight, the more variable the amino acids are.

Figure 7. MSA for finding motifs in LCYE. Black-highlighted amino acids indicate highly conserved sequence.

The lighter the highlight, the more variable the amino acids are.

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high percentage of gaps (75.2%) was due to the truncated protein of MaLCYB.c07 missing the C- terminal region. The structure of the LCYB puta-

tive protein domain can be seen in Figure 5 (a).

The presence of a domain from the NADB_Rossmann superfamily was indicated in all LCYB protein sequences. The protein members of the NADB_Rossmann superfamily have a Rossmann-fold NADPH/NADP (+) binding (NADB) domain in their three-dimensional structure [29]. A specific hit with the accession code PLN02463 was detected on MaLCYB.c09 and MbLCYB.c09 proteins, whereas no specific hit to MaLCYB.c07 and MbLCYB.c07. The accession code PLN02463 refers to lycopene beta cyclase

protein. The absence of specific hits for the MaL- CYB.c07 and MbLCYB.c07 necessitated further confirmation at the motif level.

Pairwise sequence alignment between the putative protein sequences of MaLCYE and MbLCYE showed the percentage of identity and similarity of 93.7% and 94.4%, respectively, indicating a conserved protein’s primary structure.

The structure of the LCYE protein domains can be seen in Figure 5 (b).

As with LCYB proteins, there was the domain from the NADB_Rossmann superfamily in both LCYE proteins. However, different hit, with the accession code PLN02697, was detected in these two LCYE protein sequences, which belong to lycopene epsylon cyclase protein.

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Figure 8. The alignment of three-dimensional structure of LCYB from chromosome 9 (a), LCYB from chromosome 7 (b), and LCYE (c).

Figure 9. Phylogenetic tree based on CDS (a) and protein sequences (b) of LCYB. Red dashed box indicates clade of DH-Pahang and DH-PKW CDS or protein.

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Motifs analysis was carried out with reference to Cunningham et al. [30]. For LCYB proteins, 5 motifs were tried to be identified, namely plant conserved region, di-nucleotide binding site, cyclase motif I (CMI), cyclase motif II (CMII),

and charged region. Plant conserved region is a unique motif that is only found in plant LCYB proteins, but not in cyanobacteria. The presence of this motif at the N-terminal of LCYB indicates the importance of this motif in LCYB localization in plastids [30]. The di-nucleotide binding site motif with identity of GXGXXG, is important in binding to the NADP, NAD, ADP, and FAD cofactors.

These cofactors are important in stabilizing the carbonium intermediates formed during the lycopene cyclization process [30]. Lastly, the CMI motif, CMII, and the charged region are important in catalytic function and substrate binding. Align- ment along with four LCYB protein sequences from other species to find these motifs is shown in Figure 6.

MaLCYB.c09 and MbLCYB.c09 own all motifs. This predicts that these two LCYB proteins from chromosome 9 are fully functional. On another hand, the absence of the CMI, CMII, and charged region motifs in the MaLCYB.c07 puta-

tive protein will greatly affect the catalytic function of this protein. Furthermore, the absence of the plant conserved region and di-nucleotide binding site motif in the MbLCYB.c07 putative protein may indicate differences in the structure or locali-

zation of this protein compared to functional LCYB proteins.

Moreover, we investigated the presence of five motifs in LCYE proteins, namely dinucleotide binding site, cyclase motif I (CMI), cyclase motif II (CMII), charged region, and ring switch [30, 31]. As shown in Figure 7, both LCYE proteins were indicated to have these five motifs. Similar to LCYB proteins, the dinucleotide-binding site motif is important in stabilizing the structure of LCYE, whereas the CMI, CMII, and charged region motifs are important for catalytic function and substrate binding. The ring switch motif is a region that plays a role in determining the number of rings added in the cyclization of lycopene into certain types of carotenoids [31]. Ring switch motif with identity of XLXXXX indicates the cyclization carried out by MaLCYE and MbLCYE will form α-carotene, which has one β-ring and one ε-ring.

Figure 10. Phylogenetic tree based on CDS (a) and protein sequences (b) of LCYE. Red dashed box indicates clade of DH-Pahang dan DH-PKW CDS or protein.

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Three-Dimensional structure of LCYB and LCYE putative proteins

To date, the crystal structure of any LCYB protein of any species has not been reported [32].

Through i-TASSER, three-dimensional structure prediction can be done by multiple threading alignment and iterative structural assembly based on their similarity to other better known proteins in database [20]. A comparative analysis by performing a three-dimensional structural alignment between the paralogous LCYB proteins is shown in Figure 8 (a) and 8 (b).

The RMSD value for MaLCYB.c09 and MbLCYB.c09 protein comparison was 0.596, indicating these two paralog proteins are structurally similar. The main dissimilarity was observed in the loop structure. Conversely, the RMSD value for MaLCYB.c07 and MbLCYB.c07 protein comparison was 17.203, indicating these two proteins are not structurally similar.

As with LCYB, the crystal structure of the LCYE protein of any species has not been reported. The predictive three-dimensional structural alignment for LCYE proteins in DH-Pahang and DH-PKW can be seen in Figure 8 (c).

Comparative analysis of the three-dimensional structure between MaLCYE and MbLCYE proteins was shown with an RMSD value of 1.025, indicating these two proteins are similar. The main difference lies in the loop structure.

Phylogenetic trees of LCYB and LCYE based on CDS and protein sequences

The presence of all LCYB CDS of DH-Pahang and DH-PKW in an ingroup along with the CDS of the other species, as shown in Figure 9 (a), confirmed that these CDS were correctly belong to a group of LCYB genes. CDS of MaLCYB.c09 is placed in the same clade with MbLCYB.c09; as well as for CDS of MaLCYB.c07 and MbLCYB.c07. These results indicate that the LCYB gene duplication event most likely oc- curred in the common ancestor of DH-Pahang and DH-PKW, before the two species started to sepa- rate. The long branch length that separated the MaLCYB.c07 and MbLCYB.c07 clade from other clades indicates a distant divergence of this clade.

The analysis was continued by constructing a phylogenetic tree based on LCYB protein sequences, as shown in figure 9 (b). LCYB proteins were also grouped according to their chromosomal location. MaLCYB.c09 and MbLCYB.c09 protein are in a monophyletic group along with other

LCYB from Musa troglodytarum and Ensete ven- tricosum. The distantly located clade of MaL- CYB.c07 and MbLCYB.c07 is caused by the absence of an important motif in the primary structure of MaLCYB.c07 and MbLCYB.c07 proteins.

The presence of MaLCYE and MbLCYE clade in an ingroup along with CDS of other species, as shown in figure 10 (a), confirms that these CDS were correctly belong to a group of LCYE genes.

This tree also concluded that the LCYE from Musa have common ancestor close to the clade from Elaeis guineensis, Phoenix dactylifera, and Ananas comosus.

The presence of MaLCYE and MbLCYE proteins in an ingroup along with LCYE proteins of other species, as shown in Figure 10 (b), confirms that structurally, these proteins are similar to LCYE proteins of other species. This tree also concluded that the LCYE proteins from Musa have recent common ancestor with Dendrobium catenatum, Apostasia shenzhenica, and Dioscorea cayenensis.

Conclusion

The gene structure between DH-Pahang (M.

acuminata, A Genome) and DH-PKW (M. balbi- siana, B Genome) was observed to be similar in LCYB in chromosome 9, but different in LCYB in chromosome 7 and LCYE. CAREs that respond to light dominate in all LCYB and LCYE in both spe- cies. LCYB and LCYE proteins of the two species have similarities in the appearance of the NADB_Rossmann superfamily, but different in motifs. Three-dimensional structure showed significant difference only detected between MaL- CYB.c07 and MbLCYB.c07. For future research, determining the effect of light treatment to the ex- pression of LCYB and LCYE and content of provit- amin A carotenoids in both A and B bananas genome could be an interest of future research.

Moreover, the research to enhance the production of β-carotene in these both genomes might be started from overexpressing the functional LCYB proteins or downregulating the expression of LCYE proteins.

Acknowledgment

This study was funded by Institut Teknologi Bandung Priority Research KK A 2021 for Fenny M. Dwivany.

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