Molecular Characteristics and Functional Identification of a Key Alpha-Amylase-Encoding Gene AMY11 in Musa acuminata

Alpha-amylase (AMY) plays a significant role in regulating the growth, development, and postharvest quality formation in plants. Nevertheless, little is known about the genome-wide features, expression patterns, subcellular localization, and functional regulation of AMY genes (MaAMYs) in the common starchy banana (Musa acuminata). Twelve MaAMY proteins from the banana genome database were clustered into two groups and contained a conserved catalytic domain. These MaAMYs formed collinear pairs with the AMYs of maize and rice. Three tandem gene pairs were found within the MaAMYs and are indicative of putative gene duplication events. Cis-acting elements of the MaAMY promoters were found to be involved in phytohormone, development, and stress responses. Furthermore, MaAMY02, 08, 09, and 11 were actively expressed during fruit development and ripening. Specifically, MaAMY11 showed the highest expression level at the middle and later stages of banana ripening. Subcellular localization showed that MaAMY02 and 11 were predominately found in the chloroplast, whereas MaAMY08 and 09 were primarily localized in the cytoplasm. Notably, transient attenuation of MaAMY11 expression resulted in an obvious increase in the starch content of banana fruit, while a significant decrease in starch content was confirmed through the transient overexpression of MaAMY11. Together, these results reveal new insights into the structure, evolution, and expression patterns of the MaAMY family, affirming the functional role of MaAMY11 in the starch degradation of banana fruit.


Introduction
Plant starch is the major storage carbohydrate in roots, leaves, tubers, seeds, and fruits [1,2].Starch degradation in various organs not only provides energy for seed germination, seedling growth, and development in cereals [3][4][5], but also plays a crucial role in the postharvest quality formation, including sweetness, waxiness, and softening in banana, kiwifruit, and mango fruits [6][7][8].The process of starch degradation is complex and involves the synergistic actions of many enzymes, such as α-amylase (AMY), β-amylase (BAM), α-glucosidase (GLU), starch phosphorylase (PHO), and limit dextrinase (LD) [8,9].Among these enzymes, AMY is a glycosyl hydrolase with endoglycolytic activity on the α-1,4-glycosidic linkage in starch and is one of the key enzymes in converting starch into glucose and maltose via the combined action of BAM, GLU, and LD [3,10].
AMYs are encoded by a multi-gene family that has been screened in many plants through genome data analysis: three family members have been identified in kiwifruit (Actinidia deliciosa) and Arabidopsis thaliana; six in cassava (Manihot esculenta) [10]); seven in wheat (Triticum aestivum) [11]; ten in rice (Oryza sativa) [11] and barley (Hordeum vulgare) [12]; eleven in apple (Malus domestica) [13].In vascular plants, AMYs can be divided into three subfamilies (AMY1, 2, and 3), each of which typically contains a conserved α-amylase catalytic domain (ACD, PF00128) [13].Amy1 members have a signal peptide that activates the mobilization of starch in cereal endosperms [3].Amy2 members have no signal peptide, and their function remains largely unknown despite reports of cytoplasmic localization [14].However, Amy3 members have a large N-terminal extension of 400-500 amino acids in length, which contains tandem carbohydrate-binding modules and a predicted chloroplast transit peptide.The functional role of Amy3 is in leaf starch breakdown [10].
The expression patterns and functions of AMYs have been revealed in A. thaliana and cereals.In A. thaliana, AMY1 was expressed at low levels in the leaf stalks and roots and was highly expressed in the leaves, stems, and flowers [15].Upon stress treatment, the up-regulated expression of AMY1 conferred high heat tolerance [15].Among the ten rice AMYs, most were specifically expressed during the seed germination process and in the developing seed embryo [3].In contrast to rice and A. thaliana, barley AMY expression was not found to be tissue-specific [16].In wheat grains, the expression of a late-maturity AMY gene was induced by a cool temperature shock close to physiological maturity [17].Moreover, a 20-to 230-fold increase in total AMY activity in mature grains was caused by the over-expression of wheat AMY1 [18].By contrast, the antisense suppression of an AMY1 in the rice endosperm led to delayed germination [19].In maize kernels, RNA interference (RNAi) with AMY gene expression in Aspergillus flavus led to decreased aflatoxin production [20].The RNAi-mediated suppression of rice AMY1A, 1C, 3A, 3D, and 3E led to fewer chalky grains in ripening seeds under high-temperature conditions [21].Together, the abovementioned reports imply that the expression of AMYs plays a crucial role in enhancing seed germination, grain development and maturation, and tolerance to stresses.
Around 70-80% of the dry weight of unripe banana fruits is composed of starch.During the ripening process, this starch is rapidly converted into soluble sugars by a series of starch-degrading enzymes [8,22].Early research by Bassinello et al. [23] reported that AMY activity might be involved in the early stage of starch degradation during banana fruit ripening.Subsequently, Junior et al. [24] found that, among several hydrolytic enzymes, AMY is the only enzyme able to act upon intact granules.More recently, it was shown that the transcription factor BEL1-LIKE HOMEODOMAIN 1 interacted with the MaAMY3 gene promoter to accelerate softening and ripening in banana fruit [25].However, the genome-wide systematic screening of MaAMYs associated with the starch degradation in banana fruit remains to be further researched.The genome-wide characteristics, subcellular localization, expression features, and functional regulation of the key MaAMYs are still unclear.In the current study, we identified and characterized 12 distinct MaAMY members in the banana A genome database and evaluated the evolutional features as well as the transcriptome and spatio-temporal expression patterns.Furthermore, the subcellular localization of four expressed proteins, namely, MaAMY02, 08, 09, and 11, was analyzed in Nicotiana benthamiana leaf cells.The functional role of the most highly expressed protein, MaAMY11, during ripening was determined by transient silencing and transient overexpression in banana fruit.Together, our findings enhance our understanding of the function of MaAMY11 in fruit starch degradation and provide key target genes for the genetic improvement of quality formation in common starchy bananas and other crops.

Evolutionary Relationships of AMYs from Banana and Other Plant Species
To investigate the evolutionary relationships of the MaAMYs, a total of 108 AMY protein sequences from M. acuminata, A. thaliana, Brachypodium distachyon, Capsicum annuum, H. vulgare, O. sativa, Panicum hallii, Seturia italica, Solanum tuberosum, Solanum lycopersicum, T. aestivum, Sorghum bicolor, and Zea mays were used to construct a tree (Figure 2A).All of the accession numbers for these proteins are provided in Table S3.Groups I, II, and III were present in the classification analysis of these proteins.The majority of banana MaAMYs, including eight MaAMY (MaAMY01, 02, 03, 07, 08, 09, 10, and 12) members, were found in group III.Four members, namely, MaAMY04, 05, 06, and 11, were found in group II.However, zero MaAMY members in group I were detected in the banana genome, in contrast to monocot plants such as T. aestivum and Z. mays, in which 1-2 group I members have been found (Figure 2A), implying that the banana MaAMYs have undergone different genome evolutionary processes compared with other monocot plants.Furthermore, with the exception of pepper, a Ka/Ks ratio of less than one was found in bananas and other plant species (Figure 2B), indicating that strong purifying selection had occurred in the MaAMYs.

Intergenomic and Intragenomic Collinearity Analysis
Different linear relationships among banana chromosomes were revealed.There was collinearity between MaAMY10 and MaAMY12 in the intrachromosomal regions (Figure 4A).The intragenomic collinearity of the AMYs among banana, tomato, maize, rice, and A. thaliana was further analyzed.Three MaAMY members were collinear with three rice OsAMYs and three maize ZmAMYs (Figure 4B).In contrast, MaAMYs did not exhibit collinearity with A. thaliana AtAMYs and tomato SlAMYs (Figure 4C).Notably, the AMYs were mainly collinear between banana and rice, and between banana and maize, based on the comparative genomic analysis, and these duplicated genes may have been lost or altered in different species due to evolutionary processes.
The spatio-temporal expression characteristics of the four expressed genes (MaMAY02, 08, 09, and 11) were further identified using quantitative real-time polymerase chain reaction (qRT-PCR).Following normalization, the relative expression of each gene in different organs or at different temporal stages was verified through comparison to the root and at 0 DAF, with the relative expression of each gene in the root or at 0 DAF considered "1".The qRT-PCR results showed that all four MaAMYs exhibited better agreement with the RNA-seq data (Figure 5C-J).The correlation coefficients exceeded 0.9730 to 0.9998 for both the RNA-seq and qRT-PCR data in the different organs and at various temporal stages, respectively, implying that the RNA-seq data were reliable.Moreover, a higher abundance of MaAMY11 than other MaAMYs in the fruits or during banana fruit ripening was revealed by both the transcriptome data and qRT-PCR data, suggesting that MaAMY11 may play a crucial role in driving fruit ripening.

Co-Localization of Four Expressed MaAMY Proteins
The open reading frames (ORFs) of the abovementioned expressed MaAMY02, 08, 09, and 11 were inserted into pCAMBIA1302-GFP vectors, respectively.These four recombinant constructs were transiently expressed into N. benthamiana leaves via agroinfiltration, from which four fusion proteins were generated, including MaAMY02-GFP, MaAMY08-GFP, MaAMY09-GFP, and MaAMY11-GFP.The subcellular co-localization results showed that the MaAMY02-GFP and MaAMY11-GFP proteins were localized in the chloroplast with a co-localized chloroplast marker (red fluorescent protein, RFP).The MaAMY08-GFP and MaAMY09-GFP proteins were mainly localized in the cytoplasm with a co-localized cytoplasm marker (RFP).By contrast, in the GFP-positive control (PC), fluorescence distribution across the entire cell was observed in the N. benthamiana leaf cells (Figure 6).different tissues.(G-J) Expression of MaAMY02, 08, 09, and 11 at different stages of fruit development and ripening.Data are presented as means ± standard deviations, n = 3 biological replicates.

Co-Localization of Four Expressed MaAMY Proteins
The open reading frames (ORFs) of the abovementioned expressed MaAMY02, 08, 09, and 11 were inserted into pCAMBIA1302-GFP vectors, respectively.These four recombinant constructs were transiently expressed into N. benthamiana leaves via agroinfiltration, from which four fusion proteins were generated, including MaAMY02-GFP, MaAMY08-GFP, MaAMY09-GFP, and MaAMY11-GFP.The subcellular co-localization results showed that the MaAMY02-GFP and MaAMY11-GFP proteins were localized in the chloroplast with a co-localized chloroplast marker (red fluorescent protein, RFP).The MaAMY08-GFP and MaAMY09-GFP proteins were mainly localized in the cytoplasm with a co-localized cytoplasm marker (RFP).By contrast, in the GFP-positive control (PC), fluorescence distribution across the entire cell was observed in the N. benthamiana leaf cells (Figure 6).

Banana MaAMY11 Plays a Crucial Role in Fruit Starch Degradation
The function of MaAMY11, which had the highest expression level during banana ripening, was evaluated.As shown in Figure 7A, transient silencing of MaAMY11 expression led to darker staining by iodine-potassium-iodide (I 2 -KI) in banana fruit discs compared to the controls.In the transiently silenced fruit discs, the expression level of MaAMY11 was obviously decreased (Figure 7B), but the contents of total starch, amylose, and amylopectin were obviously increased by 9.49%, 3.76%, and 5.73%, respectively, compared the empty vector (Figure 7C-E).By contrast, lighter staining by I 2 -KI was exhibited in the banana fruit discs by transiently overexpressing MaAMY11 (Figure 7F).The expression level of MaAMY11 was obviously increased in overexpressing fruit discs (Figure 7G), whereas the total starch, amylose, and amylopectin contents were significantly decreased by approximately 31.77%, 6.81%, and 24.97%, respectively (Figure 7H-J).

Banana MaAMY11 Plays a Crucial Role in Fruit Starch Degradation
The function of MaAMY11, which had the highest expression level during banana ripening, was evaluated.As shown in Figure 7A, transient silencing of MaAMY11 expression led to darker staining by iodine-potassium-iodide (I2-KI) in banana fruit discs compared to the controls.In the transiently silenced fruit discs, the expression level of MaAMY11 was obviously decreased (Figure 7B), but the contents of total starch, amylose, and amylopectin were obviously increased by 9.49%, 3.76%, and 5.73%, respectively, compared with the empty vector (Figures 7C-E).By contrast, lighter staining by I2-KI was exhibited in the banana fruit discs by transiently overexpressing MaAMY11 (Figure 7F).The expression level of MaAMY11 was obviously increased in overexpressing fruit discs (Figure 7G), whereas the total starch, amylose, and amylopectin contents were significantly decreased by approximately 31.77%, 6.81%, and 24.97%, respectively (Figures 7H-J).
We found that banana MaAMY proteins were categorized into two groups (groups II and III), with group I not present.This differs from T. aestivum and Z. mays (Figure 2A) and vascular plants [3,10,12,14], suggesting that the phylogenetic evolution of banana MaAMYs may be different from T. aestivum, Z. mays, or vascular plants.It may also be because starch biosynthesis and degradation in bananas are completed in the same generation, whereas starch biosynthesis in other monocot plants such as T. aestivum occurs in the first generation, with starch degradation completed in the next generation.In addition, we found that MaAMYs in group II demonstrated a close evolutionary relationship to orthologs AtAMY03 and OsAMY04 in A. thaliana and O. sativa.The closest orthologs to MaAMYs in group III include TaAMY06 and 13 in T. aestivum (Figure 2A).A Ka/Ks ratio of less than one was detected in all MaAMYs (Figure 2B), implying that a strong purifying selection may have occurred during the evolution of the banana MaAMYs [42].Gene duplication is an important mechanism for acquiring new genes during evolution [43].The present analysis found that banana MaAMY expansion was likely the result of tandem duplication (Figure 3A).An unbalanced distribution of MaAMYs on the chromosomes, with chromosomes 3, 4, and 5 containing three-fourths of the entire MaAMY gene family, was also found.Furthermore, the collinearity analysis identified intrachromosomal collinear pairs between MaAMY10 and MaAMY12 within the banana genome (Figure 4A), three collinear AMY gene pairs between banana and maize, and three gene pairs between banana and rice (Figure 4B).This suggests that the duplicated AMYs might have undergone divergence during the evolutionary process.Similar results have been reported in the synthetic analysis of AMYs from cassava [10].
The AMY promoters contain several cis-acting elements that are associated with the growth, development and maturation, and abiotic and biotic stresses in plants [11,44].In rice, three elements include a G-box-related element, the amylase element, and a CGACG element that regulate the high-level expression of rice Amy3D during seedling development [45].Loss-and gain-of-function studies have reported that the sugar response sequence of the rice AMY3 promoter serves as a transcriptional enhancer under sugar starvation conditions [46].In barley and wheat, AMY1 promoters contain a GARE, pyrimidine box, and TATCCAT/C box [11].In Camellia sinensis, cis-acting elements of CsAMY promoters determining the quality during the postharvest processing of tea leaves were analyzed [44].In MaAMY promoters, the phytohormone-related ABRE, ARE, CGTCA, and GARE, the stress-related LTR and MBS, and the development-and growth-related elements (MRE and O2-site) have been found.MaAMY11 was particularly enriched in phytohormone-and development-related elements (Figure 3B), which may play an important role in phytohormone-and development-related biological processes.In addition, a large number of conserved cis-acting elements, including the TATA-box and CAAT-box, were found in MaAMY gene promoter regions, which were consistent with the characteristics of the AMY gene promoters in rice [45] and cassava [10].
AMYs are expressed during seed germination and fruit development in several plant species, such as rice [3], wheat [47], potato [48], and kiwifruit [41].Among the ten rice AMYs, most are highly expressed during the seed germination process and in the developing seed embryo [3,49].In wheat, AMYs were significantly highly expressed at 6 days after anthesis, suggesting that AMY likely catalyzes the hydrolysis of starch granules [47].In kiwifruit, the AdAMY1 expression was obviously induced by ethylene and was positively correlated with starch degradation [41].In the present study, MaAMY02, 08, 09, and 11 were expressed during fruit development, and MaAMY11 was the only gene that was highly expressed during banana fruit ripening (Figure 5), implying its involvement in fruit ripening.Furthermore, the MaAMY02 and 11 proteins were localized in the chloroplast, which is consistent with previous reports in rice AMYI-1 [19] and A. thaliana AtAMY3 [48].The MaAMY08 and 09 proteins were also found to be localized in the cytoplasm (Figure 6).This finding is consistent with a previous report in potato StAMY23 [50].
Suppressed or overexpressed AMYs influence the starch metabolism in rice, potatoes, and wheat [4,19,48].In rice, the suppressed expression of AMYI-1 resulted in an obvious increase in starch accumulation in the young leaf; the overexpression of AMYI-1, 1A, 3C, and 3D resulted in a reduction in the starch content in the leaves or in the developing endosperm [19,49].Silencing potato StAMY23 expression led to higher phytoglycogen and lower resistant starch accumulation in tubers and delayed tuber sprouting [48,50].In wheat, the overexpression of TaAMY2 resulted in an absence of dormancy in the ripened grain [4], whereas the overexpression of TaAMY3 led to an increase in the total AMY activity, but the increased activity did not affect the starch content or the composition of the dry grain [51].Moreover, in A. thaliana, double-knock-out AtAMYs (AtAMY1, 2, and 3) combinations and a triple-knock-out mutant did not have a significant impact on starch degradation in the leaves [52].We were interested in whether the high expression of MaAMY11 during banana ripening has an impact on fruit starch degradation.Herein, the transient silencing of MaAMY11 expression resulted in an obvious increase in the total starch content, followed by lighter I 2 -KI staining, which corroborates previous reports in rice [19] and potato [48].However, this result differs from reports in dry wheat grain [51] and A. thaliana leaf [50].Furthermore, the transient overexpression of MaAMY11 resulted in an obvious decrease in the content of total starch in the banana fruit.This finding agrees with previous assumptions that AMY may be important for fruit starch degradation in bananas [53].In the current study, direct evidence supports that MaAMY11 plays an important role in the starch degradation of banana fruit by transient silencing or transient overexpression.

Plant Materials
The banana cultivar 'Cavendish' (M.acuminata AAA genotype) was obtained from the Banana Germplasm Nursery (19 • N, 109 • E) located in the Chinese Academy of Tropical Agricultural Sciences, Danzhou City, Hainan Province, China.The different organs (roots, leaves, and fruit) were harvested at 80 days after the emergence of the inflorescence from the pseudostem (DAF) for spatial expression analysis.Following fruit development and ripening, pulps at 0, 20, and 80 DAF, and 8 and 14 days postharvest (DPH), were selected for temporal expression analysis.Three replicates were conducted for these expression studies.

Genome-Wide Identification of Banana AMY Family Genes
The AMY family members were identified based on the banana genome database [26].AMY amino acid sequences were defined by the InterPro database [54].An HMM profile was analyzed by the Pfam database.Twelve MaAMY members were annotated, and their ACDs (PF00128) were confirmed using the Conserved Domain Database and SMART software v.8.0.The physicochemical properties of MaAMY1-MaAMY12 proteins were annotated by the ExPASy database.The secondary structures and subcellular localizations of the MaAMY proteins were clarified with SOPMA and the Cell-PLoc 2.0 web-based tool, respectively.Table S7 presents all of the bioinformatic analytical websites, databases, and software used in this study.

Motifs, Structures, and Multiple Sequence Alignment
To identify conserved motifs within the amino acid sequences, we used Motif Elicitation analysis (MEME) and set the maximum motif count to 12 [55,56].GSDS v. 2.0 was used to elucidate the exon-intron structures.In addition, for a comprehensive analysis, we performed multiple sequence alignments on the MaAMY gene family using MEGA X v. 10.1.1 software.The conserved motifs, gene structure, and multiple sequence alignment characteristics were visualized using TBtools v.1.0[57].

Phylogenetic Tree and AMY Gene Family Evolutionary Selection Pressure Analysis
The annotation data for the genomes of various plant species, including M. acuminata, B. distachyon, A. thaliana, C. annuum, O. sativa, H. vulgare, P. hallii, S. italica, S. bicolor, S. lycopersicum, S. tuberosum, Z. mays, and T. aestivum, were sourced using the banana A genome, phytozome v13 database, and ensembl plants database (Table S7).To identify relevant AMY genes across these species, we utilized the Basic Local Alignment Search Tool (BLAST) within TBtools v.1.0[54].Genes exhibiting an e-value of less than 0.00005 were deemed candidate genes [54].
The sequences and the presence of the ACD were verified using the NCBI's CD-Search tool and SMART software v. 1.A phylogenetic tree of AMY amino acid sequences from bananas and other species was constructed using MEGA X v. 10.1.1 with 1000 bootstrap replications and the neighbor-joining (NJ) method, following which the phylogenetic tree was enhanced using the iTOL website.In addition, we used the itol.toolkitpackage in R for further modifications [58].We extracted the CDS sequences of the AMYs from the various species genomes using the phytozome v13 database.The ratio of nonsynonymous (Ka) and synonymous (Ks) substitutions for these CDS sequences was calculated by KaKs Calculator v. 1.2.Finally, the results were visually analyzed and presented using the ggplot2 package in R [59].

Chromosome Localization, Gene Duplication and Collinearity, and Promoter Cis-Acting Element Analyses
The chromosomal localization of the MaAMYs was analyzed using Mapchart v. 2.3.Gene duplication and collinearity were analyzed using MCS-canX [60].Genome collinearity was visualized using Circos software v. 0.69-9.The specific loci of the MaAMYs, as well as the 2000 bp upstream regions preceding them, were designated as promoter regions.The cis-acting elements of these promoter regions were predicted by the PlantCARE website [29].TBtools software was used to determine the precise locations of these elements, and R software v. R-4.41 was used to construct a heatmap for quantifying the occurrence of each gene element [37].

Transcriptomic Analysis
RNA was extracted from the roots, leaves, fruit, and pulps of bananas at different developmental (0, 20, and 80 DAF) and ripening stages (8 and 14 DPH) using an RNAprep Pure Plant Kit (supplied by Tiangen, Beijing, China).An Illumina GAII platform was used for deep sequencing (Illumina, Inc., San Diego, CA, USA).Low-quality reads and adapter sequences were eliminated by the FastQC software and FASTX toolkit, respectively.Transcriptome assemblies were generated by Cufflinks.The gene expression levels were calculated based on the RPKM value.The DESeq package was used to screen the differentially expressed genes (DEGs).Two technical replicates and three biological replicates were evaluated in the sequencing process.According to the RPKM value of the MaAMYs, a heatmap was constructed using MeV 4.9.0 software.

Quantitative Real-Time Polymerase Chain Reaction and Statistical Analysis
The relative expression levels of the MaAMYs in roots, leaves, and fruit at different development stages and different ripening stages were performed with a SYBR ® Premix Ex Taq™ kit (TaKaRa, Shiga, Japan) on a qRT-PCR system (qTOWER3G, Analytik, Jena, Germany).The internal controls were Actin (EF672732) and UBQ2 (HQ853254).Table S8 provides the primer sequence information.The expression levels of the MaAMYs relative to UBQ2 and Actin were estimated by the 2 −∆∆CT method [61].Each sample included three replicates.

Transient Silencing or Transient Overexpression of MaAMY11 in Banana Fruit
The transient silencing vector pTRV2-MaAMY11 was constructed using the EcoR I and Kpn I enzymes (Figure S2).The transient overexpression vector pCAMBIA3300-MaAMY11 was constructed using the Kpn I and Sal I enzymes (Figure S2).The primer sequences are provided in Table S8.The constructed pCAMBIA3300-MaAMY11 and pTRV2-MaAMY11 plasmids were then transferred into the GV3101 strain.Surface-sterilized banana fruit discs at 80 DAF were infiltrated with Agrobacterium (OD 600 = 0.6) and placed on a Murashige and Skoog medium, incubated at 30 • C for 3 d, and then stained with I 2 -KI solution [8,31].The expression level of MaAMY11 was detected in the transiently silenced or transiently overexpressed fruit slices.Total starch, amylose, and amylopectin contents were detected using the method reported by Miao et al. [8,31].The experiment used triplicate biological replicates.

Figure 1 .
Figure 1.Bioinformatics analyses of the MaAMY gene family.(A) Phylogenetic evolution of the MaAMY protein family.UTR represents untranslated region.CDS represents coding sequence.(B) Motif analysis of the MaAMY gene family.(C) Gene structure analysis of the MaAMY gene family.(D) Multiple sequence alignment of MaAMY family amino acids, where red letters and blue boxes show the highly conservative positions and black letters indicate less conserved positions.Red line represents the α-amylase catalytic domain.

Figure 1 .
Figure 1.Bioinformatics analyses of the MaAMY gene family.(A) Phylogenetic evolution of the MaAMY protein family.UTR represents untranslated region.CDS represents coding sequence.(B) Motif analysis of the MaAMY gene family.(C) Gene structure analysis of the MaAMY gene family.(D) Multiple sequence alignment of MaAMY family amino acids, where red letters and blue boxes show the highly conservative positions and black letters indicate less conserved positions.Red line represents the α-amylase catalytic domain.

Figure 3 .
Figure 3. Chromosomal distribution and cis-acting element analysis of MaAMY family.(A) Chromosomal localization of 12 MaAMY gene family members.Green, blue, and orange colors represent low, medium, and high gene density in the region of the chromosome, respectively.(B) Promoter cis-acting elements of MaAMY genes; the number represents the number of cis-acting element in each MaAMY promoter.

Figure 3 .
Figure 3. Chromosomal distribution and cis-acting element analysis of MaAMY family.(A) Chromosomal localization of 12 MaAMY gene family members.Green, blue, and orange colors represent low, medium, and high gene density in the region of the chromosome, respectively.(B) Promoter cis-acting elements of MaAMY genes; the number represents the number of cis-acting element in each MaAMY promoter.

Figure 4 .
Figure 4. Intergenomic and intragenomic synteny relationship of the AMY genes among banana (Musa acuminata), tomato (Solanum lycopersicum), maize (Zea mays), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa).(A) Intergenomic synteny relationship between the MaAMY genes in the banana genome.Purple lines indicate the GC content.Red lines indicate the GC Skew.Green dots indicate the Nratio.Gray lines indicate the interchromosomal collinear relationship in the banana A genome.Blue lines indicate the intrachromosomal collinearity between MaAMY10 and MaAMY12.(B) Collinear AMY family gene pairs between tomato and banana and between banana and maize.(C) Collinear AMY family gene pairs between A. thaliana and banana and between banana and rice.Colored lines highlight the collinear gene pair.

Figure 4 .
Figure 4. Intergenomic and intragenomic synteny relationship of the AMY genes among banana (Musa acuminata), tomato (Solanum lycopersicum), maize (Zea mays), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa).(A) Intergenomic synteny relationship between the MaAMY genes in the banana genome.Purple lines indicate the GC content.Red lines indicate the GC Skew.Green dots indicate the Nratio.Gray lines indicate the interchromosomal collinear relationship in the banana A genome.Blue lines indicate the intrachromosomal collinearity between MaAMY10 and MaAMY12.(B) Collinear AMY family gene pairs between tomato and banana and between banana and maize.(C) Collinear AMY family gene pairs between A. thaliana and banana and between banana and rice.Colored lines highlight the collinear gene pair.

Figure 5 .
Figure 5. Expression profiles of MaAMY family genes in the different organs and different developmental stages by banana transcriptome and qRT-PCR.(A,B) Expression of MaAMYs in different organs and during different stages of banana fruit development and ripening.The heat map with clustering was created based on the FPKM value of the MaAMYs.Differences in gene expression changes are shown in color in the blue-red scale.(C-F) Expression of MaAMY02, 08, 09, and 11 in

Figure 5 .
Figure 5. Expression profiles of MaAMY family genes in the different organs and different developmental stages by banana transcriptome and qRT-PCR.(A,B) Expression of MaAMYs in different organs and during different stages of banana fruit development and ripening.The heat map with clustering was created based on the FPKM value of the MaAMYs.Differences in gene expression changes are shown in color in the blue-red scale.(C-F) Expression of MaAMY02, 08, 09, and 11 in different tissues.(G-J) Expression of MaAMY02, 08, 09, and 11 at different stages of fruit development and ripening.Data are presented as means ± standard deviations, n = 3 biological replicates.

Figure 6 .
Figure 6.Co-localization of MaAMY02, 08, 09, and 11 proteins.The green fluorescent protein (GFP) fluorescence is represented by green, whereas red fluorescent indicates red fluorescent proteins (RFPs).A composite image was created by merging the GFP and RFP fluorescence images,

Figure 7 .
Figure 7. Transient suppression and overexpression of the MaAMY11 gene in banana fruit discs.(A) I2-KI staining indicative of MaAMY11 suppression in banana fruit discs.(B) Expression level of MaAMY11 in MaAMY11 suppression in banana fruit discs.(C-E) Variation in total starch, amylose, and amylopectin contents following MaAMY11 suppression in banana fruit discs.(F) I2-KI staining of MaAMY11-overexpressing banana fruit discs.(G) Expression level of MaAMY11 in MaAMY11 overexpression in banana fruit discs.(H-J) Change in total starch, amylose, and amylopectin contents following MaAMY11 overexpression in banana fruit discs.Triplicate replicates were tested, and the asterisks denote statistically significant differences compared to pTRV1 + pTRV2 (empty vector control) or pCAMBIA3300 (empty vector control) (*, p < 0.05; **, p < 0.01).

Figure 7 .
Figure 7. Transient suppression and overexpression of the MaAMY11 gene in banana fruit discs.(A) I 2 -KI staining indicative of MaAMY11 suppression in banana fruit discs.(B) Expression level of MaAMY11 in MaAMY11 suppression in banana fruit discs.(C-E) Variation in total starch, amylose, and amylopectin contents following MaAMY11 suppression in banana fruit discs.(F) I 2 -KI staining of MaAMY11-overexpressing banana fruit discs.(G) Expression level of MaAMY11 in MaAMY11 overexpression in banana fruit discs.(H-J) Change in total starch, amylose, and amylopectin contents following MaAMY11 overexpression in banana fruit discs.Triplicate replicates were tested, and the asterisks denote statistically significant differences compared to pTRV1 + pTRV2 (empty vector control) or pCAMBIA3300 (empty vector control) (*, p < 0.05; **, p < 0.01).