Next Article in Journal
Charge Transfer in He+ − He → He(1s4l, l ≥ 2) − He+ Collisions in Intermediate Energy Range
Previous Article in Journal
Vascular Endothelial Growth Factor C (VEGF-C) Sensitizes Lymphatic Endothelial Cells to Oxidative-Stress-Induced Apoptosis through DNA Damage and Mitochondrial Dysfunction: Implications for Lymphedema
Previous Article in Special Issue
Physiological, Metabolic, and Transcriptomic Analyses Reveal Mechanisms of Proliferation and Somatic Embryogenesis of Litchi (Litchi chinensis Sonn.) Embryogenic Callus Promoted by D-Arginine Treatment
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 25
Issue 14
10.3390/ijms25147832
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Peiguang Sun
1,†,
Zhao Zhu
1,2,†,
Zhiqiang Jin
1,
Jianghui Xie
1,
Hongxia Miao
1,3,* and
Juhua Liu
1,3,*
1
National Key Laboratory of Tropical Crop Biological Breeding, Institute of Tropical Bioscience and Biotechnology and Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences, 4 Xueyuan Road, Haikou 571101, China
2
College of Tropical Crops, Hainan University, 58 Renmin Avenue, Haikou 571100, China
3
Hainan Key Laboratory for Protection and Utilization of Tropical Bioresources, Hainan Institute for Tropical Agricultural Resources, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2024, 25(14), 7832; https://doi.org/10.3390/ijms25147832
Submission received: 20 May 2024 / Revised: 28 June 2024 / Accepted: 12 July 2024 / Published: 17 July 2024
(This article belongs to the Special Issue Molecular Research of Tropical Fruit)
Browse Figures
Review Reports Versions Notes

Abstract

:
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.

1. 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.

2. Results

2.1. Identification and Physicochemical Characterization of MaAMY Proteins in Banana

All AMY protein sequences were screened from the banana A genome database [26]. A total of 12 AMY proteins, named MaAMY01–MaAMY12 based on their order on the chromosomes, were identified and possessed a conserved ACD (PF00128). The physicochemical characteristics of the MaAMYs were systematically analyzed (Table S1). The coding sequences (CDSs) and amino acid (aa) lengths were in the range of 285 (MaAMY01)–2841 bp (MaAMY11) and 94 (MaAMY01)–948 aa (MaAMY11), respectively. C, H, N, O, and S constituted the main elemental composition of their amino acids. Their isoelectric points and molecular weights were in the ranges of 5.20 (MaAMY12)–9.23 (MaAMY03) and 10.89 (MaAMY01)–106.66 kDa (MaAMY11), respectively. Their aliphatic indexes ranged from 67.10 (MaAMY04) to 93.19 (MaAMY01), indicating that most were thermostable. All proteins except MaAMY02, 03, 07, 08, 09, and 10 were unstable (>40), and all except MaAMY01 were hydrophilic (<0), according to the instability calculations and grand average of hydropathicity (GRAVY) scores. MaAMY01, 02, 03, and 11 were localized in the chloroplast; MaAMY04, 05, and 06 were localized in the nucleus; MaAMY07, 08, 09, and 12 were localized in the cytoplasm; and MaAMY10 was localized in the vacuole (Table S1), implying that they may be involved in different biological processes.

2.2. Motifs, Gene Structure, and Multiple Sequence Alignments of MaAMY Family Members

Within the banana genome, 12 MaAMY proteins were classified into groups I and II; MaAMY01, 02, 03, 07, 08, 09, 10, and 12 were in group I; MaAMY04, 05, 06, and 11 were in group II (Figure 1A). The MaAMY members in group I contained motifs 2–12, while the MaAMYs in group II contained conserved motif 11 (Figure 1B). The exon–intron structure of the MaAMYs was further analyzed (Figure 1C). Group I had relatively few introns, with 1–4 introns found in MaAMY01, 07, 08, 09, 10, and 12. In group II, MaAMY04, 05, 06, and 11 had 12 introns (Figure 1C), implying that the MaAMYs in group II have similar motifs and exon–intron structures. Multiple sequence alignments showed that each MaAMY protein contained an ACD (Figure 1D and Table S1), which is a hallmark of AMY proteins [3,10]. In addition, an alpha-amylase C-terminal domain or a signal peptide was found in MaAMY02, 04, 05, 06, 08, 09, 10, 11, and 12 (Table S2). Interestingly, MaAMY11 has the longest amino acid sequence in the gene families compared with bananas and other plants (Figure 1D and Figure S1).

2.3. 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.

2.4. Chromosomal Localization and Tandem Duplication of MaAMYs and Cis-acting Element Analyses of MaAMY Gene Promoters

Twelve MaAMY genes were mapped on six chromosomes in bananas. These included MaAMY01, 02, and 03 on Chr 3; MaAMY04, 05, and 06 on Chr 4; MaAMY07, 08, and 09 on Chr 5; MaAMY10 on Chr 7; MaAMY11 on Chr 8; and MaAMY12 on Chr 10 (Figure 3A). Moreover, Figure 3A revealed the discovery of three tandem gene pairs (MaAMY01, 02, and 03; MaAMY04, 05, and 06; MaAMY07 and 08) which may have undergone gene tandem duplication events.
A promoter region including a 2000 bp upstream sequence of each MaAMY gene was selected; promoter structure comparisons and the cis-elements were confirmed by the database (http://www.fruitfly.org/seq_tools/promoter.html, accessed on 20 June 2023) and PlantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 20 June 2023), respectively. Notably, these promoter regions did not contain 5′ untranslated region (UTR) introns, but all of the promoters contained transcription start sites (TSSs). The location information for the TSSs is provided in Table S4. The typical core sequences, namely, the TATA-box (9–144) and CAAT-box (8–20), were enriched in each MaAMY promoter region (Figure 3B). Cis-phytohormone-responsive, development- and growth-related, stress-responsive, light-responsive, or circadian-control elements were found in most MaAMY promoters (Figure 3B). The phytohormone-related elements ABRE, ARE, CGTCA, and GARE were identified in the MaAMY11, 09, 07, and 05 promoters. Abiotic stress-related elements, including LTR and MBS, were identified in the MaAMY07, 04, and 01 promoters. Box4, G-box, GT1-box, and I-box light-responsive-related elements were identified in the MaAMY01 and 06 promoters. The development-related elements (MRE and O2-site) existed in the MaAMY11, 09, 04, or 02 promoters.

2.5. 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.

2.6. MaAMY11 Exhibits Higher Expression than Other MaAMYs in the Fruit or during Ripening

Transcriptome data (SRX3938715, SRX3938722, SRX3938708, SRX3938709, SRX3938706, SRX3938707, and SRX3938704) from various tissues and fruits at different developmental stages were used to analyze MaAMY gene expression patterns (Tables S5 and S6). Five MaAMYs, including MaAMY02, 08, 09, 10, and 11, were expressed (reads per kilobase pair of transcripts per million reads, RPKM > 7.9) in at least two of the tested tissues at 80 days after the emergence of the inflorescence from the pseudostem (DAF) from ‘Cavendish’ (M. acuminata AAA genotype). However, the expression of MaAMY11 was high in both the leaves and fruits (RPKM > 79.6). Moreover, MaAMY01, 03, 04, 05, 06, 07, and 12 were not expressed in different organs (Figure 5A).
The temporal expression patterns of the MaAMYs were analyzed in fruit sampled at 0, 20, and 80 DAF and 8 and 14 days after harvest (DPH) (Figure 5B, Table S6). Except for MaAMY01, 03, 04, 05, 06, 07, 10, and 12, four MaAMY genes (MaAMY02, 08, 09, and 11) were expressed (RPKM > 1.9) during banana development and ripening. Among the expressed MaAMYs, MaAMY11 showed the highest transcriptional accumulation (82.7 < FPKM < 1426.7) at the middle and later stages of banana fruit ripening.
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.

2.7. 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).

2.8. 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 (Figure 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 (Figure 7H–J).

3. Discussion

Starch is an important fundamental substance for yield and quality formation in banana [26,27]. In our previous research, a series of genes associated with banana starch biosynthesis were identified [28,29,30,31]. Recently, some genes or transcription factors involved in banana starch degradation, including BAM9b [32], PHO1 [8], MYB3 [33], bHLH6 [34], MYB16L [35], APETALA2 (AP2a) [36], ERF12 [37], NAC029 [38], BEL1 [25], CCCH33-like2 [39], and C2H2 [40], were reported. AMY is an important enzyme that catalyzes the hydrolysis of fruit starch degradation [41], and the genome-wide characteristics of the MaAMY genes in banana have not yet been assessed. Herein, 12 members (MaAMY01–MaAMY12) were identified in the banana A genome database (Figure 1 and Table S1). An ACD was found in all MaAMYs, and these AMY gene members have relatively conservative motifs and exon–intron structures (Figure 1). The finding corroborates other reports in plants, such as rice [3], wheat [9], and cassava [10]. Notably, we found that MaAMY11 has the longest amino acid sequence compared with other plants (Figure S1). However, further research is necessary to determine whether MaAMY11 has a special function in multiple biological processes.
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 I2-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.

4. Materials and Methods

4.1. 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.

4.2. 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.

4.3. 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].

4.4. 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.toolkit package 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].

4.5. 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-4.4.1 was used to construct a heatmap for quantifying the occurrence of each gene element [37].

4.6. 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.

4.7. 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−ΔΔCT method [61]. Each sample included three replicates.

4.8. 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 (OD600 = 0.6) and placed on a Murashige and Skoog medium, incubated at 30°C for 3 d, and then stained with I2-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.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25147832/s1.

Author Contributions

Conceived and designed the experiments: H.M. and J.L. Performed the experiments: P.S., Z.Z. and H.M. Analyzed the data: Z.Z., H.M. and P.S. Contributed reagents/materials/analysis tools: J.X., J.L. and Z.J. Wrote the paper: H.M., P.S. and Z.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key R&D Program of China (No. 2023YFD2300801), the National Natural Science Foundation of China (NSFC, No. 32172545), the Hainan Provincial Natural Science Foundation of China (No. 321RC636 and ZDYF2023XDNY179), the Central Public-interest Scientific Institution Basal Research Fund (No. 1630052022007 and CATASCXTD202403), and the Modern Agro-industry Technology Research System (No. CARS-32).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Acknowledgments

We thank Qing Liu for language modification assistant.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Seung, D.; Smith, A.M. Starch granule initiation and morphogenesis-progress in Arabidopsis and cereals. J. Exp. Bot. 2019, 70, 771–784. [Google Scholar] [CrossRef]
  2. David, L.C.; Lee, S.K.; Bruderer, E.; Abt, M.R.; Fischer-Stettler, M.; Tschopp, M.A.; Solhaug, E.M.; Sanchez, K.; Zeeman, S.C. BETA-AMYLASE9 is a plastidial nonenzymatic regulator of leaf starch degradation. Plant Physiol. 2022, 188, 191–207. [Google Scholar] [CrossRef] [PubMed]
  3. Damaris, R.N.; Lin, Z.; Yang, P.; He, D. The rice alpha-amylase, conserved regulator of seed maturation and germination. Int. J. Mol. Sci. 2019, 20, 450. [Google Scholar] [CrossRef]
  4. Zhang, Q.; Pritchard, J.; Mieog, J.; Byrne, K.; Colgrave, M.L.; Wang, J.R.; Ral, J.F. Overexpression of a wheat alpha-amylase type 2 impact on starch metabolism and abscisic acid sensitivity during grain germination. Plant J. 2021, 108, 378–393. [Google Scholar] [CrossRef] [PubMed]
  5. Fu, J.; Pei, W.; He, L.; Ma, B.; Tang, C.; Zhu, L.; Wang, L.; Zhong, Y.; Chen, G.; Wang, Q.; et al. ZmEREB92 plays a negative role in seed germination by regulating ethylene signaling and starch mobilization in maize. PLoS Genet. 2023, 19, e1011052. [Google Scholar] [CrossRef] [PubMed]
  6. Peroni, F.H.; Koike, C.; Louro, R.P.; Purgatto, E.; do Nascimento, J.R.; Lajolo, F.M.; Cordenunsi, B.R. Mango starch degradation. ll. The binding of alpha-amylase and beta-amylase to the starch granule. J. Agric. Food Chem. 2008, 56, 7416–7421. [Google Scholar] [CrossRef] [PubMed]
  7. Nardozza, S.; Boldingh, H.L.; Osorio, S.; Höhne, M.; Wohlers, M.; Gleave, A.P.; MacRae, E.A.; Richardson, A.C.; Atkinson, R.G.; Sulpice, R.; et al. Metabolic analysis of kiwifruit (Actinidia deliciosa) berries from extreme genotypes reveals hallmarks for fruit starch metabolism. J. Exp. Box. 2013, 64, 5049–5063. [Google Scholar] [CrossRef]
  8. Miao, H.X.; Sun, P.G.; Zhu, W.N.; Liu, Q.; Zhang, J.B.; Jia, C.H.; Sun, J.M.; Zhu, Z.; Xie, J.H.; Wang, W.; et al. Exploring the function of MaPHO1 in starch degradation and its protein interactions in postharvest banana fruits. Postharvest Biol. Technol. 2024, 209, 112687. [Google Scholar] [CrossRef]
  9. Sethi, S.; Saini, J.S.; Mohan, A.; Brar, N.K.; Verma, S.; Sarao, N.K.; Gill, K.S. Comparative and evolutionary analysis of α-amylase gene across monocots and dicots. Funct. Integr. Genom. 2016, 16, 545–555. [Google Scholar] [CrossRef] [PubMed]
  10. Yang, T.; Li, H.; Li, L.; Wei, W.; Huang, Y.; Xiong, F.; Wei, M. Genome-wide characterization and expression analysis of alpha-amylase and beta-amylase genes underlying drought tolerance in cassava. BMC Genom. 2023, 24, 190. [Google Scholar] [CrossRef] [PubMed]
  11. Zhang, Q.S.; Li, C.D. Comparisons of copy number, genomic structure, and conserved motifs for α-amylase genes from barley, rice, and wheat. Front. Plant Sci. 2017, 8, 1727. [Google Scholar] [CrossRef] [PubMed]
  12. Bak-Jensen, K.S.; Laugesen, S.; Ostergaard, O.; Finnie, C.; Roepstorff, P.; Svensson, B. Spatio-temporal profiling and degradation of alpha-amylase isozymes during barley seed germination. FEBS J. 2007, 274, 2552–2565. [Google Scholar] [CrossRef]
  13. Stanley, D.; Fitzgerald, A.M.; Farnden, K.; Macrae, E.A. Characterisation of putative alpha-amylases from apple (Malus domestica) and Arabidopsis thaliana. In Proceedings of the 1st Symposium on the Alpha-Amylase Family, Smolenice Castle, Slovakia, 30 September–4 October 2002. [Google Scholar]
  14. Søgaard, M.; Svensson, B. Expression of cDNAs encoding barley alpha-amylases 1 and 2 in yeast and characterization of the secreted proteins. Gene 1990, 94, 173–179. [Google Scholar] [CrossRef] [PubMed]
  15. Doyle, E.A.; Lane, A.M.; Sides, J.M.; Mudgett, M.B.; Monroe, J.D. An alpha-amylase (At4g25000) in Arabidopsis leaves is secreted and induced by biotic and abiotic stress. Plant Cell Environ. 2007, 30, 388–398. [Google Scholar] [CrossRef] [PubMed]
  16. Karrer, E.E.; Litts, J.C.; Rodriguez, R.L. Differential expression of alpha-amylase genes in germinating rice and barley seeds. Plant Mol. Biol. 1991, 16, 797–805. [Google Scholar] [CrossRef] [PubMed]
  17. Derkx, A.P.; Mares, D.J. Late-maturity α-amylase expression in wheat is influenced by genotype, temperature and stage of grain development. Planta 2020, 251, 51. [Google Scholar] [CrossRef] [PubMed]
  18. Zhang, Q.; Pritchard, J.; Mieog, J.; Byrne, K.; Colgrave, M.L.; Wang, J.R.; Ral, J.P. Over-expression of a wheat late maturity alpha-amylase type 1 impact on starch properties during grain development and germination. Front. Plant Sci. 2022, 13, 811728. [Google Scholar] [CrossRef] [PubMed]
  19. Asatsuma, S.; Sawada, C.; Itoh, K.; Okito, M.; Kitajima, A.; Mitsui, T. Involvement of alpha-amylase I-1 in starch degradation in rice chloroplasts. Plant Cell Physiol. 2005, 46, 858–869. [Google Scholar] [CrossRef] [PubMed]
  20. Gilbert, M.K.; Majumdar, R.; Rajasekaran, K.; Chen, Z.Y.; Wei, Q.; Sickler, C.M.; Lebar, M.D.; Cary, J.W.; Frame, B.R.; Wang, K. RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels. Planta 2018, 247, 1465–1473. [Google Scholar] [CrossRef] [PubMed]
  21. Hakata, M.; Kuroda, M.; Miyashita, T.; Yamaguchi, T.; Kojima, M.; Sakakibara, H.; Mitsui, T.; Yamakawa, H. Suppression of α-amylase genes improves quality of rice grain ripened under high temperature. Plant Biotechnol. J. 2012, 10, 1110–1117. [Google Scholar] [CrossRef]
  22. Leonel, M.; Bolfarini, A.C.B.; da Silva, M.J.R.; Souza, J.M.A.; Leonel, S. Banana fruits with high content of resistant starch: Effect of genotypes and phosphorus fertilization. Int. J. Biol. Macromol. 2020, 50, 1020–1026. [Google Scholar] [CrossRef] [PubMed]
  23. Bassinello, P.Z.; Cordenunsi, B.R.; Lajolo, F.M. Amylolytic acitivity in fruits: Comparison of different substrates and methods using banana as model. J. Agric. Food Chem. 2002, 50, 5781–5786. [Google Scholar] [CrossRef] [PubMed]
  24. Junior, A.V.; do Nascimento, J.R.O.; Lajolo, F.M. Molecular cloning and characterization of an alpha-amylase occurring in the pulp of ripening bananas and its expression in Pichia pastoris. J. Agric. Food Chem. 2006, 54, 8222–8228. [Google Scholar] [CrossRef] [PubMed]
  25. Song, Z.Y.; Zhu, X.Y.; Lai, X.H.; Chen, H.C.; Wang, L.H.; Yao, Y.L.; Chen, W.X.; Li, X.P. MaBEL1 regulates banana fruit ripening by activating cell wall and starch degradation-related genes. J. Integr. Plant Biol. 2023, 65, 2036–2055. [Google Scholar] [CrossRef] [PubMed]
  26. D’Hont, A.; Denoeud, F.; Aury, J.M.; Baurens, F.C.; Carreel, F.; Garsmeur, O.; Noel, B.; Bocs, S.; Droc, G.; Rouard, M.; et al. The banana (Musa acuminata) genome and the evolution of monocotyledonous plants. Nature 2012, 488, 213–217. [Google Scholar] [CrossRef] [PubMed]
  27. Wang, Z.; Miao, H.X.; Liu, J.H.; Xu, B.Y.; Yao, X.M.; Xu, C.Y.; Zhao, S.C.; Fang, X.D.; Jia, C.H.; Wang, J.Y.; et al. Musa balbisiana genome reveals subgenome evolution and functional divergence. Nat. Plants 2019, 5, 810–821. [Google Scholar] [CrossRef] [PubMed]
  28. Miao, H.X.; Sun, P.G.; Liu, W.X.; Xu, B.Y.; Jin, Z.Q. Identification of genes encoding granule-bound starch synthase involved in amylose metabolism in banana fruit. PLoS ONE 2014, 9, e88077. [Google Scholar] [CrossRef]
  29. Miao, H.X.; Sun, P.G.; Liu, Q.; Liu, J.H.; Xu, B.Y.; Jin, Z.Q. The AGPase family proteins in banana: Genome-wide identification, phylogeny, and expression analyses reveal their involvement in the development, ripening, and abiotic/biotic stress responses. Int. J. Mol. Sci. 2017, 18, 1581. [Google Scholar] [CrossRef] [PubMed]
  30. Miao, H.X.; Sun, P.G.; Liu, Q.; Jia, C.H.; Liu, J.H.; Hu, W.; Jin, Z.Q.; Xu, B.Y. Soluble starch synthase III-1 in amylopectin metabolism of banana fruit: Characterization, expression, enzyme activity, and functional analyses. Front. Plant Sci. 2017, 8, 454. [Google Scholar] [CrossRef] [PubMed]
  31. Miao, H.X.; Sun, P.G.; Liu, Q.; Liu, J.H.; Jia, C.H.; Zhao, D.F.; Xu, B.Y.; Jin, Z.Q. Molecular identification of the key starch branching enzyme-encoding gene SBE2.3 and its interacting transcription factors in banana fruits. Hortic. Res. 2020, 7, 101. [Google Scholar] [CrossRef] [PubMed]
  32. Miao, H.X.; Sun, P.G.; Miao, Y.L.; Liu, J.H.; Zhang, J.B.; Jia, C.H.; Wang, J.Y.; Wang, Z.; Jin, Z.Q.; Xu, B.Y. Genome-wide identification and expression analysis of the β-amylase genes strongly associated with fruit development, ripening, and abiotic stress response in two banana cultivars. Front. Agr. Sci. Eng. 2016, 3, 346–356. [Google Scholar] [CrossRef]
  33. Fan, Z.Q.; Ba, L.J.; Shan, W.; Xiao, Y.Y.; Lu, W.J.; Kuang, J.F.; Chen, J.Y. A banana R2R3-MYB transcription factor MaMYB3 is involved in fruit ripening through modulation of starch degradation by repressing starch degradation-related genes and MabHLH6. Plant J. 2018, 96, 1191–1205. [Google Scholar] [CrossRef] [PubMed]
  34. Xiao, Y.Y.; Kuang, J.F.; Qi, X.N.; Ye, Y.J.; Wu, Z.X.; Chen, J.Y.; Lu, W.J. A comprehensive investigation of starch degradation process and identification of a transcriptional activator MabHLH6 during banana fruit ripening. Plant Biotechnol. J. 2018, 16, 151–164. [Google Scholar] [CrossRef] [PubMed]
  35. Jiang, G.X.; Zhang, D.D.; Li, Z.W.; Liang, H.Z.; Deng, R.F.; Su, X.G.; Jiang, Y.M.; Duan, X.W. Alternative splicing of MaMYB16L regulates starch degradation in banana fruit during ripening. J. Integr. Plant Biol. 2021, 63, 1341–1352. [Google Scholar] [CrossRef] [PubMed]
  36. Xiao, Y.Y.; Li, Y.; Ouyang, L.J.; Yin, A.G.; Xu, B.; Zhang, L.; Chen, J.Y.; Liu, J.F. A banana transcriptional repressor MaAP2a participates in fruit starch degradation during postharvest ripening. Front. Plant Sci. 2022, 13, 1036719. [Google Scholar] [CrossRef] [PubMed]
  37. Chen, H.C.; Lai, X.H.; Wang, L.H.; Li, X.P.; Chen, W.X.; Zhu, X.Y. Ethylene response factor MaERF012 modulates fruit ripening by regulating chlorophyII degradation and softening in banana. Foods 2022, 11, 3882. [Google Scholar] [CrossRef] [PubMed]
  38. Wei, W.; Yang, Y.Y.; Chen, J.Y.; Lakshmanan, P.; Kuang, J.F.; Lu, W.J.; Shan, W. MaNAC029 modulates ethylene biosynthesis and fruit quality and undergoes MaXB3-mediated proteasomal degradation during banana ripening. J. Adv. Res. 2023, 53, 33–47. [Google Scholar] [CrossRef] [PubMed]
  39. Song, Z.Y.; Chen, H.C.; Lai, X.H.; Wang, L.H.; Yao, Y.L.; Qin, J.J.; Pang, X.Q.; Zhu, H.; Chen, W.X.; Li, X.P.; et al. The zinc finger protein MaCCCH33-like2 positively regulates banana fruit ripening by modulating genes in starch and cell wall degradation. Plant Cell Physiol. 2024, 65, 49–67. [Google Scholar] [CrossRef] [PubMed]
  40. Song, Z.; Li, W.; Lai, X.; Chen, H.; Wang, L.; Chen, W.; Li, X.; Zhu, X. MaC2H2-IDD regulates fruit softening and involved in softening disorder induced by cold stress in banana. Plant J. 2024, 118, 1937–1954. [Google Scholar] [CrossRef] [PubMed]
  41. Hu, X.; Kuang, S.; Zhang, A.D.; Zhang, W.S.; Chen, M.J.; Yin, X.R.; Chen, K.S. Characterization of starch degradation related genes in postharvest kiwifruit. Int. J. Mol. Sci. 2016, 17, 2112. [Google Scholar] [CrossRef] [PubMed]
  42. Ma, C.; Hu, R.Y.; Costa, C.; Li, J.K. Genetic drift and purifying selection shaped mitochondrial genome variation in the high royal jelly-producing honeybee strain (Apis mellifera ligustica). Front. Genet. 2022, 9, 835967. [Google Scholar] [CrossRef] [PubMed]
  43. Paterson, S.; Vogwill, T.; Buckling, A.; Benmayor, R.; Spoers, A.J.; Thomson, N.R.; Quail, M.; Smith, F.; Walker, D.; Libberton, B.; et al. Antagonistic coevolution accelerates molecular evolution. Nature 2010, 464, 275–278. [Google Scholar] [CrossRef] [PubMed]
  44. Yue, C.; Cao, H.L.; Lin, H.Z.; Hu, J.; Ye, Y.J.; Li, J.M.; Hao, Z.L.; Hao, X.Y.; Sun, Y.; Yang, Y.J.; et al. Expression patterns of alpha-amylase and beta-amylase genes provide insights into the molecular mechanisms underlying the responses of tea plants (Camellia sinensis) to stress and postharvest processing treatments. Planta 2019, 250, 281–298. [Google Scholar] [CrossRef] [PubMed]
  45. Hwang, Y.S.; Karrer, E.E.; Thomas, B.R.; Chen, L.; Rodriguez, R.L. Three cis-elements required for rice alpha-amylase Amy3D expression during sugar starvation. Plant Mol. Biol. 1998, 36, 331–341. [Google Scholar] [CrossRef] [PubMed]
  46. Lu, C.A.; Lim, E.K.; Yu, S.M. Sugar response sequence in the promoter of a rice alpha-amylase gene serves as a transcriptional enhancer. J. Biol. Chem. 1998, 273, 10120–10131. [Google Scholar] [CrossRef] [PubMed]
  47. Mrva, K.; Wallwork, M.; Mares, D.J. Alpha-amylase and programmed cell death in aleurone of ripening wheat grains. J. Exp. Bot. 2006, 57, 877–885. [Google Scholar] [CrossRef] [PubMed]
  48. Hou, J.; Liu, T.; Reid, S.; Zhang, H.; Peng, X.; Sun, K.; Du, J.; Sonnewald, U.; Song, B. Silencing of alpha-amylase StAmy23 in potato tuber leads to delayed sprouting. Plant Physiol. Biochem. 2019, 139, 411–418. [Google Scholar] [CrossRef] [PubMed]
  49. Nakata, M.; Fukamatsu, Y.; Miyashita, T.; Hakata, M.; Kimura, R.; Nakata, Y.; Kuroda, M.; Yamaguchi, T.; Yamakawa, H. High temperature-induced expression of rice alpha-amylases in developing endosperm produces chalky grains. Front. Plant Sci. 2017, 8, 2089. [Google Scholar] [CrossRef] [PubMed]
  50. Hou, J.; Zhang, H.; Liu, J.; Reid, S.; Liu, T.; Xu, S.; Tian, Z.; Sonnewald, U.; Song, B.; Xie, C. Amylases StAmy23, StBAMY1 and StBAM9 regulate cold-induced sweetening of potato tubers in distinct ways. J. Exp. Bot. 2017, 68, 2317–2331. [Google Scholar] [CrossRef] [PubMed]
  51. Whan, A.; Dielen, A.S.; Mieog, J.; Bowerman, A.; Robinson, H.M.; Byrne, K.; Colgrave, M.; Larkin, P.J.; Howitt, C.A.; Morell, M.K.; et al. Engineering α-amylase levels in wheat grain suggests a highly sophisticated level of carbohydrate regulation during development. J. Exp. Bot. 2014, 65, 5443–5457. [Google Scholar] [CrossRef]
  52. Yu, T.S.; Zeeman, S.C.; Thorneycroft, D.; Fulton, D.C.; Dunstan, H.; Lue, W.L.; Hegemann, B.; Tung, S.Y.; Umemoto, T.; Chapple, A.; et al. Alpha-amylase is not required for breakdown of transitory starch in Arabidopsis leaves. J. Biol. Chen. 2005, 280, 9773–9779. [Google Scholar] [CrossRef] [PubMed]
  53. Jourda, C.; Cardi, C.; Gibert, O.; Toro, A.G.; Ricci, J.; Mbéguié-A-Mbéguié, D.; Yahiaoui, N. Lineage-specific evolutionary histories and regulation of major starch metabolism genes during banana ripening. Front. Plant Sci. 2016, 7, 1778. [Google Scholar] [CrossRef] [PubMed]
  54. Li, L.; Liu, Z.; Pan, X.; Yao, K.D.; Wang, Y.H.; Yang, T.Y.; Huang, G.H.; Liao, W.B.; Wang, C.L. Genome-wide identification and characterization of tomato fatty acid β-oxidase family genes KAT and MFP. Int. J. Mol. Sci. 2024, 25, 2273. [Google Scholar] [CrossRef] [PubMed]
  55. Yin, S.; Zhao, L.L.; Liu, J.Q.; Sun, Y.J.; Li, B.H.; Wang, L.N.; Ren, Z.H.; Chen, C.H. Pan-genome analysis of WOX gene family and function exploration of CsWOX9 in cucumber. Int. J. Mol. Sci. 2023, 24, 17568. [Google Scholar] [CrossRef] [PubMed]
  56. Türkmen, D.; Bowden, J.; Masoli, J.A.H.; Melzer, D.; Pilling, L.C. SLCO1B1 exome sequencing and statin treatment response in 64,000 UK biobank patients. Int. J. Mol. Sci. 2024, 25, 4426. [Google Scholar] [CrossRef]
  57. Chen, C.J.; Wu, Y.; Li, J.W.; Wang, X.; Zeng, Z.H.; Xu, J.; Liu, Y.L.; Feng, J.T.; Chen, H.; Feng, J.T.; et al. TBtools-II: A “one of all, all for one” bioinformatics platform for biological big-data mining. Mol. Plant 2023, 16, 1733–1742. [Google Scholar] [CrossRef] [PubMed]
  58. Wu, L.; Zhang, Y.; Guo, X.; Ning, D.L.; Zhou, X.S.; Feng, J.J.; Yuan, M.M.; Liu, S.; Guo, J.J.; Gao, Z.P.; et al. Reduction of microbial diversity in grassland soil is driven by long-term climate warming. Nat. Microbiol. 2022, 7, 1054–1062. [Google Scholar] [CrossRef] [PubMed]
  59. Gogarten, S.M.; Bhangale, T.; Conomos, M.P.; Laurie, C.A.; McHugh, C.P.; Painter, I.; Zheng, X.W.; Crosslin, D.R.; Levine, D.; Lumley, T.; et al. GWASTools: An R/Bioconductor package for quality control and analysis of genome-wide association studies. Bioinformatics 2012, 28, 3329–3331. [Google Scholar] [CrossRef] [PubMed]
  60. Wang, Y.; Tang, H.; Debarry, J.D.; Tan, X.; Li, J.; Wang, X.; Lee, T.H.; Jin, H.; Marler, B.; Guo, H.; et al. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012, 40, e49. [Google Scholar] [CrossRef] [PubMed]
  61. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
Ijms 25 07832 g001
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. 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.
Ijms 25 07832 g001
Ijms 25 07832 g002
Figure 2. Phylogenetic and Ka/Ks ratio analysis of 108 AMY proteins from banana and 11 other plant species. The phylogenetic tree was constructed with the MEGA X v. 10.1.1 software. (A) The AMYs were divided into three groups, including group I, II, and III, based on the amino acid sequences of the AMYs in banana and other plants. The MaAMYs, OsAMYs, ZmAMYs, StAMYs, TaAMYs, HvAMYs, BdAMYs, SiAMYs, SlAMYs, CaAMYs, SbAMYs, PhAMYs, and AtAMYs, represent AMYs from Musa acuminata, Oryza sativa, Zea mays, Solanum tuberosum, Triticum aestivum, Hordeum vulgare, Brachypodium distachyon, Seturia italica, Solanum lycopersicum, Capsicum annuum, Sorghum bicolor, Panicum hallii, and Arabidopsis thaliana, respectively. (B) Boxplot with scatter points of Ka/Ks values by species.
Figure 2. Phylogenetic and Ka/Ks ratio analysis of 108 AMY proteins from banana and 11 other plant species. The phylogenetic tree was constructed with the MEGA X v. 10.1.1 software. (A) The AMYs were divided into three groups, including group I, II, and III, based on the amino acid sequences of the AMYs in banana and other plants. The MaAMYs, OsAMYs, ZmAMYs, StAMYs, TaAMYs, HvAMYs, BdAMYs, SiAMYs, SlAMYs, CaAMYs, SbAMYs, PhAMYs, and AtAMYs, represent AMYs from Musa acuminata, Oryza sativa, Zea mays, Solanum tuberosum, Triticum aestivum, Hordeum vulgare, Brachypodium distachyon, Seturia italica, Solanum lycopersicum, Capsicum annuum, Sorghum bicolor, Panicum hallii, and Arabidopsis thaliana, respectively. (B) Boxplot with scatter points of Ka/Ks values by species.
Ijms 25 07832 g002
Ijms 25 07832 g003
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. 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.
Ijms 25 07832 g003
Ijms 25 07832 g004
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. 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.
Ijms 25 07832 g004
Ijms 25 07832 g005
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 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.
Ijms 25 07832 g005
Ijms 25 07832 g006
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, represented under “merge”. PC represented the positive control (pCAMBIA1302-GFP plasmid). Scale bars = 10 μm.
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, represented under “merge”. PC represented the positive control (pCAMBIA1302-GFP plasmid). Scale bars = 10 μm.
Ijms 25 07832 g006
Ijms 25 07832 g007
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. 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).
Ijms 25 07832 g007
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Sun, P.; Zhu, Z.; Jin, Z.; Xie, J.; Miao, H.; Liu, J. Molecular Characteristics and Functional Identification of a Key Alpha-Amylase-Encoding Gene AMY11 in Musa acuminata. Int. J. Mol. Sci. 2024, 25, 7832. https://doi.org/10.3390/ijms25147832

AMA Style

Sun P, Zhu Z, Jin Z, Xie J, Miao H, Liu J. Molecular Characteristics and Functional Identification of a Key Alpha-Amylase-Encoding Gene AMY11 in Musa acuminata. International Journal of Molecular Sciences. 2024; 25(14):7832. https://doi.org/10.3390/ijms25147832

Chicago/Turabian Style

Sun, Peiguang, Zhao Zhu, Zhiqiang Jin, Jianghui Xie, Hongxia Miao, and Juhua Liu. 2024. "Molecular Characteristics and Functional Identification of a Key Alpha-Amylase-Encoding Gene AMY11 in Musa acuminata" International Journal of Molecular Sciences 25, no. 14: 7832. https://doi.org/10.3390/ijms25147832

APA Style

Sun, P., Zhu, Z., Jin, Z., Xie, J., Miao, H., & Liu, J. (2024). Molecular Characteristics and Functional Identification of a Key Alpha-Amylase-Encoding Gene AMY11 in Musa acuminata. International Journal of Molecular Sciences, 25(14), 7832. https://doi.org/10.3390/ijms25147832

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop