Banana Fruit Development Research Articles

Changes in hemicellulose metabolism in banana peel during fruit development and ripening

Hemicellulose is key in determining the fate of plant cell wall in almost all growth and developmental stages. Nevertheless, there is limited knowledge regarding its involvement in the development and ripening of banana fruit. This study investigated changes in the temporal-spatial distribution of various hemicellulose components, hemicellulose content, activities of the main hydrolysis enzymes, and transcription level of the main hemicellulose-related gene families in banana peels. Both hemicellulose and xylan contents were positively correlated to the fruit firmness observed in our previous study. On the contrary, the xylanase activity was negatively correlated to xylan content and the fruit firmness. The vascular bundle cells, phloem, and cortex of bananas are abundant in xyloglucan, xylan, and mannan contents. Interestingly, the changes in the signal intensity of the CCRC-M104 antibody recognizing non-XXXG type xyloglucan are positively correlated to hemicellulose content. According to RNA-Seq analysis, xyloglucan and xylan-related genes were highly active in the early stages of growth, and the expression of MaMANs and MaXYNs increased as the fruit ripened. The abundance of plant hormonal and growth-responsive cis-acting elements was detected in the 2 kb upstream region of hemicellulose-related gene families. Interaction between hemicellulose and cell wall-specific proteins and MaKCBP1/2, MaCKG1, and MaHKL1 was found. The findings shed light on cell wall hemicellulose's role in banana fruit development and ripening, which could improve nutrition, flavor, and reduce postharvest fruit losses.

Protein expression and fine interaction mechanism of banana MuMADS1 and MaOFP1

MuMADS1 is an AGAMOUS (AG)-like MADS-box gene previously isolated from banana fruits. Previous investigation demonstrated that the K-domain of MuMADS1 interacts with MaOFP1. However, the expression characteristics of MuMADS1 and MaOFP1 proteins in banana fruits and their fine interaction mechanism need to be further investigated. In the present study, the expression characteristics of MuMADS1 and MaOFP1 proteins during banana fruits development and ripening process was investigated by Western blot. The interaction of MuMADS1 and MaOFP1 was demonstrated again by Pull down. The fine interaction mechanism of K-domain of MuMADS1 interacting with the ovate-domain and non-ovate-domain of MaOFP1 was further investigated by yeast two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assay. The result indicated that MuMADS1 was differentially expressed during Baxi (BX) and Fenjiao (FJ) fruits development and ripening process. With the progress of banana fruit development, the expression level gradually increased and reached the peak at 0 DPH, and then gradually decreased. The expression level of MaOFP1 during BX and FJ fruits development and ripening process was significantly lower than MuMADS1. Moreover, the expression level of MaOFP1 in FJ was higher than in BX. The K-domain of MuMADS1 interacted with the ovate-domain of MaOFP1. These results laid the foundation for deeply understanding the role of MuMADS1 and MaOFP1 in banana fruits and provide gene resources for banana fruit ripening regulation.

Genome-wide identification and characterization of glutathione S-transferase gene family in Musa acuminata L. AAA group and gaining an insight to their role in banana fruit development

Glutathione S-transferases are a multifunctional protein superfamily that is involved in diverse plant functions such as defense mechanisms, signaling, stress response, secondary metabolism, and plant growth and development. Although the banana whole-genome sequence is available, the distribution of GST genes on banana chromosomes, their subcellular localization, gene structure, their evolutionary relation with each other, conserved motifs, and their roles in banana are still unknown. A total of 62 full-length GST genes with the canonical thioredoxin fold have been identified belonging to nine GST classes, namely tau, phi, theta, zeta, lambda, DHAR, EF1G, GHR, and TCHQD. The 62 GST genes were distributed into 11 banana chromosomes. All the MaGSTs were majorly localized in the cytoplasm. Gene architecture showed the conservation of exon numbers in individual GST classes. Multiple Em for Motif Elicitation analyses revealed few class-specific motifs and many motifs were found in all the GST classes. Multiple sequence alignment of banana GST amino acid sequences with rice, Arabidopsis, and soybean sequences revealed the Ser and Cys as conserved catalytic residues. Gene duplication analyses showed the tandem duplication as a driving force for GST gene family expansion in banana. Cis-regulatory element analysis showed the dominance of light-responsive element followed by stress- and hormone-responsive elements. Expression profiling analyses were also done by RNA-seq data. It was observed that MaGSTs are involved in various stages of fruit development. MaGSTU1 was highly upregulated. The comprehensive and organized studies of MaGST gene family provide groundwork for further functional analysis of MaGST genes in banana at molecular level and further for plant breeding approaches.

Genome-Wide Analysis of the Banana WRKY Transcription Factor Gene Family Closely Related to Fruit Ripening and Stress.

WRKY transcription factors (TFs) play an important role in plant responses to biotic and abiotic stress as well as in plant growth and development. In the present study, bioinformatics methods were used to identify members of the WRKY transcription factor family in the Musa acuminata (DH-Pahang) genome (version 2). A total of 164 MaWRKYs were identified and phylogenetic analysis showed that MaWRKYs could be categorized into three subfamilies. Overall, the 162 MaWRKYs were distributed on 11 chromosomes, and 2 genes were not located on the chromosome. There were 31 collinear genes from segmental duplication and 7 pairs of genes from tandem duplication. RNA-sequencing was used to analyze the expression profiles of MaWRKYs in different fruit development, ripening stages, under various abiotic and biotic stressors. Most of the MaWRKYs showed a variety of expression patterns in the banana fruit development and ripening stages. Some MaWRKYs responded to abiotic stress, such as low temperature, drought, and salt stress. Most differentially expressed MaWRKYs were downregulated during banana’s response to Foc TR4 infection, which plays an important role in physiological regulation to stress. Our findings indicate that MaWRKY21 directly binds to the W-box of the MaICS promoter to decrease MaICS transcription and then reduce the enzyme activity. These studies have improved our understanding of the molecular basis for the development and stress resistance of an important banana variety.

Role of apyrase-mediated eATP signal in chilling injury of postharvest banana fruit during storage

Apyrase (APY) might regulate the homeostasis of extracellular ATP (eATP) / intracellular ATP (iATP) and mediate plant growth and defense response, but little information on the roles of APY genes in eATP-mediated chilling response of banana fruit during storage has been available. This study identified a total of 10 full-length banana APY genes, analyzed cis-elements in promoters and potential miRNA target sites and found that these APYs had highly conserved functions and might regulate energy metabolism during banana fruit development, ripening and senescence. Exogenous ATP was used to investigate the roles of APY genes in chilling injury (CI) of banana fruit stored for up to 6 d at 6 °C. Application of 1 mM ATP solution effectively reduced CI symptoms, inhibited malondialdehyde (MDA) accumulation, improved antioxidant capacity, and relatively maintained the homeostasis of iATP / eATP of banana fruit during cold storage. Compared with non-ATP-treated fruit, transcriptional levels of MaAPYs were up-regulated whereas those of nucleoside transporter genes and purine permease genes were down-regulated in ATP-treated banana fruit during cold storage, suggesting that the ATP pretreatment could maintain the relative homeostasis of iATP / eATP. The results provided the new insights into the roles of apyrase-mediated eATP signal in regulating the cold tolerance of banana fruit during storage at low temperature.

Analyses of key gene networks controlling carotenoid metabolism in Xiangfen 1 banana

BackgroundBanana fruits are rich in various high-value metabolites and play a key role in the human diet. Of these components, carotenoids have attracted considerable attention due to their physiological role and human health care functions. However, the accumulation patterns of carotenoids and genome-wide analysis of gene expression during banana fruit development have not been comprehensively evaluated.ResultsIn the present study, an integrative analysis of metabolites and transcriptome profiles in banana fruit with three different development stages was performed. A total of 11 carotenoid compounds were identified, and most of these compounds showed markedly higher abundances in mature green and/or mature fruit than in young fruit. Results were linked to the high expression of carotenoid synthesis and regulatory genes in the middle and late stages of fruit development. Co-expression network analysis revealed that 79 differentially expressed transcription factor genes may be responsible for the regulation of LCYB (lycopene β-cyclase), a key enzyme catalyzing the biosynthesis of α- and β-carotene.ConclusionsCollectively, the study provided new insights into the understanding of dynamic changes in carotenoid content and gene expression level during banana fruit development.

Genome-Wide Identification and Transcript Analysis of TCP Gene Family in Banana (Musa acuminata L.).

Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (TCP) gene family has versatile functions in diverse aspects of plants. However, less research on banana TCPs was done comprehensively. Accordingly, 48 banana TCP genes were characterized on aspects of gene structure, conserved motifs, phylogenetic relationship, and expression patterns. Members of the MaTCP gene family were unevenly distributed among 11 chromosomes and purification selection was the driving force of the MaTCP gene family. Gene duplication analysis indicated that segmental duplication is the major contributor to family expansion. Promoter analysis showed that MaTCPs might be involved in banana growth, development, and abiotic stress responses. Further, the expression of 12 MaTCPs was analyzed by real-time quantitative RT-PCR, and the protein interaction analysis showed that MaPCF10 and MaPCF13 may have an important function in banana fruit development and ripening. These results lay the foundation for further study of the functions of TCP genes in banana.

Characteristics of banana B genome MADS-box family demonstrate their roles in fruit development, ripening, and stress

MADS-box genes are critical regulators of growth and development in flowering plants. Sequencing of the Musa balbisiana (B) genome has provided a platform for the systematic analysis of the MADS-box gene family in the important banana ancestor Musa balbisiana. Seventy-seven MADS-box genes, including 18 type I and 59 type II, were strictly identified from the banana (Pisang Klutuk Wulung, PKW, 2n = 2x = 22) B genome. These genes have been preferentially placed on the banana B genome. Evolutionary analysis suggested that M. balbisiana MCM1-AGAMOUS-DEFICIENS-SRF (MbMADS) might be organized into the MIKCc, MIKC*, Mα, Mβ, and Mγ groups according to the phylogeny. MIKCc was then further categorized into 10 subfamilies according to conserved motif and gene structure analyses. The well-defined MADS-box genes highlight gene birth and death in banana. MbMADSes originated from the same ancestor as MaMADSes. Transcriptome analysis in cultivated banana (ABB) revealed that MbMADSes were conserved and differentially expressed in several organs, in various fruit developing and ripening stages, and in stress treatments, indicating the participation of these genes in fruit development, ripening, and stress responses. Of note, SEP/AGL2 and AG, as well as other several type II MADS-box genes, including the STMADS11 and TM3/SOC1 subfamilies, indicated elevated expression throughout banana fruit development, ripening, and stress treatments, indicating their new parts in controlling fruit development and ripening. According to the co-expression network analysis, MbMADS75 interacted with bZIP and seven other transcription factors to perform its function. This systematic analysis reveals fruit development, ripening, and stress candidate MbMADSes genes for additional functional studies in plants, improving our understanding of the transcriptional regulation of MbMADSes genes and providing a base for genetic modification of MADS-mediated fruit development, ripening, and stress.

Molecular identification of the key starch branching enzyme-encoding gene SBE2.3 and its interacting transcription factors in banana fruits

Starch branching enzyme (SBE) has rarely been studied in common starchy banana fruits. For the first time, we report here the molecular characterization of seven SBE (MaSBE) and six SBE (MbSBE) genes in the banana A- and B-genomes, respectively, which could be classified into three distinct subfamilies according to genome-wide identification. Systematic transcriptomic analysis revealed that six MaSBEs and six MbSBEs were expressed in the developing banana fruits of two different genotypes, BaXi Jiao (BX, AAA) and Fen Jiao (FJ, AAB), among which MaSBE2.3 and MbSBE2.3 were highly expressed. Transient silencing of MaSBE2.3 expression in banana fruit discs led to a significant decrease in its transcription, which coincides with significant reductions in total starch and amylopectin contents compared to those of empty vector controls. The suggested functional role of MaSBE2.3 in banana fruit development was corroborated by its transient overexpression in banana fruit discs, which led to significant enhancements in total starch and amylopectin contents. A number of transcription factors, including three auxin response factors (ARF2/12/24) and two MYBs (MYB3/308), that interact with the MaSBE2.3 promoter were identified by yeast one-hybrid library assays. Among these ARFs and MYBs, MaARF2/MaMYB308 and MaARF12/MaARF24/MaMYB3 were demonstrated via a luciferase reporter system to upregulate and downregulate the expression of MaSBE2.3, respectively.

Soluble Starch Synthase III-1 in Amylopectin Metabolism of Banana Fruit: Characterization, Expression, Enzyme Activity, and Functional Analyses.

Soluble starch synthase (SS) is one of the key enzymes involved in amylopectin biosynthesis in plants. However, no information is currently available about this gene family in the important fruit crop banana. Herein, we characterized the function of MaSSIII-1 in amylopectin metabolism of banana fruit and described the putative role of the other MaSS family members. Firstly, starch granules, starch and amylopectin content were found to increase during banana fruit development, but decline during storage. The SS activity started to increase later than amylopectin and starch content. Secondly, four putative SS genes were cloned and characterized from banana fruit. Among them, MaSSIII-1 showed the highest expression in banana pulp during fruit development at transcriptional levels. Further Western blot analysis suggested that the protein was gradually increased during banana fruit development, but drastically reduced during storage. This expression pattern was highly consistent with changes in starch granules, amylopectin content, and SS activity at the late phase of banana fruit development. Lastly, overexpression of MaSSIII-1 in tomato plants distinctly changed the morphology of starch granules and significantly increased the total starch accumulation, amylopectin content, and SS activity at mature-green stage in comparison to wild-type. The findings demonstrated that MaSSIII-1 is a key gene expressed in banana fruit and responsible for the active amylopectin biosynthesis, this is the first report in a fresh fruit species. Such a finding may enable the development of molecular markers for banana breeding and genetic improvement of nutritional value and functional properties of banana fruit.

Lineage-Specific Evolutionary Histories and Regulation of Major Starch Metabolism Genes during Banana Ripening.

Starch is the most widespread and abundant storage carbohydrate in plants. It is also a major feature of cultivated bananas as it accumulates to large amounts during banana fruit development before almost complete conversion to soluble sugars during ripening. Little is known about the structure of major gene families involved in banana starch metabolism and their evolution compared to other species. To identify genes involved in banana starch metabolism and investigate their evolutionary history, we analyzed six gene families playing a crucial role in plant starch biosynthesis and degradation: the ADP-glucose pyrophosphorylases (AGPases), starch synthases (SS), starch branching enzymes (SBE), debranching enzymes (DBE), α-amylases (AMY) and β-amylases (BAM). Using comparative genomics and phylogenetic approaches, these genes were classified into families and sub-families and orthology relationships with functional genes in Eudicots and in grasses were identified. In addition to known ancestral duplications shaping starch metabolism gene families, independent evolution in banana and grasses also occurred through lineage-specific whole genome duplications for specific sub-families of AGPase, SS, SBE, and BAM genes; and through gene-scale duplications for AMY genes. In particular, banana lineage duplications yielded a set of AGPase, SBE and BAM genes that were highly or specifically expressed in banana fruits. Gene expression analysis highlighted a complex transcriptional reprogramming of starch metabolism genes during ripening of banana fruits. A differential regulation of expression between banana gene duplicates was identified for SBE and BAM genes, suggesting that part of starch metabolism regulation in the fruit evolved in the banana lineage.

The Quest for Golden Bananas: Investigating Carotenoid Regulation in a Fe'i Group Musa Cultivar.

The regulation of carotenoid biosynthesis in a high-carotenoid-accumulating Fe'i group Musa cultivar, "Asupina", has been examined and compared to that of a low-carotenoid-accumulating cultivar, "Cavendish", to understand the molecular basis underlying carotenogenesis during banana fruit development. Comparisons in the accumulation of carotenoid species, expression of isoprenoid genes, and product sequestration are reported. Key differences between the cultivars include greater carotenoid cleavage dioxygenase 4 (CCD4) expression in "Cavendish" and the conversion of amyloplasts to chromoplasts during fruit ripening in "Asupina". Chromoplast development coincided with a reduction in dry matter content and fruit firmness. Chromoplasts were not observed in "Cavendish" fruits. Such information should provide important insights for future developments in the biofortification and breeding of banana.

Genome-Wide Identification and Expression Analyses of Aquaporin Gene Family during Development and Abiotic Stress in Banana.

Aquaporins (AQPs) function to selectively control the flow of water and other small molecules through biological membranes, playing crucial roles in various biological processes. However, little information is available on the AQP gene family in bananas. In this study, we identified 47 banana AQP genes based on the banana genome sequence. Evolutionary analysis of AQPs from banana, Arabidopsis, poplar, and rice indicated that banana AQPs (MaAQPs) were clustered into four subfamilies. Conserved motif analysis showed that all banana AQPs contained the typical AQP-like or major intrinsic protein (MIP) domain. Gene structure analysis suggested the majority of MaAQPs had two to four introns with a highly specific number and length for each subfamily. Expression analysis of MaAQP genes during fruit development and postharvest ripening showed that some MaAQP genes exhibited high expression levels during these stages, indicating the involvement of MaAQP genes in banana fruit development and ripening. Additionally, some MaAQP genes showed strong induction after stress treatment and therefore, may represent potential candidates for improving banana resistance to abiotic stress. Taken together, this study identified some excellent tissue-specific, fruit development- and ripening-dependent, and abiotic stress-responsive candidate MaAQP genes, which could lay a solid foundation for genetic improvement of banana cultivars.

Citrate and malate accumulation in banana fruit (Musa sp. AA) is highly affected by genotype and fruit age, but not by cultural practices

Sourness and sweetness are major drivers of consumer preference for banana fruits and are mainly linked to the presence of citrate and malate. The objectives of the present work were to determine how agro-environmental and genotypic factors affect the concentrations of citrate and malate in banana pulp during growth and postharvest ripening. Changes in citrate and malate concentrations in the pulp during the development of the fruit were investigated in relation to fruit age, fruit load, and potassium fertilization in three cultivars of dessert banana presenting contrasted acidity at the eating stage. Major differences in the pattern of citrate and malate accumulation were found in the three cultivars both during growth and postharvest ripening. The fruit growth rate was greater when the fruit load was reduced, but this treatment had no effect on the accumulation of organic acids in any of the three cultivars. A high potassium supply increased fruit growth but had no effect on organic acid accumulation in any of the three cultivars. Late harvested fruits had higher citrate and lower malate concentrations in the pulp at the eating stage. Our results showed that the concentration of organic acids in banana pulp is mainly controlled by genotype and that this may be an interesting trait to target in breeding programs to improve the organoleptic quality of new cultivars. The physiological mechanisms likely to control the accumulation of citrate and malate during banana fruit development are discussed.

The interaction of banana MADS-box protein MuMADS1 and ubiquitin-activating enzyme E-MuUBA in post-harvest banana fruit

KEY MESSAGE : The interaction of MuMADS1 and MuUBA in banana was reported, which will help us to understand the mechanism of the MADS-box gene in regulating banana fruit development and ripening. The ubiquitin-activating enzyme E1 gene fragment MuUBA was obtained from banana (Musa acuminata L.AAA) fruit by the yeast two-hybrid method using the banana MADS-box gene MuMADS1 as bait and 2-day post-harvest banana fruit cDNA library as prey. MuMADS1 interacted with MuUBA. The interaction of MuMADS1 and MuUBA in vivo was further proved by bimolecular fluorescence complementation assay. Real-time quantitative PCR evaluation of MuMADS1 and MuUBA expression patterns in banana showed that they are highly expressed in the ovule 4 stage, but present in low levels in the stem, which suggests a simultaneously differential expression action exists for both MuMADS1 and MuUBA in different tissues and developmental fruits. MuMADS1 and MuUBA expression was highly stimulated by exogenous ethylene and suppressed by 1-methylcyclopropene. These results indicated that MuMADS1 and MuUBA were co-regulated by ethylene and might play an important role in post-harvest banana fruit ripening.

Involvement of a banana MADS-box transcription factor gene in ethylene-induced fruit ripening

To investigate the regulation of MADS-box genes in banana (Musa acuminata L. AAA group cv. Brazilian) fruit development and postharvest ripening, we isolated from banana fruit a MADS-box gene designated MuMADS1. Amino acid alignment indicated MuMADS1 belongs to the AGAMOUS subfamily, and phylogenetic analysis indicates that this gene is most similar to class D MADS-box genes. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that MuMADS1 is expressed in the stamen and pistil of male and female flowers and in the rhizome, the vegetative reproductive organ of the banana plant. In preharvest banana fruit, MuMADS1 is likely expressed throughout banana fruit development. In postharvest banana ripening, MuMADS1 is associated with ethylene biosynthesis. Expression patterns of MuMADS1 during postharvest ripening as determined by real-time RT-PCR suggest that differential expression of MuMADS1 may not only be induced by ethylene biosynthesis associated with postharvest banana ripening, but also may be induced by exogenous ethylene.

Use of suppression subtractive hybridization approach to identify genes differentially expressed during early banana fruit development undergoing changes in ethylene responsiveness

Little is known about the early green developmental stage of banana fruit before the commercial harvesting stage. In this study, we demonstrate that banana fruit (cv Cavendish) grown under our pedoclimatical conditions undergoes changes in ethylene responsiveness between 40 (immature fruit unable to respond to ethylene), 60 and 90 DAF (days after flowering; early and late mature fruit able to respond to ethylene, respectively). Further, we have combined subtractive suppression hybridization (SSH) and macro-array hybridization to construct four different SSH libraries comprising a total of 3072 clones and identify 876 clones that are differentially expressed during fruit ripening. Some of these positive clones were partially sequenced to generate ESTs. Sequence analysis revealed that 163 clones on 177 (92%) presented a high similarity with different genes in the database and were related to various plant mechanisms. Northern blot analysis and real-time quantitative PCR conducted on 24 selected clones showed that the subtraction worked properly and led to get more insights into the early green developmental stages of banana. These genes will contribute to increase pools of public EST collections of banana for identification of candidate genes and the providing of molecular markers usable to improve banana fruit quality throughout conventional breeding or biotechnology approaches.

Differential expression of genes during banana fruit development, ripening and 1-MCP treatment: Presence of distinct fruit specific, ethylene induced and ethylene repressed expression

Fruit ripening in climacteric fruit is triggered by ethylene and associated with the expression of hundreds of genes that influence various ripening related changes. We have used mRNA differential display to identify 22 ripening related genes in banana of which only two have previously been characterized in banana. Transcript analyses show that six genes are down-regulated and 16 up-regulated during the course of ripening with varied patterns of transcript accumulation. Treatment with 1-methylcyclopropene inhibits ripening and represses the expression of most of the up-regulated genes indicating that their expression is directly or indirectly governed by ethylene. The expression of most of the up- and down-regulated genes is specific to fruit and not observed in other tissues. Short time ethylene treatment of banana fruit reveals the induction of four up-regulated genes within 10–30 min of ethylene treatment and the repression of two down-regulated genes within 30–60 min of ethylene treatment. Sequence analyses reveal homology to genes that are involved in diverse processes such as gene regulation/signaling, defense, softening and other unknown functions. The studies have identified several candidate genes whose promoters could be used for isolation of specific cis elements involved in fruit specific expression as well as those responsible for ethylene induction and repression.

Experiments on Banana Fruit Development

A number of synthetic auxins and auxin-antagonists were applied in lanolin emulsions to young banana fruits and their effects on fruit development and seed fertility estimated. Auxins stimulated the formation of edible pulp in both seedy and parthenocarpic bananas and induced varying degrees of seed sterility. Of the auxin antagonists tried, coumarin (contrary to a mistaken earlier report) stimulated parthenocarpy and induced seed sterility, three others were not or but slightly active, and thiourea markedly inhibited parthenocarpy. Study of the relation between size of mature fruit and seed content indicates that developing seeds stimulate the growth of pulp and that the amount of stimulation is roughly proportional to the level of parthenocarpy. Fruit development in the banana seems to fit in with Luckwill's general scheme of dual hormonal stimulus.