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Bananas are model fruits for studying starch conversion and climactericity. Starch degradation and ripening are two important biological processes that occur concomitantly in banana fruit. Ethylene biosynthesis and postharvest fruit ripening processes, i.e. starch degradation, fruit softening, and sugar accumulation, are highly correlated and thus could be controlled by a common regulatory switch. However, this switch has not been identified. In this study, we transformed red banana (Musa acuminata L.) with sense and anti-sense constructs of the MaMADS36 transcription factor gene (also MuMADS1, Ma05_g18560.1). Analysis of these lines showed that MaMADS36 interacts with 74 other proteins to form a co-expression network and could act as an important switch to regulate ethylene biosynthesis, starch degradation, softening, and sugar accumulation. Among these target genes, musa acuminata beta-amylase 9b (MaBAM9b, Ma05_t07800.1), which encodes a starch degradation enzyme, was selected to further investigate the regulatory mechanism of MaMADS36. Our findings revealed that MaMADS36 directly binds to the CA/T(r)G box of the MaBAM9b promoter to increase MaBAM9b transcription and, in turn, enzyme activity and starch degradation during ripening. These results will further our understanding of the fine regulatory mechanisms of MADS-box transcription factors in regulating fruit ripening, which can be applied to breeding programs to improve fruit shelf-life.

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.

The BAHD family is involved in different biological roles in plants, including secondary metabolite synthesis, improving abiotic/biotic stress resistance, and influencing fruit quality. However, the knowledge about BAHD in banana, an important fruit crop, is limited. In this study, 46 banana BAHD genes (MaBAHDs) were identified and divided into four groups according to phylogenetic analysis. Most of the MaBAHD genes in the same group presented similar conserved motifs and genetic structures. MaBAHD genes have similar expression patterns in two banana varieties, and more genes showed high expressions in the roots. The comprehensive MaBAHD gene expression patterns obtained from two varieties of banana showed valuable information regarding their participation in fruit development, ripening, and response to abiotic/biotic stresses, suggesting that they play key roles in these processes. The systematic analysis of MaBAHD genes offered basic insight for further gene functional assays and potential applications in genetically improving banana cultivars.

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.

Background Drought stress can severely affect plant growth and crop yield. The cloning and identification of drought-inducible promoters would be of value for genetically-based strategies to improve resistance of crops to drought. Results Previous studies showed that the MaPIP1;1 gene encoding an aquaporin is involved in the plant drought stress response. In this study, the promoter pMaPIP1;1, which lies 1362 bp upstream of the MaPIP1;1 transcriptional initiation site, was isolated from the banana genome..And the transcription start site(A) is 47 bp before the ATG. To functionally validate the promoter, various lengths of pMaPIP1;1 were deleted and fused to GUS to generate pMaPIP1;1::GUS fusion constructs that were then transformed into Arabidopsis to generate four transformants termed M-P1, M-P2, M-P3 and M-P4.Mannitol treatment was used to simulate drought conditions. All four transformants reacted well to mannitol treatment. M-P2 (− 1274 bp to − 1) showed the highest transcriptional activity among all transgenic Arabidopsis tissues, indicating that M-P2 was the core region of pMaPIP1;1. This region of the promoter also confers high levels of gene expression in response to mannitol treatment. Using M-P2 as a yeast one-hybrid bait, 23 different transcription factors or genes that interacted with MaPIP1;1 were screened. In an dual luciferase assay for complementarity verification, the transcription factor MADS3 positively regulated MaPIP1;1 transcription when combined with the banana promoter. qRT-PCR showed that MADS3 expression was similar in banana leaves and roots under drought stress. In banana plants grown in 45% soil moisture to mimic drought stress, MaPIP1;1 expression was maximized, which further demonstrated that the MADS3 transcription factor can synergize with MaPIP1;1. Conclusions Together our results revealed that MaPIP1;1 mediates molecular mechanisms associated with drought responses in banana, and will expand our understanding of how AQP gene expression is regulated. The findings lay a foundation for genetic improvement of banana drought resistance.

Background Heterozygous genomes are widespread in outcrossing and clonally propagated crops. However, the variation in heterozygosity underlying key agronomic traits and crop domestication remains largely unknown. Cassava is a staple crop in Africa and other tropical regions and has a highly heterozygous genome. Results We describe a genomic variation map from 388 resequenced genomes of cassava cultivars and wild accessions. We identify 52 loci for 23 agronomic traits through a genome-wide association study. Eighteen allelic variations in heterozygosity for nine candidate genes are significantly associated with seven key agronomic traits. We detect 81 selective sweeps with decreasing heterozygosity and nucleotide diversity, harboring 548 genes, which are enriched in multiple biological processes including growth, development, hormone metabolisms and responses, and immune-related processes. Artificial selection for decreased heterozygosity has contributed to the domestication of the large starchy storage root of cassava. Selection for homozygous GG allele in MeTIR1 during domestication contributes to increased starch content. Selection of homozygous AA allele in MeAHL17 is associated with increased storage root weight and cassava bacterial blight (CBB) susceptibility. We have verified the positive roles of MeTIR1 in increasing starch content and MeAHL17 in resistance to CBB by transient overexpression and silencing analysis. The allelic combinations in MeTIR1 and MeAHL17 may result in high starch content and resistance to CBB. Conclusions This study provides insights into allelic variation in heterozygosity associated with key agronomic traits and cassava domestication. It also offers valuable resources for the improvement of cassava and other highly heterozygous crops.

Aquaporins can improve the ability of plants to resist abiotic stresses, but the mechanism is still not completely clear. In this research, overexpression of MaPIP1;1 in banana improved tolerance to multiple stresses. The transgenic plants resulted in lower ion leakage and malondialdehyde content, while the proline, chlorophyll, soluble sugar, and abscisic acid (ABA) contents were higher. In addition, under high salt and recovery conditions, the content of Na+ and K+ is higher, also under recovery conditions, the ratio of K+/Na+ is higher. Finally, under stress conditions, the expression levels of ABA biosynthesis and response genes in the transgenic lines are higher than those of the wild type. In previous studies, we proved that the MaMADS3 could bind to the promoter region of MaPIP1;1, thereby regulating the expression of MaPIP1;1 and affecting the drought tolerance of banana plants. However, the mechanism of MaPIP1;1 gene response to stress under different adversity conditions might be regulated differently. In this study, we proved that some transcription factor genes, including MaERF14, MaDREB1G, MaMYB1R1, MaERF1/39, MabZIP53, and MaMYB22, showed similar expression patterns with MaPIP1;1 under salt or cold stresses, and their encoded proteins could bind to the promoter region of MaPIP1;1. Here we proposed a novel MaPIP1;1-mediated mechanism that enhanced salt and cold tolerance in bananas. The results of this study have enriched the stress-resistant regulatory network of aquaporins genes and are of great significance for the development of molecular breeding strategies for stress-resistant fruit crops.

Avocado (Persea americana) is a tropical fruit that has drawn great interest its oil for foods and cosmetic industries; however, avocado oil processing by-product is a potential source of edible protein. Herein, edible protein was prepared from defatted avocado meal, and it's physicochemical, functional and emulsion properties were investigated. The avocado protein showed U-shaped exhibiting strong effect of pH, and a minimum solubility being observed at pH 4.5, confirming the isoelectric point of avocado protein. Nutritionally, the avocado protein contains all the essential amino acids. Avocado protein provided higher water and oil absorption capacities, higher radical scavenging capacity but lower in-vitro digestibility compared with soy protein. Furthermore, the avocado protein as emulsifier afforded a stability oil-in-water emulsion system, resulting in a greater emulsifying stability than that of soy protein. The present results highlight the potential source of edible protein from avocado oil processing by-products for functional food ingredients.

Banana cultivars (Musa ssp.) are diploid, triploid and tetraploid hybrids derived from Musa acuminata and Musa balbisiana. We presented a high-quality draft genome assembly of M. balbisiana with 430 Mb (87%) assembled into 11 chromosomes. We identified that the recent divergence of M. acuminata (A-genome) and M. balbisiana (B-genome) occurred after lineage-specific whole-genome duplication, and that the B-genome may be more sensitive to the fractionation process compared to the A-genome. Homoeologous exchanges occurred frequently between A- and B-subgenomes in allopolyploids. Genomic variation within progenitors resulted in functional divergence of subgenomes. Global homoeologue expression dominance occurred between subgenomes of the allotriploid. Gene families related to ethylene biosynthesis and starch metabolism exhibited significant expansion at the pathway level and wide homoeologue expression dominance in the B-subgenome of the allotriploid. The independent origin of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) homoeologue gene pairs and tandem duplication-driven expansion of ACO genes in the B-subgenome contributed to rapid and major ethylene production post-harvest in allotriploid banana fruits. The findings of this study provide greater context for understanding fruit biology, and aid the development of tools for breeding optimal banana cultivars., A high-quality draft genome assembly of Musa balbisiana (the banana B-genome) is reported. Comparative genomic and gene functional analyses provide insights into the subgenome evolution and functional divergence between the A- and B-subgenomes.

Calcium-dependent protein kinases (CDPKs) play vital roles in the regulation of plant growth, development, and tolerance to various abiotic stresses. However, little information is available for this gene family in banana. In this study, 44 CDPKs were identified in banana and were classified into four groups based on phylogenetic, gene structure, and conserved motif analyses. The majority of MaCDPKs generally exhibited similar expression patterns in the different tissues. Transcriptome analyses revealed that many CDPKs showed strong transcript accumulation at the early stages of fruit development and postharvest ripening in both varieties. Interaction network and co-expression analysis further identified some CDPKs-mediated network that was potentially active at the early stages of fruit development. Comparative expression analysis suggested that the high levels of CDPK expression in FJ might be related to its fast ripening characteristic. CDPK expression following the abiotic stress treatments indicated a significant transcriptional response to osmotic, cold, and salt treatment, as well as differential expression profiles, between BX and FJ. The findings of this study elucidate the transcriptional control of CDPKs in development, ripening, and the abiotic stress response in banana. Some tissue-specific, development/ripening-dependent, and abiotic stress-responsive candidate MaCDPK genes were identified for further genetic improvement of banana.

Lots of bananas were wasted before commercialization. It is necessary to search potential industrial applications of banana. In the present study, starches from seven banana cultivars (labeled as A-G) were isolated and then characterized. These starches presented different and irregular shapes, such as sphere, long spheroid and polygonal granules. The distribution of size and analyses of average molecular weight showed more small granules in samples B, D, F and G than other samples. The amylose content varied from 22.59% to 38.40%. The crystal types of these starches were a mixture of B-type and C-type, and the relative crystallinity varied greatly. The differential scanning calorimetry (DSC) results showed that the onset temperature of gelatinization increased as follows: A B E C ≈ D F. The maximum viscosity of banana starch decreased as follows: G C D F E = B A. The in vitro digestibility test showed that the content of resistant starch was very high in banana starches. These results would be useful to the application of those starches in food and nonfood industries.

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.

Polyphenols based bioactive compounds from vegetables and fruits are known for impressive antioxidant activity. Ingestion of these antioxidants may promote human health against cardiovascular diseases and cancer. Mango is a popular tropical fruit with special taste, high nutritional value and health-enhancing metabolites. The aim was to investigate the diversity of phytochemicals between two mango cultivars of china at three stages of fruit maturity. We used ESI-QTRAP-MS/MS approach to characterize comprehensively the metabolome of two mango cultivars named Hongguifei (HGF) and Tainong (TN). HPLC was used to quantify selected catechin based phenolic compounds. Moreover, real-time qPCR was used to study the expression profiles of two key genes (ANR and LAR) involved in proanthocyanidin biosynthesis from catechins and derivatives. A total of 651 metabolites were identified, which include at least 257 phenolic compounds. Higher number of metabolites were differentially modulated in peel as compared to pulp. Overall, the relative quantities of amino acids, carbohydrates, organic acids, and other metabolites were increased in the pulp of TN cultivar. While the contents of phenolic compounds were relatively higher in HGF cultivar. Moreover, HPLC based quantification of catechin and derivatives exhibited cultivar specific variations. The ANR and LAR genes exhibited an opposite expression profile in both cultivars. Current study is the first report of numerous metabolites including catechin-based derivatives in mango fruit. These findings open novel possibilities for the use of mango as a source of bioactive compounds.

Background: The genetic basis of metabolic pathways that operate during fruit ripening needs to be understood before the nutritional value of the banana can be improved. The banana is a typical starch conversion fruit, and β-amylase is a key enzyme that may play an important role in starch degradation during the ripening process. Musa acuminata β-amylase 9b (MaBAM9b) is closely related to starch degradation. However, its exact function in starch degradation has not been demonstrated in banana. Stable genetic transformation to identify gene function is time- and energy-consuming. Thus, an efficient and rapid method is needed for functional identification. Virus-induced gene silencing (VIGS) is a reverse-genetics method based on RNA-mediated antiviral plant defense that has been used to rapidly identify gene functions in plants. The aim of this study is to optimize a VIGS system and functional elucidation of MaBAM9b in postharvest banana fruits. Results: Using 0.5% iodine-potassium-iodide (I2-KI) staining for 150 s, we found that 1:3 TRV1:TRV2-MaBAM9b cultivated at 30 mmHg for 30 s to an optical density (OD) of 0.8 at 600 nm, and kept on Murashige & Skoog (MS) media for 5 days produced the best silencing results. Under these conditions, the total starch content was greatly increased, whereas the β-amylase activity, soluble sugar content, and expression of endogenous MaBAM9b greatly decreased. Conclusions: The system described here is particularly useful for studying genes and networks involved in starch conversion in fruit, which alone would not produce a visual phenotype. This system will provide a platform for functional genomics and fruit quality improvement in the banana.

Plant aquaporins (AQPs) are subdivided into eight groups: PIPs, NIPs, GIPs, HIPs, XIPs, LIPs, TIPs, and SIPs. However, the genetic function and physiological mechanisms of the SIP group in abiotic stress response in plants remains unknown. In this study, we cloned and characterized a banana SIP group AQP gene, designated MaSIP2-1. MaSIP2-1 showed upregulation after osmotic, salt, and cold treatments. Overexpression of MaSIP2-1 in bananas resulted in smaller leaf area and lower plant height than wild-type (WT) plants under normal growth conditions. Lines over-expressing MaSIP2-1 showed increased tolerance to drought and cold stresses, demonstrating that transgenic lines had higher content of chlorophyll, proline, soluble sugar, and abscisic acid (ABA), but lower levels of ion leakage (IL), malondialdehyde (MDA), H2O2, and gibberellins (GAs), than WT after drought, cold, and recovery conditions. Moreover, transgenic lines had higher expression of ABA biosynthetic and responsive genes than WT under drought and cold conditions. Taken together, our results show that MaSIP2-1 plays a positive role in dwarfing and tolerance to drought and cold stresses through improved osmotic adjustment, less membrane injury, and changes in ABA and GA levels. Therefore, MaSIP2-1 has the potential for utilization to improve abiotic stress tolerance in crop breeding.

The basic helix-loop-helix (bHLH) proteins are a superfamily of transcription factors (TFs) that can bind to specific DNA target sites, playing a central role in a wide range of metabolic, physiological, and developmental processes in higher organisms. However, no systemic analysis of bHLH TFs has been reported in banana, a typical climacteric fruit in tropical and subtropical regions. In our study, 259 MabHLH TF genes were identified in the genome of Musa acuminata (A genome), and phylogenetic analysis indicated that these MabHLHs could be classified into 23 subfamilies with the bHLHs from rice and Arabidopsis. The amino acid sequences of the bHLH domain in all MabHLH protein sequences were quite conserved, especially Arg-12, Arg-13, Leu-23, and Leu-79. Distribution mapping results showed that 258 MabHLHs were localized on the 11 chromosomes in the M. acuminata genome. The results indicated that 40.7% of gene duplication events were located in collinear fragments, and segmental duplications might have played a key role in the expansion of MabHLHs. Moreover, the expression profiles of MabHLHs in different fruit development and ripening stages and under various abiotic and biotic stresses were investigated using available RNA-sequencing data to obtain fruit development, ripening-specific, and stress-responsive candidate genes. Finally, a co-expression network of MabHLHs was constructed by weighted gene co-expression network analysis to elucidate the MabHLHs that might participate in important metabolic biosynthesis pathways in banana during development and the response to stress. A total of 259 MabHLHs were identified, and their sequence features, conserved domains, phylogenetic relationships, chromosomal distributions, gene duplications, expression profiles, and co-expression networks were investigated. This study systematically identified the MabHLHs in the M. acuminata genome at the genome-wide level, providing important candidate genes for further functional analysis. These findings improve our understanding of the molecular basis of developmental and stress tolerance in an important banana cultivar.

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.

Drought and salt stresses often affect plant growth and crop yields. Identification of promoters involved in drought and salt stress responses is of great significance for genetic improvement of crop resistance. Our previous studies showed that aquaporin can respond to drought and salt stresses, but its promoter has not yet been reported in plants. In the present study, cis-acting elements of MaAQP family member promoters were systematically analyzed in banana. Expression of MaTIP1; 2 was induced by drought and salt stresses but not sensitive to cold stress, waterlogging stress, or mechanical damage, and its promoter contained five stress-related cis-acting elements. The MaTIP1; 2 promoter (841 bp upstream of translation initiation site) from banana (Musa acuminata L. AAA group cv. Brazilian) was isolated through genome walking polymerase chain reaction, and found to contain a TATA Box, CAAT box, ABRE element, CCGTCC box, CGTCA motif, and TCA element. Transformation of the MaTIP1; 2 promoter into Arabidopsis to assess its function indicated that it responds to both drought and salt stress treatments. These results suggest that MaTIP1; 2 utilization may improve drought and salt stresses resistance of the transgenic plants by promoting banana aquaporin expression.

Summary Fruit ripening and quality are common botanical phenomena that are closely linked and strictly regulated by transcription factors. It was previously discovered that a banana MADS‐box protein named MuMADS1 interacted with an ovate family protein named MaOFP1 to regulate banana fruit ripening. To further investigate the role of MuMADS1 and MaOFP1 in the regulation of fruit quality, a combination of genetic transformation and transcriptional characterization was used. The results indicated that the co‐expression of MuMADS1 and MaOFP1 in the ovate mutant could compensate for fruit shape and inferior qualities relating to fruit firmness, soluble solids and sugar content. The number of differentially expressed genes (DEGs) was 1395 in WT vs. ovate, with 883 up‐regulated and 512 down‐regulated genes, while the numbers of DEGs gradually decreased with the transformation of MuMADS1 and MaOFP1 into ovate. ‘Starch and sucrose metabolism’ constituted the primary metabolic pathway, and the gene numbers in this pathway were obviously different when MuMADS1 and MaOFP1 were integrated into ovate. A series of metabolic genes involved in cell wall biosynthesis were up‐regulated in the WT vs. ovate, which probably resulted in the firmer texture and lower sugar contents in the ovate fruit. These results demonstrate that MuMADS1 and MaOFP1 are coregulators of fruit quality, facilitating the dissection of the molecular mechanisms underlying fruit quality formation.

Banana (Musa acuminata) is a common starch-conversion fruit. Starch is rapidly degraded and converted to soluble sugars during postharvest banana fruit ripening. The conversion efficiency directly influences fruit firmness, sweetness, and shelf life. Glucan, water dikinase (GWD) is the first rate-limiting enzyme of starch metabolism. Musa acuminata glucan water dikinase 1 (MaGWD1) plays an important role in banana starch metabolism and its expression is regulated by transcription factors (TFs). However, the regulation of MaGWD1 by MADS-box TFs remains unclear. In this study, the molecular mechanism underlying the cooperative regulation of MaGWD1-mediated starch degradation by MaMADS36 and MaMADS55 was investigated. The results indicated that MaMADS36 interacts with MaMADS55. Both MaMADS36 and MaMADS55 could directly bind to the same position on the CA/T(r)G box within the MaGWD1 promoter. MaMADS36 and MaMADS55 cooperatively regulate banana fruit starch degradation via transcriptional control of MaGWD1.These findings improve our understanding of how TFs regulate starch degradation; this information may be used for banana fruit quality improvement and to support the development of the banana industry.

Background Abscisic acid (ABA) signaling plays a crucial role in developmental and environmental adaptation processes of plants. However, the PYL-PP2C-SnRK2 families that function as the core components of ABA signaling are not well understood in banana. Results In the present study, 24 PYL, 87 PP2C, and 11 SnRK2 genes were identified from banana, which was further supported by evolutionary relationships, conserved motif and gene structure analyses. The comprehensive transcriptomic analyses showed that banana PYL-PP2C-SnRK2 genes are involved in tissue development, fruit development and ripening, and response to abiotic stress in two cultivated varieties. Moreover, comparative expression analyses of PYL-PP2C-SnRK2 genes between BaXi Jiao (BX) and Fen Jiao (FJ) revealed that PYL-PP2C-SnRK2-mediated ABA signaling might positively regulate banana fruit ripening and tolerance to cold, salt, and osmotic stresses. Finally, interaction networks and co-expression assays demonstrated that the core components of ABA signaling were more active in FJ than in BX in response to abiotic stress, further supporting the crucial role of the genes in tolerance to abiotic stress in banana. Conclusions This study provides new insights into the complicated transcriptional control of PYL-PP2C-SnRK2 genes, improves the understanding of PYL-PP2C-SnRK2-mediated ABA signaling in the regulation of fruit development, ripening, and response to abiotic stress, and identifies some candidate genes for genetic improvement of banana. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-1093-4) contains supplementary material, which is available to authorized users.

Background: Banana (Musa spp.) is an important staple food, economic crop, and nutritional fruit worldwide. Conventional breeding has been seriously hampered by their long generation time, polyploidy, and sterility of most cultivated varieties. Establishment of an efficient regeneration and transformation system for banana is critical to its genetic improvement and functional genomics. Results: In this study, a vigorous and repeatable transformation system for banana using direct organogenesis was developed. The greatest number of shoots per explant for all five Musa varieties was obtained using Murashige and Skoog medium supplemented with 8.9 μM benzylaminopurine and 9.1 μM thidiazuron. One immature male flower could regenerate 380–456, 310–372, 200–240, 130–156, and 100–130 well-developed shoots in only 240–270 d for Gongjiao, Red banana, Rose banana, Baxi, and Xinglongnaijiao, respectively. Longitudinal sections of buds were transformed through particle bombardment combined with Agrobacterium-mediated transformation using a promoterless β-glucuronidase (GUS) reporter gene; the highest transformation efficiency was 9.81% in regenerated Gongjiao plantlets in an optimized selection medium. Transgenic plants were confirmed by a histochemical assay of GUS, polymerase chain reaction, and Southern blot. Conclusions: Our robust transformation platform successfully generated hundreds of transgenic plants. Such a platform will facilitate molecular breeding and functional genomics of banana.

Aquaporins (AQPs) transport water and other small molecules; however, their precise role in abiotic stress responses is not fully understood. In this study, we cloned and characterized the PIP2 group AQP gene, MaPIP2-7, in banana. MaPIP2-7 expression was upregulated after osmotic (mannitol), cold, and salt treatments. Overexpression of MaPIP2-7 in banana improved tolerance to multiple stresses such as drought, cold, and salt. MaPIP2-7 transgenic plants showed lower levels of malondialdehyde (MDA) and ion leakage (IL), but higher contents of chlorophyll, proline, soluble sugar, and abscisic acid (ABA) compared with wild type (WT) plants under stress and recovery conditions. Additionally, MaPIP2-7 overexpression decreased cellular contents of Na+ and K+ under salt and recovery conditions, and produced an elevated K+/Na+ ratio under recovery conditions. Finally, ABA biosynthetic and responsive genes exhibited higher expression levels in transgenic lines relative to WT under stress conditions. Taken together, our results demonstrate that MaPIP2-7 confers tolerance to drought, cold, and salt stresses by maintaining osmotic balance, reducing membrane injury, and improving ABA levels.

Ovate Family Proteins (OFPs) belong to a plant-specific transcription factor family. They have been found to have significant roles in growth and development in Arabidopsis and tomato; however, little is known regarding their role in banana. Thus, a genome-wide study of OFP genes in banana was conducted for the first time in the present study. The results demonstrated that 49 OFP family members are unequally distributed across 11 chromosomes. Phylogenetic analysis grouped these genes into two subfamilies and eight subgroups, which was confirmed by the conserved motif and gene structure analysis. Furthermore, MaOFPs genes duplicates were found to have originated from whole-genome duplication (WGD). The expression patterns of the genes in the various tissues and at different fruit development and ripening stages in the BaXi Jiao (BX) and Feng Jiao (FJ), banana cultivars were elucidated using transcriptome analysis. Using co-expression network analysis, MaOFP1 was found to interact not only with MaMADS36 but also with hormone response proteins. These findings improve our understanding of the functions of MaOFPs genes in the control of plant hormone signal transduction pathways during banana growth and ripening, which should inform the genetic improvement of important agricultural characters.

Phenylalanine ammonia lyase (PAL) is the first enzyme in the phenylpropanoid pathway. In this study, we describe the molecular characteristics of four PAL genes (MaPAL1, MaPAL2, MaPAL3, and MaPAL4) cloned from banana (Musa acuminata L. AAA group, cv. Cavendish) using a rapid amplification of cDNA ends and real time quantitative PCR. The predicted molecular masses of corresponding PALs ranged from 70.2 to 77.3 kDa and their isoelectric points were acidic. At the amino acid level, they shared a high sequence similarity with PALs in the banana DH-Pahang (AA group) genome. Phylogenetic analysis shows that the deduced amino acid sequences of MaPALs had also a high similarity with PALs of other plant species. Expression analysis by semi-quantitative reverse transcriptase PCR reveals that these genes were differentially expressed in various tissues. Enzyme activity of PAL and expression of MaPALs in bananas were induced after infection with Fusarium oxysporum f. sp cubense Tropical Race 4. Our findings suggest that MaPALs play important roles in banana resistance to F. oxysporum.

MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs that regulate targeted mRNAs by post-transcription; thus, miRNAs are considered post-transcription regulators. Although numerous miRNAs have been found in model plants, information on miRNAs in banana is limited. In this study, 139 members in 38 miRNA families were determined by sequencing the small RNA (sRNA) transcriptomes of Fusarium wilt-resistant and susceptible banana varieties, and six out of eight new miRNAs were confirmed by RT-PCR. According to the sRNA transcriptome data and qRT-PCR verification, some miRNAs were differentially expressed between Fusarium wilt-resistant and susceptible banana varieties. A total of 293 and 31 target genes were predicted based on the draft maps of banana A genome and Fusarium oxysporum (FOC1, FOC4) genomes, respectively. Two important pathogenic genes in F. oxysporum genomes, namely, feruloyl esterase gene and proline iminopeptidase gene, were targeted by banana miRNAs. These novel findings may provide a new strategy for preventing and controlling Fusarium wilt in banana.

Plant translation elongation factor 1 alpha (EF1A) is both a protein synthesis factor and an important component of plant signal transduction, immune responses, protein trafficking, and apoptosis. However, its role in plant growth and development remains unclear. Herein, a full-length EF1A gene was isolated from banana (Musa acuminata L.) fruit and termed MaEF1A. We found that MaEF1A shared a high sequence identify with respective genes in other plants and the deduced amino acid sequence contained conserved regions of GTP-EFTU, GTP-EFTU-02, and GTP-EFTU-03, as well as two tRNA binding domains and six GTP-binding sites which represent functional domains for protein biosynthesis. MaEF1A protein is mainly localized to the nucleus. MaEF1A was constitutively expressed in different banana organs including developing fruits, and the highest expression was detected in ovary 4 stage. Arabidopsis thaliana L. (ecotype Columbia) was transformed with MaEF1A and four transgenic lines were obtained. Three transgenic lines were selected for further phenotypic analyses. Our findings indicate that overexpressed MaEF1A could greatly enhance plant height, root length, and both rhachis and silique length by promoting cell expansion and elongation. These experiments suggest an important role for MaEF1A in plant growth and development.

The calcium-dependent protein kinase (CDPK) family is comprised of essential calcium sensors and responders that play important roles in response to various abiotic stresses in plants. In this study we describe the molecular characteristics of eight CDPK genes (MaCDPK1, MaCDPK2, MaCDPK3, MaCDPK4, MaCDPK5, MaCDPK6, MaCDPK7, and MaCDPK8) cloned from banana (Musa acuminate L. AAA group, cv. Cavendish) using a RACE-PCR-based strategy. The predicted molecular masses of these MaCDPKs ranged from 57.78 to 60.46 kDa and their pIs ranged from 5.89 to 8.97. At the amino acid level, they shared high sequence similarity with CDPKs in the banana DH-Pahang (AA group) genome. Phylogenetic analysis showed that the deduced amino acid sequences of MaCDPKs also had high similarity to CPKs of Arabidopsis. Expression analysis by semi-quantitative RT-PCR revealed that these genes were differently expressed in various tissues. In addition, their expression was regulated by various stress conditions, including exposure to signaling molecules, cold, salinity, drought, and Fusarium oxysporum f specialis(f. Sp) cubense Tropical Race 4 (Foc TR4) infection. MaCDPK3 responded to abiotic stresses, such as drought, cold, and salinity, while MaCDPK2 and MaCDPK4 were sensitive to Foc TR4. Our results indicate that MaCDPKs play important roles in both the development and stress responses of banana plantlets.

MicroRNAs (miRNAs) play an important role in plant resistance to pathogen infections. However, little is known about the role of miRNAs in banana Fusarium wilt, which is the most economically devastating disease in banana production. In the present study, we identified and characterized a total of 18 miR169 family members in banana (Musa acuminata L.) based on small RNA sequencing. The banana miR169 family clustered into two groups based on miRNA evolutionary analysis. Multiple sequence alignment indicated a high degree of sequence conservation in miRNA169 family members across 28 plant species. Computational target prediction algorithms were used to identify 25 targets of miR169 family members in banana. These targets were enriched in various metabolic pathways that include the following molecules: glycine, serine, threonine, pentose, glycerolipids, nucleotide sugars, starch, and sucrose. Through miRNA transcriptomic analysis, we found that ma-miR169a and ma-miR169b displayed high expression levels, whereas the other 16 ma-miR169 members exhibited low expression in the HG and Baxi banana cultivars. Further experiments indicate that there were negative relationships between ma-miR169a, ma-miR169b and their targets basing on their expression levels to Foc4 (Fusarium oxysporum f. sp. cubense tropical race 4) infection in resistant cultivars. But they were low expressed in susceptive cultivars. These results suggested that the expression levels of ma-miR169a and ma-miR169b were consistent with the resistance degree of the banana cultivars to Foc4. The analysis presented here constitutes a starting point to understand ma-miR169-mediated Fusarium wilt resistance at the transcriptional level in banana and predicts possible candidate targets for the genetic improvement of banana resistance to Foc4.

Rho-like GTPases from plants (ROPs) are plant-specific molecular switches that are crucial for plant survival when subjected to abiotic stress. We identified and characterized 17 novel ROP proteins from Musa acuminata (MaROPs) using genomic techniques. The identified MaROPs fell into three of the four previously described ROP groups (Groups II–IV), with MaROPs in each group having similar genetic structures and conserved motifs. Our transcriptomic analysis showed that the two banana genotypes tested, Fen Jiao and BaXi Jiao, had similar responses to abiotic stress: Six genes (MaROP-3b, -5a, -5c, -5f, -5g, and -6) were highly expressed in response to cold, salt, and drought stress conditions in both genotypes. Of these, MaROP5g was most highly expressed in response to salt stress. Co-localization experiments showed that the MaROP5g protein was localized at the plasma membrane. When subjected to salt stress, transgenic Arabidopsis thaliana overexpressing MaROP5g had longer primary roots and increased survival rates compared to wild-type A. thaliana. The increased salt tolerance conferred by MaROP5g might be related to reduced membrane injury and the increased cytosolic K+/Na+ ratio and Ca2+ concentration in the transgenic plants as compared to wild-type. The increased expression of salt overly sensitive (SOS)-pathway genes and calcium-signaling pathway genes in MaROP5g-overexpressing A. thaliana reflected the enhanced tolerance to salt stress by the transgenic lines in comparison to wild-type. Collectively, our results suggested that abiotic stress tolerance in banana plants might be regulated by multiple MaROPs, and that MaROP5g might enhance salt tolerance by increasing root length, improving membrane injury and ion distribution.

Starch branching enzyme (SBE), which is one of the key enzymes associated with amylopectin biosynthesis, plays important roles in variable biological processes. Despite its importance, SBE is rarely studied in the banana (Musa acuminata L.) which is a typical starchy fruit. Here, a family of ten SBE proteins (MaSBE) was firstly identified through genome-wide characterization in M. acuminata, which could be clustered into three subfamilies. Systematic transcriptome analysis revealed temporal and spatial expression variations of MaSBE genes and differential response patterns under abiotic and biotic stresses in both banana genotypes, Fen Jiao (FJ) and BaXi Jiao (BX). Moreover, MaSBE2.4 was temporally regulated during fruit development and ripening as well as in response to various abiotic/biotic stresses in both genotypes. Specifically, MaSBE2.3 expression level was higher in FJ than in BX following cold, salt, and drought stress treatments, and it was specifically induced by fungal infection in BX. Characterization of hormone- and stress-related cis-acting elements in the promoters of MaSBE genes suggests their multiple biological functions. In conclusion, our study provides new insights into the complex transcriptional characteristics of the SBE genes, and demonstrates their crucial roles in improving amylopectin biosynthesis and strengthening stress resistance in banana.

The ovate mutation has frequently been used to study changes in fruit shape but not fruit quality. A deterioration in fruit quality associated with the ovate mutation was discovered in this study. To elucidate how ovate influences the quality of fruit, we performed a proteomics analysis of the fruits of the ovate mutant (LA3543) and wild-type (“Ailsa Craig”, LA2838A) using tandem mass tag analysis. The results indicated that the ovate mutation significantly influences fruit quality in a number of ways, including by reducing the expression of 1-aminocyclopropane-1-carboxylic acid oxidase 3 (ACO3) in ethylene biosynthesis, improving firmness by reducing the amount of pectinesterase and polygalacturonase, reducing sugar accumulation by downregulating the abundance of mannan endo-1,4-β-mannosidase 4, β-galactosidase, and β-amylase, and reducing the malic acid content by downregulating the accumulation of malic enzymes and malate synthase. These findings could inform future improvements in fruit quality.

MADS-box transcription factors play important roles in organ development. In plants, most studies on MADS-box genes have mainly focused on flower development and only a few concerned fruit development and ripening. A new MADS-box gene named MaMADS7 was isolated from banana fruit by rapid amplification of cDNA ends (RACE) based on a MADS-box fragment obtained from a banana suppression subtractive hybridization (SSH) cDNA library. MaMADS7 is an AGAMOUS-like MADS-box gene that is preferentially expressed in the ovaries and fruits and in tobacco its protein product localizes to the nucleus. This study found that MaMADS7 expression can be induced by exogenous ethylene. Ectopic expression of MaMADS7 in tomato resulted in broad ripening phenotypes. The expression levels of seven ripening and quality-related genes, ACO1, ACS2, E4, E8, PG, CNR and PSY1 in MaMADS7 transgenic tomato fruits were greatly increased while the expression of the AG-like MADS-box gene TAGL1 was suppressed. Compared with the control, the contents of β-carotene, lycopene, ascorbic acid and organic acid in transformed tomato fruits were increased, while the contents of glucose and fructose were slightly decreased. MaMADS7 interacted with banana 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene 1 (MaACO1) and tomato phytoene synthase gene (LePSY1) promoters. Our results indicated that MaMADS7 plays an important role in initiating endogenous ethylene biosynthesis and fruit ripening.

Proteins encoded by MADS-box genes are important transcription factors involved in the regulation of flowering plant growth and development. Currently, no systematic information exists regarding the MADS-box family in the important tropical fruit banana. Ninety-six MADS-box genes were identified from the banana (Pahang) A genome. Phylogenetic analysis indicated that Musa acuminata MCM1-AGAMOUS- DEFICIENS-SRF (MaMADS) could be divided into MIKCc, MIKC*, Mα/β and Mγ groups. MIKCc could be further divided into 11 subfamilies, which was further supported by conserved motif and gene structure analyses. Transcriptome analysis on the Feng Jiao (FJ) and BaXi Jiao (BX) banana cultivars revealed that MaMADS genes are differentially expressed in various organs, at different fruit development and ripening stages, indicating the involvement of these genes in fruit development and ripening processes. Interactive network analysis indicated that MaMADS24 and 49 not only interacted with MaMADS proteins themselves, but also interacted with hormone-response proteins, ethylene signal transduction and biosynthesis-related proteins, starch biosynthesis proteins and metabolism-related proteins. This systematic analysis identified candidate MaMADS genes related to fruit development and ripening for further functional characterization in plants, and also provided new insights into the transcriptional regulation of MaMADS genes, facilitating the future genetic manipulation of MADS-mediated fruit development and ripening.

Sugars Will Eventually be Exported Transporters (SWEET) are a novel type of sugar transporter that plays crucial roles in multiple biological processes. From banana, for the first time, 25 SWEET genes which could be classified into four subfamilies were identified. Majority of MaSWEETs in each subfamily shared similar gene structures and conserved motifs. Comprehensive transcriptomic analysis of two banana genotypes revealed differential expression patterns of MaSWEETs in different tissues, at various stages of fruit development and ripening, and in response to abiotic and biotic stresses. More than 80% MaSWEETs were highly expressed in BaXi Jiao (BX, Musa acuminata AAA group, cv. Cavendish), in sharp contrast to Fen Jiao (FJ, M. acuminata AAB group) when pseudostem was first emerged. However, MaSWEETs in FJ showed elevated expression under cold, drought, salt, and fungal disease stresses, but not in BX. Interaction networks and co-expression assays further revealed that MaSWEET-mediated networks participate in fruit development signaling and abiotic/biotic stresses, which was strongly activated during early stage of fruit development in BX. This study provides new insights into the complex transcriptional regulation of SWEETs, as well as numerous candidate genes that promote early sugar transport to improve fruit quality and enhance stress resistance in banana.

The mitogen-activated protein kinase (MAPK) cascade, which is a major signal transduction pathway widely distributed in eukaryotes, has an important function in plant development and stress responses. However, less information is known regarding the MAPKKK and MAPKK gene families in the important fruit crop banana. In this study, 10 MAPKK and 77 MAPKKK genes were identified in the banana genome, and were classified into 4 and 3 subfamilies respectively based on phylogenetic analysis. Majority of MAPKKK and MAPKK genes in the same subfamily shared similar gene structures and conserved motifs. The comprehensive transcriptome analysis indicated that MAPKKK-MAPKK genes is involved in tissue development, fruit development and ripening, and response to abiotic stress of drought, cold and salt in two banana genotypes. Interaction networks and co-expression assays demonstrated that MAPK signaling cascade mediated network participates in multiple stress signaling, which was strongly activated in Fen Jiao (FJ). The findings of this study advance understanding of the intricately transcriptional control of MAPKKK-MAPKK genes and provide robust candidate genes for further genetic improvement of banana.

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.

The growth, development, and production of banana plants are constrained by multiple abiotic stressors. However, it remains elusive for the tolerance mechanisms of banana responding to multiple abiotic stresses. In this study, we found that Fen Jiao (FJ) was more tolerant to osmotic, cold, and salt stresses than BaXi Jiao (BX) by phenotypic and physiological analyses. Comparative transcriptomic analyses highlighted stress tolerance genes that either specifically regulated in FJ or changed more than twofold in FJ relative to BX after treatments. In total, 933, 1644, and 133 stress tolerance genes were identified after osmotic, cold, and salt treatments, respectively. Further integrated analyses found that 30 tolerance genes, including transcription factor, heat shock protein, and E3 ubiquitin protein ligase, could be commonly regulated by osmotic, cold, and salt stresses. Finally, ABA and ROS signaling networks were found to be more active in FJ than in BX under osmotic, cold, and salt treatments, which may contribute to the strong stress tolerances of FJ. Together, this study provides new insights into the tolerance mechanism of banana responding to multiple stresses, thus leading to potential applications in the genetic improvement of multiple abiotic stress tolerances in banana.

A full-length citrate synthase gene designated MaGCS was isolated from a banana fruit cDNA library, which contained a 1885 bp fragment carrying a 1542 bp open reading frame (ORF) that encodes a 513 amino acid protein. MaGCS includes the glyoxysomal targeting signal PST2, an oxaloacetic acid binding site and a citrate synthase active site. Southern blotting showed that the banana genome has two copies of MaGCS . MaGCS is constitutively expressed in all organs with high levels in the fruit and the lowest levels in the leaves. With natural ripening, the MaGCS expression pattern was similar to that of ethylene production and respiration rate. MaGCS expression could be induced by ethylene and inhibited by the ethylene receptor inhibitor 1-methylcyclopropene (1-MCP), which is in accordance with ripening-related ethylene synthesis and respiration rate, and demonstrates that MaGCS is associated with ethylene biosynthesis. Oxaloacetic acid (OA) accelerated fruit ripening, and in contrast, citric acid (CA) delayed it. The expression of MaGCS , the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene 1 ( MaACO1 , AJ223232) and the ACC synthase gene 1 ( MaACS1 , AB021906) was evaluated by real-time quantitative PCR (QPCR), which showed that the expression of MaGCS , MaACO1 , and MaACS1 were greatly improved by OA and suppressed by CA. These results indicate that MaGCS plays an important role in postharvest banana fruit ripening, and that OA and CA could affect fruit ripening. This effect on climacteric ethylene and ripening progress was due to the regulation of MaGCS , MaACO1 , and MaACS1 expression.

Three tau class MaGSTs responded to abiotic stress, MaGSTF1 and MaGSTL1 responded to signaling molecules, they may play an important role in the growth of banana plantlet. Glutathione S-transferases (GST) are multifunctional detoxification enzymes that participate in a variety of cellular processes, including stress responses. In this study, we report the molecular characteristics of five GST genes (MaGSTU1, MaGSTU2, MaGSTU3, MaGSTF1 and MaGSTL1) cloned from banana (Musa acuminate L. AAA group, cv. Cavendish) using a RACE-PCR-based strategy. The predicted molecular masses of these GSTs range from 23.4 to 27.7 kDa and their pIs are acidic. At the amino acid level, they share high sequence similarity with GSTs in the banana DH-Pahang (AA group) genome. Phylogenetic analysis showed that the deduced amino acid sequences of MaGSTs also have high similarity to GSTs of other plant species. Expression analysis by semi-quantitative RT-PCR revealed that these genes are differentially expressed in various tissues. In addition, their expression is regulated by various stress conditions, including exposure to signaling molecules, cold, salinity, drought and Fusarium oxysporum f specialis(f. Sp) cubense Tropical Race 4 (Foc TR4) infection. The expression of the tau class MaGSTs (MaGSTU1, MaGSTU2 and MaGSTU3) mainly responded to cold, salinity and drought while MaGSTF1 and MaGSTL1 expressions were upregulated by signaling molecules. Our findings suggest that MaGSTs play a key role in both development and abiotic stress responses.

Plant 14-3-3 proteins act as critical components of various cellular signaling processes and play an important role in regulating multiple physiological processes. However, less information is known about the 14-3-3 gene family in banana. In this study, 25 14-3-3 genes were identified from the banana genome. Based on the evolutionary analysis, banana 14-3-3 proteins were clustered into ε and non-ε groups. Conserved motif analysis showed that all identified banana 14-3-3 genes had the typical 14-3-3 motif. The gene structure of banana 14-3-3 genes showed distinct class-specific divergence between the ε group and the non-ε group. Most banana 14-3-3 genes showed strong transcript accumulation changes during fruit development and postharvest ripening in two banana varieties, indicating that they might be involved in regulating fruit development and ripening. Moreover, some 14-3-3 genes also showed great changes after osmotic, cold, and salt treatments in two banana varieties, suggested their potential role in regulating banana response to abiotic stress. Taken together, this systemic analysis reveals the involvement of banana 14-3-3 genes in fruit development, postharvest ripening, and response to abiotic stress and provides useful information for understanding the functions of 14-3-3 genes in banana.

The leucine zipper (bZIP) transcription factors play important roles in multiple biological processes. However, less information is available regarding the bZIP family in the important fruit crop banana. In this study, 121 bZIP transcription factor genes were identified in the banana genome. Phylogenetic analysis showed that MabZIPs were classified into 11 subfamilies. The majority of MabZIP genes in the same subfamily shared similar gene structures and conserved motifs. The comprehensive transcriptome analysis of two banana genotypes revealed the differential expression patterns of MabZIP genes in different organs, in various stages of fruit development and ripening and in responses to abiotic stresses, including drought, cold and salt. Interaction networks and co-expression assays showed that group A MabZIP-mediated networks participated in various stress signaling, which was strongly activated in Musa ABB Pisang Awak. This study provided new insights into the complicated transcriptional control of MabZIP genes and provided robust tissue-specific, development-dependent and abiotic stress-responsive candidate MabZIP genes for potential applications in the genetic improvement of banana cultivars.

The abscisic acid (ABA)-, stress-, and ripening-induced (ASR) protein is a plant-specific hydrophilic transcriptional factor involved in fruit ripening and the abiotic stress response. To date, there have been no studies on the role of ASR genes in delayed flowering time. Here, we found that the ASR from banana, designated as MaASR, was preferentially expressed in the banana female flowers from the eighth, fourth, and first cluster of the inflorescence. MaASR transgenic lines (L14 and L38) had a clear delayed-flowering phenotype. The number of rosette leaves, sepals, and pedicel trichomes in L14 and L38 was greater than in the wild type (WT) under long day (LD) conditions. The period of buds, mid-flowers, and full bloom of L14 and L38 appeared later than the WT. cDNA microarray and quantitative real-time PCR (qRT-PCR) analyses revealed that overexpression of MaASR delays flowering through reduced expression of several genes, including photoperiod pathway genes, vernalization pathway genes, gibberellic acid pathway genes, and floral integrator genes, under short days (SD) for 28 d (from vegetative to reproductive transition stage); however, the expression of the autonomous pathway genes was not affected. This study provides the first evidence of a role for ASR genes in delayed flowering time in plants.

A full-length cDNA isolated from banana (Musa acuminata L. AAA group) fruit was named MuBTB, and it contains an open reading frame encoding 354 amino acids. Sequence analysis showed that MuBTB shares high similarity with broad complex, tramtrack, and bric-a-bracs from other plant species. Reverse transcriptase PCR analysis of MuBTB spatial expression showed that MuBTB was expressed in all organs examined: root, rhizome, leaf, flower, and fruit. Real-time quantitative PCR of postharvest banana revealed that MuBTB exhibited differential expression patterns that are associated with ethylene biosynthesis. In naturally ripened bananas, expression of MuBTB was in accordance with ethylene biosynthesis. In contrast, for 1-methylcyclopropene-treated bananas, MuBTB expression levels were inhibited and remained constant. Following treatment with ethylene, MuBTB expression in banana fruit significantly increased with ethylene biosynthesis and peaked 3 days after harvest, which was 11 days earlier than that for naturally ripened banana fruits. These results suggest that MuBTB expression is induced by ethylene for regulation of postharvest banana ripening. Finally, subcellular localization assays showed that the MuBTB protein localizes to the nucleus.

Efficient transformation of lilies is required for their genetic improvement in ornamental and marketable qualities. Although Lilium longiflorum can be transformed by particle bombardment and Agrobacterium, the transformation frequency is low. In this study, we tested new Agrobacterium-mediated transformation methods using shoot segments combined with two different regeneration systems. Shoot segments were co-cultivated for 2 d with Agrobacterium tumefaciens strain AGL1/pCAS04 harboring a binary vector carrying the neomycin phosphotransferase II driven by a promoter from the maize ubiquitin gene. The effect of different concentrations of 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on regeneration was investigated. The results indicated that Murashige and Skoog (MS) medium with 4.4 μM BA and 0.5 μM α-naphthalene acetic acid was optimal for shoot formation, and the nodal stem was the best explant for shoot induction. MS medium with 9.0 μM 2,4-D and 0.4 μM BA was optimal for callus induction. The direct shoot formation method regenerated 187 plantlets per 100 explants, and 74.4% of the regenerants were positive in transgene PCR. The callus regeneration method regenerated 20 plantlets per 100 explants, and 31.5% of them were PCR positive. Southern blotting confirmed the insertion of transgene in the plant host genome. The direct shoot formation method is more than 20-fold more efficient than previously reported transformation method in this species.

Rho-like GTPases from plants (ROPs) are plant-specific molecular switches that are crucial for plant survival when subjected to abiotic stress. We identified and characterized 17 novel ROP proteins from Musa acuminata (MaROPs) using genomic techniques. The identified MaROPs fell into three of the four previously described ROP groups (Groups II&ndash, IV), with MaROPs in each group having similar genetic structures and conserved motifs. Our transcriptomic analysis showed that the two banana genotypes tested, Fen Jiao and BaXi Jiao, had similar responses to abiotic stress: Six genes (MaROP-3b, -5a, -5c, -5f, -5g, and -6) were highly expressed in response to cold, salt, and drought stress conditions in both genotypes. Of these, MaROP5g was most highly expressed in response to salt stress. Co-localization experiments showed that the MaROP5g protein was localized at the plasma membrane. When subjected to salt stress, transgenic Arabidopsis thaliana overexpressing MaROP5g had longer primary roots and increased survival rates compared to wild-type A. thaliana. The increased salt tolerance conferred by MaROP5g might be related to reduced membrane injury and the increased cytosolic K+/Na+ ratio and Ca2+ concentration in the transgenic plants as compared to wild-type. The increased expression of salt overly sensitive (SOS)-pathway genes and calcium-signaling pathway genes in MaROP5g-overexpressing A. thaliana reflected the enhanced tolerance to salt stress by the transgenic lines in comparison to wild-type. Collectively, our results suggested that abiotic stress tolerance in banana plants might be regulated by multiple MaROPs, and that MaROP5g might enhance salt tolerance by increasing root length, improving membrane injury and ion distribution.

Auxin signaling regulates various auxin-responsive genes via two types of transcriptional regulators, Auxin Response Factors (ARF) and Aux/IAA. ARF transcription factors act as critical components of auxin signaling that play important roles in modulating various biological processes. However, limited information about this gene family in fruit crops is currently available. Herein, 47 ARF genes were identified in banana based on its genome sequence. Phylogenetic analysis of the ARFs from banana, rice, and Arabidopsis suggested that the ARFs could be divided into four subgroups, among which most ARFs from the banana showed a closer relationship with those from rice than those from Arabidopsis. Conserved motif analysis showed that all identified MaARFs had typical DNA-binding and ARF domains, but 12 members lacked the dimerization domain. Gene structure analysis showed that the number of exons in MaARF genes ranged from 5 to 21, suggesting large variation amongst banana ARF genes. The comprehensive expression profiles of MaARF genes yielded useful information about their involvement in diverse tissues, different stages of fruit development and ripening, and responses to abiotic stresses in different varieties. Interaction networks and co-expression assays indicated the strong transcriptional response of banana ARFs and ARF-mediated networks in early fruit development for different varieties. Our systematic analysis of MaARFs revealed robust tissue-specific, development-dependent, and abiotic stress-responsive candidate MaARF genes for further functional assays in planta. These findings could lead to potential applications in the genetic improvement of banana cultivars, and yield new insights into the complexity of the control of MaARF gene expression at the transcriptional level. Finally, they support the hypothesis that ARFs are a crucial component of the auxin signaling pathway, which regulates a wide range of physiological processes.

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.

Mitogen-activated protein kinases (MAPKs) play important roles in signal transduction pathways responding to various stresses. In this study, we described the molecular characteristics of six MAPK genes (MaMAPK1, MaMAPK2, MaMAPK3, MaMAPK4, MaMAPK5 and MaMAPK6) cloned from banana (Musa acuminate L. AAA group, cv. Cavendish) using a RACE-PCR-based strategy. The predicted molecular masses of these MAPKs ranged from 43.0 to 70.1 kDa and their pIs ranged from 5.67 to 9.32. At the amino acid level, they shared high sequence similarity with MAPKs in the banana DH-Pahang (AA group) genome. Phylogenetic analysis showed that the deduced amino acid sequences of MaMAPKs also had high similarity with MAPKs of other plant species. These genes were expressed in nearly all tissues using semi-quantitative RT-PCR. All of the six MaMAPKs were induced by signal molecules in banana seedlings, such as ABA, Ethephon, MeJA and SA. The gene expressions of MaMAPK1, MaMAPK2, MaMAPK3, MaMAPK5 and MaMAPK6 were induced in resistant cultivar of banana after being inoculated with Fusarium Oxysporum f specialis (f. Sp) cubense Tropical Race 4 (Foc TR4). Our results suggest that MaMAPKs play a key role in both the development of banana seedlings and banana resistance to Foc TR4.

The ovate family protein named MaOFP1 was identified in banana (Musa acuminata L.AAA) fruit by a yeast two-hybrid (Y2H) method using the banana MADS-box gene MuMADS1 as bait and a 2 day postharvest (DPH) banana fruit cDNA library as prey. The interaction between MuMADS1 and MaOFP1 was further confirmed by Y2H and Bimolecular Fluorescence Complementation (BiFC) methods, which showed that the MuMADS1 K domain interacted with MaOFP1. Real-time quantitative PCR evaluation of MuMADS1 and MaOFP1 expression patterns in banana showed that they are highly expressed in 0 DPH fruit, but present in low levels in the stem, which suggests that simultaneous but different expression patterns exist for both MuMADS1 and MaOFP1 in different tissues and developing fruits. Meanwhile, MuMADS1 and MaOFP1 expression was highly stimulated and greatly suppressed, respectively, by exogenous ethylene. In contrast, MaOFP1 expression was highly stimulated while MuMADS1 was greatly suppressed by the ethylene competitor 1-methylcyclopropene (1-MCP). These results indicate that MuMADS1 and MaOFP1 are antagonistically regulated by ethylene and might play important roles in postharvest banana fruit ripening.