High-quality genome assemblies for two Australimusa bananas (Musa spp.) and insights into regulatory mechanisms of superior fiber properties.

Bananas (Musa spp.) are monocotyledonous plants with high genetic diversity in the Musaceae family that are cultivated mainly in tropical and subtropical countries. The fruits are a popular food, and the plants themselves have diverse uses. Four genetic groups (genomes) are thought to have contributed to current banana cultivars: Musa acuminata (A genome), Musa balbisiana (B genome), Musa schizocarpa (S genome), and species of the Australimusa section (T genome). However, the T genome has not been effectively explored. Here, we present the high-quality TT genomes of two representative accessions, Abaca (Musa textilis), with high-quality natural fiber, and Utafun (Musa troglodytarum, Fe'i group), with abundant ß-carotene. Both the Abaca and Utafun assemblies comprise 10 pseudochromosomes, and their total genome sizes are 613 Mb and 619 Mb, respectively. Comparative genome analysis revealed that the larger size of the T genome is likely attributable to rapid expansion and slow removal of transposons. Compared with those of Musa AA or BB accessions or sisal (Agava sisalana), Abaca fibers exhibit superior mechanical properties, mainly because of their thicker cell walls with a higher content of cellulose, lignin, and hemicellulose. Expression of MusaCesA cellulose synthesis genes peaks earlier in Abaca than in AA or BB accessions during plant development, potentially leading to earlier cellulose accumulation during secondary cell wall formation. The Abaca-specific expressed gene MusaMYB26, which is directly regulated by MusaMYB61, may be an important regulator that promotes precocious expression of secondary cell wall MusaCesAs. Furthermore, MusaWRKY2 and MusaNAC68, which appear to be involved in regulating expression of MusaLAC and MusaCAD, may at least partially explain the high accumulation of lignin in Abaca. This work contributes to a better understanding of banana domestication and the diverse genetic resources in the Musaceae family, thus providing resources for Musa genetic improvement.

Two haplotype-resolved genome assemblies for AAB allotriploid bananas provide insights into banana subgenome asymmetric evolution and Fusarium wilt control.

Bananas (Musa spp.) are one of the world's most important fruit crops and play a vital role in food security for many developing countries. Most banana cultivars are triploids derived from inter- and intraspecific hybridizations between the wild diploid ancestor species Musa acuminate (AA) and M. balbisiana (BB). We report two haplotype-resolved genome assemblies of the representative AAB-cultivated types, Plantain and Silk, and precisely characterize ancestral contributions by examining ancestry mosaics across the genome. Widespread asymmetric evolution is observed in their subgenomes, which can be linked to frequent homologous exchange events. We reveal the genetic makeup of triploid banana cultivars and verify that subgenome B is a rich source of disease resistance genes. Only 58.5% and 59.4% of Plantain and Silk genes, respectively, are present in all three haplotypes, with >50% of genes being differentially expressed alleles in different subgenomes. We observed that the number of upregulated genes in Plantain is significantly higher than that in Silk at one-week post-inoculation with Fusarium wilt tropical race 4 (Foc TR4), which confirms that Plantain can initiate defense responses faster than Silk. Additionally, we compared genomic and transcriptomic differences among the genes related to carotenoid synthesis and starch metabolism between Plantain and Silk. Our study provides resources for better understanding the genomic architecture of cultivated bananas and has important implications for Musa genetics and breeding.

Genome-wide analysis of the polyphenol oxidase gene family reveals that MaPPO1 and MaPPO6 are the main contributors to fruit browning in Musa acuminate.

Introduction: Polyphenol oxidases (PPOs), which are widely present in plants, play an important role in the growth, development, and stress responses. They can catalyze the oxidization of polyphenols and result in the browning of damaged or cut fruit, which seriously affects fruit quality and compromises the sale of fruit. In banana (Musa acuminata, AAA group), 10 PPO genes were determined based on the availability of a high-quality genome sequence, but the role of PPO genes in fruit browning remains unclear. Methods: In this study, we analyzed the physicochemical properties, gene structure, conserved structural domains, and evolutionary relationship of the PPO gene family of banana. The expression patterns were analyzed based on omics data and verified by qRT-PCR analysis. Transient expression assay in tobacco leaves was used to identify the subcellular localization of selected MaPPOs, and we analyzed the polyphenol oxidase activity using recombinant MaPPOs and transient expression assay. Results and discussion: We found that more than two-thirds of the MaPPO genes had one intron, and all contained three conserved structural domains of PPO, except MaPPO4. Phylogenetic tree analysis revealed that MaPPO genes were categorized into five groups. MaPPOs did not cluster with Rosaceae and Solanaceae, indicating distant affinities, and MaPPO6/7/8/9/10 clustered into an individual group. Transcriptome, proteome, and expression analyses showed that MaPPO1 exhibits preferential expression in fruit tissue and is highly expressed at respiratory climacteric during fruit ripening. Other examined MaPPO genes were detectable in at least five different tissues. In mature green fruit tissue, MaPPO1 and MaPPO6 were the most abundant. Furthermore, MaPPO1 and MaPPO7 localized in chloroplasts, and MaPPO6 was a chloroplast- and Endoplasmic Reticulum (ER)-localized protein, whereas MaPPO10 only localized in the ER. In addition, the enzyme activity in vivo and in vitro of the selected MaPPO protein showed that MaPPO1 had the highest PPO activity, followed by MaPPO6. These results imply that MaPPO1 and MaPPO6 are the main contributors to banana fruit browning and lay the foundation for the development of banana varieties with low fruit browning.

WRKY transcription factor MaWRKY49 positively regulates pectate lyase genes during fruit ripening of Musa acuminata.

Fruit ripening is the last phase of fruit growth and development. The initiation and progression of fruit ripening are highly modulated by a plethora of key genes, such as transcription factor (TF) genes. The WRKY gene family is a large group of TFs that play important roles in various cellular processes; nevertheless, the role of WRKY TF on fruit ripening remains enigmatic. Here, we report that a banana WRKY TF, MaWRKY49 functions in ethylene-induced fruit ripening by modulating the expression of fruit softening-related genes. We found that the expression of MaWRKY49 is highly induced by ethephon and inhibited by 1-methylcyclopropene, which is synchronous with the ripening process. Moreover, based on transcriptome data on fruit ripening, two pectate lyase (PL) genes that are involved in fruit softening were determined, and their expression pattern is also consistent with the fruit ripening process. Yeast one-hybrid and dual-luciferase assay confirmed that MaWRKY49 activated the transcription of two PL genes. In addition, transient overexpression of MaWRKY49 in banana fruits can apparently accelerate fruit ripening processs. Taken together, our findings indicate that MaWRKY49 acts as a potential modulator of fruit ripening by direct regulation of PL expression. This work contributes to developing the technology for improving the shelf-life of banana fruit.

Increased mutation efficiency of CRISPR/Cas9 genome editing in banana by optimized construct.

The CRISPR/Cas9-mediated genome editing system has been used extensively to engineer targeted mutations in a wide variety of species. Its application in banana, however, has been hindered because of the species' triploid nature and low genome editing efficiency. This has delayed the development of a DNA-free genome editing approach. In this study, we reported that the endogenous U6 promoter and banana codon-optimized Cas9 apparently increased mutation frequency in banana, and we generated a method to validate the mutation efficiency of the CRISPR/Cas9-mediated genome editing system based on transient expression in protoplasts. The activity of the MaU6c promoter was approximately four times higher than that of the OsU6a promoter in banana protoplasts. The application of this promoter and banana codon-optimized Cas9 in CRISPR/Cas9 cassette resulted in a fourfold increase in mutation efficiency compared with the previous CRISPR/Cas9 cassette for banana. Our results indicated that the optimized CRISPR/Cas9 system was effective for mutating targeted genes in banana and thus will improve the applications for basic functional genomics. These findings are relevant to future germplasm improvement and provide a foundation for developing DNA-free genome editing technology in banana.

Transcriptome and metabolome profiling provide insights into molecular mechanism of pseudostem elongation in banana.

BACKGROUND: Banana plant height is an important trait for horticultural practices and semi-dwarf cultivars show better resistance to damages by wind and rain. However, the molecular mechanisms controlling the pseudostem height remain poorly understood. Herein, we studied the molecular changes in the pseudostem of a semi-dwarf banana mutant Aifen No. 1 (Musa spp. Pisang Awak sub-group ABB) as compared to its wild-type dwarf cultivar using a combined transcriptome and metabolome approach. RESULTS: A total of 127 differentially expressed genes and 48 differentially accumulated metabolites were detected between the mutant and its wild type. Metabolites belonging to amino acid and its derivatives, flavonoids, lignans, coumarins, organic acids, and phenolic acids were up-regulated in the mutant. The transcriptome analysis showed the differential regulation of genes related to the gibberellin pathway, auxin transport, cell elongation, and cell wall modification. Based on the regulation of gibberellin and associated pathway-related genes, we discussed the involvement of gibberellins in pseudostem elongation in the mutant banana. Genes and metabolites associated with cell wall were explored and their involvement in cell extension is discussed. CONCLUSIONS: The results suggest that gibberellins and associated pathways are possibly developing the observed semi-dwarf pseudostem phenotype together with cell elongation and cell wall modification. The findings increase the understanding of the mechanisms underlying banana stem height and provide new clues for further dissection of specific gene functions.

MaMAPK3-MaICE1-MaPOD P7 pathway, a positive regulator of cold tolerance in banana.

BACKGROUND: Banana is a tropical fruit with a high economic impact worldwide. Cold stress greatly affects the development and production of banana. RESULTS: In the present study, we investigated the functions of MaMAPK3 and MaICE1 involved in cold tolerance of banana. The effect of RNAi of MaMAPK3 on Dajiao (Musa spp. 'Dajiao'; ABB Group) cold tolerance was evaluated. The leaves of the MaMAPK3 RNAi transgenic plants showed wilting and severe necrotic symptoms, while the wide-type (WT) plants remained normal after cold exposure. RNAi of MaMAPK3 significantly changed the expressions of the cold-responsive genes, and the oxidoreductase activity was significantly changed in WT plants, while no changes in transgenic plants were observed. MaICE1 interacted with MaMAPK3, and the expression level of MaICE1 was significantly decreased in MaMAPK3 RNAi transgenic plants. Over-expression of MaICE1 in Cavendish banana (Musa spp. AAA group) indicated that the cold resistance of transgenic plants was superior to that of the WT plants. The POD P7 gene was significantly up-regulated in MaICE1-overexpressing transgenic plants compared with WT plants, and the POD P7 was proved to interact with MaICE1. CONCLUSIONS: Taken together, our work provided new and solid evidence that MaMAPK3-MaICE1-MaPOD P7 pathway positively improved the cold tolerance in monocotyledon banana, shedding light on molecular breeding for the cold-tolerant banana or other agricultural species.

Overexpression of MaTPD1A impairs fruit and pollen development by modulating some regulators in Musa itinerans.

BACKGROUND: Pollen formation and development is important for crop fertility and is a key factor for hybrid development. Previous reports have indicated that Arabidopsis thaliana TAPETUM DETERMINANT1 (AtTPD1) and its rice (Oryza sativa) homolog, OsTPD1-like (OsTDL1A), are required for cell specialization and greatly affect pollen formation and development. Little is known about the role of the TPD1 homolog in banana pollen development. RESULTS: Here, we report the identification and characterization of TPD1 homologs in diploid banana (Musa itinerans) and examine their role in pollen development by overexpressing the closest homolog, MaTPD1A. MaTPD1A exhibits high expression in stamen and localizes in the plasma membrane. MaTPD1A-overexpressing plants produce no pollen grains and smaller and seedless fruit compared to wild-type plants. Transcriptome analysis showed that in plant hormone, starch and sucrose metabolism, and linolenic acid metabolism-related pathways were affected by overexpression of MaTPD1A, and the expression of several key regulators, such as PTC1 and MYB80, which are known to affect anther development, is affected in MaTPD1A-overexpressing lines. CONCLUSIONS: Our results indicate that MaTPD1A plays an important role in pollen formation and fruit development in diploid banana, possibly by affecting the expression of some key regulators of pollen development.

Fusaric acid instigates the invasion of banana by Fusarium oxysporum f. sp. cubense TR4.

Fusaric acid (FSA) is a phytotoxin produced by several Fusarium species and has been associated with plant disease development, although its role is still not well understood. Mutation of key genes in the FSA biosynthetic gene (FUB) cluster in Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) reduced the FSA production, and resulted in decreased disease symptoms and reduced fungal biomass in the host banana plants. When pretreated with FSA, both banana leaves and pseudostems exhibited increased sensitivity to Foc TR4 invasion. Banana embryogenic cell suspensions (ECSs) treated with FSA exhibited a lower rate of O2 uptake, loss of mitochondrial membrane potential, increased reactive oxygen species (ROS) accumulation, and greater nuclear condensation and cell death. Consistently, transcriptomic analysis of FSA-treated ECSs showed that FSA may induce plant cell death through regulating the expression of genes involved in mitochondrial functions. The results herein demonstrated that the FSA from Foc TR4 functions as a positive virulence factor and acts at the early stage of the disease development before the appearance of the fungal hyphae in the infected tissues.

CgGCS, Encoding a Glucosylceramide Synthase, Is Required for Growth, Conidiation and Pathogenicity in Colletotrichum gloeosporioides.

Fungal glucosylceramide plays important role in cell division, hyphal formation and growth, spore germination and the modulation of virulence and has recently been considered as target for small molecule inhibitors. In this study, we characterized CgGCS, a protein encoding a glucosylceramide synthase (GCS) in Colletotrichum gloeosporioides. Disruption of CgGCS resulted in a severe reduction of mycelial growth and defects in conidiogenesis. Sphingolipid profile analysis revealed large decreases in glucosylceramide production in the mutant strains. Pathogenicity assays indicated that the ability of the ΔCgGCS mutants to invade both tomato and mango hosts was almost lost. In addition, the expression levels of many genes, especially those related to metabolism, were shown to be affected by the mutation of CgGCS via transcriptome analysis. Overall, our results demonstrate that C. gloeosporioides glucosylceramide is an important regulatory factor in fungal growth, conidiation, and pathogenesis in hosts.

Early Cold-Induced Peroxidases and Aquaporins Are Associated With High Cold Tolerance in Dajiao (Musa spp. 'Dajiao').

Banana is an important tropical fruit with high economic value. One of the main cultivars ('Cavendish') is susceptible to low temperatures, while another closely related specie ('Dajiao') has considerably higher cold tolerance. We previously reported that some membrane proteins appear to be involved in the cold tolerance of Dajiao bananas via an antioxidation mechanism. To investigate the early cold stress response of Dajiao, here we applied comparative membrane proteomics analysis for both cold-sensitive Cavendish and cold-tolerant Dajiao bananas subjected to cold stress at 10°C for 0, 3, and 6 h. A total of 2,333 and 1,834 proteins were identified in Cavendish and Dajiao, respectively. Subsequent bioinformatics analyses showed that 692 Cavendish proteins and 524 Dajiao proteins were predicted to be membrane proteins, of which 82 and 137 differentially abundant membrane proteins (DAMPs) were found in Cavendish and Dajiao, respectively. Interestingly, the number of DAMPs with increased abundance following 3 h of cold treatment in Dajiao (80) was seven times more than that in Cavendish (11). Gene ontology molecular function analysis of DAMPs for Cavendish and Dajiao indicated that they belong to eight categories including hydrolase activity, binding, transporter activity, antioxidant activity, etc., but the number in Dajiao is twice that in Cavendish. Strikingly, we found peroxidases (PODs) and aquaporins among the protein groups whose abundance was significantly increased after 3 h of cold treatment in Dajiao. Some of the PODs and aquaporins were verified by reverse-transcription PCR, multiple reaction monitoring, and green fluorescent protein-based subcellular localization analysis, demonstrating that the global membrane proteomics data are reliable. By combining the physiological and biochemical data, we found that membrane-bound Peroxidase 52 and Peroxidase P7, and aquaporins (MaPIP1;1, MaPIP1;2, MaPIP2;4, MaPIP2;6, MaTIP1;3) are mainly involved in decreased lipid peroxidation and maintaining leaf cell water potential, which appear to be the key cellular adaptations contributing to the cold tolerance of Dajiao. This membrane proteomics study provides new insights into cold stress tolerance mechanisms of banana, toward potential applications for ultimate genetic improvement of cold tolerance in banana.

Comparative transcriptomics analysis reveals difference of key gene expression between banana and plantain in response to cold stress.

BACKGROUND: Banana and plantain (Musa spp.) comprise an important part of diets for millions of people around the globe. Low temperature is one of the key environmental stresses which greatly affects the global banana production. To understand the molecular mechanism of the cold-tolerance in plantain we used RNA-Seq based comparative transcriptomics analyses for both cold-sensitive banana and cold-tolerant plantain subjected to the cold stress for 0, 3 and 6 h. RESULTS: The cold-response genes at early stage are identified and grouped in both species by GO analysis. The results show that 10 and 68 differentially expressed genes (DEGs) are identified for 3 and 6 h of cold stress respectively in plantain, while 40 and 238 DEGs are identified respectively in banana. GO classification analyses show that the majority of DEGs identified in both banana and plantain belong to 11 categories including regulation of transcription, response to stress signal transduction, etc. A similar profile for 28 DEGs was found in both banana and plantain for 6 h of cold stress, suggesting both share some common adaptation processes in response to cold stress. There are 17 DEGs found uniquely in cold-tolerance plantain, which were involved in signal transduction, abiotic stress, copper ion equilibrium, photosynthesis and photorespiration, sugar stimulation, protein modifications etc. Twelve early responsive genes including ICE1 and MYBS3 were selected and further assessed and confirmed by qPCR in the extended time course experiments (0, 3, 6, 24 and 48 h), which revealed significant expression difference of key genes in response to cold stress, especially ICE1 and MYBS3 between cold-sensitive banana and cold-tolerant plantain. CONCLUSIONS: We found that the cold-tolerance pathway appears selectively activated by regulation of ICE1 and MYBS3 expression in plantain under different stages of cold stress. We conclude that the rapid activation and selective induction of ICE1 and MYBS3 cold tolerance pathways in plantain, along with expression of other cold-specific genes, may be one of the main reasons that plantain has higher cold resistance than banana.