Investigating the ibrutinib resistance mechanism of L528W mutation on Bruton's tyrosine kinase via molecular dynamics simulations.

Drug resistance to Bruton's Tyrosine Kinase (BTK) inhibitors presents a challenge in treating B-cell malignancies, and the mechanism behind drug resistance remains unclear. In this study, we focused on the BTK L528W mutation and investigated the underlying mechanisms of resistance to ibrutinib (including prototype and its active metabolite from, PCI-45227) using a combination of bioinformatics analysis, and molecular dynamics (MD) simulations. Protein stability of wild type (WT) BTK and L528W mutant was predicted using DUET, PoPMuSiC, and I-Mutant2.0. We performed MD simulations of six systems, apo-WT, metabolite-WT, prototype-WT and their mutants, to analyze the significant conformational and BTK-inhibitor binding affinity changes induced by the L528W mutation. Results show that the L528W mutation reduces the conformational stability of BTK compared to the WT. Principal component analysis (PCA) based free energy landscape (FEL) analysis shows that the L528W mutant ensemble tends to form more conformation clusters and exhibit higher levels of local minima than the WT counterpart. The interaction analysis reveal that the L528W mutation disrupts the strong hydrogen bond between Cys481 and inhibitors and reduces the number of hydrogen bonds between inhibitors and BTK in the L528W mutant complex structures compared to the WT. Porcupine plot analysis in association with cross-correlation analysis show the high-intensity flexible motion exhibited by the P-loop region. MM/GBSA calculations show that the L528W mutation in metabolite-BTK and prototype-BTK complexes increases binding free energy compared to the WT, with a reduction in binding affinity confirmed by per-residue energy decomposition. Specifically, the binding free energy increases from -57.86 kcal/mol to -48.26 kcal/mol for the metabolite-BTK complex and from -62.04 kcal/mol to -50.55 kcal/mol for the prototype-BTK complex. Overall, our study finds that the L528W mutation reduces BTK stability, decreases binding affinity, and leads to drug resistance and potential disease recurrence.

GoSTR, a negative modulator of stem trichome formation in cotton.

Trichomes, the outward projection of plant epidermal tissue, provide an effective defense against stress and insect pests. Although numerous genes have been identified to be involved in trichome development, the molecular mechanism for trichome cell fate determination is not well enunciated. Here, we reported GoSTR functions as a master repressor for stem trichome formation, which was isolated by map-based cloning based on a large F2 segregating population derived from a cross between TM-1 (pubescent stem) and J220 (smooth stem). Sequence alignment revealed a critical G-to-T point mutation in GoSTR's coding region that converted codon 2 from GCA (Alanine) to TCA (Serine). This mutation occurred between the majority of Gossypium hirsutum with pubescent stem (GG-haplotype) and G. barbadense with glabrous stem (TT-haplotype). Silencing of GoSTR in J220 and Hai7124 via virus-induced gene silencing resulted in the pubescent stems but no visible change in leaf trichomes, suggesting stem trichomes and leaf trichomes are genetically distinct. Yeast two-hybrid assay and luciferase complementation imaging assay showed GoSTR interacts with GoHD1 and GoHOX3, two key regulators of trichome development. Comparative transcriptomic analysis further indicated that many transcription factors such as GhMYB109, GhTTG1, and GhMYC1/GhDEL65 which function as positive regulators of trichomes were significantly upregulated in the stem from the GoSTR-silencing plant. Taken together, these results indicate that GoSTR functions as an essential negative modulator of stem trichomes and its transcripts will greatly repress trichome cell differentiation and growth. This study provided valuable insights for plant epidermal hair initiation and differentiation research.

Identification and analysis of lignin biosynthesis genes related to fruit ripening and stress response in banana (Musa acuminata L. AAA group, cv. Cavendish).

Background: Lignin is a key component of the secondary cell wall of plants, providing mechanical support and facilitating water transport as well as having important impact effects in response to a variety of biological and abiotic stresses. Results: In this study, we identified 104 genes from ten enzyme gene families related to lignin biosynthesis in Musa acuminata genome and found the number of MaCOMT gene family was the largest, while MaC3Hs had only two members. MaPALs retained the original members, and the number of Ma4CLs in lignin biosynthesis was significantly less than that of flavonoids. Segmental duplication existed in most gene families, except for MaC3Hs, and tandem duplication was the main way to expand the number of MaCOMTs. Moreover, the expression profiles of lignin biosynthesis genes during fruit development, postharvest ripening stages and under various abiotic and biological stresses were investigated using available RNA-sequencing data to obtain fruit ripening and stress response candidate genes. Finally, a co-expression network of lignin biosynthesis genes was constructed by weighted gene co-expression network analysis to elucidate the lignin biosynthesis genes that might participate in lignin biosynthesis in banana during development and in response to stresses. Conclusion: This study systematically identified the lignin biosynthesis genes in the Musa acuminata genome, providing important candidate genes for further functional analysis. The identification of the major genes involved in lignin biosynthesis in banana provides the basis for the development of strategies to improve new banana varieties tolerant to biological and abiotic stresses with high yield and high quality.

MaDREB1F confers cold and drought stress resistance through common regulation of hormone synthesis and protectant metabolite contents in banana.

Adverse environmental factors severely affect crop productivity. Improving crop resistance to multiple stressors is an important breeding goal. Although CBFs/DREB1s extensively participate in plant resistance to abiotic stress, the common mechanism underlying CBFs/DREB1s that mediate resistance to multiple stressors remains unclear. Here, we show the common mechanism for MaDREB1F conferring cold and drought stress resistance in banana. MaDREB1F encodes a dehydration-responsive element binding protein (DREB) transcription factor with nuclear localization and transcriptional activity. MaDREB1F expression is significantly induced after cold, osmotic, and salt treatments. MaDREB1F overexpression increases banana resistance to cold and drought stress by common modulation of the protectant metabolite levels of soluble sugar and proline, activating the antioxidant system, and promoting jasmonate and ethylene syntheses. Transcriptomic analysis shows that MaDREB1F activates or alleviates the repression of jasmonate and ethylene biosynthetic genes under cold and drought conditions. Moreover, MaDREB1F directly activates the promoter activities of MaAOC4 and MaACO20 for jasmonate and ethylene syntheses, respectively, under cold and drought conditions. MaDREB1F also targets the MaERF11 promoter to activate MaACO20 expression for ethylene synthesis under drought stress. Together, our findings offer new insight into the common mechanism underlying CBF/DREB1-mediated cold and drought stress resistance, which has substantial implications for engineering cold- and drought-tolerant crops.

Elucidating the role of MaBAM9b in starch degradation.

Banana is a typical starch conversion fruit. The high content of starch at harvest is quickly digested and converted to soluble sugars during the postharvest ripening process, ultimately contributing to fruit flavor. This process is regulated in a complex manner by genes and environmental factors. MaBAM9b is one of the main enzyme genes previously found by transcriptomic analysis to be highly expressed in banana fruit. However, its exact role in starch degradation remains unclear. Here, full-length MaBAM9b was isolated from banana fruit, and its subcellular localization, protein expression, and transient expression in banana fruit slices were investigated. In addition, sense and anti-sense MaBAM9b were transformed into rice (Oryza sativa L. japonica. cv. 'Nipponbare') to identify the function of MaBAM9b. MaBAM9b was 1599 bp and encoded 532 amino acids. It contained two conserved domains of PLN02803 and glycosyl hydrolase family 14 and was localized in the chloroplast. The protein expression pattern of MaBAM9b remained consistently high throughout banana fruit ripening and starch degradation. Transient overexpression or inhibition of MaBAM9b in banana fruit greatly improved or suppressed starch degradation. Genetic modification of rice indicated that overexpression of MaBAM9b greatly improved starch degradation and seed germination, while inhibition of its expression suppressed these biological processes. These results support the key role of MaBAM9b in starch degradation and provide a target gene for banana fruit quality improvement and biological breeding.

Development of the engineered "glanded plant and glandless seed" cotton.

After fiber, cottonseed is the second most important by-product of cotton production. However, high concentrations of toxic free gossypol deposited in the glands of the cottonseed greatly hamper its effective usage as food or feed. Here, we developed a cotton line with edible cottonseed by specifically silencing the endogenous expression of GoPGF in the seeds, which led to a glandless phenotype with an ultra-low gossypol content in the seeds and nearly normal gossypol in other parts of the plants. This engineered cotton maintains normal resistance to insect pests, but the gossypol content in the seeds dropped by 98%, and thus, it can be consumed directly as food. The trait of a low gossypol content in the cottonseeds was stable and heritable, while the protein, oil content, and fiber yield or quality were nearly unchanged compared to the transgenic receptor W0. In addition, comparative transcriptome analysis showed that down-regulated genes in the ovules of the glandless cotton were enriched in terpenoid biosynthesis, indicating the underlying relationship between gland formation and gossypol biosynthesis. These results pave the way for the comprehensive utilization of cotton as a fiber, oil, and feed crop in the future.

Advanced genes expression pattern greatly contributes to divergence in Verticillium wilt resistance between Gossypium barbadense and Gossupium hirsutum.

Verticillium, representing one of the world's major pathogens, causes Verticillium wilt in important woody species, ornamentals, agricultural, etc., consequently resulting in a serious decline in production and quality, especially in cotton. Gossupium hirutum and Gossypium barbadense are two kinds of widely cultivated cotton species that suffer from Verticillium wilt, while G. barbadense has much higher resistance toward it than G. hirsutum. However, the molecular mechanism regarding their divergence in Verticillium wilt resistance remains largely unknown. In the current study, G. barbadense cv. Hai7124 and G. hirsutum acc. TM-1 were compared at 0, 12, 24, 48, 72, 96, 120, and 144 h post-inoculation (hpi) utilizing high throughput RNA-Sequencing. As a result, a total of 3,549 and 4,725 differentially expressed genes (DEGs) were identified, respectively. In particular, the resistant type Hai7124 displayed an earlier and faster detection and signaling response to the Verticillium dahliae infection and demonstrated higher expression levels of defense-related genes over TM-1 with respect to transcription factors, plant hormone signal transduction, plant-pathogen interaction, and nucleotide-binding leucine-rich repeat (NLR) genes. This study provides new insights into the molecular mechanisms of divergence in Verticillium wilt resistance between G. barbadense and G. hirsutum and important candidate genes for breeding V. dahliae resistant cotton cultivars.

The Feasibility Mechanism of Nerve Interventional Thrombectomy for Occlusion of Cranial Artery M1 and M2 Segments.

This study was aimed at exploring the feasibility and clinical efficacy of nerve interventional thrombectomy (NIT) to treat occlusion of cranial artery M1 and M2 segments. 80 patients were selected and rolled into a control group (intravenous thrombolysis) and an experimental group (NIT). Patients' vascular recanalization rates following therapy were compared, and the National Institutes of Health Stroke Scale (NIHSS) was used to measure neurological function. The improvement in hemodynamics and the occurrence of adverse responses were compared. The results showed that the experimental group's recanalization rate was up to 74.23%, which was significantly greater than the control group's (P < 0.05). One week after treatment, the neurological function scores in both groups decreased, and the score in the experimental group was only 15.23, which was much lower than that in the control group (P < 0.05). The peak systolic flow rates of the basilar artery, internal carotid artery, and common carotid artery in the experimental group were 132 cm/s, 147 cm/s, and 114 cm/s, respectively, which were lower greatly than those in the control group (P < 0.05). There was no significant difference in incidence of adverse reactions between the two groups (P > 0.05). In summary, NIT showed a significant therapeutic effect on cranial artery occlusion of M1 and M2 segments, can dredge the occluded blood vessels, and effectively improve the neurological deficits of patients, showing reliable feasibility.

Population-Scale Polymorphic Short Tandem Repeat Provides an Alternative Strategy for Allele Mining in Cotton.

Short tandem repeats (STRs), which vary in size due to featuring variable numbers of repeat units, are present throughout most eukaryotic genomes. To date, few population-scale studies identifying STRs have been reported for crops. Here, we constructed a high-density polymorphic STR map by investigating polymorphic STRs from 911 Gossypium hirsutum accessions. In total, we identified 556,426 polymorphic STRs with an average length of 21.1 bp, of which 69.08% were biallelic. Moreover, 7,718 (1.39%) were identified in the exons of 6,021 genes, which were significantly enriched in transcription, ribosome biogenesis, and signal transduction. Only 5.88% of those exonic STRs altered open reading frames, of which 97.16% were trinucleotide. An alternative strategy STR-GWAS analysis revealed that 824 STRs were significantly associated with agronomic traits, including 491 novel alleles that undetectable by previous SNP-GWAS methods. For instance, a novel polymorphic STR consisting of GAACCA repeats was identified in GH_D06G1697, with its (GAACCA)5 allele increasing fiber length by 1.96-4.83% relative to the (GAACCA)4 allele. The database CottonSTRDB was further developed to facilitate use of STR datasets in breeding programs. Our study provides functional roles for STRs in influencing complex traits, an alternative strategy STR-GWAS for allele mining, and a database serving the cotton community as a valuable resource.

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

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

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

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

Genome-Wide Characterization and Analysis of R2R3-MYB Genes Related to Fruit Ripening and Stress Response in Banana (Musa acuminata L. AAA Group, cv. 'Cavendish').

MYB is an important type of transcription factor in eukaryotes. It is widely involved in a variety of biological processes and plays a role in plant morphogenesis, growth and development, primary and secondary metabolite synthesis, and other life processes. In this study, bioinformatics methods were used to identify the R2R3-MYB transcription factor family members in the whole Musa acuminata (DH-Pahang) genome, one of the wild ancestors of banana. A total of 280 MaMYBs were obtained, and phylogenetic analysis indicated that these MaMYBs could be classified into 33 clades with MYBs from Arabidopsis thaliana. The amino acid sequences of the R2 and R3 Myb-DNA binding in all MaMYB protein sequences were quite conserved, especially Arg-12, Arg-13, Leu-23, and Leu-79. Distribution mapping results showed that 277 MaMYBs were localized on the 11 chromosomes in the Musa acuminata genome. The MaMYBs were distributed unevenly across the 11 chromosomes. More than 40.0% of the MaMYBs were located in collinear fragments, and segmental duplications likely played a key role in the expansion of the MaMYBs. Moreover, the expression profiles of MaMYBs 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. Weighted gene co-expression network analysis (WGCNA) was used to analyze transcriptome data of banana from the above 11 samples. We found MaMYBs participating in important metabolic biosynthesis pathways in banana. Collectively, our results represent a comprehensive genome-wide study of the MaMYB gene family, which should be helpful in further detailed studies on MaMYBs functions related to fruit development, postharvest ripening, and the seedling response to stress in an important banana cultivar.

Resequencing of 388 cassava accessions identifies valuable loci and selection for variation in heterozygosity.

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.

Overexpression of a Banana Aquaporin Gene MaPIP1;1 Enhances Tolerance to Multiple Abiotic Stresses in Transgenic Banana and Analysis of Its Interacting Transcription Factors.

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.

Transcription factor MaMADS36 plays a central role in regulating banana fruit ripening.

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.

Identification and expression of the BAHD family during development, ripening, and stress response 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.

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

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

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

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

Genome-Wide Analysis of Basic Helix-Loop-Helix Transcription Factors to Elucidate Candidate Genes Related to Fruit Ripening and Stress in Banana (Musa acuminata L. AAA Group, cv. Cavendish).

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.