Deep RNA-seq analysis reveals key responding aspects of wild banana relative resistance to Fusarium oxysporum f. sp. cubense tropical race 4.

Tropical race 4 of Fusarium oxysporum f. sp. cubense (FocTR4) is seriously threatening the banana industry worldwide. Resistant genotypes are present in wild relatives of banana, but little is known about the genetic and molecular mechanisms driving resistance responses. In this work, through in-depth expression analysis, we compared the responses of the resistant wild relative Musa acuminata ssp. burmanicoides (WTB) with the susceptible banana cultivar "Brizilian" (CAV, as it belongs to the Cavendish subgroup) to FocTR4 infection. Our findings showed that 1196 defense-related genes in the resistant WTB were differentially expressed genes (DEGs); only 358 defense-related DEGs were detected in CAV. DEGs related to pattern recognition receptors (PRRs) and disease resistance (R genes) were found in both genotypes, indicating the onset of both basal and specific defenses to FocTR4. Genes associated with cell wall modification exhibited a more remarkable upregulation in WTB than in CAV and might be involved in resistance during penetration steps. Our data also suggested that the high resistance of WTB is quantitatively driven with larger numbers and higher expression levels of defense-related DEGs. Fine-tuning studies to understand the resistance responses of WTB at early stages should be conducted to better support banana breeding programs. Further investigations are also required to validate the role of key genes screened in this study.

Three-dimensional poly-(ε-caprolactone) nanofibrous scaffolds directly promote the cardiomyocyte differentiation of murine-induced pluripotent stem cells through Wnt/ß-catenin signaling.

BACKGROUND: Environmental factors are important for stem cell lineage specification, and increasing evidence indicates that the nanoscale geometry/topography of the extracellular matrix (ECM) directs stem cell fate. Recently, many three-dimensional (3D) biomimetic nanofibrous scaffolds resembling many characteristics of the native ECM have been used in stem cell-based myocardial tissue engineering. However, the biophysical role and underlying mechanism of 3D nanofibrous scaffolds in cardiomyocyte differentiation of induced pluripotent stem cells (iPSCs) remain unclear. RESULTS: Here, we fabricated a 3D poly-(ε-caprolactone) (PCL) nanofibrous scaffold using the electrospinning method and verified its nanotopography and porous structure by scanning electron microscopy. We seeded murine iPSCs (miPSCs) directly on the 3D PCL nanofibrous scaffold and initiated non-directed, spontaneous differentiation using the monolayer method. After the 3D PCL nanofibrous scaffold was gelatin coated, it was suitable for monolayer miPSC cultivation and cardiomyocyte differentiation. At day 15 of differentiation, miPSCs differentiated into functional cardiomyocytes on the 3D PCL nanofibrous scaffold as evidenced by positive immunostaining of cardiac-specific proteins including cardiac troponin T (cTnT) and myosin light chain 2a (MLC2a). In addition, flow cytometric analysis of cTnT-positive cells and cardiac-specific gene and protein expression of cTnT and sarcomeric alpha actinin (α-actinin) demonstrated that the cardiomyocyte differentiation of miPSCs was more efficient on the 3D PCL nanofibrous scaffold than on normal tissue culture plates (TCPs). Furthermore, early inhibition of Wnt/ß-catenin signaling by the selective antagonist Dickkopf-1 significantly reduced the activity of Wnt/ß-catenin signaling and decreased the cardiomyocyte differentiation of miPSCs cultured on the 3D PCL nanofibrous scaffold, while the early activation of Wnt/ß-catenin signaling by CHIR99021 further increased the cardiomyocyte differentiation of miPSCs. CONCLUSION: These results indicated that the electrospun 3D PCL nanofibrous scaffolds directly promoted the cardiomyocyte differentiation of miPSCs, which was mediated by the activation of the Wnt/ß-catenin signaling during the early period of differentiation. These findings highlighted the biophysical role of 3D nanofibrous scaffolds during the cardiomyocyte differentiation of miPSCs and revealed its underlying mechanism involving Wnt/ß-catenin signaling, which will be helpful in guiding future stem cell- and scaffold-based myocardium bioengineering.

De novo assembly and characterization of pericarp transcriptome and identification of candidate genes mediating fruit cracking in Litchi chinensis Sonn.

Fruit cracking has long been a topic of great concern for growers and researchers of litchi (Litchi chinensis Sonn.). To understand the molecular mechanisms underlying fruit cracking, high-throughput RNA sequencing (RNA-Seq) was first used for de novo assembly and characterization of the transcriptome of cracking pericarp of litchi. Comparative transcriptomic analyses were performed on non-cracking and cracking fruits. A total of approximately 26 million and 29 million high quality reads were obtained from the two groups of samples, and were assembled into 46,641 unigenes with an average length of 993 bp. These unigenes can be useful resources for future molecular studies of the pericarp in litchi. Furthermore, four genes (LcAQP, 1; LcPIP, 1; LcNIP, 1; LcSIP, 1) involved in water transport, five genes (LcKS, 2; LcGA2ox, 2; LcGID1, 1) involved in GA metabolism, 21 genes (LcCYP707A, 2; LcGT, 9; Lcß-Glu, 6; LcPP2C, 2; LcABI1, 1; LcABI5, 1) involved in ABA metabolism, 13 genes (LcTPC, 1; Ca2+/H+ exchanger, 3; Ca2+-ATPase, 4; LcCDPK, 2; LcCBL, 3) involved in Ca transport and 24 genes (LcPG, 5; LcEG, 1; LcPE, 3; LcEXP, 5; Lcß-Gal, 9; LcXET, 1) involved in cell wall metabolism were identified as genes that are differentially expressed in cracked fruits compared to non-cracked fruits. Our results open new doors to further understand the molecular mechanisms behind fruit cracking in litchi and other fruits, especially Sapindaceae plants.

Expression patterns, activities and carbohydrate-metabolizing regulation of sucrose phosphate synthase, sucrose synthase and neutral invertase in pineapple fruit during development and ripening.

Differences in carbohydrate contents and metabolizing-enzyme activities were monitored in apical, medial, basal and core sections of pineapple (Ananas comosus cv. Comte de paris) during fruit development and ripening. Fructose and glucose of various sections in nearly equal amounts were the predominant sugars in the fruitlets, and had obvious differences until the fruit matured. The large rise of sucrose/hexose was accompanied by dramatic changes in sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) activities. By contrast, neutral invertase (NI) activity may provide a mechanism to increase fruit sink strength by increasing hexose concentrations. Furthermore, two cDNAs of Ac-sps (accession no. GQ996582) and Ac-ni (accession no. GQ996581) were first isolated from pineapple fruits utilizing conserved amino-acid sequences. Homology alignment reveals that the amino acid sequences contain some conserved function domains. Transcription expression analysis of Ac-sps, Ac-susy and Ac-ni also indicated distinct patterns related to sugar accumulation and composition of pineapple fruits. It suggests that differential expressions of multiple gene families are necessary for sugar metabolism in various parts and developmental stages of pineapple fruit. A cycle of sucrose breakdown in the cytosol of sink tissues could be mediated through both Ac-SuSy and Ac-NI, and Ac-NI could be involved in regulating crucial steps by generating sugar signals to the cells in a temporally and spatially restricted fashion.

Cloning and expression analysis of a PISTILLATA homologous gene from pineapple (Ananas comosus L. Merr).

PISTILLATA (PI)-like genes are crucial regulators of flowering in angiosperms. A homologue of PI, designated as AcPI (Genbank accession number HQ717796), was isolated from pineapple cultivar Comte de Paris by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of AcPI is 907 bp in length and contains an open reading frame of 594 bp, which encodes a protein of 197 amino acids. The molecular weight was 2.29 kDa and the isoelectric point was 9.28. The alignment showed that AcPI had a high identity with CsPIC2 (78.6%), AoPI (77.4%), OrcPI (75.7%) and HPI2 (72.4%). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses in different tissues showed that the expression pattern of AcPI was different from the B-class genes in eudicots. AcPI was expressed in all the tissues investigated. The expression level was very low in fruit stems, bracts, leaves and sepals, high in petals and carpels, and moderate in apical meristems, flesh and stamens. The qRT-PCR analyses in different stages indicated that the expression of AcPI reached the highest level at 40 days after flower inducement, when the multiple fruit and floral organs were forming. It proved the important role of AcPI in floral organs and fruit development. The 35S::AcPI transgenic Arabidopsis plants flowered earlier and had more inflorescences or branches than wild type plants.

Involvement of abscisic acid-responsive element-binding factors in cassava (Manihot esculenta) dehydration stress response.

Cassava (Manihot esculenta) is a major staple food, animal feed and energy crop in the tropics and subtropics. It is one of the most drought-tolerant crops, however, the mechanisms of cassava drought tolerance remain unclear. Abscisic acid (ABA)-responsive element (ABRE)-binding factors (ABFs) are transcription factors that regulate expression of target genes involved in plant tolerance to drought, high salinity, and osmotic stress by binding ABRE cis-elements in the promoter regions of these genes. However, there is little information about ABF genes in cassava. A comprehensive analysis of Manihot esculenta ABFs (MeABFs) described the phylogeny, genome location, cis-acting elements, expression profiles, and regulatory relationship between these factors and Manihot esculenta betaine aldehyde dehydrogenase genes (MeBADHs). Here we conducted genome-wide searches and subsequent molecular cloning to identify seven MeABFs that are distributed unevenly across six chromosomes in cassava. These MeABFs can be clustered into three groups according to their phylogenetic relationships to their Arabidopsis (Arabidopsis thaliana) counterparts. Analysis of the 5'-upstream region of MeABFs revealed putative cis-acting elements related to hormone signaling, stress, light, and circadian clock. MeABF expression profiles displayed clear differences among leaf, stem, root, and tuberous root tissues under non-stress and drought, osmotic, or salt stress conditions. Drought stress in cassava leaves and roots, osmotic stress in tuberous roots, and salt stress in stems induced expression of the highest number of MeABFs showing significantly elevated expression. The glycine betaine (GB) content of cassava leaves also was elevated after drought, osmotic, or salt stress treatments. BADH1 is involved in GB synthesis. We show that MeBADH1 promoter sequences contained ABREs and that MeBADH1 expression correlated with MeABF expression profiles in cassava leaves after the three stress treatments. Taken together, these results suggest that in response to various dehydration stresses, MeABFs in cassava may activate transcriptional expression of MeBADH1 by binding the MeBADH1 promoter that in turn promotes GB biosynthesis and accumulation via an increase in MeBADH1 gene expression levels and MeBADH1 enzymatic activity. These responses protect cells against dehydration stresses by preserving an osmotic balance that enhances cassava tolerance to dehydration stresses.

Optimization of fermentation conditions through response surface methodology for enhanced antibacterial metabolite production by Streptomyces sp. 1-14 from cassava rhizosphere.

Streptomyces species 1-14 isolated from cassava rhizosphere soil were evaluated for their antibacterial efficacy against Fusarium oxysporum f.sp. cubense race 4 (FOC4). Of the 63 strains tested, thirteen exhibited potent antibacterial properties and were further screened against eight fungal pathogens. The strain that showed maximum inhibition against all of the test pathogens was identified by 16S rDNA sequencing as Streptomyces sp. 1-14, was selected for further studies. Through the propagation of Streptomyces sp. 1-14 in soil under simulated conditions, we found that FOC4 did not significantly influence the multiplication and survival of Streptomyces sp. 1-14, while indigenous microorganisms in the soil did significantly influence Streptomyces sp. 1-14 populations. To achieve maximum metabolite production, the growth of Streptomyces 1-14 was optimized through response surface methodology employing Plackett-Burman design, path of steepest ascent determinations and Box-Behnken design. The final optimized fermentation conditions (g/L) included: glucose, 38.877; CaCl2•2H2O, 0.161; temperature, 29.97°C; and inoculation amount, 8.93%. This optimization resulted in an antibacterial activity of 56.13% against FOC4, which was 12.33% higher than that before optimization (43.80%). The results obtained using response surface methodology to optimize the fermentation medium had a significant effect on the production of bioactive metabolites by Streptomyces sp. 1-14. Moreover, during fermentation and storage, pH, light, storage temperature, etc., must be closely monitored to reduce the formation of fermentation products with reduced antibacterial activity. This method is useful for further investigations of the production of anti-FOC4 substances, and could be used to develop bio-control agents to suppress or control banana fusarium wilt.

Expression patterns of members of the ethylene signaling-related gene families in response to dehydration stresses in cassava.

Drought is the one of the most important environment stresses that restricts crop yield worldwide. Cassava (Manihot esculenta Crantz) is an important food and energy crop that has many desirable traits such as drought, heat and low nutrients tolerance. However, the mechanisms underlying drought tolerance in cassava are unclear. Ethylene signaling pathway, from the upstream receptors to the downstream transcription factors, plays important roles in environmental stress responses during plant growth and development. In this study, we used bioinformatics approaches to identify and characterize candidate Manihot esculenta ethylene receptor genes and transcription factor genes. Using computational methods, we localized these genes on cassava chromosomes, constructed phylogenetic trees and identified stress-responsive cis-elements within their 5' upstream regions. Additionally, we measured the trehalose and proline contents in cassava fresh leaves after drought, osmotic, and salt stress treatments, and then it was found that the regulation patterns of contents of proline and trehalose in response to various dehydration stresses were differential, or even the opposite, which shows that plant may take different coping strategies to deal with different stresses, when stresses come. Furthermore, expression profiles of these genes in different organs and tissues under non-stress and abiotic stress were investigated through quantitative real-time PCR (qRT-PCR) analyses in cassava. Expression profiles exhibited clear differences among different tissues under non-stress and various dehydration stress conditions. We found that the leaf and tuberous root tissues had the greatest and least responses, respectively, to drought stress through the ethylene signaling pathway in cassava. Moreover, tuber and root tissues had the greatest and least reponses to osmotic and salt stresses through ethylene signaling in cassava, respectively. These results show that these plant tissues had differential expression levels of genes involved in ethylene signaling in response to the stresses tested. Moreover, after several gene duplication events, the spatiotemporally differential expression pattern of homologous genes in response to abiotic and biotic stresses may imply their functional diversity as a mechanism for adapting to the environment. Our data provide a framework for further research on the molecular mechanisms of cassava resistance to drought stress and provide a foundation for breeding drought-resistant new cultivars.

Transcriptome Profiling of Light-Regulated Anthocyanin Biosynthesis in the Pericarp of Litchi.

Light is a key environmental factor that affects anthocyanin biosynthesis. To enhance our understanding of the mechanisms involved in light-regulated anthocyanin biosynthesis in the pericarp of litchi, we performed transcriptomic analyses on the basis of Illumina sequencing. Fruit clusters were bagged with double-layer Kraft paper bags at 42 days after anthesis. The bags were removed after 2 weeks. Under light conditions, anthocyanins accumulated rapidly in the pericarp. RNA sequences were de novo assembled into 75,935 unigenes with an average length of 913 bp. Approximately 74.5% of unigenes (56,601) were annotated against four public protein databases. A total of 16,622 unigenes that significantly differed in terms of abundance were identified. These unigenes are implicated in light signal perception and transduction, flavonoid biosynthesis, carotenoid biosynthesis, plant hormone signal transduction, and photosynthesis. In photoreceptors, the expression levels of UV RESISTANCE LOCUS 8 (UVR8), Phototropin 2 (PHOT2), Phytochrome B (PHYB), and Phytochrome C (PHYC) increased significantly when the fruits were exposed to light. This result indicated that they likely play important roles in anthocyanin biosynthesis regulation. After analyzed digital gene expression (DGE), we found that the light signal transduction elements of COP1 and COP10 might be responsible for anthocyanin biosynthesis regulation. After the bags were removed, nearly all structural and regulatory genes, such as UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT), MYB, basic helix-loop-helix (bHLH), and WD40, involved in the anthocyanin biosynthetic pathway were upregulated. In addition to MYB-bHLH-WD40 transcription complex, ELONGATED HYPOCOTYL (HY5), NAM/ATAF/CUC (NAC), homeodomain leucine zipper proteins (ATHBs), and FAR-RED ELONGATED HYPOCOTYL (FHY) possibly participate in light-induced responses. On the basis of DGEs and qRT-PCR validation, we observed a light-induced anthocyanin biosynthesis and regulation pattern in litchi pericarp. This study enhanced our understanding of the molecular mechanisms governing light-induced anthocyanin biosynthesis in litchi pericarp.