Transcriptome, Plant Hormone, and Metabolome Analysis Reveals the Mechanism of Purple Pericarp Formation in 'Zihui' Papaya (Carica papaya L.).

The color of the pericarp is a crucial characteristic that influences the marketability of papaya fruit. Prior to ripening, normal papaya exhibits a green pericarp, whereas the cultivar 'Zihui' displays purple ring spots on the fruit tip, which significantly affects the fruit's visual appeal. To understand the mechanism behind the formation of purple pericarp, this study performed a thorough examination of the transcriptome, plant hormone, and metabolome. Based on the UPLC-ESI-MS/MS system, a total of 35 anthocyanins and 11 plant hormones were identified, with 27 anthocyanins and two plant hormones exhibiting higher levels of abundance in the purple pericarp. In the purple pericarp, 14 anthocyanin synthesis genes were up-regulated, including CHS, CHI, F3H, F3'5'H, F3'H, ANS, OMT, and CYP73A. Additionally, through co-expression network analysis, three MYBs were identified as potential key regulators of anthocyanin synthesis by controlling genes encoding anthocyanin biosynthesis. As a result, we have identified numerous key genes involved in anthocyanin synthesis and developed new insights into how the purple pericarp of papaya is formed.

Quantitative proteomic analysis reveals that antioxidation mechanisms contribute to cold tolerance in plantain (Musa paradisiaca L.; ABB Group) seedlings.

Banana and its close relative, plantain are globally important crops and there is considerable interest in optimizing their cultivation. Plantain has superior cold tolerance compared with banana and a thorough understanding of the molecular mechanisms and responses of plantain to cold stress has great potential value for developing cold tolerant banana cultivars. In this study, we used iTRAQ-based comparative proteomic analysis to investigate the temporal responses of plantain to cold stress. Plantain seedlings were exposed for 0, 6, and 24 h of cold stress at 8 °C and subsequently allowed to recover for 24 h at 28 °C. A total of 3477 plantain proteins were identified, of which 809 showed differential expression from the three treatments. The majority of differentially expressed proteins were predicted to be involved in oxidation-reduction, including oxylipin biosynthesis, whereas others were associated with photosynthesis, photorespiration, and several primary metabolic processes, such as carbohydrate metabolic process and fatty acid beta-oxidation. Western blot analysis and enzyme activity assays were performed on seven differentially expressed, cold-response candidate plantain proteins to validate the proteomics data. Similar analyses of the seven candidate proteins were performed in cold-sensitive banana to examine possible functional conservation, and to compare the results to equivalent responses between the two species. Consistent results were achieved by Western blot and enzyme activity assays, demonstrating that the quantitative proteomics data collected in this study are reliable. Our results suggest that an increase of antioxidant capacity through adapted ROS scavenging capability, reduced production of ROS, and decreased lipid peroxidation contribute to molecular mechanisms for the increased cold tolerance in plantain. To the best of our knowledge, this is the first report of a global investigation on molecular responses of plantain to cold stress by proteomic analysis.

Transcriptome profiling of resistant and susceptible Cavendish banana roots following inoculation with Fusarium oxysporum f. sp. cubense tropical race 4.

BACKGROUND: Fusarium wilt, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is considered the most lethal disease of Cavendish bananas in the world. The disease can be managed in the field by planting resistant Cavendish plants generated by somaclonal variation. However, little information is available on the genetic basis of plant resistance to Foc TR4. To a better understand the defense response of resistant banana plants to the Fusarium wilt pathogen, the transcriptome profiles in roots of resistant and susceptible Cavendish banana challenged with Foc TR4 were compared. RESULTS: RNA-seq analysis generated more than 103 million 90-bp clean pair end (PE) reads, which were assembled into 88,161 unigenes (mean size = 554 bp). Based on sequence similarity searches, 61,706 (69.99%) genes were identified, among which 21,273 and 50,410 unigenes were assigned to gene ontology (GO) categories and clusters of orthologous groups (COG), respectively. Searches in the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) mapped 33,243 (37.71%) unigenes to 119 KEGG pathways. A total of 5,008 genes were assigned to plant-pathogen interactions, including disease defense and signal transduction. Digital gene expression (DGE) analysis revealed large differences in the transcriptome profiles of the Foc TR4-resistant somaclonal variant and its susceptible wild-type. Expression patterns of genes involved in pathogen-associated molecular pattern (PAMP) recognition, activation of effector-triggered immunity (ETI), ion influx, and biosynthesis of hormones as well as pathogenesis-related (PR) genes, transcription factors, signaling/regulatory genes, cell wall modification genes and genes with other functions were analyzed and compared. The results indicated that basal defense mechanisms are involved in the recognition of PAMPs, and that high levels of defense-related transcripts may contribute to Foc TR4 resistance in banana. CONCLUSIONS: This study generated a substantial amount of banana transcript sequences and compared the defense responses against Foc TR4 between resistant and susceptible Cavendish bananas. The results contribute to the identification of candidate genes related to plant resistance in a non-model organism, banana, and help to improve the current understanding of host-pathogen interactions.

Comparative transcriptomics and genomic analyses reveal differential gene expression related to Colletotrichum brevisporum resistance in papaya (Carica papaya L.).

Colletotrichum brevisporum is an important causal pathogen of anthracnose that seriously affects the fruit quality and yield of papaya (Carica papaya L.). Although many genes and biological processes involved in anthracnose resistance have been reported in other species, the molecular mechanisms involved in the response or resistance to anthracnose in post-harvest papaya fruits remain unclear. In this study, we compared transcriptome changes in the post-harvest fruits of the anthracnose-susceptible papaya cultivar Y61 and the anthracnose-resistant cultivar G20 following C. brevisporum inoculation. More differentially expressed genes (DEGs) and differentially expressed long non-coding RNAs (DElnRNAs) were identified in G20 than in Y61, especially at 24 h post-inoculation (hpi), suggesting a prompt activation of defense responses in G20 in the first 24 h after C. brevisporum inoculation. These DEGs were mainly enriched in plant-pathogen interaction, phenylpropanoid biosynthesis/metabolism, and peroxisome and flavonoid biosynthesis pathways in both cultivars. However, in the first 24 hpi, the number of DEGs related to anthracnose resistance was greater in G20 than in Y61, and changes in their expression levels were faster in G20 than in Y61. We also identified a candidate anthracnose-resistant gene cluster, which consisted of 12 genes, 11 in G20 and Y61, in response to C. brevisporum inoculation. Moreover, 529 resistance gene analogs were identified in papaya genome, most of which responded to C. brevisporum inoculation and were genetically different between papaya cultivars and wild-type populations. The total expression dose of the resistance gene analogs may help papaya resist C. brevisporum infection. This study revealed the mechanisms underlying different anthracnose resistance between the anthracnose-resistant and anthracnose-susceptible cultivars based on gene expression, and identified some potential anthracnose resistance-related candidate genes/major regulatory factors. Our findings provided potential targets for developing novel genetic strategies to overcome anthracnose in papaya.

Quantitative Ubiquitylomic Analysis of the Dynamic Changes and Extensive Modulation of Ubiquitylation in Papaya During the Fruit Ripening Process.

Lysine ubiquitination is a highly conserved post-translational modification with diverse biological functions. However, there is little available information on lysine ubiquitination of non-histone proteins in papaya (Carica papaya L.). In total, 3,090 ubiquitination sites on 1,249 proteins with diverse localizations and functions were identified. Five conserved ubiquitinated K motifs were identified. Enrichment analysis showed that many Hsps were differentially ubiquitinated proteins (DUPs), suggesting an essential role of ubiquitination in degradation of molecular chaperone. Furthermore, 12 sugar metabolism-related enzymes were identified as DUPs, including an involvement of ubiquitination in nutrimental changes during the papaya ripening process. The ubiquitination levels of five fruit ripening-related DUPs, including one ethylene-inducible protein, two 1-aminocyclopropane-1-carboxylic acid oxidases, one endochitinase, and one cell wall invertase, were significantly changed during the ripening process. Our study extends the understanding of diverse functions for lysine ubiquitination in regulation of the papaya fruit ripening process.

Genome-wide analysis of basic helix-loop-helix transcription factors in papaya (Carica papaya L.).

The basic helix-loop-helix (bHLH) transcription factors (TFs) have been identified and functionally characterized in many plants. However, no comprehensive analysis of the bHLH family in papaya (Carica papaya L.) has been reported previously. Here, a total of 73 CpbHLHs were identified in papaya, and these genes were classified into 18 subfamilies based on phylogenetic analysis. Almost all of the CpbHLHs in the same subfamily shared similar gene structures and protein motifs according to analysis of exon/intron organizations and motif compositions. The number of exons in CpbHLHs varied from one to 10 with an average of five. The amino acid sequences of the bHLH domains were quite conservative, especially Leu-27 and Leu-63. Promoter cis-element analysis revealed that most of the CpbHLHs contained cis-elements that can respond to various biotic/abiotic stress-related events. Gene ontology (GO) analysis revealed that CpbHLHs mainly functions in protein dimerization activity and DNA-binding, and most CpbHLHs were predicted to localize in the nucleus. Abiotic stress treatment and quantitative real-time PCR (qRT-PCR) revealed some important candidate CpbHLHs that might be responsible for abiotic stress responses in papaya. These findings would lay a foundation for further investigate of the molecular functions of CpbHLHs.

Comparative Phosphoproteomics Reveals an Important Role of MKK2 in Banana (Musa spp.) Cold Signal Network.

Low temperature is one of the key environmental stresses, which greatly affects global banana production. However, little is known about the global phosphoproteomes in Musa spp. and their regulatory roles in response to cold stress. In this study, we conducted a comparative phosphoproteomic profiling of cold-sensitive Cavendish Banana and relatively cold tolerant Dajiao under cold stress. Phosphopeptide abundances of five phosphoproteins involved in MKK2 interaction network, including MKK2, HY5, CaSR, STN7 and kinesin-like protein, show a remarkable difference between Cavendish Banana and Dajiao in response to cold stress. Western blotting of MKK2 protein and its T31 phosphorylated peptide verified the phosphoproteomic results of increased T31 phosphopeptide abundance with decreased MKK2 abundance in Daojiao for a time course of cold stress. Meanwhile increased expression of MKK2 with no detectable T31 phosphorylation was found in Cavendish Banana. These results suggest that the MKK2 pathway in Dajiao, along with other cold-specific phosphoproteins, appears to be associated with the molecular mechanisms of high tolerance to cold stress in Dajiao. The results also provide new evidence that the signaling pathway of cellular MKK2 phosphorylation plays an important role in abiotic stress tolerance that likely serves as a universal plant cold tolerance mechanism.

Contamination of bananas with beauvericin and fusaric acid produced by Fusarium oxysporum f. sp. cubense.

BACKGROUND: Fusarium wilt, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of banana. Toxins produced by Foc have been proposed to play an important role during the pathogenic process. The objectives of this study were to investigate the contamination of banana with toxins produced by Foc, and to elucidate their role in pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Twenty isolates of Foc representing races 1 and 4 were isolated from diseased bananas in five Chinese provinces. Two toxins were consistently associated with Foc, fusaric acid (FA) and beauvericin (BEA). Cytotoxicity of the two toxins on banana protoplast was determined using the Alamar Blue assay. The virulence of 20 Foc isolates was further tested by inoculating tissue culture banana plantlets, and the contents of toxins determined in banana roots, pseudostems and leaves. Virulence of Foc isolates correlated well with toxin deposition in the host plant. To determine the natural occurrence of the two toxins in banana plants with Fusarium wilt symptoms, samples were collected before harvest from the pseudostems, fruit and leaves from 10 Pisang Awak 'Guangfen #1' and 10 Cavendish 'Brazilian' plants. Fusaric acid and BEA were detected in all the tissues, including the fruits. CONCLUSIONS/SIGNFICANCE: The current study provides the first investigation of toxins produced by Foc in banana. The toxins produced by Foc, and their levels of contamination of banana fruits, however, were too low to be of concern to human and animal health. Rather, these toxins appear to contribute to the pathogenicity of the fungus during infection of banana plants.

[Establishment of embryogenic cell suspension culture and plant regeneration of edible banana Musa acuminata cv. Mas (AA)].

Conventional breeding for dual resistance of disease and pest of Musa cultivars remains a difficult endeavor, as the plant is polyploidic and high in sterility. Biotechnological techniques, eg., genetic engineering, in vitro mutation breeding, or protoplast fusion, may overcome the difficulties and improve the germplasm. Establishment of a stable embryogenic cell suspension (ECS) is a prerequisite for any of the biotechnological breeding methods. In this study an embryogenic cell suspension was established from immature male flower of Musa acuminata cv. Mas (AA), a popular commercial variety of banana in the South-East Asian region. After culture for 5-6 months on callus induction media, which consisted of MS salts, different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 4.1 micromol/L biotin, 5.7 micromol/L indoleacetic acid (IAA), 5.4 micromol/L naphthaleneacetic acid (NAA), other vitamins, 87 mmol/L sucrose, and solidified with 7 g/L agarose, meristematic globules and yellow, friable embryogenic cultures were induced from the explants of 1-15th row young floral hands of immature male flowers. Of the four treatments of 2,4-D, 9 micromol/L was the most effective on the callus induction, it transformed 40.96% and 7.45% of the cultivated male floral hands into callus and embryogenic callus respectively. The explants to produce highest frequency of the embryogenic calli were floral hands of 6 to 12th rows, which generated 5.79% of the embryogenic calli. Suspension cultures were initiated from these embryogenic calli in liquid medium supplemented with 4.5 micromol/L 2, 4-D. After sieving selection of the cultures using a stainless steel metallic strainer with pore sizes of 154 microm at 15 day intervals for 3 months, homogeneous and yellow embryogenic cell suspensions, composed of single cells and small cell aggregates, were established. Based upon the growth quantity and growth rate of ECS, it was determined that the appropriate inoculum was 2.0 mL PCV ECS/30 mL medium in 100 mL flask, and the appropriate subculture cycle was 15 days. Planting of 6 months old ECS on semi-solid medium of somatic embryo induction and development (MSD) resulted in approximately 280 x 10(3) somatic embryos/mL PCV ECS. MSD contained SH macronutrients, micro-nutrients, Fe-EDTA and MS vitamins supplemented with 4.5 micromol/L biotin, 680 micromol/L glutamine, 2 mmol/L proline, 100 mg/L malt extract, 1.1 micromol/L NAA, 0.2 micromol/L zeatin, 0.5 micromol/L kinetin, 0.7 micromol/L N6-(2-isopentenyl) adenine, 29 mmol/L lactose, 130 mmol/L sucrose and solidified with 2g/L gelrite. After 3 months of maturity on MSD, 17.28% of the somatic embryos were germinated on germination media (MG), consisted of MS salt, Morel and Wetmore vitamins, 0.2 micromol/L 6-BA, 1.1 micromol/L IAA, 87 micromol/L sucrose and solidified with 2 g/L gelrite; and 14.16% of the somatic embryos could develop into normal plantlets on rooting media contained the same composition as that of MG but without auxin and cytokinin.