Weixin Chen

Evaluation of New Reference Genes in Papaya for Accurate Transcript Normalization under Different Experimental Conditions

Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions.

Molecular characterization of banana NAC transcription factors and their interactions with ethylene signalling component EIL during fruit ripening

The plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) play important roles in plant growth, development, and stress responses. However, the precise role of NAC TFs in relation to fruit ripening is poorly understood. In this study, six NAC genes, designated MaNAC1–MaNAC6, were isolated and characterized from banana fruit. Subcellular localization showed that MaNAC1–MaNAC5 proteins localized preferentially to the nucleus, while MaNAC6 was distributed throughout the entire cell. A transactivation assay in yeast demonstrated that MaNAC4 and MaNAC6, as well as their C-terminal regions, possessed trans-activation activity. Gene expression profiles in fruit with four different ripening characteristics, including natural, ethylene-induced, 1-methylcyclopropene (1-MCP)-delayed, and a combination of 1-MCP with ethylene treatment, revealed that the MaNAC genes were differentially expressed in peel and pulp during post-harvest ripening. MaNAC1 and MaNAC2 were apparently upregulated by ethylene in peel and pulp, consistent with the increase in ethylene production. In contrast, MaNAC3 in peel and pulp and MaNAC5 in peel were constitutively expressed, and transcripts of MaNAC4 in peel and pulp and MaNAC6 in peel decreased, while MaNAC5 or MaNAC6 in pulp increased slightly during fruit ripening. Furthermore, the MaNAC2 promoter was activated after ethylene application, further enhancing the involvement of MaNAC2 in fruit ripening. More importantly, yeast two-hybrid and bimolecular fluorescence complementation analyses confirmed that MaNAC1/2 physically interacted with a downstream component of ethylene signalling, ethylene insensitive 3 (EIN3)-like protein, termed MaEIL5, which was downregulated during ripening. Taken together, these results suggest that MaNACs such as MaNAC1/MaNAC2, may be involved in banana fruit ripening via interaction with ethylene signalling components.

Expression of genes associated with ethylene-signalling pathway in harvested banana fruit in response to temperature and 1-MCP treatment

BACKGROUND Little attention has been paid to characterising the ethylene-signalling pathway genes in relation to abnormal ripening of harvested banana fruit during storage at high temperature. The aim of the present study was to investigate banana fruit abnormal ripening and the expression of ten genes associated with the ethylene-signalling pathway, namely MaACS1, MaACO1, MaERS1-4 and MaEIL1-4, at high temperature. Changes in these parameters of banana fruit at high temperature in response to 1-MCP pretreatment were also investigated. RESULTS High temperature accelerated the decline in fruit firmness, increased ethylene production and inhibited degreening in banana fruit, resulting in fruit abnormal ripening. In addition, the expression of MaACS1, MaACO1, MaERS2, MaERS3, MaERS4, MaEIL1, MaEIL3 and MaEIL4 was enhanced in banana fruit stored at high temperature. However, application of 1-MCP prior to high temperature storage delayed fruit abnormal ripening and simultaneously suppressed the expression of MaACS1, MaERS2, MaERS3, MaEIL1, MaEIL3 and MaEIL4. CONCLUSION The findings of this study suggested that the expression of genes associated with the ethylene-signalling pathway might be involved in banana fruit abnormal ripening at high temperature. Application of 1-MCP suppressed the expression of genes associated with the ethylene-signalling pathway, which may be attributed at least partially to 1-MCP delaying fruit abnormal ripening at high temperature.

Isolation and characterization of ethylene response factor family genes during development, ethylene regulation and stress treatments in papaya fruit.

Ethylene response factors (ERFs) play important roles in fruit development, ripening, defense responses and stress signaling pathways. After harvest, climacteric fruit such as papaya are subject to a range of problems associated with postharvest handling and storage treatments. There have been few attempts to evaluate the role of ERFs in fruits responses to environmental stimuli. To investigate the transcriptional mechanisms underlying fruit developmental, ripening and stresses, we cloned four ERFs from papaya. The deduced amino acid sequence of CpERFs contained the conserved apetalous (AP2)/ERF domain, which shared high similarity with other reported AP2/ERF domains. The phylogeny, gene structures, and putatively conserved motifs in papaya ERF proteins were analyzed, and compared with those of Arabidopsis. Expression patterns of CpERFs were examined during fruit development, under 1-MCP treatment, ethephon treatment, biotic stress (temperature stress) and pathogen stress. CpERFs displayed differential expression patterns and expression levels under different experimental conditions. CpERF2 and CpERF3 showed a close association with fruit ripening and CpERFs had a high expression level in the earlier stages during the fruit development period. The expression of CpERFs strongly associated with stress response. These results support the role for papaya ERFs in transcriptional regulation of ripening-related or stress-respond genes and thus, in the regulation of papaya fruit-ripening processes and stress responses.

The relationship between the expression of ethylene-related genes and papaya fruit ripening disorder caused by chilling injury.

Papaya (Carica papaya L.) is sensitive to low temperature and easy to be subjected to chilling injury, which causes fruit ripening disorder. This study aimed to investigate the relationship between the expression of genes related to ethylene and fruit ripening disorder caused by chilling injury. Papaya fruits were firstly stored at 7°C and 12°C for 25 and 30 days, respectively, then treated with exogenous ethylene and followed by ripening at 25°C for 5 days. Chilling injury symptoms such as pulp water soaking were observed in fruit stored at 7°C on 20 days, whereas the coloration and softening were completely blocked after 25 days, Large differences in the changes in the expression levels of twenty two genes involved in ethylene were seen during 7°C-storage with chilling injury. Those genes with altered expression could be divided into three groups: the group of genes that were up-regulated, including ACS1/2/3, EIN2, EIN3s/EIL1, CTR1/2/3, and ERF1/3/4; the group of genes that were down-regulated, including ACO3, ETR1, CTR4, EBF2, and ERF2; and the group of genes that were un-regulated, including ACO1/2, ERS, and EBF1. The results also showed that pulp firmness had a significantly positive correlation with the expression of ACS2, ACO1, CTR1/4, EIN3a/b, and EBF1/2 in fruit without chilling injury. This positive correlation was changed to negative one in fruit after storage at 7°C for 25 days with chilling injury. The coloring index displayed significantly negative correlations with the expression levels of ACS2, ACO1/2, CTR4, EIN3a/b, ERF3 in fruit without chilling injury, but these correlations were changed into the positive ones in fruit after storage at 7°C for 25 days with chilling injury. All together, these results indicate that these genes may play important roles in the abnormal softening and coloration with chilling injury in papaya.

Control of Phytophthora nicotianae disease, induction of defense responses and genes expression of papaya fruits treated with Pseudomonas putida MGP1

BACKGROUND Biological control is a potential strategy to reduce post-harvest decay in several fruits. Little research has been carried out on the effects of endophytic bacterium on post-harvest blight caused by Phytophthora nicotianae in papaya. In this work, the biocontrol activity of Pseudomonas putida MGP1 on this disease and its possible mechanisms, including changes of defensive enzyme activities, total phenolic content and mRNA levels of two important genes, were investigated. RESULTS Fruits treated with MGP1 showed a significant lower disease index and demonstrated increases in chitinase, β-1,3-glucanase, phenylalanine ammonia-lyase, peroxidase, polyphenol oxidase and catalase activities and total phenolic content. In addition, the expression levels of pathogenesis related protein 1 gene (PR1) and non-expressor of PR1 gene (NPR1) in papaya fruits were elevated by MGP1 treatment. CONCLUSION The results indicated that papaya fruits were responsive to the endophytic bacterium Ps. putida, which could activate defensive enzymes and genes and thereby induce host disease resistance.

The Papaya Transcription Factor CpNAC1 Modulates Carotenoid Biosynthesis through Activating Phytoene Desaturase Genes CpPDS2/4 during Fruit Ripening.

Papaya fruits accumulate carotenoids during fruit ripening. Although many papaya carotenoid biosynthesis pathway genes have been identified, the transcriptional regulators of these genes have not been characterized. In this study, a NAC transcription factor, designated as CpNAC1, was characterized from papaya fruit. CpNAC1 was localized exclusively in nucleus and possessed transcriptional activation activity. Expression of carotenoid biosynthesis genes phytoene desaturases (CpPDSs) and CpNAC1 was increased during fruit ripening and by propylene treatment, which correlates well with the elevated carotenoid content in papaya. The gel mobility shift assays and transient expression analyses demonstrated that CpNAC1 directly binds to the NAC binding site (NACBS) motifs in CpPDS2/4 promoters and activates them. Collectively, these data suggest that CpNAC1 may act as a positive regulator of carotenoid biosynthesis during papaya fruit ripening possibly via transcriptional activation of CpPDSs such as CpPDS2/4.

Benzothiadiazole-Mediated Induced Resistance to Colletotrichum musae and Delayed Ripening of Harvested Banana Fruit.

Benzothiadiazole (BTH) works as a plant activator. The effects of different BTH treatments and fungicides SPORGON on fruit ripening and disease incidence were investigated. The results showed that BTH treatment significantly delayed fruit ripening, maintained fruit firmness, color, and good fruit quality, and dramatically reduced the incidence of disease. BTH effectively inhibited the invasion and development of pathogenic bacteria and controlled the occurrence of disease. BTH treatment enhanced the activities of defense-related enzymes, including chitinase, phenylalanine ammonia-lyase, peroxidase, and polyphenol oxidase, increased the content of hydrogen peroxide and total antioxidant capacity, and reduced malondialdehyde content. Cellular structure analysis after inoculation confirmed that BTH treatment effectively maintained the cell structural integrity. SPORGON did not provide benefits for delaying fruit ripening or for the resistance system, while it can control the disease only during the earlier stage and not at later stages.

Molecular cloning, characterizing, and expression analysis of CTR1 genes in harvested papaya fruit

Fruit softening is an ethylene-dependent ripening event. 1-Methylcyclopropene (1-MCP), a synthetic plant growth regulator structurally related to the natural plant hormone ethylene, is used to slow down the fruit ripening. However, inappropriate 1-MCP treatment tends to cause the elastic texture in papaya fruit. Constitutive triple response 1 (CTR1), a downstream protein of the ethylene receptors, acts as a negative regulator of ethylene signaling. To elucidate the signal transduction pathway involved in the ethylene regulation during papaya ripening and softening, four genes homologous to Arabidopsis CTR1 were isolated from papaya fruit and designated as CpCTR1–4. Their molecular and biochemical properties were characterized, and their expression patterns in papaya fruit after treatments with 1-MCP were investigated. Four CTR1-like genes differ in sequence length and molecular size, and have a relative distance relationship in the evolutionary tree analysis. However, sequences analysis showed that the C-terminus of CpCTR1–4 proteins contained the highly conserved kinase domains, including a protein kinase ATP-binding signature (IGAGSFGTVH) and a serine/threonine protein kinase active site signature (IVHRDLKSPNLLV). Their N-terminus contains the conserved motifs (CN box) that exist in all CTR1-like proteins. CpCTR1–4 proteins were predicted to be located differently in either chloroplast or nuclei or both. Gene expression analysis showed that 1-MCP treatment significantly repressed the expression of CpCTRs and high concentration of 1-MCP treatment had a more significant effect. These results suggested that CpCTR1–4 genes may play different roles in papaya fruit ripening and softening and that the rubbery fruit might be relate to the expression of CpCTRs genes.

Comparative Study of Volatile Compounds in the Fruit of Two Banana Cultivars at Different Ripening Stages

Aromatic compounds are important for fruit quality and can vary among fruit cultivars. Volatile compounds formed during the ripening of two banana cultivars, Brazilian and Fenjiao, were determined using headspace solid-phase micro-extraction (SPME) and gas chromatography coupled with mass spectrometry (GC-MS). These two cultivars exhibited different physiological characteristics during storage. Fenjiao fruit exhibited faster yellowing and softening, a higher respiration rate and greater ethylene production. Also, the soluble sugar content in Fenjiao fruit was much higher than in Brazilian fruit. In total, 62 and 59 volatile compounds were detected in Fenjiao and Brazilian fruits, respectively. The predominant volatile components isoamyl acetate, butanoic acid, 3-methyl-3-methylbutyl ester, hexanal, trans-2-hexenal and 1-hexanol varied during ripening stages. Moreover, esters were more abundant in Fenjiao, and propanoic acid 2-methylbutyl ester, and octanoic acid were only detected in Fenjiao. These compounds contribute to the unique flavors and aromas of the two cultivars.