Insights into cell wall changes during fruit softening from transgenic and naturally occurring mutants.

Excessive softening during fleshy fruit ripening leads to physical damage and infection that reduce quality and cause massive supply chain losses. Changes in cell wall (CW) metabolism, involving loosening and disassembly of the constituent macromolecules, are the main cause of softening. Several genes encoding CW metabolizing enzymes have been targeted for genetic modification to attenuate softening. At least 9 genes encoding CW-modifying proteins have increased expression during ripening. Any alteration of these genes could modify CW structure and properties and contribute to softening, but evidence for their relative importance is sparse. The results of studies with transgenic tomato (Solanum lycopersicum), the model for fleshy fruit ripening, investigations with strawberry (Fragaria spp.) and apple (Malus domestica), and results from naturally occurring textural mutants provide direct evidence of gene function and the contribution of CW biochemical modifications to fruit softening. Here we review the revised CW structure model and biochemical and structural changes in CW components during fruit softening and then focus on and integrate the results of changes in CW characteristics derived from studies on transgenic fruits and mutants. Potential strategies and future research directions to understand and control the rate of fruit softening are also discussed.

Citrus heat shock transcription factor CitHsfA7-mediated citric acid degradation in response to heat stress.

Heat stress is a major abiotic stress for plants, which can generate a range of biochemical and genetic responses. In 'Ponkan' mandarin fruit, hot air treatment (HAT) accelerates the degradation of citric acid. However, the transcriptional regulatory mechanisms of citrate degradation in response to HAT remain to be elucidated. Here, 17 heat shock transcription factor sequences were isolated, and dual-luciferase assays were employed to investigate whether the encoded proteins that could trans-activate the promoters of key genes in the GABA shunt, involved in citrate metabolism. We identified four heat shock transcription factors (CitHsfA7, CitHsfA3, CitHsfA4b and CitHsfA8) that showed trans-activation effects on CitAco3, CitIDH3 and CitGAD4, respectively. Transient expression of the CitHsfs in citrus fruits indicated that CitHsfA7 was the only factor that resulted in a significant lowering of the citric acid content, and these results were confirmed by a virus-induced gene silencing system (VIGS). Sub-cellar localization showed that CitHsfA7 is located in the nucleus and is capable of binding directly to a putative HSE in the CitAco3 promoter and enhance its expression. We proposed that the induction of CitHsfA7 transcript level contributes to citric acid degradation in citrus fruit, via modulation of CitAco3 in response to HAT.

Transcriptional regulation of fleshy fruit texture.

Fleshy fruit texture is a critically important quality characteristic of ripe fruit. Softening is an irreversible process which operates in most fleshy fruits during ripening which, together with changes in color and taste, contributes to improvements in mouthfeel and general attractiveness. Softening results mainly from the expression of genes encoding enzymes responsible for cell wall modifications but starch degradation and high levels of flavonoids can also contribute to texture change. Some fleshy fruit undergo lignification during development and post-harvest, which negatively affects eating quality. Excessive softening can also lead to physical damage and infection, particularly during transport and storage which causes severe supply chain losses. Many transcription factors (TFs) that regulate fruit texture by controlling the expression of genes involved in cell wall and starch metabolism have been characterized. Some TFs directly regulate cell wall targets, while others act as part of a broader regulatory program governing several aspects of the ripening process. In this review, we focus on advances in our understanding of the transcriptional regulatory mechanisms governing fruit textural change during fruit development, ripening and post-harvest. Potential targets for breeding and future research directions for the control of texture and quality improvement are discussed.

DNA demethylation is involved in the regulation of temperature-dependent anthocyanin accumulation in peach.

Anthocyanin biosynthesis is induced by low temperatures in a number of plants. However, in peach (cv Zhonghuashoutao), anthocyanin accumulation was observed in fruit stored at 16°C but not at or below 12°C. Fruit stored at 16°C showed elevated transcript levels of genes encoding anthocyanin biosynthetic enzymes, the transport protein glutathione S-transferase and key transcription factors. Higher transcript levels of PpPAL1/2, PpC4H, Pp4CL4/5/8, PpF3H, PpF3'H, PpDFR1/2/3 and PpANS, as well as transcription factor gene PpbHLH3, were associated with lower methylation levels in the promoter of these genes. The DNA methylation level was further highly correlated with the expression of the DNA methyltransferase genes and DNA demethylase genes. The application of DNA methylation inhibitor 5-azacytidine induced anthocyanin accumulation in peach flesh, further implicating a critical role for DNA demethylation in regulating anthocyanin accumulation in peach flesh. Our data reveal that temperature-dependent DNA demethylation is a key factor to the post-harvest temperature-dependent anthocyanin accumulation in peach flesh.

High-CO2/Hypoxia-Responsive Transcription Factors DkERF24 and DkWRKY1 Interact and Activate DkPDC2 Promoter.

Identification and functional characterization of hypoxia-responsive transcription factors is important for understanding plant responses to natural anaerobic environments and during storage and transport of fresh horticultural products. In this study, yeast one-hybrid library screening using the persimmon (Diospyros kaki) pyruvate decarboxylase (DkPDC2) promoter identified three ethylene response factor (ERF) genes (DkERF23/DkERF24/DkERF25) and four WRKY transcription factor genes (DkWRKY/DdkWRKY5/DkWRKY6/DkWRKY7) that were differentially expressed in response to high CO2 (95%, with 4% N2 and 1% oxygen) and high N2 (99% N2 and 1% oxygen). Yeast one-hybrid assays and electrophoretic mobility shift assays indicated that DkERF23, DkERF24, DkERF25, DkWRKY6, and DkWRKY7 could directly bind to the DkPDC2 promoter. Dual-luciferase assays confirmed that these transcription factors were capable of transactivating the DkPDC2 promoter. DkERF24 and DkWRKY1 in combination synergistically transactivated the DkPDC2 promoter, and yeast two-hybrid and bimolecular fluorescence complementation assays confirmed protein-protein interaction between DkERF24 and DkWRKY1. Transient overexpression of DkERF24 and DkWRKY1 separately and in combination in persimmon fruit discs was effective in maintaining insolubilization of tannins, concomitantly with the accumulation of DkPDC2 transcripts. Studies with Arabidopsis (Arabidopsis thaliana) homologs AtERF1 and AtWRKY53 indicated that similar protein-protein interactions and synergistic regulatory effects also occur with the DkPDC2 promoter. We propose that an ERF and WRKY transcription factor complex contributes to responses to hypoxia in both persimmon fruit and Arabidopsis, and the possibility that this is a general plant response requires further investigation.

High CO2/hypoxia-induced softening of persimmon fruit is modulated by DkERF8/16 and DkNAC9 complexes.

Most persimmon (Diospyros kaki) cultivars are astringent and require post-harvest deastringency treatments such as 95% CO2 (high-CO2 treatment) to make them acceptable to consumers. High-CO2 treatment can, however, also induce excessive softening, which can be reduced by adding 1-methylcyclopropene (1-MCP). Previous studies have shown that genes encoding the ETHYLENE RESPONSE FACTORS (ERFs) DkERF8/16/19 can trans-activate xyloglucan endotransglycosylase/hydrolase (DkXTH9), which encodes the cell wall-degrading enzyme associated with persimmon fruit softening. In this study, RNA-seq data between three treatments were compared, namely high-CO2, high-CO2+1-MCP, and controls. A total of 227 differentially expressed genes, including 17 transcription factors, were predicted to be related to persimmon post-deastringency softening. Dual-luciferase assays indicated that DkNAC9 activated the DkEGase1 promoter 2.64-fold. Synergistic effects on transcription of DkEGase1 that involved DkNAC9 and the previously reported DkERF8/16 were identified. Electrophoretic mobility shift assay indicated that DkNAC9 could physically bind to the DkEGase1 promoter. Bimolecular fluorescence complementation and firefly luciferase complementation imaging assays indicated protein-protein interactions between DkNAC9 and DkERF8/16. Based on these findings, we conclude that DkNAC9 is a direct transcriptional activator of DkEGase1 that can co-operate with DkERF8/16 to enhance fruit post-deastringency softening.

ETHYLENE RESPONSE FACTOR39-MYB8 complex regulates low-temperature-induced lignification of loquat fruit.

Flesh lignification is a specific chilling response that causes deterioration in the quality of stored red-fleshed loquat fruit (Eribotrya japonica) and is one aspect of wider chilling injury. APETALA2/ETHLENE RESPONSIVE FACTOR (AP2/ERF) transcription factors are important regulators of plant low-temperature responses and lignin biosynthesis. In this study, the expression and action of 27 AP2/ERF genes from the red-fleshed loquat cultivar 'Luoyangqing' were investigated in order to identify transcription factors regulating low-temperature-induced lignification. EjERF27, EjERF30, EjERF36, and EjERF39 were significantly induced by storage at 0 °C but inhibited by a low-temperature conditioning treatment (pre-storage at 5 °C for 6 days before storage at 0 °C, which reduces low-temperature-induced lignification), and their transcript levels positively correlated with flesh lignification. A dual-luciferase assay indicated that EjERF39 could transactivate the promoter of the lignin biosynthetic gene Ej4CL1, and an electrophoretic mobility shift assay confirmed that EjERF39 recognizes the DRE element in the promoter region of Ej4CL1. Furthermore, the combination of EjERF39 and the previously characterized EjMYB8 synergistically transactivated the Ej4CL1 promoter, and both transcription factors showed expression patterns correlated with lignification in postharvest treatments and red-fleshed 'Luoyangqing' and white-fleshed 'Ninghaibai' cultivars with different lignification responses. Bimolecular fluorescence complementation and luciferase complementation imaging assays confirmed direct protein-protein interaction between EjERF39 and EjMYB8. These data indicate that EjERF39 is a novel cold-responsive transcriptional activator of Ej4CL1 that forms a synergistic activator complex with EjMYB8 and contributes to loquat fruit lignification at low temperatures.

Transcriptome Analysis Identifies a Zinc Finger Protein Regulating Starch Degradation in Kiwifruit.

Ripening, including softening, is a critical factor in determining the postharvest shelf-life of fruit and is controlled by enzymes involved in cell wall metabolism, starch degradation, and hormone metabolism. Here, we used a transcriptomics-based approach to identify transcriptional regulatory components associated with texture, ethylene, and starch degradation in ripening kiwifruit (Actinidia deliciosa). Twelve differentially expressed structural genes, including seven involved in cell wall metabolism, four in ethylene biosynthesis, and one in starch degradation, and 14 transcription factors (TFs) induced by exogenous ethylene treatment and inhibited by the ethylene signaling inhibitor 1-methylcyclopropene were identified as changing in transcript levels during ripening. Moreover, analysis of the regulatory effects of differentially expressed genes identified a zinc finger TF, DNA BINDING WITH ONE FINGER (AdDof3), which showed significant transactivation on the AdBAM3L (ß-amylase) promoter. AdDof3 interacted physically with the AdBAM3L promoter, and stable overexpression of AdBAM3L resulted in lower starch content in transgenic kiwifruit leaves, suggesting that AdBAM3L is a key gene for starch degradation. Moreover, transient overexpression analysis showed that AdDof3 up-regulated AdBAM3L expression in kiwifruit. Thus, transcriptomics analysis not only allowed the prediction of some ripening-regulating genes but also facilitated the characterization of a TF, AdDof3, and a key structural gene, AdBAM3L, in starch degradation.

Tomato CRY1a plays a critical role in the regulation of phytohormone homeostasis, plant development, and carotenoid metabolism in fruits.

Blue light photoreceptors, cryptochromes (CRYs), regulate multiple aspects of plant growth and development. However, our knowledge of CRYs is predominantly based on model plant Arabidopsis at early growth stage. In this study, we elucidated functions of CRY1a gene in mature tomato (Solanum lycopersicum) plants by using cry1a mutants and CRY1a-overexpressing lines (OE-CRY1a-1 and OE-CRY1a-2). In comparison with wild-type plants, cry1a mutants are relatively tall, accumulate low biomass, and bear more fruits, whereas OE-CRY1a plants are short stature, and they not only flower lately but also bear less fruits. RNA-seq, qRT-PCR, and LC-MS/MS analysis revealed that biosynthesis of gibberellin, cytokinin, and jasmonic acid was down-regulated by CRY1a. Furthermore, DNA replication was drastically inhibited in leaves of OE-CRY1a lines, but promoted in cry1a mutants with concomitant changes in the expression of cell cycle genes. However, CRY1a positively regulated levels of soluble sugars, phytofluene, phytoene, lycopene, and ß-carotene in the fruits. The results indicate the important role of CRY1a in plant growth and have implications for molecular interventions of CRY1a aimed at improving agronomic traits.

A transcription factor network responsive to high CO2/hypoxia is involved in deastringency in persimmon fruit.

Plant responses to anaerobic environments are regulated by ethylene-response factors (ERFs) in both vegetative and productive organs, but the roles of other transcription factors (TFs) in hypoxia responses are poorly understood. In this study, eight TFs (DkbHLH1, DkMYB9/10/11, DkRH2-1, DkGT3-1, DkAN1-1, DkHSF1) were shown to be strongly up-regulated by an artificial high-CO2 atmosphere (1% O2 and 95% CO2). Dual-luciferase assays indicated that some TFs were activators of previously characterized DkERFs, including DkMYB10 for the DkERF9 promoter, DkERF18/19 and DkMYB6 for the DkERF19 promoter, and DkERF21/22 for the DkERF10 promoter. Yeast one-hybrid and cis-element mutagenesis confirmed these physical interactions with one exception. The potential roles of these TFs in persimmon fruit deastringency were analysed by investigating their transient over-expression (TOX) in persimmon fruit discs, which indicated that DkMYB6TOX, DkMYB10TOX, DkERF18TOX, and DkERF19TOX were all effective in causing insolubilization of tannins, concomitantly with the up-regulation of the corresponding genes. These results indicated that multiple TFs of different classes are responsive to high-CO2/hypoxia in fruit tissues, and that a TF-TF regulatory cascade is involved in the hypoxia responses involving the Group VII DkERF10, and DkERFs and DkMYBs.

Involvement of an ethylene response factor in chlorophyll degradation during citrus fruit degreening.

Chlorophyll degradation naturally occurs during plant senescence. However, in fruit such as citrus, it is a positive characteristic, as degreening is an important colour development contributing to fruit quality. In the present work, Citrus sinensis Osbeck, cv. Newhall fruit was used as a model for chlorophyll degradation. An ethylene response factor, CitERF13, was isolated and its transcriptional changes were closely correlated with fruit peel degreening during development or in response to ethylene. Dual-luciferase and yeast one-hybrid assays, as well as motif mutation, indicated that CitERF13 directly binds to the CitPPH promoter and enhances its activity. Transient and stable over-expression of CitERF13 resulted in rapid chlorophyll degradation in Nicotiana tabacum leaves and led to accumulation of pheophorbide (Pheide) a, a metabolite of pheophorbide hydrolase (PPH). Similar results were observed from transient transformation of CitERF13 in citrus fruit peel. Moreover, this function of CitERF13 was conserved within Arabidopsis and tomato, as the homologs AtERF17 and SlERF16 similarly acted as activators of PPH genes and accelerators of chlorophyll degradation.

Hypoxia-responsive ERFs involved in postdeastringency softening of persimmon fruit.

Removal of astringency by endogenously formed acetaldehyde, achieved by postharvest anaerobic treatment, is of critical importance for many types of persimmon fruit. Although an anaerobic environment accelerates de-astringency, it also has the deleterious effect of promoting excessive softening, reducing shelf life and marketability. Some hypoxia-responsive ethylene response factors (ERFs) participate in anaerobic de-astringency, but their role in accelerated softening was unclear. Undesirable rapid softening induced by high CO2 (95%) was ameliorated by adding the ethylene inhibitor 1-MCP (1 µL/L), resulting in reduced astringency while maintaining firmness, suggesting that CO2 -induced softening involves ethylene signalling. Among the hypoxia-responsive genes, expression of eight involved in fruit cell wall metabolism (Dkß-gal1/4, DkEGase1, DkPE1/2, DkPG1, DkXTH9/10) and three ethylene response factor genes (DkERF8/16/19) showed significant correlations with postdeastringency fruit softening. Dual-luciferase assay indicated that DkERF8/16/19 could trans-activate the DkXTH9 promoter and this interaction was abolished by a mutation introduced into the C-repeat/dehydration-responsive element of the DkXTH9 promoter, supporting the conclusion that these DkERFs bind directly to the DkXTH9 promoter and regulate this gene, which encodes an important cell wall metabolism enzyme. Some hypoxia-responsive ERF genes are involved in deastringency and softening, and this linkage was uncoupled by 1-MCP. Fruit of the Japanese cultivar 'Tonewase' provide a model for altered anaerobic response, as they lost astringency yet maintained firmness after CO2 treatment without 1-MCP and changes in cell wall enzymes and ERFs did not occur.

Transcriptomic and metabolic analyses provide new insights into chilling injury in peach fruit.

Low temperature conditioning (LTC) alleviates peach fruit chilling injury but the underlying molecular basis is poorly understood. Here, changes in transcriptome, ethylene production, flesh softening, internal browning and membrane lipids were compared in fruit maintained in constant 0 °C and LTC (pre-storage at 8 °C for 5 d before storage at 0 °C). Low temperature conditioning resulted in a higher rate of ethylene production and a more rapid flesh softening as a result of higher expression of ethylene biosynthetic genes and a series of cell wall hydrolases. Reduced internal browning of fruit was observed in LTC, with lower transcript levels of polyphenol oxidase and peroxidase, but higher lipoxygenase. Low temperature conditioning fruit also showed enhanced fatty acid content, increased desaturation, higher levels of phospholipids and a preferential biosynthesis of glucosylceramide. Genes encoding cell wall hydrolases and lipid metabolism enzymes were coexpressed with differentially expressed ethylene response factors (ERFs) and contained ERF binding elements in their promoters. In conclusion, LTC is a special case of cold acclimation which increases ethylene production and, operating through ERFs, promotes both softening and changes in lipid composition and desaturation, which may modulate membrane stability, reducing browning and contributing to alleviation of peach fruit chilling injury.

EjNAC3 transcriptionally regulates chilling-induced lignification of loquat fruit via physical interaction with an atypical CAD-like gene.

Lignin is an important component of many plant secondary cell walls. In the fruit of loquat (Eriobotrya japonica), lignification of cell walls in the fleshy tissue occurs when fruit are subjected to low-temperature storage, which is commonly used to avoid the rapid senescence that occurs at room temperature. In this study, two NAC domain genes, EjNAC3 and EjNAC4, were isolated and shown to be significantly induced at 0 °C, which was concomitant with an increase in the fruit lignification index. Lignification and expression of both EjNAC3 and EjNAC4 were inhibited by low-temperature conditioning and by heat treatment. In addition, EjNAC3 trans-activated the lignin biosynthesis-related EjCAD-like promoter, which was measured using a dual-luciferase assay. Further analysis with yeast one-hybrid and electrophoretic mobility shift assays indicated that EjNAC3 could physically bind to the promoter of the EjCAD-like gene. Thus, EjNAC3 is a direct regulator of loquat chilling-induced lignification, via regulations of EjCAD-like.

Citrus CitNAC62 cooperates with CitWRKY1 to participate in citric acid degradation via up-regulation of CitAco3.

Citric acid is the predominant organic acid of citrus fruit. Degradation of citric acid occurs during fruit development, influencing fruit acidity. Associations of CitAco3 transcripts and citric acid degradation have been reported for citrus fruit. Here, transient overexpression of CitAco3 significantly reduced the citric acid content of citrus leaves and fruits. Using dual luciferase assays, it was shown that CitNAC62 and CitWRKY1 could transactivate the promoter of CitAco3. Subcellular localization results showed that CitWRKY1 was located in the nucleus and CitNAC62 was not. Yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC) assays indicated that the two differently located transcription factors could interact with each other. Furthermore, BiFC showed that the protein-protein interaction occurred only in the nucleus, indicating the potential mobility of CitNAC62 in plant cells. A synergistic effect on citrate content was observed between CitNAC62 and CitWRKY1. Transient overexpression of CitNAC62 or CitWRKY1 led to significantly lower citrate content in citrus fruit. The combined expression of CitNAC62 and CitWRKY1 resulted in lower citrate content compared with the expression of CitNAC62 or CitWRKY1 alone. The transcript abundance of CitAco3 was consistent with the citrate content. Thus, we propose that a complex of CitWRKY1 and CitNAC62 contributes to citric acid degradation in citrus fruit, potentially via modulation of CitAco3.

DWARF overexpression induces alteration in phytohormone homeostasis, development, architecture and carotenoid accumulation in tomato.

Brassinosteroids (BRs) play a critical role in plant growth, development and stress response; however, genetic evidence for the BR-mediated integrated regulation of plant growth still remains elusive in crop species. Here, we clarified the function of DWARF (DWF), the key BR biosynthetic gene in tomato, in the regulation of plant growth and architecture, phytohormone homeostasis and fruit development by comparing wild type, d^(im), a weak allele mutant impaired in DWF, and DWF-overexpressing plants in tomato. Results showed that increases in DWF transcripts and endogenous BR level resulted in improved germination, lateral root development, CO2 assimilation and eventually plant growth as characterized by slender and compact plant architecture. However, an increase in DWF transcript down-regulated the accumulation of gibberellin, which was associated with decreases in leaf size and thickness. BRs positively regulated lateral bud outgrowth, which was associated with decreased transcript of Aux/IAA3, and the ethylene-dependent petiole bending and fruit ripening. Notably, overexpression of DWF did not significantly alter fruit yield per plant; however, increases by 57.4% and 95.3% might be estimated in fruit yield per square metre in two transgenic lines due to their compact architecture. Significantly, BR level was positively related with the carotenoid accumulation in the fruits. Taken together, our results demonstrate that BRs are actively involved in the regulation of multiple developmental processes relating to agronomical important traits.

Regulation of loquat fruit low temperature response and lignification involves interaction of heat shock factors and genes associated with lignin biosynthesis.

Transcriptional regulatory mechanisms underlying lignin metabolism have been widely studied in model plants and woody trees, as well as fruit, such as loquat (Eriobotrya japonica). Unlike the well-known NAC, MYB and AP2/ERF transcription factors, the roles of heat shock factors (HSFs) in lignin regulation have been rarely reported. Two treatments (heat treatment, HT; low temperature conditioning, LTC) were applied to alleviate low temperature-induced lignification in loquat fruit. Gene expression analysis indicated that EjHSF1 transcript abundance, in parallel with heat shock protein genes (EjHsp), was induced by HT, while expression of EjHSF3 was repressed by LTC. Using dual-luciferase assays, EjHSF1 and EjHSF3 trans-activated the promoters of EjHsp genes and lignin biosynthesis-related genes, respectively. Thus, two distinct regulatory mechanisms of EjHSF transcription factors in chilling injury-induced fruit lignification are proposed: EjHSF1 transcriptionally regulated EjHsp genes are involved in chilling tolerance, while EjHSF3 transcriptionally regulated lignin biosynthesis. Furthermore, the relations between EjHSF3 and previously characterized fruit lignification regulators, including EjAP2-1, EjMYB1 and EjMYB2, were also investigated. Yeast-two hybrid (Y2H) and biomolecular fluorescence complementation (BiFC) assays demonstrated protein-protein interaction between EjHSF3 and EjAP2-1. Thus, the involvement of EjHSF3 in fruit lignification is via both lignin biosynthetic genes and the regulator, EjAP2-1.

CitAP2.10 activation of the terpene synthase CsTPS1 is associated with the synthesis of (+)-valencene in 'Newhall' orange.

Aroma is a vital characteristic that determines the quality and commercial value of citrus fruits, and characteristic volatiles have been analyzed in different citrus species. In sweet orange, Citrus sinensis, the sesquiterpene (+)-valencene is a key volatile compound in the fruit peel. Valencene synthesis is catalyzed by the terpene synthase CsTPS1, but the transcriptional mechanisms controlling its gene expression are unknown. Here, the AP2/ERF (APETALA2/ethylene response factor) transcription factor, CitAP2.10, is characterized as a regulator of (+)-valencene synthesis. The expression pattern of CitAP2.10 was positively correlated with (+)-valencene content and CsTPS1 expression. Dual-luciferase assays indicated that CitAP2.10 could trans-activate the CsTPS1 promoter. Ethylene enhanced expression of CitAP2.10 and this effect was abolished by the ethylene antagonist 1-methylcyclopropene. The role and function of CitAP2.10 in (+)-valencene biosynthesis were confirmed using the Arabidopsis homolog (AtWRI1), which also transiently activated the CsTPS1 promoter. Furthermore, transient over-expression of CitAP2.10 triggered (+)-valencene biosynthesis in sweet orange fruit. These results indicate that CitAP2.10 regulates (+)-valencene synthesis via induction of CsTPS1 mRNA accumulation.

Two ω-3 FADs Are Associated with Peach Fruit Volatile Formation.

Aroma-related volatiles, together with sugars and acids, play an important role in determining fruit flavor quality. Characteristic volatiles of peach fruit are mainly derived from fatty acids such as linoleic acid (18:2) and linolenic acid (18:3). In the present study, six genes encoding fatty acid desaturases (FAD) were cloned, including two ω-6 FAD genes (PpFAD2, PpFAD6) and four ω-3 FAD genes (PpFAD3-1, PpFAD3-2, PpFAD7 and PpFAD8). Heterologous expression of peach FADs in tobacco plants showed that PpFAD3-1, and PpFAD3-2 significantly reduced contents of 18:2, and accumulated significant higher levels of 18:3. In the case of volatiles, transgenic plants produced lower concentrations of hexanal and higher levels of (E)-2-hexenal. Consequently, the ratio of the (E)-2-hexenal and hexanal was about 5- and 3-fold higher than that of wild type (WT) in PpFAD3-1 and PpFAD3-2 transformants, respectively. No significant changes in volatile profiles were observed in transgenic plants overexpressing the four other peach FAD genes. Real-time quantitative polymerase chain reaction (qPCR) analysis showed that ripe fruit had high PpFAD3-1 and low PpFAD3-2 transcript levels. In contrast, high PpFAD3-2 and low PpFAD3-1 transcript levels were observed in young fruit. These results indicate a temporal regulation of these two ω-3 FADs during development and ripening, influencing peach fruit volatile formation.

Characterization of Starch Degradation Related Genes in Postharvest Kiwifruit.

Starch is one of the most important storage carbohydrates in plants. Kiwifruit typically accumulate large amounts of starch during development. The fruit retain starch until commercial maturity, and its postharvest degradation is essential for consumer acceptance. The activity of genes related to starch degradation has, however, rarely been investigated. Based on the kiwifruit genome sequence and previously reported starch degradation-related genes, 17 novel genes were isolated and the relationship between their expression and starch degradation was examined using two sets of materials: ethylene-treated (100 µL/L, 20 °C; ETH) vs. control (20 °C; CK) and controlled atmosphere stored (CA, 5% CO2 + 2% O2, 0 °C) vs. normal atmosphere in cold storage (NA, 0 °C). Physiological analysis indicated that ETH accelerated starch degradation and increased soluble solids content (SSC) and soluble sugars (glucose, fructose and sucrose), while CA inhibited starch reduction compared with NA. Using these materials, expression patterns of 24 genes that may contribute to starch degradation (seven previously reported and 17 newly isolated) were analyzed. Among the 24 genes, AdAMY1, AdAGL3 and AdBAM3.1/3L/9 were significantly induced by ETH and positively correlated with starch degradation. Furthermore, these five genes were also inhibited by CA, conforming the likely involvement of these genes in starch degradation. Thus, the present study has identified the genes with potential for involvement in starch degradation in postharvest kiwifruit, which will be useful for understanding the regulation of kiwifruit starch content and metabolism.