First Report of Colletotrichum fructicola Causing Anthracnose on Paeonia lactiflora in China.

Paeonia lactiflora Pall is a traditional famous flower with long cultivated history in China, and has important medical and ornamental functions (Duan et al. 2022). In the middle of June 2022, anthracnose disease was observed nearly 25% (n=90) on P. lactiflora in Poyang County, Shangrao City, Jiangxi Province (29.00° N, 116.67° E) (Figure 1 E). The symptoms of the disease were small, round, light brown spots then grew bigger to round or irregular dark brown lesions (5 to 7 mm diameter) progressively on the leaves with disease spread (Figure 1 A). Subsequently, necrotic tissue was formed in the center and caused fade and wilt on the leaves ultimately, which reduced the medicinal and aesthetic value severely. Small pieces of diseased tissue (5 × 5 mm) were cut from the diseased junction, disinfected with 75% ethanol for 30 to 45 seconds, then 1% NaClO for 1 to 2 minutes, rinsed three times with sterile water. To identify the pathogen, tissues were placed on PDA and incubated for 3 days at 28°C. Single spore isolates were cultured on PDA, the colonies of one representative strain (SY4) were originally white with a lot of aerial mycelium after 5 to 7 days at 28°C in the incubator. The center of the colony turned greyish-white, released tiny orange-yellow particles (conidia) (Figure 1 F and 1 G), which were single, colorless, elongated ovals with rounded ends and measured 11.29 to 23.24 × 3.94 to 5.60 µm (av=15.89 µm × 4.74 µm, n=50) (Figure 1 H and 1 I). The isolate SY4 was identified to Colletotrichum fructicola based on morphological characteristics (Yang et al. 2021; Li et al. 2022b). For further molecular identification, the rDNA-ITS, actin gene (ACT), glyceraldehyde-3-phosphatedehydrogenase (GAPDH), chitin synthase (CHS) and calmodulin gene (CAL) genes were amplified and sequenced with primers of ITS1/ITS4 (Gardes et al. 1993), ACT-512F/ACT-783R, GDF/GDR (Templeton et al. 1992), CHS-79F/CHS-345R (Carbone et al. 1999) and CL1C/ CL2C (Weir et al. 2012) respectively. The accession numbers in GenBank were OP523977 (ITS-rDNA), OP547618 (ACT), OP605733 (GAPDH), OP605732 (CHS), and OP605731 (CAL). The BLAST analysis revealed that these sequences were identical more than 99% with those of C. fructicola (GenBank accession Nos. MZ437948.1, MN525803.1, MN525860.1, MZ13360.1 and ON188684.1) (Figure 2). To confirm pathogenicity, the leaves were cleaned with 75% ethanol, rinsed with sterile water. After the leaf surface was dried naturally, 20 leaves were pricked at two symmetrical places on either side of the main veins of the leaf with a sterilized inoculum needle (2.0 mm in diameter), half of the wounded leaves were inoculated with 20 µL spore suspension (1.0 × 106 spores/mL) (Figure 1 C and 1 D), while the other half were inoculated with sterile water as controls (Figure 1 B). Inoculated leaves were grown for 5 days in an incubator at 28 °C and above 90% relative humidity, repeated three times. The results demonstrated that the wounded leaves with C. fructicola showed the same signs of wilting with the original disease leaves, while control leaves remained healthy. The same fungus was reisolated from the diseased leaves which confirmed with Koch's postulates. The same fungus was re-isolated from the diseased leaves while it was not isolated from control leaves, confirmed with Koch's postulates. In China, it had been reported that C. fructicola caused anthracnose on Persea americana (Li et al. 2022a) and Myrica rubra (Li et al. 2022b). To the best of our knowledge, this is the first report of anthracnose on P. lactiflora caused by C. fructicola in China. The results will help to develop effective control strategies for anthracnose on P. lactiflora.

Hot Water Treatment Improves Date Drying and Maintains Phytochemicals and Fruit Quality Characteristics of Date Palm (Phoenix dactylifera).

Fresh date fruits (cvs. Hillawi and Khadrawi) were harvested at the khalal stage and treated with hot water treatment (HWT) for different time durations (control, HWT-1 min, HWT-3 min, HWT-5 min, and HWT-7 min) to investigate the physicochemical characteristics, phytochemical properties, and sensory attributes. The results revealed that both date cultivars took less time to reach the tamar stage in response to HWT-7 min compared to control. However, Hillawi date fruit showed a higher fruit ripening index (75%) at HWT-3 min, while Khadrawi fruit had a higher ripening index (80%) at HWT-5 min than untreated fruit (10%). Higher weight loss and lower moisture contents were observed in Hillawi (25%) and Khadrawi (20%) date fruit as the immersion period increased in both cultivars. Moreover, soluble solid content was higher in Hillawi (11.77° Brix) in response to HWT-3 min and Khadrawi (10.02° Brix) date fruit immersed in HWT-5 min in contrast with the control group, whereas significantly lower levels of titratable acidity and ascorbic acid content were observed in Hillawi (0.162%, 0.67 mg/100 g) and Khadrawi (0.206%, 0.73 mg/100 g) date fruit in response to HWT (HWT-1 min, HWT-3 min, HWT-5 min, and HWT-7 min) than untreated fruit. Furthermore, noticeably higher levels of reducing sugar (69.83%, 57.01%), total sugar (34.47%, 31.14%), glucose (36.84%, 29.42%), fructose (33.99%, 27.61%), and sucrose (3.16%, 1.33%) were found in hot water-treated Hillawi (immersed for 3-min) and Khadrawi (immersed for 5-min) date fruit, respectively. In addition, total phenolic content, total flavonoids, total antioxidants, and total tannins were substantially superior in date fruits subjected to HWT-3 min (in Hillawi, 128 mg GAE/100 g, 61.78%, 20.18 mg CEQ/100 g) and HWT-5 min (in Khadrawi, 139.43 mg GAE/100 g, 72.84%, and 18.48 mg CEQ/100 g) compared to control. Overall, sensory attributes were recorded to be higher in Hillawi and Khadrawi date fruit after treatment for 3 min and 5 min, respectively. Our findings suggest that HWT is a promising technique that can be adopted commercially to improve fruit ripening and preserved nutritional quality of dates after harvest.

First Report of Black Spot Disease Caused by Alternaria alternata on Persimmon Fruit in China.

Sweet persimmon is native to Japan and valued for its fruit, which are high in sugar and vitamins. In October 2021, symptoms were observed on persimmon (Diospyros kaki L. cv. Yangfeng) fruits in cold storage room in Suiping county, Henan Province (32.59 °N, 15 113.37 °E). Initially, small circular dark-brown spots were visible on the fruit rind, turning into irregular sunken dark areas, and eventually rotting 15% of 200 fruits after four weeks of cold storage (10°C, 95% relative humidity). To isolate the causal agent, 10 fruits of symptomatic tissues (4 mm2) were surface-sterilized in 2% sodium hypochlorite (NaOCl) for 1 minute, washed three times in sterile distilled water, then aseptically transferred to potato dextrose agar (PDA) and incubated for 7 days at 25°C. Fungal colonies were isolated from plant tissue, and on three colonies of similar morphology, single-spore isolation was performed. On PDA, the isolates produced circular colonies of fluffy aerial mycelia, gray-brown in the center with gray-white margins. Conidia were dark brown, obclavate or pyriform, with 0 to 3 longitudinal septa and 1 to 5 transverse septa, and a size range of 19.2 - 35.1 × 7.9 - 14.6 µm (n=100). Conidiophores were olivaceous, septate, straight, or bent, with a length of 18 - 60 × 1 - 3 µm (n=100). These morphological characteristics identify the isolates as Alternaria alternata (Simmons. 2007). Genomic DNA was extracted from a representative isolate YX and re-isolated strain Re-YX by cetyltrimethylammonium bromide (CTAB). The primers of ITS1/4, Alt-F/R, GPD-F/R, EF1/2, EPG-F/R (Chen et al. 2022), RPB2-5F/7cR (Liu et al. 1999), and H3-1a/1b (Lousie et al. 1995) were used to amplify the partial internal transcribed spacer (ITS) region, Alternaria major allergen (Alt a1), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF), endo-polygalacturonase (endoPG), RNA polymerase second largest subunit (RPB2) and Histone 3 (His3), respectively. GenBank accession No of ITS, Alt a1, GAPDH, TEF, endoPG, RPB2, His3 were ON182066, ON160008 to ON160013 for YX and OP559163, OP575313 to OP575318 for Re-YX respectively. Sequence data of Alternaria spp. were downloaded from GenBank and the BLAST analysis showed 99%-100% homology between various A. alternata strains (ITS: MT498268; Alt a1: MF381763; GAPDH: KY814638; TEF: MW981281; endoPG: KJ146866; RPB2: MN649031; His3: MH824346). A phylogenetic analysis based on ITS, Alt a1, GAPDH, TEF, and RPB2 sequences using MEGA7 (Molecular Evolutionary Genetics Analysis) revealed that the isolate YX and Re-YX were clustered in A. alternata clade (Demers M. 2022). For the pathogenicity test, seven-day-old cultures were used to create spores suspensions (5.0 × 105 spores/mL) of each of the three isolates. Ten µL aliquots from each isolate were inoculated onto ten needle-wounded persimmon fruits; ten additional fruits were inoculated with water only to serve as controls. The pathogenicity test was three replications. Fruits were deposited in a climate box at 25°C, 95% relative humidity. Seven days post-inoculation, the wounded fruit treated with spore suspensions displayed black spot symptoms similar to the symptoms on the original fruit. There were no symptoms on the control fruits. The strain Re-YX was re-isolated from the symptomatic tissue of inoculated fruits and its identity confirmed using the morphological and molecular methods previously mentioned, fulfilling Koch's postulates. The persimmon fruit rot caused by A. alternata had been reported in Turkey and Spain (Kurt et al., 2010, Palou et al., 2012). According to our knowledge, this is the first report of black spot disease on persimmon fruits caused by A. alternata in China. The disease could infect persimmon fruits during cold storage, so more control methods should be developed to prevent postharvest disease of persimmon in the future.

Alleviatory effects of salicylic acid on postharvest softening and cell wall degradation of 'Jinshayou' pummelo (Citrus maxima Merr.): A comparative physiological and transcriptomic analysis.

The regulatory mechanisms underlying the salicylic acid (SA)-mediated inhibition of senescence in pummelo fruit, the largest known citrus variety, remain unclear. Herein, postharvest 0.3% SA treatment was demonstrated to delay postharvest 'Jinshayou' pummelo senescence, as evidenced by the inhibitions in firmness loss, electrolyte leakage increase, and color change. Using comparative transcriptomic data, a total of 4367, 3769, and 1659 DEGs were identified between CK0 and CK60, CK0 and SA60, and CK60 and SA60, respectively. Further GO analysis revealed that DEGs were mainly implicated in the processes of cell wall modification and phenylpropanoid pathway during fruit senescence. More importantly, postharvest exogenous 0.3% SA treatment was observed to inhibit CWDEs activities and their encoding gene expression, retain higher protopectin, cellulose, and hemicelluloses contents, as well as reduce WSP content, thus maintaining cell wall structure. These findings collectively indicated that postharvest SA treatment was a green and useful preservative for alleviating fruit senescence and prolonging the storage life of harvested 'Jiashayou' pummelo fruit.

Comparative transcriptomic and metabolomic analyses reveal the delaying effect of naringin on postharvest decay in citrus fruit.

Introduction: Naringin exhibits antioxidant capacity and can partially inhibit pathogens in many horticultural products, such as citrus fruit; however, the effects of naringin on the storage quality and mechanisms that regulate senescence in citrus fruit have not been comprehensively analyzed. Methods and results: In this study, exogenous naringin treatment was found to significantly delay citrus fruit disease, decreasing the H2O2 content, increasing the antioxidant capacity and maintaining the quality of the fruit. Metabolomic analysis of citrus peel indicated the vast majority (325) of metabolites belonging to flavonoids. Moreover, the auraptene, butin, naringenin, and luteolin derivative levels within the phenylpropanoid pathway were significantly higher in the naringin-treated fruit than in the control fruit. Transcriptomic analysis also revealed that twelve genes in the phenylpropanoid and flavonoid biosynthesis pathways were significantly upregulated. Further analysis with a co-expression network revealed significant correlation between these differential genes and metabolites. Additionally, MYC and WRKY, screened from the MAPK signaling pathway, may contribute to naringin-induced disease resistance. Conclusion: In conclusion, naringin treatment can efficiently delay decay and maintain the quality of citrus fruit, mainly by promoting metabolites accumulation, and upregulating differentially expressed genes in phenylpropanoid and flavonoid biosynthesis pathway. This study provides a better understanding of the regulatory mechanisms through which naringin delays citrus fruit decay and maintains fruit quality.

Exogenous melatonin treatment affects ascorbic acid metabolism in postharvest 'Jinyan' kiwifruit.

Ascorbic acid (AsA) is an important nutritious substance in fruits, and it also can maintain the biological activity of fruits during storage. This research investigated the effect of exogenous melatonin (MT) on AsA metabolism in postharvest kiwifruit. Our results indicated that exogenous MT delayed the decrease of fruit firmness and titratable acid (TA), inhibited the increase of soluble solids content (SSC), reduced the respiration rate and ethylene production, and maintained a higher AsA content in kiwifruit during storage. The high expression of L-galactose pathway key genes in the early storage and regeneration genes in the later storage maintained the AsA content in postharvest kiwifruit. MT treatment enhanced the expression levels of AsA biosynthesis (AcGME2, AcGalDH, and AcGalLDH) and regeneration (AcGR, AcDHAR, and AcMDHAR1) genes. Meanwhile, the expression of the degradation gene AcAO was inhibited in MT-treated kiwifruits.

First Report of Postharvest Fruit Rot on Citrus reticulata Blanco Caused by Fusarium concentricum in China.

Nanfeng tangerine (Citrus reticulata Blanco) is highly regarded for its nutritional and economic value. In January 2022, an unknown fruit rot was observed on Nanfeng tangerine fruits harvested from Nanfeng County (27.22 °N, 116.53 °E), Fuzhou City, Jiangxi Province after 70 days in storage (25 °C, 90% relative humidity). The disease mostly started from the pedicel or a wound. Symptoms initiated with dark brown lesions that rapidly expanded between the fruit center and pulp capsule causing total fruit rot. The surface of symptomatic fruit was sterilized with 75% ethanol for 30 s and 2% NaClO for 30 s. Small diseased tissue pieces (2 mm2) between diseased and healthy tissues were placed on potato dextrose agar (PDA) and put in an incubator (25 ± 1 °C) for 3 days. The representative isolate NFMJ-1 was subcultured onto PDA using single-spore purification. Colonies on PDA were light yellow to white, with abundant flocculent aerial hyphae. Microconidia were oval, obovoid to allantoid, 0 septate, occasionally 1 septate, 4.07 to 17.53 × 1.69 to 3.56 µm (average=7.40 µm × 2.55 µm, n=50). Macroconidia were slender, with a beaked apical cell and a foot-shaped basal cell, 3 to 5 septate, 22.99 to 81.12 × 2.34 to 3.81 µm (average=45.04 µm × 3.12 µm, n=50). According to morphological characteristics, the isolate was tentatively identified as Fusarium sp. (Leslie and Summerell 2006). To confirm the identification, the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF), RNA polymerase II second largest subunit (RPB2), beta-tubulin gene (TUB2), and calmodulin gene (CaM) sequences were amplified with primers ITS1/ITS4 (Gardes et al. 1993), TEF1/TEF2 (O'Donnell et al. 2010), RPB2-5f2/RPB2-7cr (Liu et al. 1999), Bt2a/Bt2b (Glass and Donaldson 1995), and CL1C/CL2C (Weir et al. 2012), respectively. The obtained sequences (ON184033, ON212051, ON212052, ON212053, ON212054) showed homology with F. concentricum ITS (MW016417.1; 514/514 bp), TEF (MK609902.1; 667/667 bp), RPB2 (LC631461.1; 941/972 bp), TUB2 (MT942588.1; 331/337 bp), and CaM (MK609916.1; 558/597 bp). A phylogenetic analysis of concatenated ITS-RPB2-TEF sequences was performed by MEGA7.0 with the maximum likelihood and Kimura 2-parameter model, revealing that the isolate was placed in the F. concentricum clade. To confirm pathogenicity, 36 healthy tangerine fruits were surface sterilized with 75% alcohol, then 18 disinfected fruits were wounded with sterile needles and 18 remained unwounded. Half of the wounded and un-wounded fruits were inoculated with 10 µL of a conidial suspension (1.0 × 106 conidia/ml) of isolate NFMJ-1 cultured for 7 days on PDA. Half of the wounded and un-wounded fruits were mock-inoculated with sterile water as controls. After incubation in an incubator (25 ± 1°C, 90% relative humidity) for 7 days, the wounded fruits inoculated with F. concentricum showed similar symptoms to the original diseased fruits, while the mock-inoculated fruits were asymptomatic. The pathogenicity test was repeated three times. The pathogen was re-isolated from the wound-inoculated fruits and identified as F. concentricum by morphological and molecular analysis, completing Koch's postulates. F. concentricum has been reported as a pathogen of Podocarpus macrophyllus (Dong et al. 2022), Capsicum annuum (Wang et al. 2013) and Zea mays (Du et al. 2020) in China. This is the first report of fruit rot caused by F. concentricum on Citrus reticulata in China. Appropriate prevention and control measure of the pathogen need to be developed to preserve marketability of this economically important citrus fruit.

Lignin Biosynthesis Pathway and Redox Balance Act Synergistically in Conferring Resistance against Penicillium italicum Infection in 7-Demethoxytylophorine-Treated Navel Orange.

7-Demethoxytylophorine (DEM), a natural water-soluble phenanthroindolizidine alkaloid, has a great potential for in vitro suppression of Penicillium italicum growth. In the present study, we investigated the ability of DEM to confer resistance against P. italicum in harvested "Newhall" navel orange and the underlying mechanism. Results from the in vivo experiment showed that DEM treatment delayed blue mold development. The water-soaked lesion diameter in 40 mg L-1 DEM-treated fruit was 35.2% lower than that in the control after 96 h. Moreover, the decrease in peel firmness loss and increase in electrolyte leakage, superoxide anion (O2•-) production, and malondialdehyde (MDA) content were significantly inhibited by DEM treatment. Hydrogen peroxide (H2O2) burst in DEM-treated fruit at the early stage of P. italicum infection contributed to the conferred resistance by increasing the activities of lignin biosynthesis-related enzymes, along with the expressions of their encoding genes, resulting in lignin accumulation. The DEM-treated fruit maintained an elevated antioxidant capacity, as evidenced by high levels of ascorbic acid and glutathione content, and enhanced or upregulated the activities and gene expression levels of APX, GR, MDHAR, DHAR, GPX, and GST, thereby maintaining ROS homeostasis and reducing postharvest blue mold. Collectively, the results in the present study revealed a control mechanism in which DEM treatment conferred the resistance against P. italicum infection in harvested "Newhall" navel orange fruit by activating lignin biosynthesis and maintaining the redox balance.

Apple polyphenols delay postharvest senescence and quality deterioration of 'Jinshayou' pummelo fruit during storage.

Introduction: Apple polyphenols (AP), derived from the peel of mature-green apples, are widely used as natural plant-derived preservatives in the postharvest preservation of numerous horticultural products. Methods: The goal of this research was to investigate how AP (at 0.5% and 1.0%) influences senescence-related physiological parameters and antioxidant capacity of 'Jinshayou' pummelo fruits stored at 20°C for 90 d. Results: The treating pummelo fruit with AP could effectively retard the loss of green color and internal nutritional quality, resulting in higher levels of total soluble solid (TSS) content, titratable acidity (TA) content and pericarp firmness, thus maintaining the overall quality. Concurrently, AP treatment promoted the increases in ascorbic acid, reduced glutathione, total phenols (TP) and total flavonoids (TF) contents, increased the scavenging rates of 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and hydroxyl radical (•OH), and enhanced the activities of superoxide dismutase (SOD), catalase, peroxidase, ascorbate peroxidase (APX), and glutathione reductase (GR) as well as their encoding genes expression (CmSOD, CmCAT, CmPOD, CmAPX, and CmGR), reducing the increases in electrolyte leakage, malondialdehyde content and hydrogen peroxide level, resulting in lower fruit decay rate and weight loss rate. The storage quality of 'Jinshayou' pummelo fruit was found to be maintained best with a 1.0% AP concentration. Conclusion: AP treatment can be regarded as a promising and effective preservative of delaying quality deterioration and improving antioxidant capacity of 'Jinshayou' pummelo fruit during storage at room temperature.

Biocontrol of Cladosporium cladosporioides of mango fruit with Bacillus atrophaeus TE7 and effects on storage quality.

The purpose of this study was to evaluate the control effect of Bacillus atrophaeus TE7 on Cladosporium cladosporioides of mango fruit and how it effects quality attributes during 'Tainong' mango fruit storage. The results showed that strain TE7 had inhibition ability with the biocontrol efficacy of 85.56%. Furthermore, strain TE7 could produce lipopeptide substance, iturin A, and surfactants, which inhibited the growth and development of C. cladosporioides. Moreover, strain TE7 had the ability of improving the activities of defense response-related enzyme in mangoes. The changes of peel color, flesh firmness, contents of total soluble solids (TSS), titratable acid (TA), and ascorbic acid (Vc) were significantly delayed by strain TE7. The results demonstrated that B. atrophaeus TE7 could be applied as a biocontrol agent for the pathogen C. cladosporioides of mango fruit.

Effect of ethanol fumigation on pericarp browning associated with phenol metabolism, storage quality, and antioxidant systems of wampee fruit during cold storage.

Wampee fruit is a popular fruit cultivar in South-East Asia due to its high levels of nutrients and antioxidants; however, pericarp browning leads to a short storage life with great economic loss during years. The purpose of this work was to determine whether postharvest ethanol fumigation affected pericarp browning development of wampee fruit during 12 days of storage at 8 ± 0.5°C, and if so, how it is related to phenol metabolism and how it affects quality attributes and antioxidant systems during storage. After fruits were fumigated with 100, 300, 500, and 800 µl/L for 5 hr at 22 ± 0.5°C, ethanol significantly reduced the development of pericarp browning by increasing total phenolics (TP) content and decreasing the activity of polyphenol oxidase (PPO), especially in 500 µl/L ethanol treatment. Additionally, ethanol delayed the losses in fruit firmness (FF), soluble solid content (SSC), and titratable acidity (TA), retarded weight loss and accumulation of malondialdehyde (MDA) content and maintained relatively high contents of ascorbic acid (AsA), total flavonoids (TF), and total antioxidant capacity (TAC) and activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). In conclusion, results demonstrated that postharvest ethanol fumigation in wampee fruit has ability to reduce pericarp browning development by regulating phenol metabolism and enhancing antioxidant systems.

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.

EjAP2-1, an AP2/ERF gene, is a novel regulator of fruit lignification induced by chilling injury, via interaction with EjMYB transcription factors.

Lignin biosynthesis is regulated by many transcription factors, such as those of the MYB and NAC families. However, the roles of AP2/ERF transcription factors in lignin biosynthesis have been rarely investigated. Eighteen EjAP2/ERF genes were isolated from loquat fruit (Eriobotrya japonica), which undergoes postharvest lignification during low temperature storage. Among these, expression of EjAP2-1, a transcriptional repressor, was negatively correlated with fruit lignification. The dual-luciferase assay indicated that EjAP2-1 could trans-repress activities of promoters of lignin biosynthesis genes from both Arabidopsis and loquat. However, EjAP2-1 did not interact with the target promoters (Ej4CL1). Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays indicated protein-protein interactions between EjAP2-1 and lignin biosynthesis-related EjMYB1 and EjMYB2. Furthermore, repression effects on the Ej4CL1 promoter were observed with the combination of EjAP2-1 and EjMYB1 or EjMYB2, while EjAP2-1 with the EAR motif mutated (mEjAP2-1) lost such repression, although mEjAP2-1 still interacted with EjMYB protein. Based on these results, it is proposed that EjAP2-1 is an indirect transcriptional repressor on lignin biosynthesis, and the repression effects were manifested by EAR motifs and were conducted via protein-protein interaction with EjMYBs.

Activator- and repressor-type MYB transcription factors are involved in chilling injury induced flesh lignification in loquat via their interactions with the phenylpropanoid pathway.

Lignin biosynthesis and its transcriptional regulatory networks have been studied in model plants and woody trees. However, lignification also occurs in some fleshy fruit and has rarely been considered in this way. Loquat ( Eriobotrya japonica ) is one such convenient tissue for exploring the transcription factors involved in regulating fruit flesh lignification. Firmness and lignin content of 'Luoyangqing' loquat were fund to increase during low-temperature storage as a typical symptom of chilling injury, while heat treatment (HT) and low-temperature conditioning (LTC) effectively alleviated them. Two novel EjMYB genes, EjMYB1 and EjMYB2, were isolated and were found to be localized in the nucleus. These genes responded differently to low temperature, with EjMYB1 induced and EjMYB2 inhibited at 0 °C. They also showed different temperature responses under HT and LTC conditions, and may be responsible for different regulation of flesh lignification at the transcriptional level. Transactivation assays indicated that EjMYB1 and EjMYB2 are a transcriptional activator and repressor, respectively. EjMYB1 activated promoters of both Arabidopsis and loquat lignin biosynthesis genes, while EjMYB2 countered the inductive effects of EjMYB1. This finding was also supported by transient overexpression in tobacco. Regulation of lignification by EjMYB1 and EjMYB2 is likely to be achieved via their competitive interaction with AC elements in the promoter region of lignin biosynthesis genes such as Ej4CL1.