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Loquat fruit accumulates lignin in its flesh when undergoing chilling injury during postharvest storage, making it a suitable model for the study of flesh lignification. Transcriptional regulation of lignin biosynthesis is principally controlled by the NAC-MYB transcriptional cascade in model plants. Previous research has demonstrated that EjMYB8 activates lignin biosynthesis through direct interaction with the promoter of Ej4CL1. However, the classic NAC-MYB gene regulation network has not been established. Here, the MADS-box gene EjAGL65 was discovered by screening a cDNA library using the EjMYB8 promoter as bait in yeast. A phylogenetic analysis and structural comparisons revealed that EjAGL65 belongs to the Mδ subgroup of the MADS-box family, whose members have not been reported as being involved in the regulation of lignin deposition. EjAGL65 transcription was downregulated at 0°C compared to 5°C, indicating a negative correlation with the change of lignin content. A dual-luciferase assay indicated that EjAGL65 is capable of inhibiting the promoter activity of EjMYB8 in vivo. These results showed that the Mδ MADS-box gene EjAGL65 transcriptionally regulates EjMYB8 during postharvest chilling induced flesh lignification, which differs from the classical regulation model of lignin biosynthesis that has been illustrated for developmental lignin accumulation.

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 lowtemperature 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. [ABSTRACT FROM AUTHOR]

Lignin is important for plant secondary cell wall formation and participates in resistance to various biotic and abiotic stresses. Loquat undergoes lignification not only in vegetative tissues but also in flesh of postharvest fruit, which adversely affects consumer acceptance. Thus, researches on lignin biosynthesis and regulation are important to understand loquat fruit lignification. In loquat, a gene encoding an enzyme in the lignin biosynthesis pathway, Ej4CL1, was reported to be regulated by transcription factors, including EjMYB1, EjMYB2, EjMYB8, and EjAP2-1, knowledge of this process is still limited. With the aim of identifying novel transcriptional factors controlling lignin biosynthesis in loquat, the promoter of Ej4CL1 was utilized to screen a cDNA library by yeast one hybrid assay. A novel R2R3 MYB, named EjODO1, was identified. Real-time PCR analyses indicated that EjODO1 is highly expressed in lignified stems and roots. During fruit development, expression of EjODO1 decreased along with the reduction of lignin content and became undetectable in mature ripe fruit. Thus, EjODO1 is likely to be involved in lignification of vegetative organs and early fruit development but not in mature fruit or postharvest lignification. Dual-luciferase assay indicated that EjODO1 could trans-activate promoters of lignin biosynthesis genes, such as EjPAL1, Ej4CL1, and Ej4CL5 and transient overexpression of EjODO1 triggered lignin biosynthesis. These results indicate a role for EjODO1 in regulating lignin biosynthesis in loquat which is different from the previously characterized transcription factors. [ABSTRACT FROM AUTHOR]

Loquat (Eriobotrya japonica) is a model fruit for investigating flesh lignification during storage and response to chilling injury. However, the investigations of enzymes and coding genes and loquat fruit lignification under low-temperature storage are still limited. Here, the activity and transcript levels of up-stream enzymes of the phenylpropanoid pathway, including L-phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:coenzyme A ligase (4CL), were investigated. The results indicated that activity of these enzymes was positively correlated with loquat fruit lignification and suppression of these increases by heat treatment (HT) and lowtemperature conditioning (LTC) significantly alleviated loquat fruit lignification. Coding genes for these enzymes were subsequently isolated based on information from an RNA-seq database and expression of Ej4CL1 was found to be the most responsive to low temperature and inhibition by HT and LTC treatment, whereas the other genes were less responsive to these treatments. Furthermore, function of Ej4CL1 was analyzed by transient overexpression in tobacco leaves, where it stimulated lignin accumulation. Ej4CL1 may be a key candidate that involved in CI-related loquat fruit lignification. [ABSTRACT FROM AUTHOR]