Fruit Lignification Research Articles

XYLEM NAC DOMAIN 1 (EjXND1) relieves cold-induced lignification by negatively regulating the EjHB1-EjPRX12 module in loquat fruit

IntroductionLignin is a principal constituent of the secondary cell wall, which plays a role in both plant growth and defensing against stress, such as low temperature and pest infestation. Additionally, it also accumulates in fleshy fruits and negatively affects fruit quality. Red-fleshed loquat is temperature sensitive and exhibits cold-induced lignification. A number of technologies have been developed, for example, Low Temperature Conditioning (LTC) treatment, which has been applied in order to relieve the symptom of cold injury. ObjectivesThe present study seeks to elucidate the regulatory mechanism underlying cold-induced lignification in loquat fruit. MethodsThe target genes were isolated through the analysis of transcriptome. The gene function was analyzed by transient transgenic method in tobacco leaves and loquat fruit, respectively, as well as stable overexpression in liverwort. The regulatory mechanism study was achieved by in vitro protein–protein interaction assays, dual-luciferase assay, and EMSA. ResultsIn the present study, the Xylem NAC Domain transcription factor EjXND1 was identified as a repressor of loquat fruit lignification. It was demonstrated that EjXND1 could interact with the characterized lignin activator EjHB1, resulting in a diminution of the activation of EjHB1 on EjPRX12 promoter. Furthermore, two highly methylated regions were identified in the promoter of EjXDN1. One of these regions exhibited a negative correlation between methylation level and EjXND1 expression. Additionally, it was shown that hypermethylation of this region weaken the binding affinity of EjXND1 activators to its promoter. ConclusionThe EjXND1 plays a role in modified Low Temperature Conditioning (mLTC) treatment that alleviates cold-induced lignification in red-fleshed loquat fruit by targeting the EjHB1-EjPRX12 module and EjXND1 is regulated by the dynamic of DNA methylation level in the promoter.

Overexpression of SlALC Increases Drought and Salt Tolerance and Affects Fruit Dehiscence in Tomatoes.

The bHLH transcription factors are important plant regulators against abiotic stress and involved in plant growth and development. In this study, SlALC, a gene coding for a prototypical DNA-binding protein in the bHLH family, was isolated, and SlALC-overexpression tomato (SlALC-OE) plants were generated by Agrobacterium-mediated genetic transformation. SlALC transgenic lines manifested higher osmotic stress tolerance than the wild-type plants, estimated by higher relative water content and lower water loss rate, higher chlorophyll, reducing sugar, starch, proline, soluble protein contents, antioxidant enzyme activities, and lower MDA and reactive oxygen species contents in the leaves. In SlALC-OE lines, there were more significant alterations in the expression of genes associated with stress. Furthermore, SlALC-OE fruits were more vulnerable to dehiscence, with higher water content, reduced lignin content, SOD/POD/PAL enzyme activity, and lower phenolic compound concentrations, all of which corresponded to decreased expression of lignin biosynthetic genes. Moreover, the dual luciferase reporter test revealed that SlTAGL1 inhibits SlALC expression. This study revealed that SlALC may play a role in controlling plant tolerance to drought and salt stress, as well as fruit lignification, which influences fruit dehiscence. The findings of this study have established a foundation for tomato tolerance breeding and fruit quality improvement.

PpWRKY65 mediates peach (Prunus persica) fruit lignification in both ambient temperature storage and chilling injury condition

The lignification process exerts an adverse impact on the peach fruit quality. The identification of a texture-related gene module was achieved through weighted gene co-expression network analysis (WGCNA) based on transcriptomic analysis of two peach fruit varieties, ‘Baili’ and ‘Hongli’. Notably, the WRKY transcription factor PpWRKY65 played a crucial role in regulating lignin biosynthesis in peach fruit. Analyzing the correlation among the expression level of PpWRKY65, the expression level of genes related to lignin synthesis, and the lignin content of peach fruit both under ambient temperature and chilling injury condition, it was found that PpWRKY65 negatively regulated the accumulation of peach fruit lignin by inhibiting the expression of genes related to lignin synthesis. Moreover, the lignin content was significantly lower in transient PpWRKY65-overexpressing fruit, compared to the control. Concurrently, the expressions of lignin biosynthesis genes (Pp4CL7, PpCAD1, PpCOMT, PpF5H) were decreased in fruit overexpressing PpWRKY65.

Genome-wide analysis of the PME gene family reveals its role in suppressing fruit lignification in pear

In pears, the presence of stone cells adversely affects fruit quality. Pectin methylesterase (PME) plays various roles in plant biology, including lignin biosynthesis. However, only a limited fraction has been functionally characterized, and the distribution and function of PME in many Rosaceae trees remain unexplored. In this study, we identified 396 putative PME family candidate genes, with 81 in Pyrus bretschneideri, 92 in Malus domestica, 62 in Fragaria vesca, 65 in Prunus mume, 15 in Pyrus communis, and 81 in Pyrus pyrifolia. Leveraging insights from model plants, we categorized PME family genes into four groups. Additionally, the evolution of the PME gene family was shaped by various gene duplication events, primarily dispersed duplication, influenced by purifying selection. A specific gene, Pbr031522.1, designated PbPME35, emerged as a candidate associated with lignin biosynthesis in pear fruits, supported by RNA-seq data. The role of PbPME35 in repressing lignification was validated through its overexpression in pear callus and Arabidopsis. Overall, our findings highlight the ability of PbPME35 to reduce lignin content in pear fruit by downregulating the expression levels of lignin biosynthesis genes. These findings provide new insights into the characteristics of PME genes and their role in regulating lignification in pear fruits.

Tissue-specific transcriptomics reveals a central role of <i>CcNST1</i> in regulating the fruit lignification pattern in <i>Camellia chekiangoleosa</i>, a woody oil-crop

Fruit lignification is of significant economic importance because it affects the quality of fruit and the production of seed oil. The specified lignification pattern in<italic> Camellia</italic> <italic>chekiangoleosa</italic> fruits plays critical roles in its seed oil yield, but little is known about how this lignification process is regulated. Here, we report on a comprehensive tissue-specific transcriptomics analysis conducted for <italic>C.</italic> <italic>chekiangoleosa</italic> fruit. By mining the differentially expressed genes, we found that lignin biosynthesis and transcriptional regulation pathways were significantly enriched in the lignified tissues. The homolog of NST-like transcription factor, <italic>CcNST1</italic>, was highly expressed in lignified seed coat and endocarp tissues; transgenic analyses of <italic>CcNST1</italic> in <italic>Arabidopsis</italic> and hybrid poplar revealed the enhanced lignification levels of various tissues. Gene expression analysis of the transgenic lines uncovered potential downstream genes involved in the regulation of lignin biosynthesis. This work provides a valuable gene expression resource and identified the pivotal role of <italic>CcNST1</italic> in regulating the lignin biosynthesis underlying fruit lignification.

Transcriptomic Analysis of Hardened ‘Suli’ Pear (Pyrus bretschneideri Rehd) for Identification of Key Genes for Lignin Biosynthesis and Accumulation

Fruit hardening, one of the physiological disorders influencing the quality of pear, is usually accompanied by fruit lignification. Nonetheless, the mechanism by which lignin accumulates in hardened pear fruit is still unknown. Here, Transcriptome analysis of normal fruits (NFs) and hardened fruits (HFs) of ‘Suli’ pear (Pyrus bretschneideri Rehd) was used to examine the molecular mechanism of fruit hardening. We found that hardened fruits (HFs), especially those in the top region within 35–180 days after flowering (DAF), possessed a prominently higher lignin level than normal fruits (NFs). Subsequently, transcriptome sequencing of fruit at 35 DAF identified 4391 [HF shoulder region (HF_S) vs. NF top region (NF_T)], 3849 (HF_T vs. NF_T) and 408 (HF_T vs. HF_S) differentially expressed genes. Among them, we clarified 26 unigenes that encode 10 enzymes as candidate genes participating in lignin biosynthesis. Thus, high expression of the lignin biosynthetic gene impels lignin to accumulate in HFs. Some transcription factors were evaluated to link with lignin formation and subsequently analysed. In addition, the genes participating in flavonoid/proanthocyanidin biosynthesis dropped from the HF_T to the HF_S, suggesting that the flavonoid metabolic pathway was reduced to promote intermediate metabolites to be converted to the lignin biosynthetic pathway. To conclude, this study lays a solid theoretical foundation and provides reference data for investigating the mechanism by which lignin accumulates in hardened pear fruit.

The regulation of cell wall lignification and lignin biosynthesis during pigmentation of winter jujube

Fruit lignification is due to lignin deposition in the cell wall during cell development. However, there are few studies on the regulation of cell wall lignification and lignin biosynthesis during fruit pigmentation. In this study, we investigated the regulation of cell wall lignification and lignin biosynthesis during pigmentation of winter jujube. The cellulose content decreased, while the lignin content increased in the winter jujube pericarp during pigmentation. Safranin O-fast green staining showed that the cellulose content was higher in the cell wall of winter jujube prior to pigmentation, whereas the lignin in the cell wall increased after pigmentation. The thickness of the epidermal cells decreased with pericarp pigmentation. A combined metabolomics and transcriptomics analysis showed that guaiacyl-syringyl (G-S) lignin was the main lignin type in the pericarp of winter jujube, and F5H (LOC107424406) and CCR (LOC107420974) were preliminarily identified as the key genes modulating lignin biosynthesis in winter jujube. Seventeen MYB and six NAC transcription factors (TFs) with potential regulation of lignin biosynthesis were screened out based on phylogenetic analysis. Three MYB and two NAC TFs were selected as candidate genes and further studied in detail. Arabidopsis ectopic expression and winter jujube pericarp injection of the candidate genes indicated that the MYB activator (LOC107425254) and the MYB repressor (LOC107415078) control lignin biosynthesis by regulating CCR and F5H, while the NAC (LOC107435239) TF promotes F5H expression and positively regulates lignin biosynthesis. These findings revealed the lignin biosynthetic pathway and associated genes during pigmentation of winter jujube pericarp and provide a basis for further research on lignin regulation.

CcBLH6, a bell-like homeodomain-containing transcription factor, regulates the fruit lignification pattern.

CcBLH6 is a bell-like homeodomain-containing transcription factor that plays an important role of lignin biosynthesis in the control of fruit lignification pattern in Camellia chekiangoleosa. The fruit of Camellia chekiangoleosa has a unique lignification pattern that features with a thick pericarp containing a low level of lignification. Yet the fruit lignification pattern and the regulatory network of responsible gene transcription are poorly understood. Here, we characterized a bell-like homeodomain-containing (BLH) transcription factor from C. chekiangoleosa, CcBLH6, in the control of fruit lignification. CcBLH6 expression was highly correlated with the unique lignification pattern during fruit development. The ectopic expression of CcBLH6 promoted the lignification process of stem and root in Arabidopsis. We found that expression of genes related to lignin biosynthesis and its transcriptional regulation was altered in transgenic lines. In a Camellia callus-transformation system, overexpression of CcBLH6 greatly enhanced the expression of genes related to lignin biosynthesis and its transcriptional regulation was altered in transgenic lines. In the callus-transformation system, overexpression of CcBLH6 greatly enhanced the lignification of parenchymal cells, and the regulation of several genes involved in lignin accumulation was largely consistent between Arabidopsis and Camellia. Our study reveals a positive role of CcBLH6 in the regulation of lignin biosynthesis during fruit lignification in Camellia.

Influence of Algal Bio-fertilizers on Fruit Lignification Time Interval, DNA Concentration and Agro-Morphological Characters in Okra (Abelmoschus Esculentus Moench)

Experiment was set up in May to September, 2019 to evaluate the impact of algal bio-fertilizers on fruit lignification time interval, total genomic DNA contents, growth attributes as well as the yield components of three okra (Abelmoschus esculentus) (NHAE-47-4, NHAe-54-4 and Agwu early) genotypes sourced from NIHORT, Ibadan, Oyo State, Nigeria. The bio-fertilizers used for the study were a combination of cow dung + algae, poultry droppings + algae, Green Compost + algae and only soil as control. The test crops were sown into 50 x 30 x 25 cm3 perforated plastic buckets containing 5 kg of sterilized sandy loam soil and applied with the bio-fertilizers at 500 g each. The bio-fertilizers were prepared by using a combination of algae and organic manures. The experiment was a 3 x 4 experiment set up in completely randomized design (CRD) with 3 replications. Factor one was the Okra (Abelmoschus esculentus) genotypes with three levels (NHAe-47-4; NHAe-54-4 and Agwu early) while factor two was the different bio-fertilizers used with four levels (Cow dung + Algae; Polutry droopings + Algae; Green compost manure + Algae and soil as control). This gave a total treatment combination of 12 which was replicated thrice to give a total of 36 experimental units. Data for yield components were collected at maturity for days to flower initiation, number of seeds per pod, number of pods per plants and fresh fruit weight. Data generated from the study were collated and subjected to statistical analysis using the analysis of variance (ANOVA) procedures and significant treatment means were further separated using the Fishers’ least significance difference test at 5 percent probability level. The results shows that genomic DNA contents varies from 134.100 ng/µl in Agwu early in control plot to 175.600 ng/µl in NHAe -54-4 treated with Poultry droppings +Algae. Fruit lignification time interval was significantly (p &lt;0.05) influenced by the bio-fertilizers and varied between 3 days 6 minutes in Agwu early in the control plots to 8 days and 23 hours in NHAe 47-4 treated with Poultry dropping + Alage. Plant height varied (p &lt;0.05) significantly and ranged from 14.13 cm in Agwu early in control plot to 44.34 cm in NHAe-47-4 treated with Poultry droppings + Algae while number of fruits per plant also varied (p &lt;0.05) significantly and ranged between 12.57 fruits per plant in Agwu early in control plots to 24.30 fruits per plant in NHAe-47-4 treated with Poultry droppings + Algae. Hence, the study advocated for the use of poultry droppings + Algal bio-fertilizer in prolonging the fruit lignification time in okra for planned harvesting, increased income and mechanization of okra harvesting.

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.

Integrative Analysis of the Core Fruit Lignification Toolbox in Pear Reveals Targets for Fruit Quality Bioengineering.

Stone cell content is an important factor affecting pear fruit flavor. Lignin, a major component of pear stone cells, hinders the quality and value of commercial fruit. The completion of the Chinese white pear (Pyrus bretschneideri) genome sequence provides an opportunity to perform integrative analysis of the genes encoding the eleven protein families (i.e., PAL, C4H, 4CL, HCT, C3H, CSE, CCoAOMT, CCR, F5H, COMT, and CAD) in the phenylpropanoid pathway. Here, a systematic study based on expression patterns and phylogenetic analyses was performed to identify the members of each gene family potentially involved in the lignification in the Chinese white pear. The phylogenetic analysis suggested that 35 P. bretschneideri genes belong to bona fide lignification clade members. Compared to other plants, some multigene families are expanded by tandem gene duplication, such as HCT, C3H, COMT, and CCR. RNA sequencing was used to study the expression patterns of the genes in different tissues, including leaf, petal, bud, sepal, ovary, stem, and fruit. Eighteen genes presented a high expression in fruit, indicating that these genes may be involved in the biosynthesis of lignin in pear fruit. Similarly to what has been observed for Populus trichocarpa, a bimolecular fluorescence complementation (BiFC) experiment indicated that P. bretschneideri C3H and C4H might also interact with each other to regulate monolignol biosynthesis in P. bretschneideri, ultimately affecting the stone cell content in pear fruits. The identification of the major genes involved in lignin biosynthesis in pear fruits provides the basis for the development of strategies to improve fruit quality.

Ternary complex EjbHLH1-EjMYB2-EjAP2-1 retards low temperature-induced flesh lignification in loquat fruit

Many transcription factors (TFs), including NACs and MYBs, are involved in regulation of lignin biosynthesis during plant development and in responses to biotic and abiotic stresses. The lignin biosynthesis gene Ej4CL1 has been identified as a target for cold-induced TFs. We isolated a bHLH gene from loquat, EjbHLH1, the expression of which was negatively correlated with cold-induced fruit lignification. During low temperature storage (0 °C), EjbHLH1 transcripts were stable but accumulated during low-temperature conditioning (LTC) treatment, an acclimation process that reduces lignification during subsequent storage at 0 °C. Dual luciferase assays showed EjbHLH1 could repress Ej4CL1 promoter, but yeast one hybrid assay indicated EjbHLH1 is not able to bind to the Ej4CL1 promoter. Bimolecular fluorescence complementation (BIFC) indicated that EjbHLH1 could interact with EjAP2-1 and EjMYB2, two previously characterized fruit lignification related transcription factors and firefly luciferase complementation imaging assay indicated EjbHLH1, EjMYB2 and EjAP2-1 could form a ternary complex which enhanced repression of transcription from the Ej4CL1 promoter, reducing lignification at 0 °C.

EjHAT1 Participates in Heat Alleviation of Loquat Fruit Lignification by Suppressing the Promoter Activity of Key Lignin Monomer Synthesis Gene EjCAD5.

Texture attributes such as firmness and lignification are important for fruit quality. Lignification has been widely studied in model plants and energy crops, but fruit lignification has rarely been investigated, despite having an adverse effect on fruit quality and consumer preference. Chilling-induced loquat fruit lignification that occurs after harvest can be alleviated by heat treatment (HT) applied prior to low temperature storage. Enzyme activity assay showed that HT treatment could retard the low temperature-induced increase in cinnamyl alcohol dehydrogenase (CAD) activity. Transcript analysis and substrate activity assays of recombinant CAD proteins highlighted the key role of EjCAD5 in chilling-induced lignin biosynthesis. A novel homeobox-leucine zipper protein ( EjHAT1) was identified as a negative regulator of EjCAD5. Therefore, the effect of HT treatment on lignification may be partially due to the suppression of the EjCAD5 promoter activity by EjHAT1.

EjMYB4 is a transcriptional activator of 4-Coumarate:coenzyme A ligase involved in lignin biosynthesis in loquat (Eriobotrya japonica)

Lignin is one of the major structural compounds for plants and the transcriptional regulatory mechanisms governing its production have been extensively studied in model plants. However, the underlying mechanisms may differ between organs and information is still limited for fruit lignification. Loquat accumulates lignin not only in vegetative organs but also in fruit flesh. To gain insight into the transcriptional regulation of loquat lignification, yeast one hybrid library screening was conducted with the 4-Coumarate:coenzyme A ligase (Ej4CL1) promoter and four proteins (EjNF-YC1, EjNF-YC2, EjMYB4 and EjGTE1) were identified with binding affinity. Dual-luciferase assays showed that EjMYB4 and EjGTE1 selectively trans-activated promoters of lignin biosynthesis genes from both loquat and Arabidopsis, while EjNF-YC1 and EjNF-YC2 didn’t have the trans-activation effects. Gene expression analysis indicated that EjMYB4 was higher expressed in vegetative tissues compared with reproductive tissues, whereas EjGTE1 expression was not correlated with lignin content in different tissues. Subcellular localization results indicated that EjMYB4 was located mainly in the nucleus and electrophoretic mobility-shift assay further validated that EjMYB4 directly binds to AC-element of Ej4CL1 promoter. EjMYB4 transient over-expression in tobacco leaves led to an increase in lignin content and up-regulation of tobacco endogenous lignin related genes. These results revealed the function of EjMYB4 in regulating lignin biosynthesis in loquat.

Label-free visualization of fruit lignification: Raman molecular imaging of loquat lignified cells

BackgroundFlesh lignification, leading to increased fruit firmness, has been reported in several kinds of fruit. Understanding the mechanisms underlying fruit lignification is important to optimize the postharvest storage strategies and reduce the quality deterioration of postharvest fruit. Especially cellular level investigation of lignin deposition in fruits provides novel insight for deciphering the mechanisms underlying fruit lignification. The primary objective of this study was to establish a procedure of using Raman microspectroscopy technique to depict fruit lignification at the cell level.ResultsLignified cells, a special kind of cells contained high lignin content, were found abundantly scattered in red-fleshed ‘Luoyangqing’ loquat. Whereas these special lignified cells were barely detected in ‘Baisha’ loquat flesh. Dominant Raman bands of lignified cells were found primarily attributed to lignin (1664, 1628, 1603, 1467, and 1272 cm−1), cellulose (1383, 1124 and 1098 cm−1) and pectin (852 and 1740 cm−1). The band intensity correlation analysis indicated the peak at 1335 cm−1 assigned to either lignin or cellulose in previous works was related to lignin for the lignified cells. Multi-peaks Gaussian fitting successfully resolved the overlapped fingerprint peaks of lignin in 1550–1700 cm−1 into three independent peaks, which were assigned to different functional groups of lignin. Furthermore, the spatially resolved Raman images of lignified cells were generated, indicating that lignin and cellulose saturated the whole lignified cells, pectin mainly located in the cell corner, and the parenchyma cells contained little lignin. In addition, both phloroglucinol-HCl staining and autofluorescence analysis confirmed the results of lignin distribution of Raman microscopic analysis.ConclusionsA procedure for the simultaneous visualization of the main components of the flesh cells without labeling by high-resolution Raman microspectroscopy has been established. With Raman microscopic imaging technique, we can add a microscopic level to cell compositions, essential for a detailed molecular understanding of loquat lignification. Such method can be further used to chemically monitor the textural changes during the ripening process or postharvest storage of other fruits and vegetables.

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.

Involvement of PAL, C4H, and 4CL in Chilling Injury-induced Flesh Lignification of Loquat Fruit

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 low-temperature 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.

EjODO1, a MYB Transcription Factor, Regulating Lignin Biosynthesis in Developing Loquat (Eriobotrya japonica) Fruit.

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.

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.

EjMYB8 Transcriptionally Regulates Flesh Lignification in Loquat Fruit.

Transcriptional regulatory mechanisms underlying lignin metabolism have been widely studied in model plants and woody trees, but seldom in fruits such as loquat, which undergo lignification. Here, twelve EjMYB genes, designed as EjMYB3-14, were isolated based on RNA-seq. Gene expression indicated that EjMYB8 and EjMYB9 were significantly induced in fruit with higher lignin content resulting from storage at low temperature (0°C), while two treatments (low temperature conditioning, LTC; heat treatment, HT) both alleviated fruit lignification and inhibited EjMYB8 and EjMYB9 expression. Dual-luciferase assays indicated that EjMYB8, but not EjMYB9, could trans-activate promoters of lignin-related genes EjPAL1, Ej4CL1 and Ej4CL5. Yeast one-hybrid assay indicated that EjMYB8 physically bind to Ej4CL1 promoter. Furthermore, the putative functions of EjMYB8 were verified using transient over-expression in both N. tabacum and loquat leaves, which increased lignin content. Moreover, combination of EjMYB8 and previously isolated EjMYB1 generated strong trans-activation effects on the Ej4CL1 promoter, indicating that EjMYB8 is a novel regulator of loquat fruit lignification.