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Malic acid is the major organic acid in loquat fruit, contributing to the sourness of fruit and affecting fruit flavor. However, the transcriptional regulation of malic acid in loquat is not well understood. Here, we discovered a MADS-box transcription factor (TF), EjAGL18, that regulated malic acid accumulation in loquat. EjAGL18 is a nucleus-localized TF without transcriptional activity. The expression of EjAGL18 increased during fruit ripening, opposite to the accumulation pattern of malic acid in loquat. The transient overexpression of EjAGL18 in loquat fruit downregulated malic acid accumulation and the transcriptional level of the tonoplast dicarboxylate transporter EjtDT1 . Conversely, silencing EjAGL18 in loquat fruit upregulated the malic acid content and EjtDT1 expression level. Dual-luciferase assays and yeast one-hybrid experiments further confirmed that EjAGL18 could bind to the promoter of EjtDT1 and repress its transcriptions. Furthermore, the transient overexpression of EjtDT1 in loquat fruit increased the malic acid content. These results revealed that EjAGL18 negatively regulates malic acid content by repressing EjtDT1 in loquat. This study broadens the understanding of the MADS-box TF's regulatory mechanisms in malic acid and provides new insights into fruit flavor improvement in loquat.

Changes in both lignin biosynthesis and DNA methylation have been reported to be associated with chilling stress in plants. When stored at low temperatures, red-fleshed loquat is prone to lignification, with increased lignin content and fruit firmness, which has deleterious effects on taste and eating quality. Here, we found that 5 °C storage mitigated the increasing firmness and lignin content of red-fleshed 'Dahongpao' ('DHP') loquat fruit that occurred during 0 °C storage. EjNAC5 was identified by integrating RNA sequencing with whole-genome bisulfite sequencing analysis of 'DHP' loquat fruit. The transcript levels of EjNAC5 were positively correlated with changes in firmness and negatively correlated with changes in DNA methylation level of a differentially methylated region in the EjNAC5 promoter. In white-fleshed 'Baisha' ('BS') loquat fruit, which do not undergo chilling-induced lignification at 0 °C, the transcripts of EjNAC5 remained low and the methylation level of the differentially methylated region in the EjNAC5 promoter was higher, compared with 'DHP' loquat fruit. Transient overexpression of EjNAC5 in loquat fruit and stable overexpression in Arabidopsis and liverwort led to an increase in lignin content. Furthermore, EjNAC5 interacts with EjERF39 and EjHB1 and activates the transcription of Ej4CL1 and EjPRX12 genes involved in lignin biosynthesis. This regulatory network involves different transcription factors from those involved in the lignification pathway. Our study indicates that EjNAC5 promoter methylation modulates EjNAC5 transcript levels and identifies novel EjNAC5-EjERF39-Ej4CL1 and EjNAC5-EjHB1-EjPRX12 regulatory modules involved in chilling induced-lignification., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Loquat (Eriobotrya japonica Lindl.) is a popular fruit and medicinal plant. Proanthocyanidins (PAs), as one of the main types of flavonoids, are the key components of loquat fruit quality and medicinal properties. However, the identification of transcription factors (TFs) involved in PA accumulation in loquat remains limited. R2R3-MYB TFs play key regulatory role in PA accumulation in plants. In this study, 190 R2R3-MYB TFs were identified in loquat genome. Combined with transcriptome data, R2R3-MYB TF EjMYB5 involved in PA accumulation in loquat was isolated. EjMYB5 was transcriptional activator localized to nucleus. Expression of EjMYB5 was closely related to PA accumulation in loquat fruits. Heterogenous overexpression of EjMYB5 in tomato (Solanum lycopersicum) inhibited anthocyanin accumulation and promoted PA accumulation. Additionally, transient overexpression of EjMYB5 in tobacco (Nicotiana benthamiana) leaves promoted PA accumulation by upregulating flavonoid biosynthesis genes (NtDFR, NtANS, and NtLAR). Transcriptome analysis of EjMYB5-overexpressing tomato fruits suggested that EjMYB5 was involved in several biological pathways, including lipid metabolism, MAPK signaling, phenylpropanoid biosynthesis, and flavonoid biosynthesis. Collectively, our findings provided basic data for further analysis the function of R2R3-MYB TFs in loquat, and revealed that EjMYB5 functioned as PA accumulation in loquat., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aneuploidy generally has severe phenotypic consequences. However, the molecular basis for this has been focused on single chromosomal dosage changes. It is not clear how the karyotype of complex aneuploidies affects gene expression. Here, we identified six different double-trisomy loquat strains from Q24 progenies of triploid loquat. The differences and similarities of the transcriptional responses of different double trisomy loquat strains were studied systematically via RNA-seq. The global modulation of gene expression indicated that both cis and trans-effects coordinately regulated gene expression in aneuploid loquat to some extent, and this coordinated regulation was determined by different gene functional groups. Aneuploidy can induce specific transcriptional responses on loquat chromosomes. The differentially expressed genes exhibited regional gene expression dysregulation domains along chromosomes. Furthermore, Aneuploidy could also promote the expression of genes with moderate and high in loquats. Our results provide new insights into the genome-wide transcriptional effects of karyotypes with complex aneuploidies., Competing Interests: Declaration of competing interest The manuscript is approved by all authors for publication and no conflict of interest exits in the submission. We would like to declare that the work carried out by all authors listed is original and has not been published in any form previously. All authors listed have contributed significantly to the work and agree to be in the author list., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The chloroplast genomes of wild loquat can help to determine their place in the history of evolution. Here, we sequenced and assembled two novel wild loquat's chloroplast genomes, one is Eriobotrya elliptica, and the other is an unidentified wild loquat, which we named "YN-1". Their sizes are 159,471 bp and 159,399 bp, respectively. We also assembled a cultivated loquat named 'JFZ', its chloroplast genome size is 159,156 bp. A comparative study was conducted with six distinct species of loquats, including five wild loquats and one cultivated loquat. The results showed that both E. elliptica and "YN-1" have 127 genes, one gene more than E. fragrans, which is psbK. Regions trnF-GAA-ndhJ, petG-trnP-UGG, and rpl32-trnL-UAG were found to exhibit high variability. It was discovered that there was a positive selection on rpl22 and rps12. RNA editing analysis found several chilling stress-specific RNA editing sites, especially in rpl2 gene. Phylogenetic analysis results showed that "YN-1" is closely related to E. elliptica, E. obovata and E. henryi., (© 2024. The Author(s).)

Background: Plant-specific TIFY proteins are widely found in terrestrial plants and play important roles in plant adversity responses. Although the genome of loquat at the chromosome level has been published, studies on the TIFY family in loquat are lacking. Therefore, the EjTIFY gene family was bioinformatically analyzed by constructing a phylogenetic tree, chromosomal localization, gene structure, and adversity expression profiling in this study., Results: Twenty-six EjTIFY genes were identified and categorized into four subfamilies (ZML, JAZ, PPD, and TIFY) based on their structural domains. Twenty-four EjTIFY genes were irregularly distributed on 11 of the 17 chromosomes, and the remaining two genes were distributed in fragments. We identified 15 covariate TIFY gene pairs in the loquat genome, 13 of which were involved in large-scale interchromosomal segmental duplication events, and two of which were involved in tandem duplication events. Many abiotic stress cis-elements were widely present in the promoter region. Analysis of the Ka/Ks ratio showed that the paralogous homologs of the EjTIFY family were mainly subjected to purifying selection. Analysis of the RNA-seq data revealed that a total of five differentially expressed genes (DEGs) were expressed in the shoots under gibberellin treatment, whereas only one gene was significantly differentially expressed in the leaves; under both low-temperature and high-temperature stresses, there were significantly differentially expressed genes, and the EjJAZ15 gene was significantly upregulated under both low- and high-temperature stress. RNA-seq and qRT-PCR expression analysis under salt stress conditions revealed that EjJAZ2, EjJAZ4, and EjJAZ9 responded to salt stress in loquat plants, which promoted resistance to salt stress through the JA pathway. The response model of the TIFY genes in the jasmonic acid pathway under salt stress in loquat was systematically summarized., Conclusions: These results provide a theoretical basis for exploring the characteristics and functions of additional EjTIFY genes in the future. This study also provides a theoretical basis for further research on breeding for salt stress resistance in loquat. RT-qPCR analysis revealed that the expression of one of the three EjTIFY genes increased and the expression of two decreased under salt stress conditions, suggesting that EjTIFY exhibited different expression patterns under salt stress conditions., (© 2024. The Author(s).)

The loquat ( Eriobotrya japonica L.) is a special evergreen tree, and its fruit is of high medical and health value as well as having stable market demand around the world. In recent years, research on the accumulation of nutrients in loquat fruit, such as carotenoids, flavonoids, and terpenoids, has become a hotspot. The SBP-box gene family encodes transcription factors involved in plant growth and development. However, there has been no report on the SBP-box gene family in the loquat genome and their functions in carotenoid biosynthesis and fruit ripening. In this study, we identified 28 EjSBP genes in the loquat genome, which were unevenly distributed on 12 chromosomes. We also systematically investigated the phylogenetic relationship, collinearity, gene structure, conserved motifs, and cis -elements of EjSBP proteins. Most EjSBP genes showed high expression in the root, stem, leaf, and inflorescence, while only five EjSBP genes were highly expressed in the fruit. Gene expression analysis revealed eight differentially expressed EjSBP genes between yellow- and white-fleshed fruits, suggesting that the EjSBP genes play important roles in loquat fruit development at the breaker stage. Notably, EjSBP01 and EjSBP19 exhibited completely opposite expression patterns between white- and yellow-fleshed fruits during fruit development, and showed a close relationship with SlCnr involved in carotenoid biosynthesis and fruit ripening, indicating that these two genes may participate in the synthesis and accumulation of carotenoids in loquat fruit. In summary, this study provides comprehensive information about the SBP-box gene family in the loquat, and identified two EjSBP genes as candidates involved in carotenoid synthesis and accumulation during loquat fruit development.

The newly released 'Snow White' (SW), a white-fleshed loquat ( Eriobotrya japonica Lindl.) cultivar, holds promise for commercial production. However, the specifics of the phenolic composition in white-fleshed loquats, along with the antioxidant substances and their regulatory mechanisms, are not yet fully understood. In this study, we examined the dynamic changes in the phenolic compounds, enzyme activities, antioxidant capacity, and gene expression patterns of SW during the key stages of fruit development and ripening. A total of 18 phenolic compounds were identified in SW, with chlorogenic acid, neochlorogenic acid, and coniferyl alcohol being the most predominant. SW demonstrated a stronger antioxidant capacity in the early stages of development, largely due to total phenolics and flavonoids. Neochlorogenic acid may be the most significant antioxidant contributor in loquat. A decline in enzyme activities corresponded with fruit softening. Different genes within a multigene family played distinct roles in the synthesis of phenolics. C4H1 , 4CL2 , 4CL9 , HCT , CCoAOMT5 , F5H , COMT1 , CAD6 , and POD42 were implicated in the regulation of neochlorogenic acid synthesis and accumulation. Consequently, these findings enhance our understanding of phenolic metabolism and offer fresh perspectives on the development of germplasm resources for white-fleshed loquats.

The Rosaceae family includes several deciduous woody species whose flower development extends over two consecutive growing seasons with a winter dormant period in between. Loquat (Eriobotrya japonica Lindl.) belongs to this family, but it is an evergreen species whose flower bud initiation and flowering occur within the same growing year. Vegetative growth dominates from spring to late summer when terminal buds bloom as panicles. Thus, its floral buds do not undergo winter dormancy until flowering, but a summer heat period of dormancy is required for floral bud differentiation, and that is why we used loquat to study the mechanism by which this summer rest period contributes to floral differentiation of Rosaceae species. As for the deciduous species, the bud transition to the generative stage is initiated by the floral integrator genes. There is evidence that combinations of environmental signals and internal cues (plant hormones) control the expression of TFL1, but the mechanism by which this gene regulates its expression in loquat needs to be clarified for a better understanding of its floral initiation and seasonal growth cycles. Under high temperatures (>25ºC) after floral bud inductive period, EjTFL1 expression decreases during meristem transition to the reproductive stage, and the promoters of flowering (EjAP1 and EjLFY) increase, indicating that the floral bud differentiation is affected by high temperatures. Monitoring the apical meristem of loquat in June-August of two consecutive years under ambient and thermal controlled conditions showed that under lower temperatures (<25ºC) during the same period, shoot apex did not stop growing and a higher EjTFL1 expression was recorded, preventing the bud to flower. Likewise, temperature directly affects ABA content in the meristem paralleling EjTFL1 expression, suggesting signaling cascades could converge to refine the expression of EjTFL1 under specific conditions (Tª<25ºC) during the floral transition stage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Sorbitol is an important signaling molecule in fruit trees. Here, we observed that sorbitol increased during flower bud differentiation (FBD) in loquat (Eriobotrya japonica Lindl.). Transcriptomic analysis suggested that bud formation was associated with the expression of the MADS-box transcription factor (TF) family gene, EjCAL. RNA fluorescence in situ hybridization showed that EjCAL was enriched in flower primordia but hardly detected in the shoot apical meristem. Heterologous expression of EjCAL in Nicotiana benthamiana plants resulted in early FBD. Yeast-one-hybrid analysis identified the ERF12 TF as a binding partner of the EjCAL promoter. Chromatin immunoprecipitation-PCR confirmed that EjERF12 binds to the EjCAL promoter, and β-glucuronidase activity assays indicated that EjERF12 regulates EjCAL expression. Spraying loquat trees with sorbitol promoted flower bud formation and was associated with increased expression of EjERF12 and EjCAL. Furthermore, we identified EjUF3GaT1 as a target gene of EjCAL and its expression was activated by EjCAL. Function characterization via overexpression and RNAi reveals that EjUF3GaT1 is a biosynthetic gene of flavonoid hyperoside. The concentration of the flavonoid hyperoside mirrored that of sorbitol during FBD and exogenous hyperoside treatment also promoted loquat bud formation. We identified a mechanism whereby EjCAL might regulate hyperoside biosynthesis and confirmed the involvement of EjCAL in flower bud formation in planta. Together, these results provide insight into bud formation in loquat and may be used in efforts to increase yield., (© 2022 Institute of Botany, Chinese Academy of Sciences.)

Loquat fruit is vulnerable to chilling injury (CI) under long-term low temperature storage. The application of calciumchloride (CaCl 2 ) can alleviate CI in loquat fruit, however, the molecular mechanisms remain unclear. In this study, the loquat fruit were immersed in 1 % CaCl 2 solution for 10 min and then stored at 1 ± 1 ℃ for 35 d. The results showed that CaCl 2 treatment suppressed the increases in internal browning index, electrolyte leakage, and malonaldehyde content in loquat fruit under chilling stress. The internal browning index in CaCl 2 -treated loquat fruit was 33.34 % lower than that in the control at the end of storage. Meanwhile, CaCl 2 treatment delayed the phospholipids degradation, and retained high level of unsaturated/saturated fatty acid ratio, which was 15.21 % higher than that in the control fruit at the end of storage. The CaCl 2 treatment also decreased the enzymatic activities and gene expressions of phospholipase D (PLD), phospholipase C (PLC), and lipoxygenase (LOX) in loquat fruit during cold storage. Moreover, a calmodulin-binding transcription activator (CAMTA) transcription factor (TF), EjCAMTA5, was cloned from loquat fruit. The EjCAMTA5 expression was up-regulated by cold stress and CaCl 2 treatment. Further investigation revealed that EjCAMTA5 directly bound to the promoters of EjPLC6-like and EjLOX5 to repress their transcription. Taken together, these findings implied that CaCl 2 application could activate the EjCAMTA5-mediated transcriptional repression of EjPLC6-like and EjLOX5 genes, which contributed to delaying the degradation of membrane lipid and maintaining the integrity of cell membrane, thereby inhibiting the occurrence of browning in loquat fruit under chilling stress., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Fruit size is an important fruit quality trait that influences the production and commodity values of loquats ( Eriobotrya japonica Lindl.). The Small Auxin Upregulated RNA ( SAUR ) gene family has proven to play a vital role in the fruit development of many plant species. However, it has not been comprehensively studied in a genome-wide manner in loquats, and its role in regulating fruit size remains unknown. In this study, we identified 95 EjSAUR genes in the loquat genome. Tandem duplication and segmental duplication contributed to the expansion of this gene family in loquats. Phylogenetic analysis grouped the SAURs from Arabidopsis , rice, and loquat into nine clusters. By analyzing the transcriptome profiles in different tissues and at different fruit developmental stages and comparing two sister lines with contrasting fruit sizes, as well as by functional predictions, a candidate gene ( EjSAUR22 ) highly expressed in expanding fruits was selected for further functional investigation. A combination of Indoleacetic acid (IAA) treatment and virus-induced gene silencing revealed that EjSAUR22 was not only responsive to auxin, but also played a role in regulating cell size and fruit expansion. The findings from our study provide a solid foundation for understanding the molecular mechanisms controlling fruit size in loquats, and also provide potential targets for manipulation of fruit size to accelerate loquat breeding.

Previous studies indicated that extensive genetic variations could be generated due to polyploidy, which is considered to be closely associated with the manifestation of polyploid heterosis. Our previous studies confirmed that triploid loquats demonstrated significant heterosis, other than the ploidy effect, but the underlying mechanisms are largely unknown. This study aimed to overcome the narrow genetic distance of loquats, increase the genetic variation level of triploid loquats, and systematically illuminate the heterosis mechanisms of triploid loquats derived from two cross combinations. Here, inter-simple sequence repeats (ISSRs) and simple sequence repeats (SSRs) were adopted for evaluating the genetic diversity, and transcriptome sequencing (RNA-Seq) was performed to investigate gene expression as well as pathway changes in the triploids. We found that extensive genetic variations were produced during the formation of triploid loquats. The polymorphism ratios of ISSRs and SSRs were 43.75% and 19.32%, respectively, and almost all their markers had a PIC value higher than 0.5, suggesting that both ISSRs and SSRs could work well in loquat assisted breeding. Furthermore, our results revealed that by broadening the genetic distance between the parents, genetic variations in triploids could be promoted. Additionally, RNA-Seq results suggested that numerous genes differentially expressed between the triploids and parents were screened out. Moreover, KEGG analyses revealed that "photosynthetic efficiency" and "glyco-metabolism" were significantly changed in triploid loquats compared with the parents, which was consistent with the results of physiological indicator analyses, leaf micro-structure observations, and qRT-PCR validation. Collectively, our results suggested that extensive genetic variations occurred in the triploids and that the changes in the "photosynthetic efficiency" as well as "glyco-metabolism" of triploids might have further resulted in heterosis manifestation in the triploid loquats.

Loquat (Eriobotrya japonica) have originated in southeastern China and spread as a cultivated plant worldwide. Many of the loquat genetic resources collected internationally are of unknown origin, and their genetic background requires clarification. This study analyzed the genetic diversity of 95 accessions by using Rad-Seq SNP markers. Data analysis broadly classified loquat into three groups: (1) Japanese and Chinese cultivars and some Japanese strains (wild plants that are not used for commercial cultivation), (2) Vietnamese, Israeli, Greek, USA, and Mexican cultivars and strains, and (3) other Japanese strains. Group 2 is cultivated mostly outside of East Asia and was clearly distinct from the other groups, indicating that varieties of unknown origin with genetic backgrounds different from those of Japanese and Chinese cultivars may have been introduced to Mediterranean countries and North America. Because Japanese and Chinese cultivars belong to group 1, the current Japanese cultivars are derived from genetic resources brought from China. Some of group 1 may have been introduced to Japan before excellent varieties were developed in China, while group 3 may have been indigenous to Japan that have not been introduced by human activities, or may have been brought to Japan by human activities from China., (© 2022. The Author(s).)

Loquat fruit are susceptible to chilling injuries induced by postharvest storage at low temperature. The major symptoms are increased lignin content and flesh firmness, which cause a leathery texture. Pretreatment with methyl jasmonate (MeJA) can alleviate this low-temperature-induced lignification, but the mechanism is not understood. In this study, we characterized a novel class III peroxidase, EjPRX12, and studied its relationship to lignification. Transcript levels of EjPRX12 were attenuated following MeJA pretreatment, consistent with the reduced lignin content in fruit. In vitro enzyme activity assay indicated that EjPRX12 polymerized sinapyl alcohol, and overexpression of EjPRX12 in Arabidopsis promoted lignin accumulation, indicating that it plays a functional role in lignin polymerization. We also identified an HD-ZIP transcription factor, EjHB1, repressed by MeJA pretreatment, which directly bound to and significantly activated the EjPRX12 promoter. Overexpression of EjHB1 in Arabidopsis promoted lignin accumulation with induced expression of lignin-related genes, especially AtPRX64. Furthermore, a JAZ-interacting repressor, EjbHLH14, was characterized, and it is proposed that MeJA pretreatment caused EjbHLH14 to be released to repress the expression of EjHB1. These results identified a novel regulatory pathway involving EjbHLH14-EjHB1-EjPRX12 and revealed the molecular mechanism whereby MeJA alleviated lignification of loquat fruit at low temperature., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Whole chloroplast genome (cpDNA) sequence is becoming widely used in the phylogenetic studies of plant and species identification, but in most cases the cpDNA were acquired from silica gel dried fresh leaves. So far few reports have been available to describe cpDNA acquisition from crude drugs derived from plant materials, the DNA of which usually was seriously damaged during their processing. In this study, we retrieved cpDNA from the commonly used crude drug Eriobotryae Folium (Pipaye in Chinese, which is the dried leaves of Eriobotrya japonica, PPY) using genome skimming technique., Results: We successfully recovered cpDNA sequences and rDNA sequences from the crude drug PPY, and bioinformatics analysis showed a high overall consistency between the cpDNA obtained from the crude drugs and fresh samples. In the ML tree, each species formed distinct monophyletic clades based on cpDNA sequence data, while the phylogenetic relationships between Eriobotrya species were poorly resolved based on ITS and ITS2., Conclusion: Our results demonstrate that both cpDNA and ITS/ITS2 are effective for identifying PPY and its counterfeits derived from distantly related species (i.e. Dillenia turbinata and Magnolia grandiflora), but cpDNA is more effective for distinguishing the counterfeits derived from the close relatives of Eriobotrya japonica, suggesting the potential of genome skimming for retrieving cpDNA from crude drugs used in Traditional Chinese Medicine for their identification., (© 2021. The Author(s).)

This study aimed to explore the effects of recombinant serine protease treatment on the development of post-harvest loquat diseases, fruit quality, and disease resistance enzyme activities. It also sought to analyze differential genes expression using RNA-seq technology. Transcriptomics analysis revealed 708 and 398 differentially expressed genes (DEGs) in loquat fruits treated with serine protease for 24 and 48 h. Furthermore, 2198 DEGs were obtained between 24 and 48 h after treatment. The genes encoding JAZ, MYC2 and ERF in the plant signal transduction pathway were significantly up-regulated. The resistance-related genes, such as PPO, PAL, TLP, WRKY, and transcription factors were also significantly up-regulated. These results indicated that the recombinant serine protease can induce plant signal transduction pathway in loquat fruit. The expression of some resistance-related genes enhanced the disease resistance of loquat fruit and revealed the molecular mechanism of loquat fruit resistance induced by recombinant serine protease., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The WRKY gene family, which is one of the largest transcription factor (TF) families, plays an important role in numerous aspects of plant growth and development, especially in various stress responses. However, the functional roles of the WRKY gene family in loquat are relatively unknown. In this study, a novel WRKY gene, EjWRKY17, was characterized from Eriobotrya japonica, which was significantly upregulated in leaves by melatonin treatment during drought stress. The EjWRKY17 protein, belonging to group II of the WRKY family, was localized in the nucleus. The results indicated that overexpression of EjWRKY17 increased cotyledon greening and root elongation in transgenic Arabidopsis lines under abscisic acid (ABA) treatment. Meanwhile, overexpression of EjWRKY17 led to enhanced drought tolerance in transgenic lines, which was supported by the lower water loss, limited electrolyte leakage, and lower levels of reactive oxygen species (ROS) and malondialdehyde (MDA). Further investigations showed that overexpression of EjWRKY17 promoted ABA-mediated stomatal closure and remarkably up-regulated ABA biosynthesis and stress-related gene expression in transgenic lines under drought stress. Overall, our findings reveal that EjWRKY17 possibly acts as a positive regulator in ABA-regulated drought tolerance.

Whole-genome duplication (WGD) plays important roles in plant evolution and function, yet little is known about how WGD underlies metabolic diversification of natural products that bear significant medicinal properties, especially in nonmodel trees. Here, we reveal how WGD laid the foundation for co-option and differentiation of medicinally important ursane triterpene pathway duplicates, generating distinct chemotypes between species and between developmental stages in the apple tribe. After generating chromosome-level assemblies of a widely cultivated loquat variety and Gillenia trifoliata, we define differentially evolved, duplicated gene pathways and date the WGD in the apple tribe at 13.5 to 27.1 Mya, much more recent than previously thought. We then functionally characterize contrasting metabolic pathways responsible for major triterpene biosynthesis in G. trifoliata and loquat, which pre- and postdate the Maleae WGD, respectively. Our work mechanistically details the metabolic diversity that arose post-WGD and provides insights into the genomic basis of medicinal properties of loquat, which has been used in both traditional and modern medicines., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Crop domestication and evolution represent key fields of plant and genetics research. Here, we re-sequenced and analyzed whole genome data from 51 wild accessions and 53 representative cultivars of Eriobotrya japonica, an important semi-subtropical fruit crop. Population genomics analysis suggested that modern cultivated E. japonica experienced a two-staged domestication fitting the "marginality model," being initially domesticated in west-northern Hubei province from a mono-phylogenetic wild progenitor, then refined mainly in Jiangsu, Zhejiang and Fujian provinces of China. Cultivated E. japonica has experienced little reduction in genome-wide nucleotide polymorphism compared with wild forms. Genes responsible for sugar biosynthesis were enriched in regions harboring putative selective sweeps. An approach based on co-clustering into gene families and evaluating chromosome colinearity of orthologous and paralogous genes was used to identify convergent/parallel selective sweeps among different crops. Specifically, more than one hundred of orthologs and paralogs undergoing selective sweeps were identified between loquat, apple and peach, among which 14 encoded "UDP glycosyltransferase 1." In sum, the study not only provided valuable information for breeding of E. japonica, but also enriched knowledge of crop domestication., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Organic acids and sugars are the primary components that determine the quality and flavor of loquat fruits. In the present study, major organic acids, sugar content, enzyme activities, and the expression of related genes were analyzed during fruit development in two loquat cultivars, 'JieFangZhong' (JFZ) and 'BaiLi' (BL). Our results showed that the sugar content increased during fruit development in the two cultivars; however, the organic acid content dramatically decreased in the later stages of fruit development. The differences in organic acid and sugar content between the two cultivars primarily occured in the late stage of fruit development and the related enzymes showed dynamic changes in activies during development. Phosphoenolpyruvate carboxylase (PEPC) and mNAD malic dehydrogenase (mNAD-MDH) showed higher activities in JFZ at 95 days after flowering (DAF) than in BL. However, NADP-dependent malic enzyme (NADP-ME) activity was the lowest at 95 DAF in both JFZ and BL with BL showing higher activity compared with JFZ. At 125 DAF, the activity of fructokinase (FRK) was significantly higher in JFZ than in BL. The activity of sucrose synthase (SUSY) in the sucrose cleavage direction (SS-C) was low at early stages of fruit development and increased at 125 DAF. SS-C activity was higher in JFZ than in BL. vAI and sucrose phosphate synthase (SPS) activities were similar in the two both cultivars and increased with fruit development. RNA-sequencing was performed to determine the candidate genes for organic acid and sugar metabolism. Our results showed that the differentially expressed genes (DEGs) with the greated fold changes in the later stages of fruit development between the two cultivars were phosphoenolpyruvate carboxylase 2 (PEPC2), mNAD-malate dehydrogenase (mNAD-MDH), cytosolic NADP-ME (cyNADP-ME2), aluminum-activated malate transporter (ALMT9), subunit A of vacuolar H+-ATPase (VHA-A), vacuolar H+-PPase (VHP1), NAD-sorbitol dehydrogenase (NAD-SDH), fructokinase (FK), sucrose synthase in sucrose cleavage (SS-C), sucrose-phosphate synthase 1 (SPS1), neutral invertase (NI), and vacuolar acid invertase (vAI). The expression of 12 key DEGs was validated by quantitative reverese transcription PCR (RT-qPCR). Our findings will help understand the molecular mechanism of organic acid and sugar formation in loquat, which will aid in breeding high-quality loquat cultivars., Competing Interests: I have declared that no competing interests exist.

Floral initiation plays a critical role for reproductive success in plants, especially fruit trees. However, little information is known on the mechanism of the initiation in loquat (Eriobotrya japonica Lindl.). Here, we used transcriptomic, expression and functional analysis to investigate the candidate genes in floral initiation in loquat. Comparative transcriptome analysis showed differentially expressed genes (DEGs) were mainly enriched in the metabolic pathways of plant hormone signal transduction. The DEGs were mainly involved in the gibberellin, auxin, cytokinin, abscisic acid, salicylic acid and ethylene signaling pathways. Meanwhile, some transcription factors, including MADS-box (MCM1, AGAMOUS, DEFICIENS and SRF), MYB (Myeloblastosis), TCP (TEOSINTE BRANCHED 1, CYCLOIDEA and PCF1), WOX (WUSCHEL-related homeobox) and WRKY (WRKY DNA-binding protein), were significantly differentially expressed. Among these key DEGs, we confirmed that an AGL17 ortholog EjAGL17 was significantly upregulated at the flower bud transition stage. Phylogenetic tree analysis revealed that EjAGL17 was grouped into an AGL17 clade of MADS-box transcription factors. Protein sequence alignment showed that EjAGL17 included a distinctive C-terminal domain. Subcellular localization of EjAGL17 was found only in the nucleus. Expression levels of EjAGL17 reached the highest at the development stage of flower bud transition. Moreover, ectopic expression of EjAGL17 in Arabidopsis significantly exhibited early flowering. Our study provides abundant resources of candidate genes for studying the mechanisms underlying the floral initiation in loquat and other Rosaceae species., Competing Interests: The authors have declared that no competing interests exist.

Loquat (Eriobotrya japonica) fruit marketability is affected by the incidence and severity of purple spot (PS), a pre-harvest physiological disorder showing an evident skin discoloration with depressed surface. Despite its impact in limiting the cultivation and economic potential of loquat, the etiology of this disorder is still poorly understood. To this end, our study aimed to investigate and disclose possible mechanisms underlying PS development. The intensity and severity of PS in three loquat cultivars ('Morphitiki', 'Karantoki' and 'Obusa') was phenotypically monitored during successive on-tree fruit developmental stages. 'Obusa' fruits harvested at commercial maturity stage showed the highest incidence of purple spot (58.6%), while 'Morphitiki' fruits did not show any symptoms. 'Karantoki' fruits demonstrated an intermediate response, with 31.3% of the fruit being affected. Thereafter, fruits with 30-50% PS severity were selected and used for further analysis; peel tissue was removed from both symptomatic and asymptomatic tissue of the same fruit for all examined cultivars. 'Karantoki' fruit with PS were characterized by the highest accumulation of total soluble sugars, sucrose, glucose and fructose contents, while the concentration of these primary metabolites was the lowest in asymptomatic fruit of 'Obusa', exception made for the sucrose. The incidence of PS was also transcriptionally investigated by assessing the mRNA profile of important genes involved in polyphenolic (PAL1, PAL2 and PPO1) and carbohydrate (CWI2, CWI3, SPS1, SPS2, NI2, NI3, SuSy, HXK, FRK and VI) pathway. The enhanced expression levels of CWI3 and VI genes in symptomatic fruit of the highly susceptible cultivar 'Obusa' highlight a cultivar-specific type of response. Notably, SuSy registered significantly suppressed levels in symptomatic tissue of both 'Obusa' and 'Karantoki'. To what extent PPO is associated with PS incidence and whether the etiology of the disorder can be assigned to an oxidative process triggered and coordinated by its action need to be further elucidated. The aforementioned genes are suggested to be further examined as potential markers towards a more sophisticated and informed characterization of purple spot detection in loquat fruit., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

In this study, third-generation full-length (FL) transcriptome sequencing was performed of loquat using single-molecule real-time(SMRT) sequencing from the pooled cDNA of embryos of young loquat fruit under different low temperatures (three biological replicates for treatments of 1°C, -1°C, and -3°C, for 12 h or 24 h) and the control group(three biological replicates for treatments of room temperature), Illumina sequencing was used to correct FL transcriptome sequences. A total of 3 PacBio Iso-Seq libraries (1-2 kb, 2-3 kb and 3-6 kb) and 21 Illumina transcriptome libraries were constructed, a total of 13.41 Gb of clean reads were generated, which included 215,636 reads of insert (ROIs) and 121,654 FL, non-chimaric (FLNC) reads. Transcript clustering analysis of the FLNC reads revealed 76,586 consensus isoforms, and a total of 12,520 high-quality transcript sequences corrected with non-FL sequences were used for subsequent analysis. After the redundant reads were removed, 38,435 transcripts were obtained. A total of 27,905 coding DNA sequences (CDSs) were identified, and 407 long non-coding RNAs (lncRNAs) were ultimately predicted. Additionally, 24,832 simple sequence repeats (SSRs) were identified, and a total of 1,295 alternative splicing (AS) events were predicted. Furthermore, 37,993 transcripts were annotated in eight functional databases. This is the first study to perform SMRT sequencing of the FL transcriptome of loquat. The obtained transcriptomic data are conducive for further exploration of the mechanism of loquat freezing injury and thus serve as an important theoretical basis for generating new loquat material and for identifying new ways to improve loquat cold resistance., Competing Interests: NO authors have competing interests.

Flower development is a vital developmental process in the life cycle of woody perennials, especially fruit trees. Herein, we used transcriptomic, proteomic, and hormone analyses to investigate the key candidate genes/proteins in loquat ( Eriobotrya japonica ) at the stages of flower bud differentiation (FBD), floral bud elongation (FBE), and floral anthesis (FA). Comparative transcriptome analysis showed that differentially expressed genes (DEGs) were mainly enriched in metabolic pathways of hormone signal transduction and starch and sucrose metabolism. Importantly, the DEGs of hormone signal transduction were significantly involved in the signaling pathways of auxin, gibberellins (GAs), cytokinin, ethylene, abscisic acid (ABA), jasmonic acid, and salicylic acid. Meanwhile, key floral integrator genes FLOWERING LOCUS T ( FT ) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 ( SOC1 ) and floral meristem identity genes SQUAMOSA PROMOTER BINDING LIKE ( SPL ), LEAFY ( LFY ), APETALA1 ( AP1 ), and AP2 were significantly upregulated at the FBD stage. However, key floral organ identity genes AGAMOUS ( AG ), AP3 , and PISTILLATA ( PI ) were significantly upregulated at the stages of FBE and FA. Furthermore, transcription factors (TFs) such as bHLH (basic helix-loop-helix), NAC (no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF1/2) and cup-shaped cotyledon (CUC2)), MYB&#95;related (myeloblastosis&#95;related), ERF (ethylene response factor), and C2H2 ( cysteine-2/histidine-2 ) were also significantly differentially expressed. Accordingly, comparative proteomic analysis of differentially accumulated proteins (DAPs) and combined enrichment of DEGs and DAPs showed that starch and sucrose metabolism was also significantly enriched. Concentrations of GA 3 and zeatin were high before the FA stage, but ABA concentration remained high at the FA stage. Our results provide abundant sequence resources for clarifying the underlying mechanisms of the flower development in loquat.

Loquat (Eriobotrya japonica Lindl.) is divided into yellow- and white-fleshed based on the difference in fruit color, and the variations in carotenoids accumulation are considered as the main reasons for this difference. Using RNA-seq technology, a transcriptome analysis was carried out on the flesh and peel of 'Baiyu' fruit during four different fruit development stages. A total of 172.53 Gb clean reads with an average of 6.33 Gb reads were detected for each library, and the percentage of Q30 was higher than 90.84%. We identified 16 carotenogenic and 13 plastid-lipid-associated protein (PAP) genes through RNA-seq. Of these, five carotenogenic and four PAP related genes exhibited remarkable differences in the expression patterns. Carotenoids biosynthetic genes, including DXS, PSY1 and VDE displayed higher expression levels in peel than that in the flesh. However, carotenoids decomposition gene, such as NCDE1, exhibited higher expression in flesh than that in the peel. Notably, all differentially expressed PAP genes showed higher expression levels in peel than flesh. We inferred that the differential accumulation of carotenoids in flesh and peel of 'Baiyu' is caused by the up- or down-regulation of the carotenogenic and PAP related genes. The functional analysis of these important genes will provide valuable information about underlying molecular mechanism of carotenoids accumulation in loquat., Competing Interests: The authors have declared that no competing interests exist.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Parthenocarpy can be induced by gibberellin (GA) treatment in plants. The fruits of the loquat exhibit many seeds. GA treatment can induce the development of seedless fruit and increase fruit quality during production. However, the molecular mechanism of fruit setting under GA treatment is still unclear., Objective: Our aim was to explore GA-induced parthenocarpy in triploid loquat by proteome analysis to identify the differentially expressed proteins., Methods: A proteome analysis was performed using TMT protein labeling and LC-MS/MS in triploid loquat., Results: A total of 7290 protein groups were identified in the two stages of fruit setting. The quantitative results showed that 923 differentially expressed proteins (DEPs) were isolated, which were enriched in five pathways: ribosome, citrate cycle (TCA cycle), pentose phosphate, carbon metabolism, and carbon fixation. Twenty-four DEPs were annotated as putative key regulatory proteins involved in fruit setting, which were related to the auxin response, gibberellin metabolism, ethylene synthesis, and cell division. In addition, thirty-five DEPs were involved in the formation of the cell wall, which might be downstream proteins involved in cell proliferation during fruit setting., Conclusion: Our report reveals new insight into the protein dynamics of loquat fruit setting induced by GA treatment via the analysis of proteome profiles and provides a reference for other Rosaceae species.

Background: The loquat (Eriobotrya japonica) is a species of flowering plant in the family Rosaceae that is widely cultivated in Asian, European, and African countries. It blossoms in the winter and ripens in the early summer. The genome of loquat has to date not been published, which limits the study of molecular biology in this cultivated species. Here, we used the third-generation sequencing technology of Nanopore and Hi-C technology to sequence the genome of Eriobotrya., Findings: We generated 100.10 Gb of long reads using Oxford Nanopore sequencing technologies. Three types of Illumina high-throughput sequencing data, including genome short reads (47.42 Gb), transcriptome short reads (11.06 Gb), and Hi-C short reads (67.25 Gb), were also generated to help construct the loquat genome. All data were assembled into a 760.1-Mb genome assembly. The contigs were mapped to chromosomes by using Hi-C technology based on the contacts between contigs, and then a genome was assembled exhibiting 17 chromosomes and a scaffold N50 length of 39.7 Mb. A total of 45,743 protein-coding genes were annotated in the Eriobotrya genome, and we investigated the phylogenetic relationships between the Eriobotrya and 6 other Rosaceae species. Eriobotrya shows a close relationship with Malus and Pyrus, with the divergence time of Eriobotrya and Malus being 6.76 million years ago. Furthermore, chromosome rearrangement was found in Eriobotrya and Malus., Conclusions: We constructed the first high-quality chromosome-level Eriobotrya genome using Illumina, Nanopore, and Hi-C technologies. This work provides a valuable reference genome for molecular studies of the loquat and provides new insight into chromosome evolution in this species., (© The Author(s) 2020. Published by Oxford University Press.)

APETALA3: (AP3) encodes a floral homeotic class B-function MADS-box protein and plays crucial roles in petal and stamen development. To better understand the functional roles of AP3 orthologs in Eriobotrya, we isolated and identified an AP3 ortholog, referred to as EjAP3, from Eriobotrya japonica. Analyses of protein sequence and phylogenetic tree showed that the EjAP3 was assigned to the rosids euAP3 lineage and included a distinctive PI-derived and euAP3 motifs at the C-terminal domain. Subcellular localization of EjAP3 was determined to be in the nucleus. Expression analysis suggested that EjAP3 expression was restricted only in petals and stamens, but not in sepals and carpels. Importantly, during the floral development, EjAP3 expression level was the highest at the stage of visible floral bud. Furthermore, ectopic expression of EjAP3 in Arabidopsis ap3-3 mutant rescued the second whorl petals and the third whorl stamens. The expression pattern and function characterization of EjAP3 contribute to better understand the roles of AP3 orthologs in Eriobotrya., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In the model species Arabidopsis thaliana , FRIGIDA ( FRI ) is a key regulator of flowering time and can inhibit flowering without vernalization. However, little information is available on the function in the Rosaceae family. Loquat ( Eriobotrya japonica ) belongs to the family Rosaceae and is a distinctive species, in which flowering can be induced without vernalization, followed by blooming in late-autumn or winter. To investigate the functional roles of FRI orthologs in this non-vernalization species, we isolated an FRI ortholog, dubbed as EjFRI , from loquat. Analyses of the phylogenetic tree and protein sequence alignment showed that EjFRI is assigned to eurosids I FRI lineage. Expression analysis revealed that the highest expression level of EjFRI was after flower initiation. Meanwhile, EjFRI was widely expressed in different tissues. Subcellular localization of EjFRI was only detected to be in the nucleus. Ectopic expression of EjFRI in wild-type Arabidopsis delayed flowering time. The expression levels of EjFRI in transgenic wild-type Arabidopsis were significantly higher than those of nontransgenic wild-type lines. However, the expression levels of AtFRI showed no significant difference between transgenic and nontransgenic wild-type lines. Furthermore, the upregulated AtFLC expression in the transgenic lines indicated that EjFRI functioned similarly to the AtFRI of the model plant Arabidopsis. Our study provides a foundation to further explore the characterization of EjFRI , and also contributes to illuminating the molecular mechanism about flowering in loquat.

The age pathway is important for regulating flower bud initiation in flowering plants. The major regulators in this pathway are miR156 and SPL transcription factors. To date, SPL genes have been identified in many species of plants. Loquat, as a woody fruit tree of Rosaceae, is unique in flowering time as it blooms in winter. However, the study of its SPL homologous genes on the regulation mechanism of flowering time is still limited. In this study, four SPL homologs- EjSPL3 , EjSPL4 , EjSPL5, and EjSPL9 -are cloned from loquat, and phylogenetic analysis showed that they share a high sequence similarity with the homologues from other plants, including a highly conserved SQUAMOSA promoter binding protein (SBP)-box domain. EjSPL3, EjSPL4, EjSPL5 are localized in the cytoplasm and nucleus, and EjSPL9 is localized only in the nucleus. EjSPL4, EjSPL5, and EjSPL9 can significantly activate the promoters of EjSOC1-1 , EjLFY-1, and EjAP1-1 ; overexpression of EjSPL3 , EjSPL4 , EjSPL5, and EjSPL9 in wild-type Arabidopsis thaliana can promote flowering obviously, and downstream flowering genes expression were upregulated. Our work indicated that the EjSPL3, EjSPL4, EjSPL5, and EjSPL9 transcription factors are speculated to likely participate in flower bud differentiation and other developmental processes in loquat. These findings are helpful to analyze the flowering regulation mechanism of loquat and provide reference for the study of the flowering mechanism of other woody fruit trees.

Flowering plants have evolved different flowering habits to sustain long-term reproduction. Most woody trees experience dormancy and then bloom in the warm spring, but loquat blooms in the cold autumn and winter. To explore its mechanism of flowering regulation, we cloned two SHORT VEGETATIVE PHASE (SVP) homologous genes from 'Jiefanzhong' loquat ( Eriobotrya japonica Lindl.), namely, EjSVP1 and EjSVP2 . Sequence analysis revealed that the EjSVPs were typical MADS-box transcription factors and exhibited a close genetic relationship with other plant SVP/DORMANCY-ASSOCIATED MADS-BOX (DAM) proteins. The temporal and spatial expression patterns showed that EjSVP1 and EjSVP2 were mainly expressed in the shoot apical meristem (SAM) after the initiation of flowering; after reaching their highest level, they gradually decreased with the development of the flower until they could not be detected. EjSVP1 expression levels were relatively high in young tissues, and EjSVP2 expression levels were relatively high in young to mature transformed tissues. Interestingly, EjSVP2 showed relatively high expression levels in various flower tissues. We analyzed the EjSVP promoter regions and found that they did not contain the C-repeat/dehydration-responsive element. Finally, we overexpressed the EjSVPs in wild-type Arabidopsis thaliana Col-0 and found no significant changes in the number of rosette leaves of Arabidopsis thaliana ; however, overexpression of EjSVP2 affected the formation of Arabidopsis thaliana flower organs. In conclusion, EjSVPs were found to play an active role in the development of loquat flowering. These findings may provide a reference for exploring the regulation mechanisms of loquat flowering and the dormancy mechanisms of other plants.

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., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

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.

Plastids are sites for carotenoid biosynthesis and accumulation, but detailed information on fruit plastid development and its relation to carotenoid accumulation remains largely unclear. Here, using Baisha (BS; white-fleshed) and Luoyangqing (LYQ; red-fleshed) loquat ( Eriobotrya japonica ), a detailed microscopic analysis of plastid development during fruit ripening was carried out. In peel cells, chloroplasts turned into smaller chromoplasts in both cultivars, and the quantity of plastids in LYQ increased by one-half during fruit ripening. The average number of chromoplasts per peel cell in fully ripe fruit was similar between the two cultivars, but LYQ peel cell plastids were 20% larger and had a higher colour density, associated with the presence of larger plastoglobules. In flesh cells, chromoplasts could be observed only in LYQ during the middle and late stages of ripening, and the quantity on a per-cell basis was higher than that in peel cells, but the size of chromoplasts was smaller. It was concluded that chromoplasts are derived from the direct conversion of chloroplasts to chromoplasts in the peel, and from de novo differentiation of proplastids into chromoplasts in flesh. The relationship between plastid development and carotenoid accumulation is discussed.

The expression levels of many genes and the related proteins change and regulate physiological and metabolic processes that help the plant survive harsh environmental conditions under cold stress. Damage due to cold and freezing conditions often causes dynamic loss of loquat fruits in cultivated parts of northern China. To illustrate the mechanism of cold tolerance in the loquat, we combined the transcriptomic analysis with isobaric tags for relative and absolute quantification (iTRAQ) and RNA sequencing (RNA-Seq) data from loquat leaves under 4 °C treatment. The results showed 122,081 genes and 1210 differentially expressed genes (DEGs), while only 4582 proteins and 300 differential proteins (DEPs) were identified. Functional annotation and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis indicated that metabolic pathways and biosynthesis of secondary metabolites were the two most common pathways in transcriptional and translational processes in this study. Comparison analysis of the transcriptomic and proteomic profiles, only 27 of 3620 genes were found to be shared both in DEGs and DEPs. Further validation with Real-Time Quantitative RT-PCR analysis showed that the genes expression of NADP-dependent D-sorbitol-6-phosphate dehydrogenase, anthocyanin synthase and phenylalanine ammonia-lyase were consistent with the pattern of transcriptome profile, which suggested that these three genes might play vital roles in cold tolerance in loquat., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Triploid loquat (Eriobotrya japonica (Thunb.) Lindl.) has greater vigor than their respective diploid and tetraploid parents, but the molecular basis of this triploid heterosis remains unclear. Recent studies have suggested that DNA methylation is involved in heterosis, which is a recognized method of suppressing gene expression. However, our previous studies revealed a trend of increased DNA methylation in triploid loquat hybrids compared to their parents. To elucidate the mechanism of triploid loquat heterosis, we investigated the levels and regulation of relative gene expression between hybrid and parental lines using RNA-Seq technology. We found that gene expression in the hybrid lines was down-regulated and gene expression analysis revealed that approximately 94.56 and 86.97% were expressed additively in triploid-A and triploid-B, respectively. Analyses of the allele-specific gene expression in the hybrids revealed significantly more Longquan-1 alleles were preferentially expressed in the two hybrid lines. Further analysis of cis- and trans-regulatory effects showed that gene expression variation between parental alleles is largely attributable to cis-regulatory variation in triploid loquat and analyses of genes belonging to cis-regulatory variation showed that 88-90% of cis genes contributed to an additive expression pattern. Taken together, our results suggest that gene expression variation in triploid loquat fundamentally cis-regulated may play a dominant role in triploid loquat heterosis.

We aim to overcome the unclear origin of the loquat and elucidate the heterosis mechanism of the triploid loquat. Here we investigated the genetic and epigenetic variations between the triploid plant and its parental lines using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified fragment length polymorphism (MSAP) analyses. We show that in addition to genetic variations, extensive DNA methylation variation occurred during the formation process of triploid loquat, with the triploid hybrid having increased DNA methylation compared to the parents. Furthermore, a correlation existed between genetic variation and DNA methylation remodeling, suggesting that genome instability may lead to DNA methylation variation or vice versa. Sequence analysis of the MSAP bands revealed that over 53% of them overlap with protein-coding genes, which may indicate a functional role of the differential DNA methylation in gene regulation and hence heterosis phenotypes. Consistent with this, the genetic and epigenetic alterations were associated closely to the heterosis phenotypes of triploid loquat, and this association varied for different traits. Our results suggested that the formation of triploid is accompanied by extensive genetic and DNA methylation variation, and these changes contribute to the heterosis phenotypes of the triploid loquats from the two cross lines.

Targeted mass spectrometric methods such as selected/multiple reaction monitoring (SRM/MRM) have found intense application in protein detection and quantification which competes with classical immunoaffinity techniques. It provides a universal procedure to develop a fast, highly specific, sensitive, accurate, and cheap methodology for targeted detection and quantification of proteins based on the direct analysis of their surrogate peptides typically generated by tryptic digestion. This methodology can be advantageously applied in the field of plant proteomics and particularly for non-model species since immunoreagents are scarcely available. Here, we describe the issues to take into consideration in order to develop a MRM method to detect and quantify isoforms of the thylakoid-bound protein polyphenol oxidase from the non-model and database underrepresented species Eriobotrya japonica Lindl.

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., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Main Conclusion: EjFT1 and EjFT2 genes were isolated and sequenced from leaves of loquat. EjFT1 is involved in bud sprouting and leaf development, and EjFT2 in floral bud induction. Loquat [Eriobotrya japonica (Thunb.) Lindl.] is an evergreen species belonging to the family Rosaceae, such as apple and pear, whose reproductive development, in contrast with these species, is a continuous process that is not interrupted by winter dormancy. Thus, the study of the mechanism of flowering in loquat has the potential to uncover the environmental and genetic networks that trigger flowering more accurately, contributing for a better understanding of the Rosaceae floral process. As a first step toward understanding the molecular mechanisms controlling flowering, extensive defoliation and defruiting assays, together with molecular studies of the key FLOWERING LOCUS T (FT) gene, were carried out. FT exhibited two peaks of expression in leaves, the first one in early to mid-May, the second one in mid-June. Two FT genes, EjFT1 and EjFT2, were isolated and sequenced and studied their expression. Expression of EjFT1 and EjFT2 peaks in mid-May, at bud sprouting. EjFT2 expression peaks again in mid-June, coinciding with the floral bud inductive period. Thus, when all leaves of the tree were continuously removed from early to late May vegetative apex differentiated into panicle, but when defoliation was performed from early to late June apex did not differentiate. On the other hand, fruit removal advanced EjFT1 expression in old leaves and the sooner the fruit detached, the sooner the bud sprouted. Accordingly, results strongly suggest that EjFT1 might be related to bud sprouting and leaf development, while EjFT2 might be involved in floral bud induction. An integrative model for FT functions in loquat is discussed.

Loquat (Eriobotrya japonica Lindl.) is an important subtropical, commercial fruit in China. It blossoms during autumn and winter in most areas of China and its fruitlets usually suffer from freezing stress. However, studies about the mechanisms underlying freezing stress in loquat are very limited. The gene expression profiles of loquat fruitlets subjected to freezing (G2 library) versus non-treated ones (G1 library) were investigated using Illumina sequencing technology to elucidate the molecular mechanisms and identify the genes that play vital roles in the freezing stress response. The results showed that approximately 157.63 million reads in total were obtained from freeze-treated and non-treated loquat fruitlets. These reads were assembled into 87,379 unigenes with an average length of 710 bp and an N50 of 1,200 bp. After comparing the profiles obtained from the G1 and G2 libraries, 2,892 differentially expressed genes were identified, of which 1,883 were up-regulated and 1,009 were down-regulated in the treated samples compared to non-treated ones. These unigenes showed significant differences in expression for carbohydrate transport and metabolism, amino acid metabolism, energy metabolism, and lipid metabolism, which are involved in defense against freezing stress. Glycolysis/gluconeogenesis was one of the most significantly regulated pathways. Freezing also significantly damaged the membrane system of loquat fruitlets, and several defense mechanisms were induced. Some selected genes related to low temperature resistance were validated by quantitative real-time PCR (qRT-PCR). The results revealed many genes and pathways that are part of freezing resistance processes and expand our understanding of the complex molecular events involved in freezing stress.

Background: Carotenoids are the main colouring substances found in orange-fleshed loquat fruits. The aim of this study was to unravel the carotenoid biosynthetic pathway of loquat fruit (cv. 'Obusa') in peel and flesh tissue during distinct on-tree developmental stages through a targeted analytical and molecular approach., Results: Substantial changes regarding colour parameters, both between peel and flesh and among the different developmental stages, were monitored, concomitant with a significant increment in carotenoid content. Key genes and individual compounds that are implicated in the carotenoid biosynthetic pathway were further dissected with the employment of molecular (RT-qPCR) and advanced analytical techniques (LC-MS). Results revealed significant differences in carotenoid composition between peel and flesh. Thirty-two carotenoids were found in the peel, while only eighteen carotenoids were identified in the flesh. Trans-lutein and trans-β-carotene were the major carotenoids in the peel; the content of the former decreased with the progress of ripening, while the latter registered a 7.2-fold increase. However, carotenoid profiling of loquat flesh indicated trans-β-cryptoxanthin, followed by trans-β-carotene and 5,8-epoxy-β-carotene to be the most predominant carotenoids. High amounts of trans-β-carotene in both tissues were supported by significant induction in a chromoplast-specific lycopene β-cyclase (CYCB) transcript levels. PSY1, ZDS, CYCB and BCH were up-regulated and CRTISO, LCYE, ECH and VDE were down-regulated in most of the developmental stages compared with the immature stage in both peel and flesh tissue. Overall, differential regulation of expression levels with the progress of on-tree fruit development was more evident in the middle and downstream genes of carotenoid biosynthetic pathway., Conclusions: Carotenoid composition is greatly affected during on-tree loquat development with striking differences between peel and flesh tissue. A link between gene up- or down-regulation during the developmental stages of the loquat fruit, and how their expression affects carotenoid content per tissue (peel or flesh) was established.

Background: The process of crop domestication has long been a major area of research to gain insights into the history of human civilization and to understand the process of evolution. Loquat (Eriobotrya japonica Lindl.) is one of the typical subtropical fruit trees, which was domesticated in China at least 2000 years ago. In the present study, we re-sequenced the genome of nine wild loquat accessions collected from wide geographical range and 10 representative cultivated loquat cultivars by using RAD-tag tacit to exploit the molecular footprints of domestication., Results: We obtained 26.4 Gb clean sequencing data from 19 loquat accessions, with an average of 32.64 M reads per genotype. We identified more than 80,000 SNPs distributed throughout the loquat genome. The SNP density and numbers were slightly higher in the wild loquat populations than that in the cultivated populations. All cultivars were clustered together by structure, phylogenetic and PCA analyses., Conclusion: The modern loquat cultivars have experienced a non-significant genetic bottleneck during domestication, and originated from a single domesticated event. Moreover, our study revealed that Hubei province of China is probably the origin center of cultivated loquat.

In order to gain insights into the controversial ripening behavior of loquat fruits, in the present study we have analyzed the expression of three genes related to ethylene biosynthesis (ACS1, ACO1 and ACO2), two ethylene receptors (ERS1a and ERS1b), one signal transduction component (CTR1) and one transcription factor (EIL1) in peel and pulp of loquat fruit during natural ripening and also in fruits treated with ethylene (10μLL -1 ) and 1-MCP (10μLL -1 ), an ethylene action inhibitor. In fruits attached to or detached from the tree, a slight increase in ethylene production was detected at the yellow stage, but the respiration rate declined progressively during ripening. Accumulation of transcripts of ethylene biosynthetic genes did not correlate with changes in ethylene production, since the maximum accumulation of ACS1 and ACO1 mRNA was detected in fully coloured fruits. Expression of ethylene receptor and signaling genes followed a different pattern in peel and pulp tissues. After fruit detachment and incubation at 20°C for up to 6days, ACS1 mRNA slightly increased, ACO1 experienced a substantial increment and ACO2 declined. In the peel, these changes were advanced by exogenous ethylene and partially inhibited by 1-MCP. In the pulp, 1-MCP repressed most of the changes in the expression of biosynthetic genes, while ethylene had almost no effects. Expression of ethylene perception and signaling genes was barely affected by ethylene or 1-MCP. Collectively, a differential transcriptional regulation of ethylene biosynthetic genes operates in peel and pulp, and support the notion of non-climacteric ripening in loquat fruits. Ethylene action, however, appears to be required to sustain or maintain the expression of specific genes., (Copyright © 2016. Published by Elsevier GmbH.)

Volatile benzenoids, including methyl p-methoxybenzoate, p-anisaldehyde, and p-anisalcohol, are responsible for the sweet and characteristic fragrance of loquat (Eriobotrya japonica, Rosaceae) flowers. Although the full pathway of volatile benzenoid synthesis has yet to be elucidated, their chemical structures suggest that O-methyltransferases are present in loquat and function in the methylation of the para-OH groups. In the present study, we used RNA-sequencing to identify four loquat genes (EjOMT1, EjOMT2, EjOMT3, and EjOMT4) that encode O-methyltransferases. We found that EjOMT1 was highly expressed in floral tissues, with an expression pattern that coincided with changes in intracellular volatile benzenoids during flower development. Recombinant EjOMT1 protein expressed in Escherichia coli showed the highest activity towards guaiacol with a K m value of 35 μM. Furthermore, the protein also showed lesser activities towards guaiacol-type benzenoids including eugenol, isoeugenol, vanillin, and ferulic acid, in addition to much weaker activities towards catechol and p-hydroxybenzenoid derivatives. However, no activity was shown towards phenylpropenes without m-methoxy substitution, t-anol and chavicol. Taken together, our findings indicate that EjOMT1 has a broad substrate specificity towards compounds with both para-OH and meta-OCH 3 groups, unlike previously characterized O-methyltransferases for volatile benzenoid/phenylpropanoid biosynthesis., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Loquat ( Eriobotrya japonica Lindl.) is an important non-climacteric fruit and rich in essential nutrients such as minerals and carotenoids. During fruit development and ripening, thousands of the differentially expressed genes (DEGs) from various metabolic pathways cause a series of physiological and biochemical changes. To better understand the underlying mechanism of fruit development, the Solexa/Illumina RNA-seq high-throughput sequencing was used to evaluate the global changes of gene transcription levels. More than 51,610,234 high quality reads from ten runs of fruit development were sequenced and assembled into 48,838 unigenes. Among 3256 DEGs, 2304 unigenes could be annotated to the Gene Ontology database. These DEGs were distributed into 119 pathways described in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A large number of DEGs were involved in carbohydrate metabolism, hormone signaling, and cell-wall degradation. The real-time reverse transcription (qRT)-PCR analyses revealed that several genes related to cell expansion, auxin signaling and ethylene response were differentially expressed during fruit development. Other members of transcription factor families were also identified. There were 952 DEGs considered as novel genes with no annotation in any databases. These unigenes will serve as an invaluable genetic resource for loquat molecular breeding and postharvest storage., Competing Interests: The authors declare no conflict of interest.

Main Conclusion: p -Methoxybenzoic acid carboxyl methyltransferase (MBMT) was isolated from loquat flowers. MBMT displayed high similarity to jasmonic acid carboxyl methyltransferases, but exhibited high catalytic activity to form methyl p -methoxybenzoate from p -methoxybenzoic acid. Volatile benzenoids impart the characteristic fragrance of loquat (Eriobotrya japonica) flowers. Here, we report that loquat produces methyl p-methoxybenzoate, along with other benzenoids, as the flowers bloom. Although the adaxial side of flower petals is covered with hairy trichomes, the trichomes are not the site of volatile benzenoid formation. Here we identified four carboxyl methyltransferase (EjMT1 to EjMT4) genes from loquat and functionally characterized EjMT1 which we found to encode a p-methoxybenzoic acid carboxyl methyltransferase (MBMT); an enzyme capable of converting p-methoxybenzoic acid to methyl p-methoxybenzoate via methylation of the carboxyl group. We found that transcript levels of MBMT continually increased throughout the flower development with peak expression occurring in fully opened flowers. Recombinant MBMT protein expressed in Escherichia coli showed the highest substrate preference toward p-methoxybenzoic acid with an apparent K m value of 137.3 µM. In contrast to benzoic acid carboxyl methyltransferase (BAMT) and benzoic acid/salicylic acid carboxyl methyltransferase, MBMT also displayed activity towards both benzoic acid and jasmonic acid. Phylogenetic analysis revealed that loquat MBMT forms a monophyletic group with jasmonic acid carboxyl methyltransferases (JMTs) from other plant species. Our results suggest that plant enzymes with same BAMT activity have evolved independently.

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., (© 2016 John Wiley & Sons Ltd.)