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Guijie Li,1,* Yujiao Cheng,1,* Tenghui Zhang,2 Yingzhuo Li,3 Leng Han,1 Guolu Liang4 1Citrus Research Institute, Southwest University, Chongqing, People’s Republic of China; 2Chengdu Centre Testing International Group Co., Ltd., Chengdu, People’s Republic of China; 3Chongqing Beibei Agricultural and Rural Committee, Chongqing, People’s Republic of China; 4College of Horticulture and Landscape Architecture, Southwest University, Chongqing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Leng HanCitrus Research Institute, Southwest University, No. 15 Ganjucun, Xiema, Beibei District, Chongqing, 400712, People’s Republic of ChinaEmail hanleng5@cric.cnGuolu LiangCollege of Horticulture and Landscape Architecture, Southwest University, No. 2 Tiansheng Road, Beibei District, Chongqing, 400715, People’s Republic of ChinaEmail lianggl@swu.edu.cnPurpose: Citrus essential oils are widely used for aromatherapy and the alternative treatment of chronic diseases. Beyond the aroma substances, they are known to contain bioactive nonvolatile components; however, little knowledge has been gained about nonvolatiles in the essential oil of pomelo (Citrus grandis Osbeck), the largest citrus fruit. The purpose of this study was to analyze the nonvolatile oxygenated heterocyclic compounds (OHCs) of pomelo essential oils and evaluate their in vitro antioxidant activities for further development.Methods: Cold-pressed essential oil (CPEO) and distilled essential oil (DEO) were obtained from the peel of the Liangping pomelo cultivar. High-performance liquid chromatography (HPLC) coupled with a photodiode array and fluorescence detection method was developed to identify and quantify the OHCs of the two essential oils. Ferric reducing antioxidant power and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide (PTIO) radical scavenging assays were used to determine the antioxidative capabilities.Results: Thirteen OHCs were identified in CPEO. Coumarins such as meranzin (2.0 mmol L− 1) and furanocoumarins such as isoimperatorin (1.3 mmol L− 1) composed the majority of nonvolatiles in CPEO. These OHCs were characterized by high proportion (58%) of side chain epoxides. Five OHCs, namely, auraptenol, 6ʹ,7ʹ-dihydroxybergamottin (6ʹ,7ʹ-DHB), imperatorin, isoimperatorin and 8-geranyloxypsoralen were first identified in pomelo CPEO. Eight OHCs were detected at trace amounts in pomelo DEO. Antioxidant assays showed that CPEO was multiple times more potent than DEO regarding the total reducing power and radical scavenging capacity. Clearance of PTIO, a stable reactive oxygen species, followed slow kinetics.Conclusion: Coumarins and furanocoumarins, two families of OHCs, constituted most of the nonvolatile components in CPEO. The nonvolatiles contributed significantly to the in vitro antioxidant activity of CPEO. Pomelo CPEO showed good prospects as a potential long-lasting natural antioxidant.Keywords: volatile oil, cold expression, hydrodistillation, psoralen, epoxides, fluorescence, radical scavenging, pummelo, Citrus maxima

Background Aneuploidy, a condition caused by an imbalance between the relative dosages of chromosomes, generally produces a novel phenotype specific to the molecular karyotype. Few techniques are currently available for detecting the molecular karyotypes of aneuploids in plants. Results Based on this imbalance in chromosome dosage, a new approach (referred to as ‘SSR-qPCR’) combining simple sequence repeat (SSR) markers and quantitative real-time PCR (qPCR) has been developed and utilized to detect some common aneuploids irrespective of heterozygosity. We screened 17 specific SSR markers covering all loquat linkage groups and redesigned 6 pairs of primers for SSR markers that can detect loquat chromosome aneuploidies. The SSR-qPCR detection results obtained for hybrid progeny and open-pollination progeny of triploid loquat showed diagnostic accuracies of 88.9% and 62.5%, respectively, compared with the chromosome preparation results. Conclusion SSR-qPCR can detect loquat aneuploids and be used to construct the entire molecular karyotypes of aneuploid individuals. Therefore, this method offers a novel alternative for the detection of chromosome aneuploidies.

Previous observation in our laboratory found that growth vigor of triploid loquats was greater than in diploids and tetraploids despite the existence of ploidy effects, but it is still lack of systematic studies (i.e., histological, physiological). In addition, the underlying mechanisms of the heterosis are largely unknown due to the fact that the origin and genetic pedigree are unclear. In this study, we sought to further confirm the heterosis of triploid loquat systematically and elucidate the relationship between heterosis and circadian rhythms in triploid loquat by using trees with defined genetic pedigree. Two sets of triploid loquat were generated by crossing a tetraploid loquat with two wild diploid loquats (GC-1 and GC-23 from Guizhou Province, China (GC). Heterosis was systematically evaluated by comparing various histological, physiological and disease resistance metrics to the parental types. The results showed that the two sets of triploid loquat generated here exhibited pronounced degree of heterosis in all three categories and this was consistent with our previous observation. To determine if heterosis was also affected by the circadian clock, three circadian clock genes (EjLHY, EjTOC1 and EjGIGANTEA) were screened out, and the expression level of three circadian clock genes and 7 clock output genes were also analyzed. Phylogenetic analysis of three circadian genes showed that they were highly homologous with any other species in Rosaceae family. QRT-PCR analysis of the three circadian clock genes showed that all of them expressed above-high-parent (AHP) in almost all of the hybrids. Surprisingly, the 7 clock output genes also demonstrated AHP expression pattern in almost all the hybrids, and this was consistent with the results from our histological, physiological and disease resistance analysis. Our results indicated that triploid loquat exhibits pronounced heterosis compared with the diploid and tetraploid loquats, and the triploid loquat heterosis may be related to the altered amplitude of circadian rhythms.

Background Clementine mandarin (Citrus clementina Hort. ex Tan.) is one of the most famous and widely grown citrus cultivars worldwide. Variations in relation to the composition and distribution of repetitive DNA sequences that dominate greatly in eukaryote genomes are considered to be species-, genome-, or even chromosome-specific. Repetitive DNA-based fluorescence in situ hybridization (FISH) is a powerful tool for molecular cytogenetic study. However, to date few studies have involved in the repetitive elements and cytogenetic karyotype of Clementine. Results A graph-based similarity sequence read clustering methodology was performed to analyze the repetitive DNA families in the Clementine genome. The bioinformatics analysis showed that repetitive DNAs constitute 41.95% of the Clementine genome, and the majority of repetitive elements are retrotransposons and satellite DNAs. Sequential multicolor FISH using a probe mix that contained CL17, four satellite DNAs, two rDNAs and an oligonucleotide of (TTTAGGG)3 was performed with Clementine somatic metaphase chromosomes. An integrated karyotype of Clementine was established based on unequivocal and reproducible chromosome discriminations. The distribution patterns of these probes in several Citrus, Poncirus and Fortunella species were summarized through extensive FISH analyses. Polymorphism and heterozygosity were commonly observed in the three genera. Some asymmetrical FISH loci in Clementine were in agreement with its hybrid origin. Conclusions The composition and abundance of repetitive elements in the Clementine genome were reanalyzed. Multicolor FISH-based karyotyping provided direct visual proof of the heterozygous nature of Clementine chromosomes with conspicuous asymmetrical FISH hybridization signals. We detected some similar and variable distribution patterns of repetitive DNAs in Citrus, Poncirus, and Fortunella, which revealed notable conservation among these genera, as well as obvious polymorphism and heterozygosity, indicating the potential utility of these repetitive element markers for the study of taxonomic, phylogenetic and evolutionary relationships in the future. Electronic supplementary material The online version of this article (10.1186/s12870-019-1676-3) contains supplementary material, which is available to authorized users.

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.

A triploid was obtained from loquat (Eriobotrya japonica cv. ‘Ninghaibai’) (2n = 2x = 34) × bronze loquat (Eriobotrya deflexa, 2n = 2x = 34). The leaves of the triploid had no petioles, similar to the female parent, and its stipules were obvious and dark green, similar to those of the male parent. There were signals of male parent DNA on ten chromosomes of the triploid according to genomic in situ hybridization (GISH) when DNA of bronze loquat was used as probe; the diploid hybrid also had ten chromosomes with signals of male parent DNA. All five selected simple sequence repeats (SSR) primers amplified specific loci of both parents. In particular, primer CH04c06 amplified both specific loci of the female parent but only amplified one specific locus of the male parent. The results of GISH and SSR marker analysis indicated that the 2n gamete which formed the triploid was from first-division restitution or pre-meiotic doubling of the female parent cv. ‘Ninghaibai’. The allotriploid can be used to breed seedless loquat germplasm with different flowering times from loquat directly or to breed new germplasm containing a few genomic components from bronze loquat, such as monosomic alien addition lines, monosomic alien substitution lines, and introgression lines. This is the first report of allotriploid in the genus Eriobotrya and it will provide a basis for breeding new loquat germplasms by distant hybridization and provide an ideal material for breeding new loquat varieties with different flowering time or time to maturity.

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_related (myeloblastosis_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 GA3 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.

Carrizo citrange [Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf., CC] is one of the most widely used rootstocks in citriculture worldwide, but its cytogenetic study has been hampered by its inherent small size, morphological similarity to mitotic chromosomes, and lack of accessible cytological landmarks. In our previous study, a spontaneously occurring tetraploid CC seedling was discovered. The main goals of this study were to elucidate the chromosome constitution and construct the karyotypes of diploid CC rootstock and its corresponding spontaneously occurring tetraploid. To accomplish these, the chromosomal characteristics were investigated by sequential multicolor fluorescence in situ hybridization (FISH) with eight properly labeled repetitive DNA sequences, including a centromere-like repeat, four satellite repeats, two rDNAs, and an oligonucleotide of telomeric (TTTAGGG) n repeat. The results nicely demonstrated that these repetitive DNAs are reliable cytogenetic markers that collectively facilitate simultaneous and unequivocal identification of homologous chromosome pairs. Based on chromosome size and morphology together with FISH patterns of repetitive DNAs, an integrated karyotype of CC rootstock was constructed, consisting of 2n = 2x = 12m (1sat) + 6sm with karyotype asymmetry degree being divided into 2B category. Cytogenetically speaking, the variable and asymmetric distribution patterns of these repetitive DNAs were fully confirmed the hybrid nature of CC rootstock. In addition, comparative distribution patterns and chromosomal localizations of these repetitive DNAs convincingly showed that this tetraploid CC material arose from somatic chromosome doubling of diploid CC rootstock. This study revealed, for the first time, the integrated karyotype and chromosomal characteristics of this important citrus rootstock as well as its spontaneously occurring tetraploid plant. Furthermore, this study is a good prospective model for study species with morphologically indistinguishable small chromosomes.

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.

Few reports have been seen on the segregation of polyploids and on the relationship between the segregation of SSR loci and that of the morphological traits in polyploid progeny. This paper attempted to gain an insight into the segregation of the hexaploid tobacco progeny and to understand the correlation of the segregation of SSR loci with that of the flower morphological traits. The segregation was evaluated with both SSR markers and morphological traits of the flowers. Twenty pairs of SSR primers were screened and a total of 42 SSR loci were identified. Chi-square analysis showed that the segregation ratio of the SSR loci in the aneuploid progeny was between 1:1 and 2:1. Six morphological traits of flowers, including the lengths of the flower and the calyx, the widths of the calyx, the corolla tube and the corolla, and the coloring of the flower, from 180 progeny and 2 progenitor plants, were determined and Chi-square analyzed. All traits were in consistent also with a segregation ratio of 1:1 to 2:1 except the width of the calyx.

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.

Double-flower Eriobotrya japonica, of which one phenotype is homeotic transformation of sepals into petals, is a new germplasm for revealing the molecular mechanisms underlying the floral organ transformation. Herein, we analyzed the sequence, expression pattern and functional characterization of EjPI, which encoded a B-class floral homeotic protein referred to as PISTILLATA ortholog, from genetically cognate single-flower and double-flower E. japonica. Phylogenetic analysis suggested that the EjPI gene was assigned to the rosids PI/GLO lineage. Analysis of protein sequence alignments showed that EjPI has typical domains of M, I, K, and C, and includes a distinctive PI motif at the C-terminal region. Compared with asterids PI/GLO lineage, the K1 and K3 subdomains of EjPI both contain a single amino acid difference. Subcellular localization of EjPI was determined to be in the nucleus. Expression pattern analysis revealed that EjPI expressed not only in petals, filament, and anther in single-flower E. japonica, but also in petaloid sepals in double-flower E. japonica. Meanwhile, there were high correlation between EjPI transcript level and petaloid area within a sepal. Furthermore, 35S::EjPI transgenic wild-type Arabidopsis caused the homeotic transformation of the first whorl sepals into petaloid sepals. Ectopic expression of EjPI in transgenic pi-1 mutant Arabidopsis rescued normal petals and stamens. These results suggest expression pattern of EjPI is associated with the formation of petaloid sepal. Our study provides the potential application of EjPI for biotechnical engineering to create petaloid sepals or regulate floral organ identity in angiosperms.

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.

Asteraceae is the largest family of angiosperms, comprising approximately 24,000 species. Molecular genetic studies of Asteraceae are essential for understanding plant diversity. Chrysanthemum morifolium is the most industrially important ornamental species in Asteraceae. Most cultivars of C. morifolium are autohexaploid and self-incompatible. These properties are major obstacles to the genetic analysis and modern breeding of C. morifolium. Furthermore, high genome heterogeneity complicates molecular biological analyses. In this study, we developed a model strain in the genus Chrysanthemum. C. seticuspe is a diploid species with a similar flowering property and morphology to C. morifolium and can be subjected to Agrobacterium-mediated transformation. We isolated a natural self-compatible mutant of C. seticuspe and established a pure line through repeated selfing and selection. The resultant strain, named Gojo-0, was favorable for genetic analyses, including isolation of natural and induced mutants, and facilitated molecular biological analysis, including whole genome sequencing, owing to the simplicity and homogeneity of its genome. Interspecific hybridization with Chrysanthemum species was possible, enabling molecular genetic analysis of natural interspecific variations. The accumulation of research results and resources using Gojo-0 as a platform is expected to promote molecular genetic studies on the genus Chrysanthemum and the genetic improvement of chrysanthemum cultivars.

Huanglongbing (HLB) is an extremely destructive and lethal disease of citrus worldwide, presumably caused by phloem-limited bacteria, Candidatus Liberibacter asiaticus (CLas). The widespread invasiveness of the HLB pathogen and lack of natural HLB-resistant citrus cultivars have underscored the need for identifying tolerant citrus genotypes to support the current citrus industry's survival and potentially to lead to future natural HLB resistance. In this study, transverse sections of leaf lamina and midribs were examined with light and epifluorescence microscopy to determine anatomical characteristics that underlie HLB-tolerant mechanisms operating among "Bearss" lemon, "LB8-9" Sugar Belle® mandarin, and its sibling trees compared with HLB-sensitive "Valencia" sweet orange. The common anatomical aberrations observed in all CLas-infected varieties are as follows: phloem necrosis, hypertrophic phloem parenchyma cells, phloem plugging with abundant callose depositions, phloem collapse with cell wall distortion and thickening, excessive starch accumulation, and sometimes even cambium degeneration. Anatomical distribution of starch accumulation even extended to tracheid elements. Although there were physical, morphological, and pathological similarities in the examined foliage, internal structural preservation in "Bearss" lemon and "LB8-9" Sugar Belle® mandarin was superior compared with HLB-sensitive "Valencia" sweet orange and siblings of "LB8-9" Sugar Belle® mandarin. Intriguingly, there was substantial phloem regeneration in the tolerant types that may compensate for the dysfunctional phloem, in comparison with the sensitive selections. The lower levels of phloem disruption, together with greater phloem regeneration, are two key elements that contribute to HLB tolerance in diverse citrus cultivars.

Blood orange [Citrus sinensis (L.) Osbeck] has been increasingly appreciated by consumers worldwide owing to its brilliant red color, abundant anthocyanin and other health-promoting compounds. However, there is still relatively little known about its cytogenetic characteristics, probably because of the small size and similar morphology of metaphase chromosomes and the paucity of chromosomal landmarks. In our previous study, a naturally occurring tetraploid blood orange plant was obtained via seedling screening. Before this tetraploid germplasm can be manipulated into a citrus triploid seedless breeding program, it is of great importance to determine its chromosome characterization and composition. In the present study, an integrated karyotype of blood orange was constructed using sequential multicolor fluorescence in situ hybridization (FISH) with four satellite repeats, two ribosomal DNAs (rDNAs), a centromere-like repeat and an oligonucleotide of telomere repeat (TTTAGGG)3 as probes. Satellite repeats were preferentially located at the terminal regions of the chromosomes of blood orange. Individual somatic chromosome pairs of blood orange were unambiguously identified by repetitive DNA-based multicolor FISH. These probes proved to be effective chromosomal landmarks. The karyotype was formulated as 2n = 2x = 18 = 16m+2sm (1sat) with the karyotype asymmetry degree belonging to 2B. The chromosomal distribution pattern of these repetitive DNAs in this spontaneously occurring tetraploid was identical to that of the diploid, but the tetraploid carried twice the number of hybridization sites as the diploid, indicating a possible pathway involving the spontaneous duplication of chromosome sets in nucellar cells. Our work may facilitate the molecular cytogenetic study of blood orange and provide chromosomal characterization for the future utilization of this tetraploid germplasm in the service of seedless breeding programs.

This study investigated the effects of melatonin (MT) treatment on the edible qualities and lignification in loquat fruit during cold storage (4 °C) for 25 days. The results exhibited that 50 μM MT treatment effectively maintained the external quality of loquat fruit by reducing weight loss and firmness. MT treatment suppressed the decline of sucrose while slowing the changes in glucose and fructose contents. MT application to loquat fruit also increased the contents of phenolic compounds (chlorogenic acid and neochlorogenic acid) and organic acids (malic acid, oxalic acid, and tartaric acid), thereby maintaining the flavor and nutritional quality of loquat fruit. Compared to untreated fruit, MT treatment reduced the accumulation of malondialdehyde (MDA) and improved antioxidant capacity in loquat fruit, as indicated by higher ABTS, DPPH, and FRAP. Furthermore, MT treatment heavily regulated lignin biosynthesis-related enzyme activities during cold storage, including phenylalanine ammonia-lyase (PAL), 4-coumarate: coenzyme A ligase (4CL), cinnamate dehydrogenase (CAD), and peroxidase (POD). Our results suggest that MT treatment could be an effective approach to alleviate lignification and maintaining the nutrition quality of loquat fruit during cold storage.

Summary Potato tuber is a high yielding food crop known for its high levels of starch accumulation but only negligible levels of triacylglycerol (TAG). In this study, we evaluated the potential for lipid production in potato tubers by simultaneously introducing three transgenes, including WRINKLED 1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and OLEOSIN under the transcriptional control of tuber‐specific (patatin) and constitutive (CaMV‐35S) promoters. This coordinated metabolic engineering approach resulted in over a 100‐fold increase in TAG accumulation to levels up to 3.3% of tuber dry weight (DW). Phospholipids and galactolipids were also found to be significantly increased in the potato tuber. The increase of lipids in these transgenic tubers was accompanied by a significant reduction in starch content and an increase in soluble sugars. Microscopic examination revealed that starch granules in the transgenic tubers had more irregular shapes and surface indentations when compared with the relatively smooth surfaces of wild‐type starch granules. Ultrastructural examination of lipid droplets showed their close proximity to endoplasmic reticulum and mitochondria, which may indicate a dynamic interaction with these organelles during the processes of lipid biosynthesis and turnover. Increases in lipid levels were also observed in the transgenic potato leaves, likely due to the constitutive expression of DGAT1 and incomplete tuber specificity of the patatin promoter. This study represents an important proof‐of‐concept demonstration of oil increase in tubers and provides a model system to further study carbon reallocation during development of nonphotosynthetic underground storage organs.

Drought limits the growth and development of loquat (Eriobotrya japonica Lindl.) seedlings. Although melatonin (MT) is a naturally occurring small molecule in plants and has been linked with response to drought stress, the underlying mechanism remains largely unknown. The protective effects and regulatory mechanisms of MT on the regulation of drought stress in grafted loquat seedlings were analyzed in this study. The physiological results showed that MT effectively mitigated chlorophyll degradation and malondialdehyde (MDA) accumulation in the loquat leaves under drought stress. In addition, MT improved the photosynthetic efficiency and starch content of grafted loquat seedlings. Transcriptome results revealed that MT regulated genes involved in the Ca2+ signal transduction (cyclic nucleotide gated channel, CNGCs and cam modulin/ calmodulin-like protein, CAM/CMLs), starch and sucrose metabolism (sucrose synthase, SuS5 and SuS6), and plant hormone signal transduction (ABA receptor, PYL4; protein phosphatase 2C, PP2Cs; auxin/indole acetic acid protein, AUX/IAAs and ethylene-responsive transcription factor 1B, ERF1B), and the transcription factors such as WRKYs, NACs, ERFs, and bHLHs. We highlighted the analysis on phytohormone signaling pathways and their possible crosstalk. The results also demonstrated that MT increased the endogenous MT, IAA and CTK contents, while decreasing the ABA content under drought condition. Taken together, these results provided a new perspective on the role of MT in drought stress and a network for gene function analysis in MT-mediated signaling pathways.

The protective effects of the topical application of orange peel cold-pressed oil (CPO), a rich polymethoxyflavones (PMFs) source, on ultraviolet B (UVB)-induced skin injury were studied. Depilated KM mice were irradiated by UVB at one minimal erythemal dose every day and the treated groups were smeared with CPO before radiation. After two weeks we found that CPO helped to maintain the dermal structure and enhanced serum levels and skin tissue expression of superoxide dismutase, catalase and glutathione peroxidase. In serum CPO lowered interleukin-6/-1β and tumor necrosis factor α levels; in skin it decreased the levels of type III collagen and H2O2 but increased hydroxyproline and type I collagen. Moreover, the skin expression of matrix metalloproteinases 2/9 was downregulated, whereas the expression of tissue inhibitor of metalloproteinases 1/2 was upregulated. The above effects followed a dose-dependent manner. Liquid chromatography analysis of irradiated skin tissue showed partial decomposition of PMFs into 5-hydroxy-PMFs.

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.

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.

Understanding the fundamental nature of a molecular process or a biological pathway is often a catalyst for the development of new technologies in biology. Indeed, studies from late 1990s to early 2000s have uncovered multiple overlapping but functionally distinct RNA silencing pathways in plants, including the posttranscriptional microRNA and small interfering RNA pathways and the transcriptional RNA-directed DNA methylation pathway. These findings have in turn been exploited for developing artificial RNA silencing technologies such as hairpin RNA, artificial microRNA, intrinsic direct repeat, 3' UTR inverted repeat, artificial trans-acting siRNA, and virus-induced gene silencing technologies. Some of these RNA silencing technologies, such as the hairpin RNA technology, have already been widely used for genetic improvement of crop plants in agriculture. For horticultural plants, RNA silencing technologies have been used to increase disease and pest resistance, alter plant architecture and flowering time, improve commercial traits of fruits and flowers, enhance nutritional values, remove toxic compounds and allergens, and develop high-value industrial products. In this article we aim to provide an overview of the RNA silencing pathways in plants, summarize the existing RNA silencing technologies, and review the current progress in applying these technologies for the improvement of agricultural crops particularly horticultural crops.