Genome-wide identification and expression analysis of the Eriobotrya japonica TIFY gene family reveals its functional diversity under abiotic stress conditions.

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.

Warm temperature during floral bud transition turns off EjTFL1 gene expression and promotes flowering in Loquat (Eriobotrya japonica Lindl.).

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.

Sorbitol induces flower bud formation via the MADS-box transcription factor EjCAL in loquat.

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 ß-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.

Genome-Wide Identification and Expression Analysis of the SBP-Box Gene Family in Loquat Fruit Development.

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.

Dynamic Changes of Phenolic Composition, Antioxidant Capacity, and Gene Expression in 'Snow White' Loquat (Eriobotrya japonica Lindl.) Fruit throughout Development and Ripening.

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.

CaCl2 mitigates chilling injury in loquat fruit via the CAMTA5-mediated transcriptional repression of membrane lipid degradation genes.

Loquat fruit is vulnerable to chilling injury (CI) under long-term low temperature storage. The application of calciumchloride (CaCl2) can alleviate CI in loquat fruit, however, the molecular mechanisms remain unclear. In this study, the loquat fruit were immersed in 1 % CaCl2 solution for 10 min and then stored at 1 ± 1 ℃ for 35 d. The results showed that CaCl2 treatment suppressed the increases in internal browning index, electrolyte leakage, and malonaldehyde content in loquat fruit under chilling stress. The internal browning index in CaCl2-treated loquat fruit was 33.34 % lower than that in the control at the end of storage. Meanwhile, CaCl2 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 CaCl2 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 CaCl2 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 CaCl2 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.

Genome-Wide Analysis of SAUR Gene Family Identifies a Candidate Associated with Fruit Size in Loquat (Eriobotrya japonica Lindl.).

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.

Photosynthetic Efficiency and Glyco-Metabolism Changes in Artificial Triploid Loquats Contribute to Heterosis Manifestation.

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.

Genetic diversity of loquat (Eriobotrya japonica) revealed using RAD-Seq SNP markers.

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.

An EjbHLH14-EjHB1-EjPRX12 module is involved in methyl jasmonate alleviation of chilling-induced lignin deposition in loquat fruit.

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.

Chloroplast genome sequencing based on genome skimming for identification of Eriobotryae Folium.

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.

Physiological and transcriptomics analysis of the effect of recombinant serine protease on the preservation of loquat.

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.

A WRKY Transcription Factor, EjWRKY17, from Eriobotrya japonica Enhances Drought Tolerance in Transgenic Arabidopsis.

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.

Polyploidy underlies co-option and diversification of biosynthetic triterpene pathways in the apple tribe.

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.

Comparative transcriptome analysis reveals key genes potentially related to organic acid and sugar accumulation in loquat.

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.

Loquat (Eriobotrya japonica (Thunb.) Lindl) population genomics suggests a two-staged domestication and identifies genes showing convergence/parallel selective sweeps with apple or peach.

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.

Comparative transcriptome analysis of flower bud transition and functional characterization of EjAGL17 involved in regulating floral initiation in loquat.

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.

Dissection of the incidence and severity of purple spot physiological disorder in loquat fruit through a physiological and molecular approach.

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.

Single-molecule real-time sequencing of the full-length transcriptome of loquat under low-temperature stress.

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.

An Integrative Analysis of Transcriptome, Proteome and Hormones Reveals Key Differentially Expressed Genes and Metabolic Pathways Involved in Flower Development in Loquat.

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.