Proteome analysis provides new insight into major proteins involved in gibberellin-induced fruit setting in triploid loquat (Eriobotrya japonica).

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.

Homeotic transformation from stamen to petal in Eriobotrya japonica is associated with hormone signal transduction and reduction of the transcriptional activity of EjAG.

Double-flower loquat (Eriobotrya japonica) is a new germplasm with homeotic transformation of stamen into petal in whorl 3. However, little information is available on the molecular mechanism of this transformation. Herein, we analyzed the transcriptome, candidate genes and endogenous hormones to investigate the mechanisms underlying this homeotic transformation. Some transcription factors, such as MADS-box, TCP and MYB, were significantly differentially expressed. Importantly, we confirmed that one of these (DN39625_c0_g1), which encoded a C-class floral homeotic protein referred to as AGAMOUS ortholog (EjAG), was significantly downregulated. Subcellular localization of EjAG was found to be in the nucleus. Ectopic expression of EjAG rescued the development of stamens and carpels from the double-flower phenotype in an Arabidopsis ag mutant, suggesting that EjAG expression is associated with double-flower formation. Meanwhile, enrichment analyses showed that the differentially expressed genes (DEGs) were mainly involved in the metabolic pathways of hormone signal transduction. The DEGs of auxin, gibberellin A (GA) and cytokinin signaling pathways were mainly upregulated. However, the DEGs of abscisic acid (ABA) and the ethylene signaling pathway were mainly downregulated. Accordingly, the concentrations of indoleacetic acid, kinetin and GA3 were high at the petaloid stamen stage, but the ABA concentration remained low. The identified genes and pathways provide abundant sequence resources for studying the mechanisms underlying the homeotic transformation in loquat and other Rosaceae species.

The Role of EjSVPs in Flower Initiation in Eriobotrya japonica.

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.

Functional characterization of GI and CO homologs from Eriobotrya deflexa Nakai forma koshunensis.

KEY MESSAGE: The first report of the cloning and characterization of the flowering time-regulating genes GI and CO homologs from loquat. Flowering time is critical for successful reproduction in plants. In fruit trees, it can also influence the fruit yield and quality. In the previous work, we cloned the important florigen one EdFT and two EdFDs from wild loquat (Eriobotrya deflexa Nakai forma koshunensis); however, the upstream transcription factors are still unknown. The photoperiod pathway genes GIGANTEA (GI) and CONSTANS (CO) have been reported to mainly regulate FT expression in model plants. In this work, we first cloned photoperiod pathway orthologs EdGI and EdCO from E. deflexa Nakai f. koshunensis. Phylogenetic analysis showed they are highly conserved to those from Arabidopsis. They are mainly expressed in the leaves. The EdGI and EdCO were localized in the nucleus. Their expression showed in photoperiodic regulation, while the EdCO transcripts reached the peak at different periods from that of CO in Arabidopsis. Moreover, EdCO significantly activated the EdFT promoter activity. In the transgenic Arabidopsis, downstream-flowering genes like FT and AP1 were obviously upregulated, and consequently resulted in early-flowering phenotype compared to the wild type. These data revealed that the EdGI and EdCO may play a similar role as GI and CO in Arabidopsis, and regulate flower initiation in loquat.

EjNAC3 transcriptionally regulates chilling-induced lignification of loquat fruit via physical interaction with an atypical CAD-like gene.

Lignin is an important component of many plant secondary cell walls. In the fruit of loquat (Eriobotrya japonica), lignification of cell walls in the fleshy tissue occurs when fruit are subjected to low-temperature storage, which is commonly used to avoid the rapid senescence that occurs at room temperature. In this study, two NAC domain genes, EjNAC3 and EjNAC4, were isolated and shown to be significantly induced at 0 °C, which was concomitant with an increase in the fruit lignification index. Lignification and expression of both EjNAC3 and EjNAC4 were inhibited by low-temperature conditioning and by heat treatment. In addition, EjNAC3 trans-activated the lignin biosynthesis-related EjCAD-like promoter, which was measured using a dual-luciferase assay. Further analysis with yeast one-hybrid and electrophoretic mobility shift assays indicated that EjNAC3 could physically bind to the promoter of the EjCAD-like gene. Thus, EjNAC3 is a direct regulator of loquat chilling-induced lignification, via regulations of EjCAD-like.

Bud sprouting and floral induction and expression of FT in loquat [Eriobotrya japonica (Thunb.) Lindl.].

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.

Adaptive biochemical and physiological responses of Eriobotrya japonica to fluoride air pollution.

The biochemical and physiological effects of fluoride were investigated in loquat trees (Eriobotrya japonica) grown in the vicinity of a phosphate fertilizer plant in Tunisia. Photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (E), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were assessed; along with photosynthetic pigments, lipid peroxidation, electrolytic leakage (EL) and total phenolic contents in foliage and roots of trees at different distances from the phosphate fertilizer plant. All assessed parameters showed significant discrepancies in comparison with unpolluted sites. Obtained results showed high oxidative stress indices including H2O2, lipid peroxidation, and EL, SOD, CAT and GPx activities and proline contents in leaves and roots at the polluted sites as compared to control. In contrast, leaf Pn, Gs, E and photosynthetic pigment contents were low as compared to the control. These results indicate that even though antioxidant responses increased near the factory, adverse effects on physiology were pronounced.

Comparative transcriptome profiling of freezing stress responses in loquat (Eriobotrya japonica) fruitlets.

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.

Loquat fruit ripening is associated with root depletion. Nutritional and hormonal control.

In woody species, it is known that there is a competition for nutrients, water and carbohydrates between root and fruit-shoot systems, however the influence of root development on fruit quality has received little attention. This research aims to identify the network of mechanisms involved in loquat (Eriobotrya japonica Lindl.) fruit ripening in connection with root activity. The study includes root growth rate measurements paralleling the ongoing fruit developmental stages, photosynthate translocation to the root by using (13)CO2 tracing, and nitrogen fractions (N-NH4(+), N-NO3(-), and N-proteinaceous) as well as their upward translocation to the fruit. The role of hormones (IAA, zeatin and ABA) in regulating the responses is also addressed. The experiment was conducted during two consecutive years on adult and 3-year-old loquat trees from early fruit developmental stage (10% of final size, 701 BBCH scale) to fully developed fruit colour (809 BBCH scale). This approach revealed that root development depends on the growing fruit sink strength, which reduces carbohydrates translocation to the roots and prevents them for further elongation. A nitrate accumulation in roots during the active fruit growth period takes place, which also contributes to slowing elongation and paralleled reduced ammonium and proteinaceous nitrogen concentrations. Concomitantly, the concentration of IAA and zeatin were lowest while that of ABA was highest when root exhibited minimum elongation. The depletion in zeatin and nitrogen supply by the roots paralleling the high ABA transport to the fruit allowed for colour break. These results suggest that loquat fruit changes colour by reducing root growth, as fruit increases sugars and ABA concentrations and reduces nitrogen and zeatin concentrations.

Molecular evidence for natural hybridization between wild loquat (Eriobotrya japonica) and its relative E. prinoides.

BACKGROUND: Interspecific hybridization has long been recognized as a pivotal process in plant evolution and speciation. It occurs fairly common in the genera of the subtribe Pyrinae. In Eriobotrya, a small tree genus of Pyrinae, E. prinoides var. daduheensis has been recognized as either a variety of E. prinoides, a natural hybrid between E. prinoides and E. japonica, or a variety of E. japonica. However, to date, there has been no convincing evidence on its status. RESULTS: Four nuclear genes and two chloroplast regions were sequenced in 89 individuals of these three Eriobotrya taxa from two locations where they coexist. A few fixed nucleotide substitutions or gaps were found in each of the investigated nuclear and chloroplast loci between E. japonica and E. prinoides. Of the 35 individuals of E. prinoides var. daduheensis, 33 showed nucleotide additivity of E. japonica and E. prinoides in at least one nuclear gene, and 10 of them harboured nucleotide additivity at all the four nuclear genes. Most haplotypes of E. prinoides var. daduheensis were also shared with those of E. japonica and E. prinoides. In the two chloroplast regions, 28 and 7 individuals were identical with E. japonica and E. prinoides, respectively. CONCLUSIONS: Our study provides compelling evidence for a hybrid status for E. prinoides var. daduheensis. Most hybrid individuals are later-generation hybrids. Both E. japonica and E. prinoides can serve as female parent. Differential adaptation might maintain the species boundary of E. prinoides and E. japonica in the face of hybridization and potential introgression.

Involvement of multiple types of dehydrins in the freezing response in loquat (Eriobotrya japonica).

Dehydrins (DHNs) are a family of plant proteins typically induced in response to stress conditions that cause cellular dehydration, such as low temperatures, high salinity, and drought. Loquat (Eriobotrya japonica) is a perennial fruit crop that blossoms during winter. Loquat fruitlets are frequently injured by freezing. To evaluate the role of the EjDHNs in freezing resistance in loquat fruitlets, two cultivars of loquat, the freezing-sensitive 'Ninghaibai' (FS-NHB) and the freezing-tolerant 'Jiajiao' (FT-JJ), were analyzed under induced freezing stress. Freezing stress led to obvious accumulation of reactive oxygen species and considerable lipid peroxidation in membranes during the treatment period. Both these phenomena were more pronounced in 'FS-NHB' than in 'FS-JJ.' Immunogold labeling of dehydrin protein was performed. DHN proteins were found to be concentrated mainly in the vicinity of the plasma membrane, and the density of the immunogold labeling was significantly higher after freezing treatment, especially in the more freezing-tolerant cultivar 'FT-JJ.' Seven DHNs, showing four different structure types, were obtained from loquat fruitlets and used to study the characteristics of different EjDHN proteins. These DHN proteins are all highly hydrophilic, but they differ significantly in size, ranging from 188 to 475 amino acids, and in biochemical properties, such as theoretical pI, aliphatic index, and instability index. Freezing treatment resulted in up-regulation of the expression levels of all seven EjDHNs, regardless of structure type. The accumulation of the transcripts of these EjDHN genes was much more pronounced in 'FT-JJ' than in 'FS-NHB.' Altogether, this study provides evidence that EjDHNs are involved in the cryoprotection of the plasma membrane during freeze-induced dehydration in loquat fruitlets.

Postharvest physiology and technology of loquat (Eriobotrya japonica Lindl.) fruit.

Loquat (Eriobotrya japonica Lindl.) is a subtropical evergreen tree whose fruit is consumed both fresh and processed. Loquat fruit is a good source of minerals and carotenoids, while the kernel is rich in protein and carbohydrates. It has been considered a non-climacteric fruit, but there is evidence that some cultivars have a ripening pattern similar to that of climacteric fruits. The fruit has a short postharvest life at ambient temperatures and is susceptible to physical and mechanical damage, loss of moisture and nutrients, and decay. Low-temperature storage extends the shelf life of loquat fruit, but some cultivars are severely affected by chilling injury and flesh browning during cold storage. Purple spot, browning and leatheriness are major postharvest disorders. The shelf life of loquat can be extended by modified or controlled atmosphere storage as well as by postharvest treatment with 1-methyl cyclopropene or methyl jasmonate.

Generalist passerine pollination of a winter-flowering fruit tree in central China.

BACKGROUND AND AIMS: Winter-flowering plants outside the tropics may experience a shortage of pollinator service, given that insect activity is largely limited by low temperature. Birds can be alternative pollinators for these plants, but experimental evidence for the pollination role of birds in winter-flowering plants is scarce. METHODS: Pollinator visitation to the loquat, Eriobotrya japonica (Rosaceae), was observed across the flowering season from November to January for two years in central China. Self- and cross-hand pollination was conducted in the field to investigate self-compatibility and pollen limitation. In addition, inflorescences were covered by bird cages and nylon mesh nets to exclude birds and all animal pollinators, respectively, to investigate the pollination role of birds in seed production. RESULTS: Self-fertilization in the loquat yielded few seeds. In early winter insect visit frequency was relatively higher, while in late winter insect pollinators were absent and two passerine birds (Pycnonotus sinensis and Zosterops japonicus) became the major floral visitors. However, seed-set of open-pollinated flowers did not differ between early and late winter. Exclusion of bird visitation greatly reduced seed-set, indicating that passerine birds were important pollinators for the loquat in late winter. The whitish perigynous flowers reward passerines with relatively large volumes of dilute nectar. Our observation on the loquat and other Rosaceae species suggested that perigyny might be related to bird pollination but the association needs further study. CONCLUSIONS: These findings suggest that floral traits and phenology would be favoured to attract bird pollinators in cold weather, in which insect activity is limited.

Neuroprotective effects of Eriobotrya japonica against ß-amyloid-induced oxidative stress and memory impairment.

The generation of oxygen free radicals and oxidative damage is believed to be involved in the pathogenesis of neurodegenerative disorders. Eriobotrya japonica has been used to treat several diseases in East Asia. In this study, we investigated the protective effect of an E. japonica extract against Aß peptide-induced oxidative stress. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay demonstrated that the E. japonica extract scavenged approximately 40% of DPPH radicals. Also, treatment of the E. japonica extract inhibited Aß(1-42)-mediated neuronal cell death. Furthermore, treatment of E. japonica extract efficiently suppressed the increase in intracellular ROS triggered by the Aß(1-42) peptide. Importantly, mice pre-treated with the E. japonica extract showed restoration of alternation behavior and reversal of Aß(1-42)-induced memory impairment. Consequently, the E. japonica extract substantially inhibited the increase in lipid peroxidation and restored superoxide dismutase activity. These results suggest that E. japonica protects from oxidative stress and cognitive deficits induced by the Aß peptide.

Ethylene signal transduction elements involved in chilling injury in non-climacteric loquat fruit.

Loquat (Eriobotrya japonica Lindl.) is a subtropical fruit, with some cultivars such as 'Luoyangqing' (LYQ) susceptible to chilling injury (CI), while others such as 'Baisha' (BS) are resistant. Although loquats are non-climacteric, modulation of ethylene has an effect on ripening-related post-harvest CI. Therefore the role of ethylene signalling in the development of CI was investigated in fruit of both the LYQ and BS cultivars. Three ethylene receptor genes, one CTR1-like gene, and one EIN3-like gene were isolated and characterized in ripening fruit. All of these genes were expressed differentially within and between fruit of the two cultivars. Transcripts either declined over fruit development (EjERS1a in both cultivars and EjEIL1 in LYQ) or showed an increase in the middle stages of fruit development before declining (EjETR1, EjERS1b, and EjCTR1 in both cultivars and EjEIL1 in BS). The main cultivar differences were in levels rather than in patterns of expression during post-harvest storage. EjETR1, EjCTR1, and EjEIL1 genes showed increased expression in response to low temperature and this was particularly notable for EjETR1, and EjEIL1 during CI development in LYQ fruit. The genes were also differentially responsive to ethylene treatment, 1-methycyclopropene (1-MCP) and low temperature conditioning, confirming a role for ethylene in regulation of CI in loquat fruit.

Effects of farmyard manure and chemical fertilizers on the nutritional status of the loquat trees.

The nutritional status of the loquat trees was investigated using cattle manure and commercial fertilizers for three years. The farmyard manure increased N, P, K, Mg, Fe and Zn contents of the leaves. No significant difference was found between the fertilizer types for trunk growth. Yield efficiency was nearly doubled by application of farmyard manure. Fertilizers did not affect the weight and shape of the fruits; however, commercial fertilizers led the lower total acidity in fruits. It was concluded that the loquat trees grown in sandy soils could fulfill their principal nutrient requirements for growth and commercial yield with application of farmyard manure.