The Role of MaWRKY70 in Regulating Lipoxygenase Gene Transcription during Chilling Injury Development in Banana Fruit.

Banana is a typical cold-sensitive fruit; it is prone to chilling injury (CI), resulting in a quality deterioration and commodity reduction. However, the molecular mechanism underlying CI development is unclear. In this study, cold storage (7 °C for 5 days) was used to induce CI symptoms in bananas. As compared with the control storage (22 °C for 5 days), cold storage increased the CI index and cell membrane permeability. Moreover, we found that the expression levels of the WRKY transcription factor MaWRKY70 were increased consistently with the progression of CI development. A subcellular localization assay revealed that MaWRKY70 was localized in the nucleus. Transcriptional activation analyses showed that MaWRKY70 processed a transactivation ability. Further, an electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter (DLR) assays showed that MaWRKY70 was directly bound to the W-box motifs in the promoters of four lipoxygenase (LOX) genes associated with membrane lipid degradation and activated their transcription. Collectively, these findings demonstrate that MaWRKY70 activates the transcription of MaLOXs, thereby acting as a possible positive modulator of postharvest CI development in banana fruit.

Tanshinone IIA Alleviates Traumatic Brain Injury by Reducing Ischemia‒Reperfusion via the miR-124-5p/FoxO1 Axis.

Background: Cerebral ischemia-reperfusion injury is a common complication of ischemic stroke that affects the prognosis of patients with ischemic stroke. The lipid-soluble diterpene Tanshinone IIA, which was isolated from Salvia miltiorrhiza, has been indicated to reduce cerebral ischemic injury. In this study, we investigated the molecular mechanism of Tanshinone IIA in alleviating reperfusion-induced brain injury. Methods: Middle cerebral artery occlusion animal models were established, and neurological scores, tetrazolium chloride staining, brain volume quantification, wet and dry brain water content measurement, Nissl staining, enzyme-linked immunosorbent assay, flow cytometry, western blotting, and reverse transcription-quantitative polymerase chain reaction were performed. The viability of cells was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assays, while cell damage was measured by lactate dehydrogenase release in the in vitro oxygen glucose deprivation model. In addition, enzyme-linked immunosorbent assay, flow cytometry, western blotting, and reverse transcription-quantitative polymerase chain reaction were used to evaluate the therapeutic effect of Tanshinone IIA on ischemia/reperfusion (I/R) induced brain injury, as well as its effects on the inflammatory response and neuronal apoptosis, in vivo and in vitro. Furthermore, this study validated the targeting relationship between miR-124-5p and FoxO1 using a dual luciferase assay. Finally, we examined the role of Tanshinone IIA in brain injury from a molecular perspective by inhibiting miR-124-5p or increasing FoxO1 levels. Results: After treatment with Tanshinone IIA in middle cerebral artery occlusion-reperfusion (MCAO/R) rats, the volume of cerebral infarction was reduced, the water content of the brain was decreased, the nerve function of the rats was significantly improved, and the cell damage was significantly reduced. In addition, Tanshinone IIA effectively inhibited the I/R-induced inflammatory response and neuronal apoptosis, that is, it inhibited the expression of inflammatory cytokines IL-1ß, IL-6, TNF-α, decreased the expression of apoptotic protein Bax and Cleaved-caspase-3, and promoted the expression of antiapoptotic protein Bcl-2. In vitro oxygen-glucose deprivation/reoxygenation (OGD/R) cell model, Tanshinone IIA also inhibited the expression of inflammatory factors in neuronal cells and inhibited the occurrence of neuronal apoptosis. In addition, Tanshinone IIA promoted the expression of miR-124-5p. Transfection of miR-124-5p mimic has the same therapeutic effect as Tanshinone IIA and positive therapeutic effect on OGD cells, while transfection of miR-124-5p inhibitor has the opposite effect. The targeting of miR-124-5p negatively regulates FoxO1 expression. Inhibition of miR-124-5p or overexpression of FoxO1 can weaken the inhibitory effect of Tanshinone IIA on brain injury induced by I/R, while inhibition of miR-124-5p and overexpression of FoxO1 can further weaken the effect of Tanshinone IIA. Conclusion: Tanshinone IIA alleviates ischemic-reperfusion brain injury by inhibiting neuroinflammation through the miR-124-5p/FoxO1 axis. This finding provides a theoretical basis for mechanistic research on cerebral ischemia-reperfusion injury.

miR-497-5p promoted neuronal injury in ischemic stroke by inhibiting the BDNF/TrkB/PI3K/Akt pathway.

The aim of this study was to investigate the molecular mechanism by which miR-497-5p regulates neuronal injury after ischemic stroke through the BDNF/TrkB/Akt signaling pathway. PC12 cells were used to construct a stroke injury model by oxygen-glucose deprivation/reoxygenation (OGD/R). The expression level of miR-497-5p was measured by RT-qPCR. CCK-8 kit was used to detect cell viability. Cell apoptosis and reactive oxygen species (ROS) were detected by flow cytometry. MDA and SOD detection kits were used to detect MDA content and SOD activity. A double luciferase reporter system was used to verify the targeting relationship between miR-497-5p and BDNF. The expression of BDNF, TrkB, p-TrkB, Akt and p-Akt was detected by Western blot. We have found that miR-497-5p expression was inhibited after treatment with OGD/R. Simultaneously, cell apoptosis, MDA content and ROS were upregulated, while cell viability and SOD were significantly decreased in PC12 cells. The effects of OGD/R on PC12 cells were reversed with the downregulation of miR-497-5p. A double luciferase reporter assay demonstrated that miR-497-5p negatively targets BDNF. BDNF inhibited cell apoptosis and oxidative stress injury in PC12 cells. These findings suggest that miR-497-5p aggravates neuronal injury in experimental model of ischemic stroke by inhibiting the BDNF/TrkB/PI3K/Akt signaling pathway.

Comprehensive metabolome and transcriptome analyses demonstrate divergent anthocyanin and carotenoid accumulation in fruits of wild and cultivated loquats.

Eriobotrya is an evergreen fruit tree native to South-West China and adjacent countries. There are more than 26 loquat species known in this genus, while E. japonica is the only species yet domesticated to produce fresh fruits from late spring to early summer. Fruits of cultivated loquat are usually orange colored, in contrast to the red color of fruits of wild E. henryi (EH). However, the mechanisms of fruit pigment formation during loquat evolution are yet to be elucidated. To understand these, targeted carotenoid and anthocyanin metabolomics as well as transcriptomics analyses were carried out in this study. The results showed that ß-carotene, violaxanthin palmitate and rubixanthin laurate, totally accounted for over 60% of the colored carotenoids, were the major carotenoids in peel of the orange colored 'Jiefangzhong' (JFZ) fruits. Total carotenoids content in JFZ is about 10 times to that of EH, and the expression levels of PSY, ZDS and ZEP in JFZ were 10.69 to 23.26 folds to that in EH at ripen stage. Cyanidin-3-O-galactoside and pelargonidin-3-O-galactoside were the predominant anthocyanins enriched in EH peel. On the contrary, both of them were almost undetectable in JFZ, and the transcript levels of F3H, F3'H, ANS, CHS and CHI in EH were 4.39 to 73.12 folds higher than that in JFZ during fruit pigmentation. In summary, abundant carotenoid deposition in JFZ peel is well correlated with the strong expression of PSY, ZDS and ZEP, while the accumulation of anthocyanin metabolites in EH peel is tightly associated with the notably upregulated expressions of F3H, F3'H, ANS, CHS and CHI. This study was the first to demonstrate the metabolic background of how fruit pigmentations evolved from wild to cultivated loquat species, and provided gene targets for further breeding of more colorful loquat fruits via manipulation of carotenoids and anthocyanin biosynthesis.

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.

Integration of genomics, transcriptomics and metabolomics identifies candidate loci underlying fruit weight in loquat.

Fruit weight is an integral part of fruit-quality traits and directly influences commodity values and economic returns of fruit crops. Despite its importance, the molecular mechanisms underlying fruit weight remain understudied, especially for perennial fruit tree crops such as cultivated loquat (Eriobotrya japonica Lindl.). Auxin is known to regulate fruit development, whereas its role and metabolism in fruit development remain obscure in loquat. In this study, we applied a multi-omics approach, integrating whole-genome resequencing-based quantitative trait locus (QTL) mapping with an F1 population, population genomics analysis using germplasm accessions, transcriptome analysis, and metabolic profiling to identify the genomic regions potentially associated with fruit weight in loquat. We identified three major loci associated with fruit weight, supported by both QTL mapping and comparative genomic analysis between small- and big-fruited loquat cultivars. Comparison between two genotypes with contrasting fruit weight performance through transcriptomic and metabolic profiling revealed an important role of auxin in regulating fruit development, especially at the fruit enlarging stage. The multi-omics approach identified two homologs of ETHYLENE INSENSITIVE 4 (EjEIN4) and TORNADO 1 (EjTRN1) as promising candidates controlling fruit weight. Moreover, three single nucleotide polymorphism (SNP) markers were closely associated with fruit weight. Results from this study provided insights from multiple perspectives into the genetic and metabolic controls of fruit weight in loquat. The candidate genomic regions, genes, and sequence variants will facilitate understanding the molecular basis of fruit weight and lay a foundation for future breeding and manipulation of fruit weight in loquat.

Gibberellin Induced Transcriptome Profiles Reveal Gene Regulation of Loquat Flowering.

Flowering is an integral part of the life cycle of flowering plants, which is essential for plant survival and crop production. Most woody fruit trees such as apples and pears bloom in spring, but loquat blooms in autumn and winter. Gibberellin (GA) plays a key role in the regulation of plant flower formation. In this study, we sprayed loquat plants with exogenous GA3, which resulted in vigorous vegetative growth rather than floral bud formation. We then performed a comprehensive RNA-seq analysis on GA3-treated and control-treated leaves and buds over three time periods to observe the effects of exogenous GA3 application on floral initiation and development. The results showed that 111 differentially expressed genes (DEGs) and 563 DEGs were down-regulated, and 151 DEGs and 506 DEGs were up-regulated in buds and leaves, respectively, upon treatment with GA3. Among those that are homologs of the DELLA-mediated GA signal pathway genes, some may be involved in the positive regulation of flower development, including EjWRKY75, EjFT, EjSOC1, EjAGL24, EjSPL, EjLFY, EjFUL, and EjAP1; while some may be involved in the negative regulation of flower development, including EjDELLA, EjMYC3, EjWRKY12, and EjWRKY13. Finally, by analyzing the co-expression of DEGs and key floral genes EjSOC1s, EjLFYs, EjFULs, EjAP1s, 330 candidate genes that may be involved in the regulation of loquat flowering were screened. These genes belong to 74 gene families, including Cyclin_C, Histone, Kinesin, Lipase_GDSL, MYB, P450, Pkinase, Tubulin, and ZF-HD_dimer gene families. These findings provide new insights into the regulation mechanism of loquat flowering.

EjBZR1 represses fruit enlargement by binding to the EjCYP90 promoter in loquat.

Loquat (Eriobotrya japonica) is a subtropical tree that bears fruit that ripens during late spring. Fruit size is one of the dominant factors inhibiting the large-scale production of this fruit crop. To date, little is known about fruit size regulation. In this study, we first discovered that cell size is more important to fruit size than cell number in loquat and that the expression of the EjBZR1 gene is negatively correlated with cell and fruit size. Virus-induced gene silencing (VIGS) of EjBZR1 led to larger cells and fruits in loquat, while its overexpression reduced cell and plant size in Arabidopsis. Moreover, both the suppression and overexpression of EjBZR1 inhibited the expression of brassinosteroid (BR) biosynthesis genes, especially that of EjCYP90A. Further experiments indicated that EjCYP90A, a cytochrome P450 gene, is a fruit growth activator, while EjBZR1 binds to the BRRE (CGTGTG) motif of the EjCYP90A promoter to repress its expression and fruit cell enlargement. Overall, our results demonstrate a possible pathway by which EjBZR1 directly targets EjCYP90A and thereby affects BR biosynthesis, which influences cell expansion and, consequently, fruit size. These findings help to elucidate the molecular functions of BZR1 in fruit growth and thus highlight a useful genetic improvement that can lead to increased crop yields by repressing gene expression.

Ectopic expression of Torenia fournieri TCP8 and TCP13 alters the leaf and petal phenotypes in Arabidopsis thaliana.

Teosinte branched1/cycloidea/proliferating cell factor (TCP) transcription factors (TFs) are essential for regulating plant developmental processes, which is still largely unknown in Torenia fournieri (T. fournieri), a widely used horticultural flower. In this study, we used a de novo transcriptome assembly method to predict the TCP transcription factors in T. fournieri. In total, 15 out of 21 predicted T. fournieri TCPs (TfTCPs) were isolated and verified with Sanger sequencing. Phylogenetic analysis showed that these 15 TfTCPs could be classified into two major classes. Most of these TfTCPs were expressed in floral buds, flowers, or leaves, suggesting an important role in developmental regulation in these tissues. Moreover, TfTCP8 and TfTCP13, the homologues of the Arabidopsis thaliana TCP5-like transcription factor, were able to bind to the conserved Class II TCP binding motifs and are localized to the nucleus, indicating that TfTCP8 and TfTCP13 act as transcriptional regulators. In agreement with the overexpression phenotype of AtTCP5, ectopic expression of TfTCP8 and TfTCP13 resulted in narrow leaves and the small petal phenotype in Arabidopsis, suggesting that these two TfTCPs potentially regulate leaf or flower shape in T. fournieri.

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.

Method for fast staining and obtaining high-magnification and high-resolution cell images of Nicotiana benthamiana.

As tools of plant molecular biology, fluorescence microscopy and Nicotiana benthamiana have been used frequently to study the structure and function of plant cells. However, it is difficult to obtain ideal micrographs; for example, the images are typically unclear, the inner cell structure cannot be observed under a high-power lens by fluorescence microscopy, etc. Here, we describe a method for observing the cell structure of N. benthamiana. This method significantly improves imaging by fluorescence microscopy and allows clear images to be obtained under a high-power lens. This method is easy to perform with good stability, and the stomatal structure, nucleus, nucleolus, chloroplast and other organelles in N. benthamiana cells as well as protein localizations and the locations of protein-protein interactions have been observed clearly. Furthermore, compared with traditional methods, fluorescent dye more efficiently dyes cells with this method. The applicability of this method was verified by performing confocal scanning laser microscopy (CSLM), and CSLM imaging was greatly improved. Thus, our results provided a method to visualize the subcellular structures of live cells in the leaves of N. benthamiana by greatly improving imaging under a fluorescence microscope and provided new insights and references for the study of cell structures and functions in other plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00931-5.

EjRAV1/2 Delay Flowering Through Transcriptional Repression of EjFTs and EjSOC1s in Loquat.

Most species in Rosaceae usually need to undergo several years of juvenile phase before the initiation of flowering. After 4-6 years' juvenile phase, cultivated loquat (Eriobotrya japonica), a species in Rosaceae, enters the reproductive phase, blooms in the autumn and sets fruits during the winter. However, the mechanisms of the transition from a seedling to an adult tree remain obscure in loquat. The regulation networks controlling seasonal flowering are also largely unknown. Here, we report two RELATED TO ABI3 AND VP1 (RAV) homologs controlling juvenility and seasonal flowering in loquat. The expressions of EjRAV1/2 were relatively high during the juvenile or vegetative phase and low at the adult or reproductive phase. Overexpression of the two EjRAVs in Arabidopsis prolonged (about threefold) the juvenile period by repressing the expressions of flowering activator genes. Additionally, the transformed plants produced more lateral branches than the wild type plants. Molecular assays revealed that the nucleus localized EjRAVs could bind to the CAACA motif of the promoters of flower signal integrators, EjFT1/2, to repress their expression levels. These findings suggest that EjRAVs play critical roles in maintaining juvenility and repressing flower initiation in the early life cycle of loquat as well as in regulating seasonal flowering. Results from this study not only shed light on the control and maintenance of the juvenile phase, but also provided potential targets for manipulation of flowering time and accelerated breeding in loquat.

Sodium valproate combined with levetiracetam in pediatric epilepsy and its influence on NSE, IL-6, hs-CRP and electroencephalogram improvement.

Efficacy of sodium valproate combined with levetiracetam (LEV) in pediatric epilepsy and its influence on neuron-specific enolase (NSE), interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hs-CRP) as well as electroencephalogram (EEG) improvement were studied. Patients (n=100) with pediatric epilepsy admitted to and treated in Xiantao First People's Hospital Affiliated to Yangtze University from December 2015 to 2018 were enrolled in this study and randomly divided into observation group (n=50) and control group (n=50). Sodium valproate was administered in the control group, and the treatment with LEV was combined with sodium valproate in the observation group. After 12 weeks the cognitive function of patients was assessed using the Mini-Mental State Examination (MMSE) scale, Montreal cognitive assessment (MoCA) scale and Wechsler Memory Scale-Revised in China (WMS-RC). The quality of life (QOL) of patients was evaluated with the QOL in epilepsy-31 inventory (QOLIE-31) scale and Barthel Index, and blood was drawn from the patients to detect the neurological function indicators [NSE and glial fibrillary acidic protein (GFAP)] and inflammatory indicators (IL-6, IL-2 and hs-CRP). After treatment, the incidence rates of adverse reactions notably declined in the observation group (P<0.05), and the improvement in the cognitive function in the observation group were both superior to those in the control group (P<0.05). Observation group had lowered content of NSE, GFAP, IL-6, hs-CRP and IL-2 (P<0.05), and α wave was markedly decreased, but θ and δ waves were notably increased in the observation group (P<0.05). In the treatment of pediatric epilepsy, sodium valproate combined with LEV produces better efficacy, fewer adverse reactions, significantly improves patients' QOL and notably lowers the content of NSE, IL-6 and hs-CRP with notable EEG improvement, so it is a safe and reliable treatment that is worth popularization.

EjTFL1 Genes Promote Growth but Inhibit Flower Bud Differentiation in Loquat.

TERMINAL FLOWER1 (TFL1), a key factor belonging to the phosphatidyl ethanolamine-binding protein (PEBP) family, controls flowering time and inflorescence architecture in some plants. However, the role of TFL1 in loquat remains unknown. In this study, we cloned two TFL1-like genes (EjTFL1-1 and EjTFL1-2) with conserved deduced amino acid sequences from cultivated loquat (Eriobotrya japonica Lindl.). First, we determined that flower bud differentiation occurs at the end of June and early July, and then comprehensively analyzed the temporal and spatial expression patterns of these EjTFL1s during loquat growth and development. We observed the contrasting expression trends for EjTFL1s and EjAP1s (APETALA 1) in shoot apices, and EjTFL1s were mainly expressed in young tissues. In addition, short-day and exogenous GA3 treatments promoted the expression of EjTFL1s, and no flower bud differentiation was observed after these treatments in loquat. Moreover, EjTFL1s were localized to the cytoplasm and nucleus, and both interacted with another flowering transcription factor, EjFD, in the nucleus, and EjTFL1s-EjFD complex significantly repressed the promoter activity of EjAP1-1. The two EjTFL1s were overexpressed in wild-type Arabidopsis thaliana Col-0, which delayed flowering time, promoted stem elongation, increased the number of branches, and also affected flower and silique phenotypes. In conclusion, our results suggested that EjTFL1-1 and EjTFL1-2 do not show the same pattern of expression whereas both are able of inhibiting flower bud differentiation and promoting vegetative growth in loquat by integrating GA3 and photoperiod signals. These findings provide useful clues for analyzing the flowering regulatory network of loquat and provide meaningful references for flowering regulation research of other woody fruit trees.

The Role of EjSOC1s in Flower Initiation in Eriobotrya japonica.

The MADS-box transcription factor SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) integrates environmental and endogenous signals to promote flowering in Arabidopsis. However, the role of SOC1 homologs in regulating flowering time in fruit trees remains unclear. To better understand the molecular mechanism of flowering regulation in loquat (Eriobotrya japonica Lindl.), two SOC1 homologs (EjSOC1-1 and EjSOC1-2) were identified and characterized in this work. Sequence analysis showed that EjSOC1-1 and EjSOC1-2 have conserved MADS-box and K-box domains. EjSOC1-1 and EjSOC1-2 were clearly expressed in vegetative organs, and high expression was detected in flower buds. As observed in paraffin-embedded sections, expression of the downstream flowering genes EjAP1s and EjLFYs started to increase at the end of June, a time when flower bud differentiation occurs. Additionally, high expression of EjSOC1-1 and EjSOC1-2 began 10 days earlier than that of EjAP1s and EjLFYs in shoot apical meristem (SAM). EjSOC1-1 and EjSOC1-2 were inhibited by short-day (SD) conditions and exogenous GA3, and flower bud differentiation did not occur after these treatments. EjSOC1-1 and EjSOC1-2 were found to be localized to the nucleus. Moreover, ectopic overexpression of EjSOC1-1 and EjSOC1-2 in wild-type Arabidopsis promoted early flowering, and overexpression of both was able to rescue the late flowering phenotype of the soc1-2 mutant. In conclusion, the results suggest that cultivated loquat flower bud differentiation in southern China begins in late June to early July and that EjSOC1-1 and EjSOC1-2 participate in the induction of flower initiation. These findings provide new insight into the artificial regulation of flowering time in fruit trees.

Selection of the optimal reference genes for expression analyses in different materials of Eriobotrya japonica.

BACKGROUND: Loquat (Eriobotrya japonica) is a subtropical tree bearing fruit that ripens during late spring and early summer, which is the off-season for fruit production. The specific flowering habit of loquat, which starts in fall and ends in winter, has attracted an increasing number of researchers who believe that it may represent an ideal model for studying flowering shift adaptations to climate change in Rosaceae. These studies require an understanding of gene expression patterns within the fruit and other tissues of this plant. Although ACTINs (ACTs) have previously been used as reference genes (RGs) for gene expression studies in loquats, a comprehensive analysis of whether these RGs are optimal for normalizing RT-qPCR data has not been performed. RESULTS: In this study, 11 candidate RGs (RIBOSOMAL-LIKE PROTEIN4 (RPL4), RIBOSOMAL-LIKE PROTEIN18 (RPL18), Histone H3.3 (HIS3), Alpha-tubulin-3 (TUA3), S-Adenosyl Methionine Decarboxylase (SAMDC), TIP41-like Family Protein (TIP41), (UDP)-glucose Pyrophosphorylase (UGPase), 18S ribosomal RNA (18S), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), Plasma Intrinsic Protein 2 (PIP2) and ACTIN(ACT)) were assessed to determine their expression stability in 23 samples from different tissues or organs of loquat. Integrated expression stability evaluations using five computational statistical methods (GeNorm, NormFinder, ΔCt, BestKeeper, and RefFinder) suggested that a RG set, including RPL4, RPL18, HIS3 and TUA3, was the most stable one across all of the tested loquat samples. The expression pattern of EjCDKB1;2 in the tested loquat tissues normalized to the selected RG set demonstrated its reliability. CONCLUSIONS: This study reveals the reliable RGs for accurate normalization of gene expression in loquat. In addition, our findings demonstrate an efficient system for identifying the most effective RGs for different organs, which may be applied to related rosaceous crops.

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.

The Role of EjSPL3, EjSPL4, EjSPL5, and EjSPL9 in Regulating Flowering in Loquat (Eriobotrya japonica Lindl.).

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

Differential gene expression between the vigorous and dwarf litchi cultivars based on RNA-Seq transcriptome analysis.

Litchi (Litchi chinesis Sonn.) is the most economically significant member of Sapindaceae family, especially in sub-tropical regions. However, its tall tree body often brings many inconveniences to production management. In order to modify the tree size or growth for productivity optimization and simplifying management, it is urgent to reveal the dwarf mechanism of litchi for dwarfing rootstocks or cultivar breeding. However, to date, the mechanisms on litchi dwarfism is still poor known. In the present study, transcriptome profiling were performed on L. chinensis cv. 'Feizixiao' (FZX, vigorous cultivar) and 'Ziniangxi' (ZNX, dwarf cultivar). A total of 55,810 unigenes were obtained, and 9,190 unigenes were differentially expressed between vigorous and dwarf litchi samples. Gene functional enrichment analysis indicated that the differentially expressed unigenes (DEGs) were related to phytohormone metabolism and signal transduction, and energy metabolism pathways. In particular, GA2ox were only up-regulated in ZNX samples, indicating GA might play an important role in regulating huge difference between vigorous and dwarf litchi cultivars. In addition, the 35S::LcGA2ox transgenic tobacco plants were dwarf and had smaller leaves or branches than wild type plants. Our study provided a series of candidate genes to reveal the mechanism of litchi dwarf.

Two LcbHLH Transcription Factors Interacting with LcMYB1 in Regulating Late Structural Genes of Anthocyanin Biosynthesis in Nicotiana and Litchi chinensis During Anthocyanin Accumulation.

Anthocyanin biosynthesis requires the MYB-bHLH-WD40 protein complex to activate the late biosynthetic genes. LcMYB1 was thought to act as key regulator in anthocyanin biosynthesis of litchi. However, basic helix-loop-helix proteins (bHLHs) as partners have not been identified yet. The present study describes the functional characterization of three litchi bHLH candidate anthocyanin regulators, LcbHLH1, LcbHLH2, and LcbHLH3. Although these three litchi bHLHs phylogenetically clustered with bHLH proteins involved in anthcoyanin biosynthesis in other plant, only LcbHLH1 and LcbHLH3 were found to localize in the nucleus and physically interact with LcMYB1. The transcription levels of all these bHLHs were not coordinated with anthocyanin accumulation in different tissues and during development. However, when co-infiltrated with LcMYB1, both LcbHLH1 and LcbHLH3 enhanced anthocyanin accumulation in tobacco leaves with LcbHLH3 being the best inducer. Significant accumulation of anthocyanins in leaves transformed with the combination of LcMYB1 and LcbHLH3 were noticed, and this was associated with the up-regulation of two tobacco endogenous bHLH regulators, NtAn1a and NtAn1b, and late structural genes, like NtDFR and NtANS. Significant activity of the ANS promoter was observed in transient expression assays either with LcMYB1-LcbHLH1 or LcMYB1-LcbHLH3, while only minute activity was detected after transformation with only LcMYB1. In contrast, no activity was measured after induction with the combination of LcbHLH2 and LcMYB1. Higher DFR expression was also oberseved in paralleling with higher anthocyanins in co-transformed lines. LcbHLH1 and LcbHLH3 are essential partner of LcMYB1 in regulating the anthocyanin production in tobacco and probably also in litchi. The LcMYB1-LcbHLH complex enhanced anthocyanin accumulation may associate with activating the transcription of DFR and ANS.