Genome-wide identification and expression analysis of carotenoid cleavage oxygenase genes in Litchi (Litchi chinensis Sonn.).

BACKGROUND: Carotenoid cleavage oxygenases (CCOs) include the carotenoid cleavage dioxygenase (CCD) and 9-cis-epoxycarotenoid (NCED), which can catalize carotenoid to form various apocarotenoids and their derivatives, has been found that play important role in the plant world. But little information of CCO gene family has been reported in litchi (Litchi chinensis Sonn.) till date. RESULTS: In this study, a total of 15 LcCCO genes in litchi were identified based on genome wide lever. Phylogeny analysis showed that LcCCO genes could be classified into six subfamilies (CCD1, CCD4, CCD7, CCD8, CCD-like, and NCED), which gene structure, domain and motifs exhibited similar distribution patterns in the same subfamilies. MiRNA target site prediction found that there were 32 miRNA target sites in 13 (86.7%) LcCCO genes. Cis-elements analysis showed that the largest groups of elements were light response related, following was plant hormones, stress and plant development related. Expression pattern analysis revealed that LcCCD4, LcNCED1, and LcNCED2 might be involving with peel coloration, LcCCDlike-b might be an important factor deciding fruit flavor, LcNCED2 and LcNCED3 might be related to flower control, LcNCED1 and LcNCED2 might function in fruitlet abscission, LcCCD4a1, LcCCD4a2, LcCCD1, LcCCD4, LcNCED1, and LcNCED2 might participate in postharvest storage of litchi. CONCLUSION: Herein, Genome-wide analysis of the LcCCO genes was conducted in litchi to investigate their structure features and potential functions. These valuable and expectable information of LcCCO genes supplying in this study will offer further more possibility to promote quality improvement and breeding of litchi and further function investigation of this gene family in plant.

Development of molecular markers based on the promoter difference of LcFT1 to discriminate easy- and difficult-flowering litchi germplasm resources and its application in crossbreeding.

BACKGROUND: Litchi is a well-known subtropical fruit crop. However, irregular bearing attributed to unstable flowering is a major ongoing problem for the development of the litchi industry. In a previous study, our laboratory proved that litchi flowering was induced by low temperature and that a FLOWERING LOCUS T (FT) homologue gene named LcFT1 played a pivotal role in this process. The present study aimed to understand the natural variation in FT among litchi germplasm resources and designed markers to verify easy- and difficult-flowering litchi germplasms. A grafting experiment was also carried out to explore whether it could shorten the seedling stage of litchi seedlings. RESULTS: Two types of LcFT1 promoter existed in different litchi germplasm resources, and we named them the 'easy-flowering type of LcFT1 promoter' and 'difficult-flowering type of LcFT1 promoter', which resulted in three different LcFT1 genotypes of litchi germplasm resources, including the homozygous easy-flowering type of the LcFT1 genotype, homozygous difficult-flowering type of the LcFT1 genotype and heterozygous LcFT1 genotype of litchi germplasm resources. The homozygous easy-flowering type of the LcFT1 genotype and heterozygous LcFT1 genotype of the litchi germplasm resources completed their floral induction more easily than the homozygous difficult-flowering type of the LcFT1 genotype of litchi germplasm resources. Herein, we designed two kinds of efficient molecular markers based on the difference in LcFT1 promoter sequences and applied them to identify of the easy- and difficult-flowering litchi germplasm resources. These two kinds of molecular markers were capable of clearly distinguishing the easy- from difficult-flowering litchi germplasm resources at the seedling stage and provided the same results. Meanwhile, grafting the scion of seedlings to the annual branches of adult litchi trees could significantly shorten the seedling stage. CONCLUSIONS: Understanding the flowering characteristics of litchi germplasm resources is essential for easy-flowering litchi breeding. In the present study, molecular markers provide a rapid and accurate approach for identifying the flowering characteristics. The application of these molecular markers not only significantly shortened the artificial crossbreeding cycle of easy-flowering litchi cultivars but also greatly saved manpower, material resources and land.

Coupling of microRNA-directed phased small interfering RNA generation from long noncoding genes with alternative splicing and alternative polyadenylation in small RNA-mediated gene silencing.

MicroRNAs (miRNAs) and phased small interfering RNAs (phasiRNAs) play vital regulatory roles in plant growth and development. Little is known about these small RNAs in litchi (Litchi chinensis), an economically important fruit crop widely cultivated in Southeast Asia. We profiled the litchi small RNA population with various deep-sequencing techniques and in-depth bioinformatic analyses. The genome-wide identification of miRNAs, their target genes, and phasiRNA-generating (PHAS) genes/loci showed that the function of miR482/2118 has expanded, relative to its canonical function. We also discovered that, for 29 PHAS loci, miRNA-mediated phasiRNA production was coupled with alternative splicing (AS) and alternative polyadenylation (APA). Most of these loci encoded long noncoding RNAs. An miR482/2118 targeted locus gave rise to four main transcript isoforms through AS/APA, and diverse phasiRNAs generated from these isoforms appeared to target long terminal repeat (LTR) retrotransposons and other unrelated genes. This coupling enables phasiRNA production from different exons of noncoding PHAS genes and yields diverse phasiRNA populations, both broadening and altering the range of downstream phasiRNA-regulated genes. Our results reveal the diversity of miRNA and phasiRNA in litchi, and demonstrate AS/APA as a new layer of regulation in small RNA-mediated gene silencing.

Genome-Wide Identification and Expression Analysis of m6A Writers, Erasers, and Readers in Litchi (Litchi chinensis Sonn.).

N6-methyladenosine (m6A) RNA modification is the most prevalent type of RNA methylation and plays a pivotal role in the development of plants. However, knowledge of the m6A modification in litchi remains limited. In this study, a complete analysis of m6A writers, erasers, and readers in litchi was performed and 31 litchi m6A regulatory genes were identified in total, including 7 m6A writers, 12 m6A erases, and 12 readers. Phylogeny analysis showed that all three of the kinds of litchi m6A regulatory proteins could be divided into three groups; domains and motifs exhibited similar patterns in the same group. MiRNA target site prediction showed that 77 miRNA target sites were located in 25 (80.6%) litchi m6A regulatory genes. Cis-elements analysis exhibited that litchi m6A regulatory genes were mainly responsive to light and plant hormones, followed by environmental stress and plant development. Expression analysis revealed litchi m6A regulatory genes might play an important role during the peel coloration and fruit abscission of litchi. This study provided valuable and expectable information of litchi m6A regulatory genes and their potential epigenetic regulation mechanism in litchi.

Genome-wide identification of myeloblastosis gene family and its response to cadmium stress in Ipomoea aquatica.

The myeloblastosis (MYB) proteins perform key functions in mediating cadmium (Cd) tolerance of plants. Ipomoea aquatica has strong adaptability to Cd Stress, while the roles of the I. aquatica MYB gene family with respect to Cd stress are still unclear. Here, we identified a total of 183 MYB genes in the I. aquatica genome (laMYB), which were classified into 66 1R-type IaMYB, 112 2R-type IaMYB, four 3R-type IaMYB, and one 4R-type IaMYB based on the number of the MYB repeat in each gene. The analysis of phylogenetic tree indicated that most of IaMYB genes are associated with the diverse biological processes including defense, development and metabolism. Analysis of sequence features showed that the IaMYB genes within identical subfamily have the similar patterns of the motif distributions and gene structures. Analysis of gene duplication events revealed that the dispersed duplication (DSD) and whole-genome duplication (WGD) modes play vital roles in the expansion of the IaMYB gene family. Expression profiling manifests that approximately 20% of IaMYB genes had significant role in the roots of I. aquatica under Cd stress. Promoter profiling implied that the differentially expressed genes might be induced by environmental factors or inherent hormones and thereby execute their function in Cd response. Remarkably, the 2R-type IaMYB157 with abundant light-responsive element G-box and ABA-responsive element ABRE in its promoter region exhibited very strong response to Cd stress. Taken together, our findings provide an important candidate IaMYB gene for further deciphering the molecular regulatory mechanism in plant with respect to Cd stress.

Circ-Sirt1 inhibits proliferation, induces apoptosis, and ameliorates inflammation in human rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a chronic inflammatory disease related to abnormal activation of fibroblast-like synovium cells (FLS) with apoptosis, inflammation, and oxidative damage. Circular RNA Sirt1 (circ-Sirt1) is an abundant circRNA, exerts the function in inhibiting inflammation. However, little is known about the roles of circ-Sirt1, if any, in RA. The present study aimed to investigate the biological roles and mechanism of circ-Sirt1 on cell inflammation in RA-FLS MH7A cell line. This study showed circ-Sirt1 inhibited the proliferation and induced apoptosis of MH7A cells. Overexpression of circ-Sirt1 decreased of the levels of interleukin (IL)-1ß and IL-6, tumour necrosis factor (TNF)-α, and matrix matalloproteinases (MMP)-1 and MMP-3 in MH7A cells. In addition, overexpression of circ-Sirt1 increased the expression of Sirt1, Nrf2, HO-1, IκBα, GCLC and GCLM, and decreased the ratio of acetylated NF-κB to normal NF-κB, and the expression of AP-1, COX-2 and HMGB1. Moreover, the expression of Keap1 and the ratio of acetylated NF-κB to normal NF-κB were partially increased and the Nrf2 and Sirt1 were partially reduced by siSirt1. Additionally, circ-Sirt1 overexpression promoted the activation of Sirt1 signal pathways by upregulating miR-132. In conclusion, the protective effect of Circ-Sirt1 on MH7A depends on inhibiting cell proliferation, promoting apoptosis and miR-132-mediated Sirt1 pathway to reduce inflammation.

24-nt reproductive phasiRNAs are broadly present in angiosperms.

Small RNAs are key regulators in plant growth and development. One subclass, phased siRNAs (phasiRNAs) require a trigger microRNA for their biogenesis. In grasses, two pathways yield abundant phasiRNAs during anther development; miR2275 triggers one class, 24-nt phasiRNAs, coincident with meiosis, while a second class of 21-nt phasiRNAs are present in premeiotic anthers. Here we report that the 24-nt phasiRNA pathway is widely present in flowering plants, indicating that 24-nt reproductive phasiRNAs likely originated with the evolutionary emergence of anthers. Deep comparative genomic analyses demonstrated that this miR2275/24-nt phasiRNA pathway is widely present in eudicots plants, however, it is absent in legumes and in the model plant Arabidopsis, demonstrating a dynamic evolutionary history of this pathway. In Solanaceae species, 24-nt phasiRNAs were observed, but the miR2275 trigger is missing and some loci displaying 12-nt phasing. Both the miR2275-triggered and Solanaceae 24-nt phasiRNAs are enriched in meiotic stages, implicating these phasiRNAs in anther and/or pollen development, a spatiotemporal pattern consistent in all angiosperm lineages that deploy them.

Genome-wide digital transcript analysis of putative fruitlet abscission related genes regulated by ethephon in litchi.

The high level of physiological fruitlet abscission in litchi (Litchi chinensis Sonn.) causes severe yield loss. Cell separation occurs at the fruit abscission zone (FAZ) and can be triggered by ethylene. However, a deep knowledge of the molecular events occurring in the FAZ is still unknown. Here, genome-wide digital transcript abundance (DTA) analysis of putative fruit abscission related genes regulated by ethephon in litchi were studied. More than 81 million high quality reads from seven ethephon treated and untreated control libraries were obtained by high-throughput sequencing. Through DTA profile analysis in combination with Gene Ontology and KEGG pathway enrichment analyses, a total of 2730 statistically significant candidate genes were involved in the ethephon-promoted litchi fruitlet abscission. Of these, there were 1867 early-responsive genes whose expressions were up- or down-regulated from 0 to 1 d after treatment. The most affected genes included those related to ethylene biosynthesis and signaling, auxin transport and signaling, transcription factors (TFs), protein ubiquitination, ROS response, calcium signal transduction, and cell wall modification. These genes could be clustered into four groups and 13 subgroups according to their similar expression patterns. qRT-PCR displayed the expression pattern of 41 selected candidate genes, which proved the accuracy of our DTA data. Ethephon treatment significantly increased fruit abscission and ethylene production of fruitlet. The possible molecular events to control the ethephon-promoted litchi fruitlet abscission were prompted out. The increased ethylene evolution in fruitlet would suppress the synthesis and polar transport of auxin and trigger abscission signaling. To the best of our knowledge, it is the first time to monitor the gene expression profile occurring in the FAZ-enriched pedicel during litchi fruit abscission induced by ethephon on the genome-wide level. This study will contribute to a better understanding for the molecular regulatory mechanism of fruit abscission in litchi.