S-locus F-Box Protein as Pollen S determinant Targets Non-self S-RNase underlying Self-Incompatibility in Citrus.

Self-incompatibility (SI) is a crucial mechanism that prevents self-fertilization and inbreeding in flowering plants. Citrus exhibits SI regulated by a polymorphic S-locus containing an S-RNase gene and multiple S-locus F-box (SLF) genes. It has been documented that S-RNase functions as the pistil S determinant, but there is no direct evidence that the SLFs closely linked with S-RNase function as pollen S determinants in Citrus. This study assembled the genomes of two pummelo (Citrus grandis) plants and obtained three novel complete and well-annotated S-haplotypes and isolated 36 SLF or SLF-like alleles on the S-loci. Phylogenetic analysis of 138 SLFs revealed that the SLFs were classified into 12 types, including six types with divergent or missing alleles. Furthermore, transformation experiments verified that the conserved S6-SLF7a protein can lead the transition of SI to self-compatibility (SC) by recognizing non-self S8-RNase in 'Mini-Citrus' plants (S7S8 and S8S29, Fortunella hindsii), a model plant for citrus gene function studies. In vitro assays demonstrated interactions between SLFs of different S haplotypes and the Skp1-Cullin1-F-box subunit CgSSK1 protein. This study provides direct evidence that SLF controls the pollen function in Citrus, demonstrating its role in the 'non-self-recognition' SI system.

A bZIP transcription factor (CiFD) regulates drought- and low-temperature-induced flowering by alternative splicing in citrus.

Drought and low temperature are two key environmental factors that induce adult citrus flowering. However, the underlying regulation mechanism is poorly understood. The bZIP transcription factor FD is a key component of the florigen activation complex (FAC) which is composed of FLOWERING LOCUS T (FT), FD, and 14-3-3 proteins. In this study, isolation and characterization of CiFD in citrus found that there was alternative splicing (AS) of CiFD, forming two different proteins (CiFDα and CiFDß). Further investigation found that their expression patterns were similar in different tissues of citrus, but the subcellular localization and transcriptional activity were different. Overexpression of the CiFD DNA sequence (CiFD-DNA), CiFDα, or CiFDß in tobacco and citrus showed early flowering, and CiFD-DNA transgenic plants were the earliest, followed by CiFDß and CiFDα. Interestingly, CiFDα and CiFDß were induced by low temperature and drought, respectively. Further analysis showed that CiFDα can form a FAC complex with CiFT, Ci14-3-3, and then bind to the citrus APETALA1 (CiAP1) promoter and promote its expression. However, CiFDß can directly bind to the CiAP1 promoter independently of CiFT and Ci14-3-3. These results showed that CiFDß can form a more direct and simplified pathway that is independent of the FAC complex to regulate drought-induced flowering through AS. In addition, a bHLH transcription factor (CibHLH96) binds to CiFD promoter and promotes the expression of CiFD under drought condition. Transgenic analysis found that CibHLH96 can promote flowering in transgenic tobacco. These results suggest that CiFD is involved in drought- and low-temperature-induced citrus flowering through different regulatory patterns.

Treatment of gastric hepatoid adenocarcinoma with pembrolizumab and bevacizumab combination chemotherapy: A case report.

BACKGROUND: Gastric hepatoid adenocarcinoma (GHA) is a rare and aggressive cancer that is characterized by foci with features of both hepatocellular differentiation and adenomatous differentiation. However, there is currently no standard treatment for this disease, which has a poor prognosis. CASE SUMMARY: A 72-year-old male with a body mass index of 20.9 was diagnosed with GHA with perigastric lymph node and liver metastasis. He underwent first-line chemotherapy but that failed. Pembrolizumab and bevacizumab with chemotherapy were used in the second-line treatment. The progression-free survival and overall survival were 14 mo and 16 mo, respectively, after treatment. In addition, the main adverse reaction was tolerable. The patient did not die of tumor progression. CONCLUSION: The combination of pembrolizumab and bevacizumab with chemotherapy is an effective and safe regimen for GHA and may be recommended as a new choice for GHA treatment. Further studies should evaluate this treatment in a larger cohort or a randomized controlled trial.

Transcriptomes and DNA methylomes in apomictic cells delineate nucellar embryogenesis initiation in citrus.

Citrus nucellar poly-embryony (NPE) is a mode of sporophytic apomixis that asexual embryos formed in the seed through adventitious embryogenesis from the somatic nucellar cells. NPE allows clonal propagation of rootstocks, but it impedes citrus cross breeding. To understand the cellular processes involved in NPE initiation, we profiled the transcriptomes and DNA methylomes in laser microdissection captured citrus apomictic cells. In apomictic cells, ribosome biogenesis and protein degradation were activated, whereas auxin polar transport was repressed. Reactive oxygen species (ROS) accumulated in the poly-embryonic ovules, and response to oxidative stress was provoked. The global DNA methylation level, especially that of CHH context, was decreased, whereas the methylation level of the NPE-controlling key gene CitRWP was increased. A C2H2 domain-containing transcription factor gene and CitRWP co-expressed specifically in apomictic cells may coordinate to initiate NPE. The activated embryogenic development and callose deposition processes indicated embryogenic fate of nucellar embryo initial (NEI) cells. In our working model for citrus NPE initiation, DNA hyper-methylation may activate transcription of CitRWP, which increases C2H2 expression and ROS accumulation, triggers epigenetic regulation and regulates cell fate transition and NEI cell identity in the apomictic cells.

LncRNA PRNCR1 rs1456315 and CCAT2 rs6983267 Polymorphisms on 8q24 Associated with Lung Cancer.

BACKGROUND: Long noncoding RNA single nucleotide polymorphisms (lncRNA-SNPs) PCAT1 rs710886, PRNCR1 rs1456315 and CCAT2 rs6983267 on 8q24 region present generalizability in the susceptibility to multiple cancers, however, the influence of rs710886, rs1456315 and rs6983267 on lung cancer has not been assessed. The aim of this study was to investigate associations between three lncRNA-SNPs and lung cancer. METHODS: A case-control study was performed on 438 patients with lung cancer and 456 healthy controls in the Han population from southern China. The collected samples were genotyped by the TaqMan genotyping, and the association with clinical characteristics, including age, gender, drinking status, smoking status, pathological types and clinical stages were analyzed. And the SNP function prediction was based on lncRNASNP2, RNAfold and GTEx. RESULTS: The rs1456315 T allele increased the risk of lung cancer [OR=1.95, 95% CI (1.58-2.43), P=0.003] compared to the rs1456315 C allele, and rs1456315 significantly increased the risk of lung cancer in the dominant model [OR=1.86, 95% CI (1.16-3.00), P=0.002]. The rs6983267 G allele, compared with the T allele, increased the risk of lung cancer [OR=1.29, 95% CI (1.07-1.57), P=0.007], and rs6983267 was identified as a risk factor for lung cancer [OR=1.28, 95% CI (1.06-1.55), P=0.003] in the additive model. Both rs1456315 and rs6983267 demonstrated significance after adjusting for the smoking status, drinking status and age. The structure prediction found rs6983267 and rs1456315 influence the secondary structure of its lncRNA. The results from lncRNASNP2 indicated that rs6983267 and rs1456315 change gain/loss target of miRNAs. CONCLUSION: PRNCR1 rs1456315 and CCAT2 rs6983267 on 8q24 region are significantly associated with lung cancer in the Han population of southern China and alter the potential biological function in bioinformatic analysis, and the results further extended generalism of the susceptibility of cancer-associated lncRNA-SNPs to lung cancer and underlying mechanism involved in lung cancer.

Comparative analysis of the transcriptome, methylome, and metabolome during pollen abortion of a seedless citrus mutant.

KEY MESSAGE: Pollen abortion could be mainly attributed to abnormal meiosis in the mutant. Multiomics analysis uncovered significant epigenetic variations between the mutant and its wild type during the pollen abortion process. Male sterility caused by aborted pollen can result in seedless fruit. A seedless Ponkan mandarin mutant (bud sport) was used to compare the transcriptome, methylome, and metabolome with its progenitor to understand the mechanism of citrus pollen abortion. Cytological observations showed that the anther of the mutant could form microspore mother cells, although the microspores failed to develop fertile pollen at the anther dehiscence stage. Based on pollen phenotypic analysis, pollen abortion could be mainly attributed to abnormal meiosis in the mutant. A transcriptome analysis uncovered the molecular mechanisms underlying pollen abortion between the mutant and its wild type. A total of 5421 differentially expressed genes were identified, and some of these genes were involved in the meiosis, hormone biosynthesis and signaling, carbohydrate, and flavonoid pathways. A total of 50,845 differentially methylated regions corresponding to 15,426 differentially methylated genes in the genic region were found between the mutant and its wild type by the methylome analysis. The expression level of these genes was negatively correlated with their methylation level, especially in the promoter regions. In addition, 197 differential metabolites were identified between the mutant and its wild type based on the metabolome analysis. The transcription and metabolome analysis further indicated that the expression of genes in the flavonoid, carbohydrate, and hormone metabolic pathways was significantly modulated in the pollen of the mutant. These results indicated that demethylation may alleviate the silencing of carbohydrate genes in the mutant, resulting in excessive starch and sugar hydrolysis and thereby causing pollen abortion in the mutant.

Genome-scale mRNA and small RNA transcriptomic insights into initiation of citrus apomixis.

Nucellar embryony (NE) is an adventitious form of apomixis common in citrus, wherein asexual embryos initiate directly from nucellar cells surrounding the embryo sac. NE enables the fixation of desirable agronomic traits and the production of clonal offspring of virus-free rootstock, but impedes progress in hybrid breeding. In spite of the great importance of NE in citrus breeding and commercial production, little is understood about the underlying molecular mechanisms. In this study, the stages of nucellar embryo initiation (NEI) were determined for two polyembryonic citrus cultivars via histological observation. To explore the genes and regulatory pathways involved in NEI, we performed mRNA-seq and sRNA-seq analyses of ovules immediately prior to and at stages during NEI in the two pairs of cultivars. A total of 305 differentially expressed genes (DEGs) were identified between the poly- and monoembryonic ovules. Gene ontology (GO) analysis revealed that several processes are significantly enriched based on DEGs. In particular, response to stress, and especially response to oxidative stress, was over-represented in polyembryonic ovules. Nearly 150 miRNAs, comprising ~90 conserved and ~60 novel miRNAs, were identified in the ovules of either cultivar pair. Only two differentially expressed miRNAs (DEMs) were identified, of which the novel miRN23-5p was repressed whereas the targets accumulated in the polyembryonic ovules. This integrated study on the transcriptional and post-transcriptional regulatory profiles between poly- and monoembryonic citrus ovules provides new insights into the mechanism of NE, which should contribute to revealing the regulatory mechanisms of plant apomixis.

A Review of Auxin Response Factors (ARFs) in Plants.

Auxin is a key regulator of virtually every aspect of plant growth and development from embryogenesis to senescence. Previous studies have indicated that auxin regulates these processes by controlling gene expression via a family of functionally distinct DNA-binding auxin response factors (ARFs). ARFs are likely components that confer specificity to auxin response through selection of target genes as transcription factors. They bind to auxin response DNA elements (AuxRE) in the promoters of auxin-regulated genes and either activate or repress transcription of these genes depending on a specific domain in the middle of the protein. Genetic studies have implicated various ARFs in distinct developmental processes through loss-of-function mutant analysis. Recent advances have provided information on the regulation of ARF gene expression, the role of ARFs in growth and developmental processes, protein-protein interactions of ARFs and target genes regulated by ARFs in plants. In particular, protein interaction and structural studies of ARF proteins have yielded novel insights into the molecular basis of auxin-regulated transcription. These results provide the foundation for predicting the contributions of ARF genes to the biology of other plants.

Maximum parsimony based resolution of inter-species phylogenetic relationships in Citrus L. (Rutaceae) using ITS of rDNA.

The present study aims to analyse phylogenetic relationships, using internal transcribed spacer sequence data of ribosomal DNA (rDNA), across 24 Citrus species and close relatives by the evaluation of several parameters such as nucleotide substitution (r), nucleotide diversity (π) and the estimated values of transition/transversion bias (R). The observed results indicated the presence of a wide divergence pattern of rDNA in subfamily Aurantioideae. Maximum parsimony (MP) analysis inferred divergence pattern in the Citrus genus. We observed seven strongly supported clades among the subfamily Aurantioideae. We postulate that the present investigation provides a more robust topology of Citrus and its close relatives, which can significantly prove as an additional support to resolve the phylogenetic relationships in Citrus genera. Therefore, sequences of noncoding regions should exhibit more phylogenetically informative sites than the coding regions do, which is in accordance with the present study.