ACC SYNTHASE4 inhibits gibberellin biosynthesis and FLOWERING LOCUS T expression during citrus flowering.

Flowering is an essential process in fruit trees. Flower number and timing have a substantial impact on the yield and maturity of fruit. Ethylene and gibberellin (GA) play vital roles in flowering, but the mechanism of coordinated regulation of flowering in woody plants by GA and ethylene is still unclear. In this study, a lemon (Citrus limon L. Burm) 1-aminocyclopropane-1-carboxylic acid synthase gene (CiACS4) was overexpressed in Nicotiana tabacum and resulted in late flowering and increased flower number. Further transformation of citrus revealed that ethylene and starch content increased, and soluble sugar content decreased in 35S:CiACS4 lemon. Inhibition of CiACS4 in lemon resulted in effects opposite to that of 35S:CiACS4 in transgenic plants. Overexpression of the CiACS4-interacting protein ETHYLENE RESPONSE FACTOR3 (CiERF3) in N. tabacum resulted in delayed flowering and more flowers. Further experiments revealed that the CiACS4-CiERF3 complex can bind the promoters of FLOWERING LOCUS T (CiFT) and GOLDEN2-LIKE (CiFE) and suppress their expression. Moreover, overexpression of CiFE in N. tabacum led to early flowering and decreased flowers, and ethylene, starch, and soluble sugar contents were opposite to those in 35S:CiACS4 transgenic plants. Interestingly, CiFE also bound the promoter of CiFT. Additionally, GA3 and 1-aminocyclopropanecarboxylic acid (ACC) treatments delayed flowering in adult citrus, and treatment with GA and ethylene inhibitors increased flower number. ACC treatment also inhibited the expression of CiFT and CiFE. This study provides a theoretical basis for the application of ethylene to regulate flower number and mitigate the impacts of extreme weather on citrus yield due to delayed flowering.

Citrus ACC synthase CiACS4 regulates plant height by inhibiting gibberellin biosynthesis.

Dwarfism is an agronomic trait that has substantial effects on crop yield, lodging resistance, planting density, and a high harvest index. Ethylene plays an important role in plant growth and development, including the determination of plant height. However, the mechanism by which ethylene regulates plant height, especially in woody plants, remains unclear. In this study, a 1-aminocyclopropane-1-carboxylic acid synthase (ACC) gene (ACS), which is involved in ethylene biosynthesis, was isolated from lemon (Citrus limon L. Burm) and named CiACS4. Overexpression of CiACS4 resulted in a dwarf phenotype in Nicotiana tabacum and lemon and increased ethylene release and decreased gibberellin (GA) content in transgenic plants. Inhibition of CiACS4 expression in transgenic citrus significantly increased plant height compared with the controls. Yeast two-hybrid assays revealed that CiACS4 interacted with an ethylene response factor (ERF), CiERF3. Further experiments revealed that the CiACS4-CiERF3 complex can bind to the promoters of 2 citrus GA20-oxidase genes, CiGA20ox1 and CiGA20ox2, and suppress their expression. In addition, another ERF transcription factor, CiERF023, identified using yeast one-hybrid assays, promoted CiACS4 expression by binding to its promoter. Overexpression of CiERF023 in N. tabacum caused a dwarfing phenotype. CiACS4, CiERF3, and CiERF023 expression was inhibited and induced by GA3 and ACC treatments, respectively. These results suggest that the CiACS4-CiERF3 complex may be involved in the regulation of plant height by regulating CiGA20ox1 and CiGA20ox2 expression levels in citrus.

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.

Drought and low temperature-induced NF-YA1 activates FT expression to promote citrus flowering.

Flower induction in adult citrus is mainly regulated by drought and low temperatures. However, the mechanism of FLOWERING LOCUS T regulation of citrus flowering (CiFT) under two flower-inductive stimuli remains largely unclear. In this study, a citrus transcription factor, nuclear factor YA (CiNF-YA1), was found to specifically bind to the CiFT promoter by forming a complex with CiNF-YB2 and CiNF-YC2 to activate CiFT expression. CiNF-YA1 was induced in juvenile citrus by low temperature and drought treatments. Overexpression of CiNF-YA1 increased drought susceptibility in transgenic citrus, whereas suppression of CiNF-YA1 enhanced drought tolerance in silenced citrus plants. Furthermore, a GOLDEN2 - LIKE protein (CiFE) that interacts with CiFT protein was also isolated. Further experimental evidence showed that CiFE binds to the citrus LEAFY (CiLFY) promoter and activates its expression. In addition, the expressions of CiNF-YA1 and CiFE showed a seasonal increase during the floral induction period and were induced by artificial drought and low-temperature treatments at which floral induction occurred. These results indicate that CiNF-YA1 may activate CiFT expression in response to drought and low temperatures by binding to the CiFT promoter. CiFT then forms a complex with CiFE to activate CiLFY, thereby promoting the flowering of adult citrus.

Mobility of FLOWERING LOCUS T protein as a systemic signal in trifoliate orange and its low accumulation in grafted juvenile scions.

The long juvenile period of perennial woody plants is a major constraint in breeding programs. FLOWERING LOCUS T (FT) protein is an important mobile florigen signal that induces plant flowering. However, whether FT can be transported in woody plants to shorten the juvenile period is unknown, and its transport mechanism remains unclear. In this study, trifoliate orange FT (ToFT) and Arabidopsis FT (AtFT, which has been confirmed to be transportable in Arabidopsis) as a control were transformed into tomato and trifoliate orange, and early flowering was induced in the transgenic plants. Long-distance and two-way (upward and downward) transmission of ToFT and AtFT proteins was confirmed in both tomato and trifoliate orange using grafting and western blot analysis. However, rootstocks of transgenic trifoliate orange could not induce flowering in grafted wild-type juvenile scions because of the low accumulation of total FT protein in the grafted scions. It was further confirmed that endogenous ToFT protein was reduced in trifoliate orange, and the accumulation of the transported ToFT and AtFT proteins was lower than that in grafted juvenile tomato scions. Furthermore, the trifoliate orange FT-INTERACTING PROTEIN1 homolog (ToFTIP1) was isolated by yeast two-hybrid analysis. The FTIP1 homolog may regulate FT transport by interacting with FT in tomato and trifoliate orange. Our findings suggest that FT transport may be conserved between the tomato model and woody plants. However, in woody plants, the transported FT protein did not accumulate in significant amounts in the grafted wild-type juvenile scions and induce the scions to flower.

Citrus FRIGIDA cooperates with its interaction partner dehydrin to regulate drought tolerance.

Drought is a major environmental stress that severely affects plant growth and crop productivity. FRIGIDA (FRI) is a key regulator of flowering time and drought tolerance in model plants. However, little is known regarding its functions in woody plants, including citrus. Thus, we explored the functional role of the citrus FRI ortholog (CiFRI) under drought. Drought treatment induced CiFRI expression. CiFRI overexpression enhanced drought tolerance in transgenic Arabidopsis and citrus, while CiFRI suppression increased drought susceptibility in citrus. Moreover, transcriptomic profiling under drought conditions suggested that CiFRI overexpression altered the expression of numerous genes involved in the stress response, hormone biosynthesis, and signal transduction. Mechanistic studies revealed that citrus dehydrin likely protects CiFRI from stress-induced degradation, thereby enhancing plant drought tolerance. In addition, a citrus brassinazole-resistant (BZR) transcription factor family member (CiBZR1) directly binds to the CiFRI promoter to activate its expression under drought conditions. CiBZR1 also enhanced drought tolerance in transgenic Arabidopsis and citrus. These findings further our understanding of the molecular mechanisms underlying the CiFRI-mediated drought stress response in citrus.

Functional analysis of a PISTILLATA-like gene CcMADS20 involved in floral organs specification in citrus.

PISTILLATA (PI), as a member of MADS-box transcription factor, plays an important role in petal and stamen specification in Arabidopsis. However, little is known about PI-like genes in citrus. To understand the molecular mechanism of PI during the developmental process of citrus flower, a PI-like gene CcMADS20 was isolated from Citrus Clemantina. Sequence alignment and phylogenetic analysis revealed that CcMADS20 had relatively high similarity with PI-like homolog and was classified in the core dicotyledonous group. The temporal and spatial expression analyses showed that CcMADS20 was specifically expressed in petal and stamen of citrus flower, which was consistent with PI expression pattern in Arabidopsis. Protein interaction revealed that CcMADS20 could form heterodimer with AP3-like proteins. Furthermore, ectopic overexpression of CcMADS20 in Arabidopsis resulted in transformation of sepals into petal-like structure, as observed in other plants overexpressing a functional PI-like homolog. Additionally, promoter fragments of CcMADS20 were also cloned in the representative 21 citrus varieties. Interestingly, four types of promoters were discovered in these citrus varieties, resulting from two stable insert/deletion fragments (Locus1 and Locus2). The homo/hetero-zygosity of promoter alleles in each variety was strongly related to the evolutionary origin of citrus. Four promoters activity analysis indicated that Locus1 presence inhibited CcMADS20 transcriptional activity and Locus2 presence promoted its transcriptional activity. These findings suggested that CcMADS20 determines petal and stamen development during the evolutionary process of citrus and four promoters discovered, as effective genetic markers, are valuable for citrus breeding practices.

Transcriptomic and physiological analysis reveals interplay between salicylic acid and drought stress in citrus tree floral initiation.

MAIN CONCLUSION: Salicylic acid (SA) and drought stress promote more flowering in sweet orange. The physiological response and molecular mechanism underlying stress-induced floral initiation were discovered by transcriptome profiling. Numerous flowering-regulated genes were identified, and ectopically expressed CsLIP2A promotes early flowering in Arabidopsis. Floral initiation is a critical developmental mechanism associated with external factors, and citrus flowering is mainly regulated by drought stress. However, little is known about the intricate regulatory network involved in stress-induced flowering in citrus. To understand the molecular mechanism of floral initiation in citrus, flower induction was performed on potted Citrus sinensis trees under the combined treatment of salicylic acid (SA) and drought (DR). Physiological analysis revealed that SA treatment significantly normalized the drastic effect of drought stress by increasing antioxidant enzyme activities (SOD, POD, and CAT), relative leaf water content, total chlorophyll, and proline contents and promoting more flowering than drought treatment. Analysis of transcriptome changes in leaves from different treatments showed that 1135, 2728 and 957 differentially expressed genes (DEGs) were revealed in response to DR, SD (SA + DR), and SA (SA + well water) treatments in comparison with the well watered plants, respectively. A total of 2415, 2318 and 1933 DEGs were expressed in DR, SD, and SA in comparison with water recovery, respectively. Some key flowering genes were more highly expressed in SA-treated drought plants than in DR-treated plants. GO enrichment revealed that SA treatment enhances the regulation and growth of meristem activity under drought conditions, but no such a pathway was found to be highly enriched in the control. Furthermore, we focused on various hormones, sugars, starch metabolism, and biosynthesis-related genes. The KEGG analysis demonstrated that DEGs enriched in starch sucrose metabolism and hormonal signal transduction pathways probably account for stress-induced floral initiation in citrus. In addition, a citrus LIPOYLTRANSFERSAE 2A homologous (LIP2A) gene was upregulated by SD treatment. Ectopic expression of CsLIP2A exhibited early flowering in transgenic Arabidopsis. Taken together, this study provides new insight that contributes to citrus tree floral initiation under the SA-drought scenario as well as an excellent reference for stress-induced floral initiation in woody trees.

High-Density Genetic Map Construction and Identification of QTLs Controlling Leaf Abscission Trait in Poncirus trifoliata.

A high-density genetic linkage map is essential for genetic and genomic studies including QTL mapping, genome assembly, and comparative genomic analysis. Here, we constructed a citrus high-density linkage map using SSR and SNP markers, which are evenly distributed across the citrus genome. The integrated linkage map contains 4163 markers with an average distance of 1.12 cM. The female and male linkage maps contain 1478 and 2976 markers with genetic lengths of 1093.90 cM and 1227.03 cM, respectively. Meanwhile, a genetic map comparison demonstrates that the linear order of common markers is highly conserved between the clementine mandarin and Poncirus trifoliata. Based on this high-density integrated citrus genetic map and two years of deciduous phenotypic data, two loci conferring leaf abscission phenotypic variation were detected on scaffold 1 (including 36 genes) and scaffold 8 (including 107 genes) using association analysis. Moreover, the expression patterns of 30 candidate genes were investigated under cold stress conditions because cold temperature is closely linked with the deciduous trait. The developed high-density genetic map will facilitate QTL mapping and genomic studies, and the localization of the leaf abscission deciduous trait will be valuable for understanding the mechanism of this deciduous trait and citrus breeding.

Genome-Wide Identification and Expression Profiling of the WOX Gene Family in Citrus sinensis and Functional Analysis of a CsWUS Member.

WUSCHEL-related homeobox (WOX) transcription factors (TFs) are well known for their role in plant development but are rarely studied in citrus. In this study, we identified 11 putative genes from the sweet orange genome and divided the citrus WOX genes into three clades (modern/WUSCHEL(WUS), intermediate, and ancient). Subsequently, we performed syntenic relationship, intron-exon organization, motif composition, and cis-element analysis. Co-expression analysis based on RNA-seq and tissue-specific expression patterns revealed that CsWOX gene expression has multiple intrinsic functions. CsWUS homolog of AtWUS functions as a transcriptional activator and binds to specific DNA. Overexpression of CsWUS in tobacco revealed dramatic phenotypic changes, including malformed leaves and reduced gynoecia with no seed development. Silencing of CsWUS in lemon using the virus-induced gene silencing (VIGS) system implied the involvement of CsWUS in cells of the plant stem. In addition, CsWUS was found to interact with CsCYCD3, an ortholog in Arabidopsis (AtCYCD3,1). Yeast one-hybrid screening and dual luciferase activity revealed that two TFs (CsRAP2.12 and CsHB22) bind to the promoter of CsWUS and regulate its expression. Altogether, these results extend our knowledge of the WOX gene family along with CsWUS function and provide valuable findings for future study on development regulation and comprehensive data of WOX members in citrus.

A MADS-Box Gene CiMADS43 Is Involved in Citrus Flowering and Leaf Development through Interaction with CiAGL9.

MADS-box genes are involved in various developmental processes including vegetative development, flower architecture, flowering, pollen formation, seed and fruit development. However, the function of most MADS-box genes and their regulation mechanism are still unclear in woody plants compared with model plants. In this study, a MADS-box gene (CiMADS43) was identified in citrus. Phylogenetic and sequence analysis showed that CiMADS43 is a GOA-like Bsister MADS-box gene. It was localized in the nucleus and as a transcriptional activator. Overexpression of CiMADS43 promoted early flowering and leaves curling in transgenic Arabidopsis. Besides, overexpression or knockout of CiMADS43 also showed leaf curl phenotype in citrus similar to that of CiMADS43 overexpressed in Arabidopsis. Protein-protein interaction found that a SEPALLATA (SEP)-like protein (CiAGL9) interacted with CiMADS43 protein. Interestingly, CiAGL9 also can bind to the CiMADS43 promoter and promote its transcription. Expression analysis also showed that these two genes were closely related to seasonal flowering and the development of the leaf in citrus. Our findings revealed the multifunctional roles of CiMADS43 in the vegetative and reproductive development of citrus. These results will facilitate our understanding of the evolution and molecular mechanisms of MADS-box genes in citrus.

Two citrus KNAT-like genes, CsKN1 and CsKN2, are involved in the regulation of spring shoot development in sweet orange.

Shoot-tip abortion is a very common phenomenon in some perennial woody plants and it affects the height, architecture, and branch orientation of trees; however, little is currently known about the underlying mechanisms. In this study, we identified a gene in sweet orange (Citrus sinensis) encoding a KNAT-like protein (CsKN1) and found high expression in the shoot apical meristem (SAM). Overexpression of CsKN1 in transgenic plants prolonged the vegetative growth of SAMs, whilst silencing resulted in either the loss or inhibition of SAMs. Yeast two-hybrid analysis revealed that CsKN1 interacted with another citrus KNAT-like protein (CsKN2), and overexpression of CsKN2 in lemon and tobacco caused an extreme multiple-meristem phenotype. Overexpression of CsKN1 and CsKN2 in transgenic plants resulted in the differential expression of numerous genes related to hormone biosynthesis and signaling. Yeast one-hybrid analysis revealed that the CsKN1-CsKN2 complex can bind to the promoter of citrus floral meristem gene LEAFY (CsLFY) and inhibit its expression. These results indicated that CsKN1 might prolong the vegetative growth period of SAMs by delaying flowering. In addition, an ethylene-responsive factor (CsERF) was found to bind to the CsKN1 promoter and suppresses its transcription. Overexpression of CsERF in Arabidopsis increased the contents of ethylene and reactive oxygen species, which might induce the occurrence of shoot-tip abscission. On the basis of our results, we conclude that CsKN1 and CsKN2 might work cooperatively to regulate the shoot-tip abscission process in spring shoots of sweet orange.

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.

HD-ZIP I Transcription Factor (PtHB13) Negatively Regulates Citrus Flowering through Binding to FLOWERING LOCUS C Promoter.

For floral induction in adult citrus, low temperature is one of the most important environmental factors. FLOWERING LOCUS C (FLC) plays a very important role in low-temperature-induced Arabidopsis flowering by repressed FLC expression under exposure to prolonged low-temperature conditions. However, little is known about the FLC regulation mechanism in perennial woody plants such as citrus. In this study, the functions of citrus FLC homolog (PtFLC) were investigated by ectopic expression in Arabidopsis. Transcription factor of homeodomain leucine zipper I (HD-ZIP I) as an upstream regulator of PtFLC was identified by yeast one-hybrid screen to regulate its transcription. The HD-ZIP I transcription factor was highly homologous to Arabidopsis ATHB13 and thus was named PtHB13. Ectopically expressed PtHB13 inhibited flowering in transgenic Arabidopsis. Furthermore, the expression of PtFLC and PtHB13 showed a seasonal change during the floral induction period and was also affected by low temperature. Thus, we propose that PtHB13 binds to PtFLC promoter to regulate its activity during the citrus floral induction process.

Genome-Wide Identification and Characterization of SQUAMOSA-Promoter-Binding Protein (SBP) Genes Involved in the Flowering Development of Citrus Clementina.

SQUAMOSA-promoter binding protein (SBP)-box genes encode a family of plant-specific transcription factors that play vital roles in plant growth and development. In this study, 15 SBP-box genes were identified and isolated from Citrus clementina (CclSBPs), where 10 of these genes were predicted to be putative targets of Citrus clementina microRNA156 (CclmiR156). The 15 CclSBP genes could be classified into six groups based on phylogenetic analysis, diverse intron⁻exon structure, and motif prediction, similar to the SQUAMOSA promoter binding protein-like (SPL) gene family of Populus trichocarpa and Arabidopsis thaliana. Furthermore, CclSBPs classified into a group/subgroup have similar gene structures and conserved motifs, implying their functional redundancy. Tissue-specific expression analysis of CclSBPs demonstrated their diversified expression patterns. To further explore the potential role of CclSBPs during floral inductive water deficits, the dynamic changes of the 15 CclSBPs were investigated during floral inductive water deficits, and the results showed that some CclSBPs were associated with floral induction. Among these genes, CclSBP6 was not homologous to the Arabidopsis SBP-box gene family, and CclSBP7 was regulated by being alternatively spliced. Therefore, CclSBP6 and CclSBP7 were genetically transformed in Arabidopsis. Overexpression of the two genes changed the flowering time of Arabidopsis.

Identification of the Genetic Variation and Gene Exchange between Citrus Trifoliata and Citrus Clementina.

To identify the genetic variation between Citrus trifoliata and Citrus clementina, we performed genome resequencing on the two citrus species. Compared with the citrus reference genome, a total of 9,449,204 single-nucleotide polymorphisms (SNPs) and 846,615 insertion/deletion polymorphisms (InDels) were identified in the two citrus species, while 1,868,115 (19.77%) of the SNPs and 190,199 (22.47%) of the InDels from the two citrus species were located in the genic regions. Meanwhile, a total of 8,091,407 specific SNPs and 692,654 specific InDels were identified in the two citrus genotypes, yielding an average of 27.32 SNPs/kb and 2.34 InDels/kb. We identified and characterized the patterns of gene exchanges in the grafted citrus plants by using specific genetic variation from genome resequencing. A total of 4396 transporting genes across graft junctions was identified. Some specific genetic variation and mobile genes was also confirmed by Sanger sequencing. Furthermore, these mobile genes could move directionally or bidirectionally between the scions and the rootstocks. In addition, a total of 1581 and 2577 differentially expressed genes were found in the scions and the rootstocks after grafting compared with the control, respectively. These genetic variations provide fundamental information on the genetic basis of important traits between C. trifoliata and C. clementina, as the transport of genes would be applicable to horticulture crops.

Genome-wide genetic variation and comparison of fruit-associated traits between kumquat (Citrus japonica) and Clementine mandarin (Citrus clementina).

KEY MESSAGE: The comprehensive genetic variation of two citrus species were analyzed at genome and transcriptome level. A total of 1090 differentially expressed genes were found during fruit development by RNA-sequencing. Fruit size (fruit equatorial diameter) and weight (fresh weight) are the two most important components determining yield and consumer acceptability for many horticultural crops. However, little is known about the genetic control of these traits. Here, we performed whole-genome resequencing to reveal the comprehensive genetic variation of the fruit development between kumquat (Citrus japonica) and Clementine mandarin (Citrus clementina). In total, 5,865,235 single-nucleotide polymorphisms (SNPs) and 414,447 insertions/deletions (InDels) were identified in the two citrus species. Based on integrative analysis of genome and transcriptome of fruit, 640,801 SNPs and 20,733 InDels were identified. The features, genomic distribution, functional effect, and other characteristics of these genetic variations were explored. RNA-sequencing identified 1090 differentially expressed genes (DEGs) during fruit development of kumquat and Clementine mandarin. Gene Ontology revealed that these genes were involved in various molecular functional and biological processes. In addition, the genetic variation of 939 DEGs and 74 multiple fruit development pathway genes from previous reports were also identified. A global survey identified 24,237 specific alternative splicing events in the two citrus species and showed that intron retention is the most prevalent pattern of alternative splicing. These genome variation data provide a foundation for further exploration of citrus diversity and gene-phenotype relationships and for future research on molecular breeding to improve kumquat, Clementine mandarin and related species.

Transcriptome-wide identification and functional prediction of novel and flowering-related circular RNAs from trifoliate orange (Poncirus trifoliata L. Raf.).

MAIN CONCLUSION: A total of 558 potential circular RNAs (circRNAs) were identified in citrus, and these were analyzed and compared. One hundred seventy-six differentially expressed circRNAs were identified in two genotypes of trifoliate orange. Circular RNAs (circRNAs) play diverse roles in transcriptional control and microRNA (miRNA) function. However, little information is known about circRNAs in citrus. To identify citrus circRNAs and investigate their functional roles, high-throughput sequencing of precocious trifoliate orange (an early-flowering trifoliate orange mutant, Poncirus trifoliata L. Raf.) and its wild type was performed. A total of 558 potential circRNAs were identified by bioinformatic analysis, and 86.02% of these were sense-overlapping circRNAs. Their sequence features, alternative circularization, and other characteristics were investigated in this study. Compared with the wild type, 176 circRNAs were identified as differentially expressed circRNAs, 61 were significantly up-regulated and 115 were down-regulated in precocious trifoliate orange, indicating that they may play an important role in the early flowering process. Alternative circularization and differential expression of some circRNAs were verified by Sanger sequencing and real-time polymerase chain reaction. The functions of differentially expressed circRNAs and their host genes were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. We found that many differentially expressed circRNAs had abundant miRNA binding sites: 29 circRNAs were found to act as the 16 miRNA targets. Overall, these results will help to reveal the biological functions of circRNAs in growth and development of citrus.

MicroRNA-Mediated Gene Silencing in Plant Defense and Viral Counter-Defense.

MicroRNAs (miRNAs) are non-coding RNAs of approximately 20-24 nucleotides in length that serve as central regulators of eukaryotic gene expression by targeting mRNAs for cleavage or translational repression. In plants, miRNAs are associated with numerous regulatory pathways in growth and development processes, and defensive responses in plant-pathogen interactions. Recently, significant progress has been made in understanding miRNA-mediated gene silencing and how viruses counter this defense mechanism. Here, we summarize the current knowledge and recent advances in understanding the roles of miRNAs involved in the plant defense against viruses and viral counter-defense. We also document the application of miRNAs in plant antiviral defense. This review discusses the current understanding of the mechanisms of miRNA-mediated gene silencing and provides insights on the never-ending arms race between plants and viruses.