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.

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.

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.

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.

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.

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.

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.

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.