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.

Shoot tip abortion is a very common phenomenon in some perennial woody plants that affects the height, architecture, and branch orientation of trees. To date, little is known about the mechanism of shoot tip abortion. In this study, a sweet orange gene encoding a KNAT-like protein (CsKN1) was identified and showed high expression in the shoot apical meristem (SAM). Overexpression of CsKN1 prolonged the vegetative growth of SAM, and silencing of CsKN1 resulted in the loss or inhibition of SAM in transgenic plants. 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 extremely multiple meristem phenotype. Overexpression of CsKN1 and CsKN2 resulted in the differential expression of numerous hormone biosynthesis and signaling genes in transgenic plants. Further evidence suggested that CsKN1 might prolong the vegetative growth period of SAM by inhibiting LEAFY. In addition, an ethylene responsive factor (CsERF) was found to bind to the CsKN1 promoter and suppresses its transcription. CsERF enhances ethylene and reactive oxygen species contents and may induce the occurrence of shoot tip abscission. Thus, we conclude that CsKN1 and CsKN2 may work cooperatively to regulate the shoot tip abscission process of sweet orange spring shoots.

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.

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.

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.