Citrus flavonoids are considered as the important secondary metabolites because of their biological and pharmacological activities. Chalcone synthase (CHS) is a key enzyme that catalyses the first committed step in the flavonoid biosynthetic pathway. CHS genes have been isolated and characterized in many plants. Previous studies indicated that CHS is a gene superfamily. In citrus, the number of CHS members and their contribution to the production of flavonoids remains a mystery. In our previous study, the copies of CitCHS2 gene were found in different citrus species and the sequences are highly conserved, but the flavonoid content varied significantly among those species. From seventy-seven CHS and CHS-like gene sequences, ten CHS members were selected as candidates according to the features of their sequences. Among these candidates, expression was detected from only three genes. A predicted CHS sequence was identified as a novel CHS gene. The structure analysis showed that the gene structure of this novel CHS is very similar to other CHS genes. All three CHS genes were highly conserved and had a basic structure that included one intron and two exons, although they had different expression patterns in different tissues and developmental stages. These genes also presented different sensitivities to methyl jasmonate (MeJA) treatment. In transgenic plants, the expression of CHS genes was significantly correlated with the production of flavonoids. The three CHS genes contributed differently to the production of flavonoids. Our study indicated that CitCHS is a gene superfamily including at least three functional members. The expression levels of the CHS genes are highly correlated to the biosynthesis of flavonoids. The CHS enzyme is dynamically produced from several CHS genes, and the production of total flavonoids is regulated by the overall expression of CHS family genes.

Limonoids produced by citrus are a group of highly bioactive secondary metabolites which provide health benefits for humans. Currently there is a lack of information derived from research on the genetic mechanisms controlling the biosynthesis of limonoids, which has limited the improvement of citrus for high production of limonoids. In this study, the transcriptome sequences of leaves, phloems and seeds of pummelo (Citrus grandis (L.) Osbeck) at different development stages with variances in limonoids contents were used for digital gene expression profiling (DGE) analysis in order to identify the genes corresponding to the biosynthesis of limonoids. Pair-wise comparison of transcriptional profiles between different tissues identified 924 differentially expressed genes commonly shared between them. Expression pattern analysis suggested that 382 genes from three conjunctive groups of K-means clustering could be possibly related to the biosynthesis of limonoids. Correlation analysis with the samples from different genotypes, and different developing tissues of the citrus revealed that the expression of 15 candidate genes were highly correlated with the contents of limonoids. Among them, the cytochrome P450s (CYP450s) and transcriptional factor MYB demonstrated significantly high correlation coefficients, which indicated the importance of those genes on the biosynthesis of limonoids. CiOSC gene encoding the critical enzyme oxidosqualene cyclase (OSC) for biosynthesis of the precursor of triterpene scaffolds was found positively corresponding to the accumulation of limonoids during the development of seeds. Suppressing the expression of CiOSC with VIGS (Virus-induced gene silencing) demonstrated that the level of gene silencing was significantly correlated to the reduction of limonoids contents. The results indicated that the CiOSC gene plays a pivotal role in biosynthesis of limonoids.

Background: Limonoids are major bioactive compounds that are produced by the triterpenoid metabolic pathway. The detailed biochemical process of limonoid biosynthesis and the mechanism of its molecular regulation remain elusive. The identification of transcription factors that regulate limonoid biosynthetic pathways is very important for understanding the underlying regulatory mechanisms. This information could also provide tools for manipulating biosynthesis genes to modulate limonoid production. Results: In this study, the CiMYB42 transcription factor was isolated to identify its role in limonoid biosynthesis. Multiple alignment analysis and phylogenetic analysis demonstrated that CiMYB42 is a typical R2R3MYB transcription factor that shares high similarity of its amino acid sequence with AtMYB42. Limonoids contents were higher in Citrus sinensis and Citrus grandis than in other species. Limonoid accumulation during leaf development also showed diverse trends in different genotypes. The expression of CiMYB42 was significantly related to the limonoid content and the expression of CiOSC in some citrus accessions. The overexpression of CiMYB42 in sweet orange resulted in significant accumulation of limonin, whereas the downregulation of CiMYB42 by RNAi resulted in a dwarf phenotype and less nomilin accumulation. Furthermore, the results of a yeast one-hybrid assay and EMSA indicated that CiMYB42 binds exclusively to the TTGTTG sequence (type II MYB core) in the promoter of CiOSC. Together, these results suggest that CiMYB42 positively regulates limonoid biosynthesis by regulating the expression of CiOSC by binding to the TTGTTG sequence (type II MYB core) of its promoter. Conclusions: CiMYB42 is an important transcription activator involved in limonoid biosynthesis that regulates the expression of CiOSC by binding to the TTGTTG sequence (type II MYB core).

The most damaging citrus diseases are Huanglongbing (HLB) and citrus canker, which are caused by Candidatus Liberibacter asiaticus (CaLas) and Xanthomonas citri pv. citri (Xcc), respectively. Endolysins from bacteriophages are a possible option for disease resistance in plant breeding. Here, we report improvement of citrus resistance to HLB and citrus canker using the LasLYS1 and LasLYS2 endolysins from CaLas. LasLYS2 demonstrated bactericidal efficacy against several Rhizobiaceae bacteria and Xcc, according to inhibition zone analyses. The two genes, driven by a strong promoter from Cauliflower mosaic virus, 35S, were integrated into Carrizo citrange via Agrobacterium-mediated transformation. More than 2 years of greenhouse testing indicated that LasLYS2 provided substantial and long-lasting resistance to HLB, allowing transgenic plants to retain low CaLas titers and no obvious symptoms while also clearing CaLas from infected plants in the long term. LasLYS2 transgenic plants with improved HLB resistance also showed resistance to Xcc, indicating that LasLYS2 had dual resistance to HLB and citrus canker. A microbiome study of transgenic plants revealed that the endolysins repressed Xanthomonadaceae and Rhizobiaceae populations in roots while increasing Burkholderiaceae and Rhodanobacteraceae populations, which might boost the citrus defense response, according to transcriptome analysis. We also found that Lyz domain 2 is the key bactericidal motif of LasLYS1 and LasLYS2. Four endolysins with potential resistance to HLB and citrus canker were found based on the structures of LasLYS1 and LasLYS2. Overall, the work shed light on the mechanisms of resistance of CaLas-derived endolysins, providing insights for designing endolysins to develop broad-spectrum disease resistance in citrus.