Objective Functions and expressions of cinnamoyl-CoA reductase genes (CmCCRs) associated with the lignin biosynthesis and juice vesicle granulation in Citrus maxima were investigated.

Method Bioinformatics, gene cloning, subcellular localization, and fluorescence quantitative PCR were applied to select and clone CmCCRs from C. maxima Sanhongmiyou. Expressions of the genes in the juice sacs at different fruit development stages of Sanhongmiyou and Liuyuezaoyihao pomelos were examined. Lignin content in the sacs was determined using the acetyl bromide method.

Results From the RNA-Seq data on Sanhongmiyou juice sacs, 5 CmCCRs were identified. Since the lignin content in the mature juice sacs was shown to correlate with the gene transcription, CmCCR1 was supposed to be involved in the lignin formation, and thus further analyzed. The ORF of CmCCR1 was 1,017 bp long. The stable hydrophilic protein had a molecular weight of 37.5 kDa and a pI of 7.06 containing an NAD(P)H/NAD(P)(+) binding (NADB) domain and three shared common motifs of CcCCR1 in sweet orange with similar distribution patterns. Phylogenetically, it closely related to CcCCR1 and belonged to the dicotyledonous plants. The gene was located on the endoplasmic reticulum same as predicted previously by software. The expression of CmCCR1 in the Sanhongmiyou juice sacs increased significantly at maturation, but that of Liuyuezaoyihao remained relatively low throughout the entire developmental stages at significantly lower levels. However, when the Liuyuezaoyihao pomelo juice sacs were transiently transformed with CmCCR1, a significantly elevated expression of the gene resulted. Moreover, the phloroglucinol-stained sacs turned pink in color indicating lignin accumulation in them.

Conclusion  CmCCR1 was postulated to regulate the biosynthesis of secondary metabolites (e.g., lignin) and the granulation of juice vesicles in pomelos.