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Zhiyong Hu, Qing Liu, Meilian Tan, Hualin Yi, and Xiuxin Deng

In this article, we describe a novel biological phenomenon that the tips of the juice sac of citrus hybrid HRB turned brown and became tough like many “brown thorns”; HRB is a triploid hybrid regenerated from the cross of diploid tangerine BDZ (Citrus reticulata Blanco cv. Huanongbendizao) with an allotetraploid somatic hybrid HR [Hamlin sweet orange (Citrus sinensis Osbeck) + rough lemon (Citrus jambhiri Lush)]. Histochemical analysis indicated that the “brown thorn” of HRB resulted from lignin deposition. The juice sac of HRB had 4.6- and 3.8-fold content of lignin of its parents tangerine and HR, respectively. Microscopy observation of the cross-sections of the juice sac verified that the cell wall of the “brown thorn” was lignified. Moreover, the cell walls near the “brown thorns” were obviously thickened. Analysis of enzyme activity indicated that the peroxidase (POD) activity of HRB was significantly higher than its parents. Quantitative real-time polymerase chain reaction analysis showed that the transcript abundance of the cinnamate 4-hydroxylase (C4H) gene in HRB was 6.5- and 3.4-fold of its parent BDZ and HR, respectively, but the POD gene transcript abundance was lower than its parent with 1/2-fold of BDZ and 1/7-fold of HR. These facts led to the conclusion that POD and C4H were the key regulating factors for lignin biosynthesis in juice sacs of the hybrid HRB. The POD gene, one member of the POD family, with transcript abundance lower than its parent indicated that this POD isoenzyme was not the regulating factor of lignin biosynthesis, and further study should be carried out to determine which POD isoenzyme is the key factor for lignin biosynthesis.