Corky split vein can develop under long-term boron deficient conditions in Citrus sinensis L. Osbeck cv. Newhall. This symptom only occurs in the upper rather than the lower epidermis of old leaves. Our previous study demonstrated that vascular hypertrophy was involved in the symptoms, and the 3rd developmental stage of corky split vein (BD3) was the critical stage for phenotype formation. Here, we performed an intensive study on the BD3 vein and its control sample (CK3 vein). A lignin test demonstrated that the lignin content in BD3 vein was approximately 1.7 times more than the CK3 vein. Anatomical investigation of the corky split vein indicated that the upper epidermis was destroyed by overgrowing vascular cells, and the increased lignin may contribute to vascular cell differentiation and wounding-induced lignification. In a subsequent small RNA sequencing of the BD3 and CK3 veins, 99 known miRNAs and 22 novel miRNAs were identified. Comparative profiling of these miRNAs demonstrated that the 57 known miRNAs and all novel miRNAs exhibited significant expression differences between the two small RNAs libraries of the BD3 and CK3 veins. Associated with our corresponding digital gene expression data, we propose that the decreased expression of two miRNAs, csi-miR156b and csi-miR164, which leads to the up-regulation of their target genes, SPLs (csi-miR156b-targeted) and CUC2 (csi-miR164-targeted), may promote vascular cell division and orderless stage transition in old leaves.
Keywords: Anatomy; Cell division; Corky split vein; Lignification; Orderless stage transition; miRNAome.