ASYMMETRIC LEAVES2 repression confers lobed leaves by regulating lignin biosynthesis of leaf veins in Betula pendula

Lobed leaves are a desirable phenotypic trait in conditions of high irradiance and environmental stresses. Betula pendula 'Dalecarlica', an intraspecific variation of B. pendula, shows a unique leaf shape with deeply lobed leaves in contrast to the serrated ovate leaves of B.pendula. The leaf vein pattern is critically important for the formation of leaf lobes. However, the underlying regulatory mechanisms remain elusive. In this study, we investigated a gene regulatory network using RNA-seq data from different development stages of midveins in B.pendula 'Dalecarlica'. We identified BpAS2 as a key negative regulator in vein development of lobed leaves. RNA-seq analysis revealed that genes related to lignin biosynthesis were significantly upregulated in bpas2 mutant plants created using the CRISPR/Cas9 genome editing system. Loss-of-function of BpAS2 gene results in overdeveloped veins in lobed leaves. Furthermore, ChIP assay confirms that BpAS2 physically binds the promoter of BpKNAT1, and represses its gene expression of BpKNAT1 which is a molecular regulator of leaf dissection and lignin biosynthesis. Our results indicate that BpAS2 represses BpKNAT1 to downregulate lignin biosynthesis, thereby, inhibiting the development of leaf vein patterns and the formation of lobed leaves.