External dsRNA Downregulates Anthocyanin Biosynthesis-Related Genes and Affects Anthocyanin Accumulation in Arabidopsis thaliana

Abstract

Exogenous application of double-stranded RNAs (dsRNAs) and small-interfering RNAs (siRNAs) to plant surfaces has emerged as a promising method for regulation of essential genes in plant pathogens and for plant disease protection. Yet, regulation of plant endogenous genes via external RNA treatments has not been sufficiently investigated. In this study, we targeted the genes of chalcone synthase (CHS), the key enzyme in the flavonoid/anthocyanin biosynthesis pathway, and two transcriptional factors, MYBL2 and ANAC032, negatively regulating anthocyanin biosynthesis in <i>Arabidopsis</i>. Direct foliar application of <i>AtCHS</i>-specific dsRNAs and siRNAs resulted in an efficient downregulation of the <i>AtCHS</i> gene and suppressed anthocyanin accumulation in <i>A. thaliana</i> under anthocyanin biosynthesis-modulating conditions. Targeting the <i>AtMYBL2</i> and <i>AtANAC032</i> genes by foliar dsRNA treatments markedly reduced their mRNA levels and led to a pronounced upregulation of the <i>AtCHS</i> gene. The content of anthocyanins was increased after treatment with <i>AtMYBL2-</i>dsRNA. Laser scanning microscopy showed a passage of Cy3-labeled <i>AtCHS</i>-dsRNA into the <i>A. thaliana</i> leaf vessels, leaf parenchyma cells, and stomata, indicating the dsRNA uptake and spreading into leaf tissues and plant individual cells. Together, these data show that exogenous dsRNAs were capable of downregulating <i>Arabidopsis</i> genes and induced relevant biochemical changes, which may have applications in plant biotechnology and gene functional studies.