HEMA RNAi silencing reveals a control mechanism of ALA biosynthesis on Mg chelatase and Fe chelatase

Abstract

Glutamyl-tRNA reductase (GluTR) is encoded by HEMA in higher plants and catalyzes in plastids the initial enzymatic step of tetrapyrrole biosynthesis eventually leading to heme and chlorophyll. GluTR activity is subjected to a complex regulation on multiple expression levels. An ethanol-inducible HEMA-RNA-interference (RNAi) gene construct was introduced into the tobacco genome to study the primary effects of low GluTR content on the tetrapyrrole biosynthetic pathway. During the first days of induced HEMA silencing the chlorophyll and heme contents were diminished in young leaves. HEMA mRNA and GluTR protein content were also strongly reduced. However, expression analyses revealed that none of the other tetrapyrrole biosynthesis genes were affected on the transcriptional level in a nine days period after HEMA inactivation. Previously generated transgenic tobacco lines with RNAi silenced expression of the glutamate 1-semialdehyde aminotransferase (GSA) gene did also not display changes of transcripts from selected genes of tetrapyrrole biosynthesis and photosynthesis. Although the transcript levels were not decreased after inactivation of HEMA and GSA-expression, enzyme activities for Mg chelatase and Fe chelatase were lower, which occurred in parallel to the loss of chlorophyll and heme content. Posttranslational modification of enzymes downstream of ALA-biosynthesis is proposed as a regulatory mechanism to adjust the flux through tetrapyrrole biosynthesis in balance to supply of ALA.