NmrA acts as a positive regulator of nitrate assimilation in Phaeodactylum tricornutum

Abstract

Nitrogen, a major element, is generally considered as one of the limiting factors for the growth of marine diatoms. Nitrogen availability influences not only nitrate assimilation but also urea cycle in diatoms, while the regulatory mechanism of nitrogen metabolism is still unknown. Nitrogen metabolite repression (NMR) is the most widely known regulatory circuit in fungal nitrogen metabolism, and NmrA is a negative regulator involved in NMR. We identified 6 putative NmrA encoding genes in Phaeodactylum tricornutum, a model pennate diatom, and phylogenetic analyses showed that the 6 NmrA isoforms were divided into three distinct evolutionary clades: bacterium (J37667, J46721), fungus (J31822) and animal (J50525, J45041 and J49854), reflecting the mosaic genome in diatoms which originated from secondary endosymbiotic event. Two highly transcriptionally nitrate-sensitive NmrA, J37667 (PtNmrA1) and J50525 (PtNmrA2), which both contained the characteristic nucleotide-binding glycine-rich motif and localized in cytoplasm and endoplasmic reticulum lumen respectively, were knockdown by RNA interference in P. tricornutum. Although the growth was not different, nitrate utilization was inhibited by PtNmrA knockdowns. Transcript levels of genes involved in transport and reduction of NO3− and NO2−, and in urea cycle were downregulated, while plastidial and mitochondrial GS/GOGAT, and enzymes related with the regeneration of ammonium and glutamate in mitochondria were strongly transcriptionally upregulated. The results suggested knockdown of NmrA decreased the nitrate utilization but enhanced mitochondrial nitrogen regeneration to satisfy nitrogen demands elsewhere in the cell, and PtNmrA might serve as a positive regulator of nitrate assimilation in the diatom.