Regulation of nitrate reductase expression in leaves by nitrate and nitrogen metabolism is completely overridden when sugars fall below a critical level

Abstract

ABSTRACTThese experiments use Nia30(145), a tobacco nia1nia2 double null mutant transformed with a NIA2 construct, to define when sugar supply plays the dominating role in the regulation of nitrate reductase (NIA) expression. The null alleles of Nia30(145) are transcribed and translated to produce non‐functional NIA transcript and NIA protein, providing an endogenous reporter system to track NIA expression at the transcript and protein level. The re‐introduced NIA2 construct is expressed at low efficiency, providing a background in which the response to changes in sugar status is not complicated by simultaneous changes in the rate of nitrate assimilation and the levels of nitrate and glutamine. In an alternating light–dark regime, Nia30(145) contained high levels of nitrate and low levels of glutamine and other amino acids. This drives constitutive overexpression of NIA. After transfer of Nia30(145) to continuous darkness, nitrate remains high and glutamine low, but the NIA transcript level and NIA protein decreased significantly within 24 h and were undetectable from 48 h onwards. The decrease of the NIA transcript level was fully reversed and the decrease of NIA protein was partly reversed when leaves were detached from the pre‐darkened plants and supplied with sucrose in the dark. The decrease was not reversed by nitrate or cytokinin. The NIA transcript disappeared when the leaf sugar content fell below 4 μmol hexose equivalents g−1 FW, and recovered when sugars rose above 8 μmol hexose equivalents g−1 FW. It is concluded that low sugar represses NIA, completely overriding signals derived from nitrate and nitrogen metabolism.