Isolation, characterization and cDNA cloning of nicotianamine synthase from barley

Abstract

Basic cellular processes such as electron transport in photosynthesis and respiration require the precise control of iron homeostasis. To mobilize iron, plants have evolved at least two different strategies. The nonproteinogenous amino acid nicotianamine which is synthesized from three molecules of S-adenosyl-l-methionine, is an essential component of both pathways. This compound is missing in the tomato mutant chloronerva, which exhibits severe defects in the regulation of iron metabolism. We report the purification and partial characterization of the nicotianamine synthase from barley roots as well as the cloning of two corresponding gene sequences. The function of the gene sequence has been verified by overexpression in Escherichia coli. Further confirmation comes from reduction of the nicotianamine content and the exhibition of a chloronerva-like phenotype due to the expression of heterologous antisense constructs in transgenic tobacco plants. The native enzyme with an apparent M r of < 105 000 probably represents a trimer of S-adenosyl-l-methionine-binding subunits. A comparison with the recently cloned chloronerva gene of tomato reveals striking sequence homology, providing support for the suggestion that the destruction of the nicotianamine synthase encoding gene is the molecular basis of the tomato mutation.Keywords: antisense constructs; chloronerva mutation; gene isolation; Hordeum vulgare; iron metabolism.Iron is essential for fundamental cellular processes such as electron transfer in photosynthesis, respiration, nitrogen fixation as well as DNA synthesis [1]. Excessive accumulation causes severe damage to cellular components due to the formation of highly reactive hydroxyl radicals by the Fenton reaction [2]. Thus, the precise control of iron homeostasis is a basic prerequisite for cellular function. According to WHO data the health of more than three billion people worldwide is affected by iron deficient diet. Crop plants with a higher iron content, for example in the endosperm of cereals, could contribute to the improvement of this situation. In soil iron is mainly found as stable Fe(III) compounds with low solubility at neutral pH [1,3]. Therefore, plants have evolved special mechanisms of iron acquisition, classified into two strategies [4]. Strategy I plants, including dicots and nongraminaceous monocots, facilitate iron uptake mainly by increased acidification of the rhizosphere due to enhanced proton extrusion and the reduction of Fe(III) to Fe(II) by an inducible plasma membrane-bound reductase. In contrast, graminaceous monocots (strategy II plants) release phytosiderophores of the mugineic acid family into the rhizosphere. These compounds act as chelators of ferric ions and are taken up by root cells as Fe(III)-phytosiderophore complexes.The nonproteinogenous amino acid nicotianamine (NA) is found in all multicellular plants [5] and is considered to be a key component for both strategies of iron acquisition (Fig.1). In strategy I plants NA might function as a chelator of iron in symplastic...

Recycling of Methylthioadenosine Is Essential for Normal Vascular Development and Reproduction in Arabidopsis

“…Barley (Hordeum vulgare) NA synthase is product inhibited by MTA (K i = 5 mM; Herbik, 1997). We suspect that the Arabidopsis enzyme is similarly affected, given its amino acid sequence identity (46%) and similar domains (Herbik et al, 1999).…”

Nitric Oxide Contributes to Cadmium Toxicity in Arabidopsis by Promoting Cadmium Accumulation in Roots and by Up-Regulating Genes Related to Iron Uptake

“…The mean 6 SE of four independent experiments is represented. Means with a same letter are not significantly different at P = 0.05. nicotianamine (NA), a ubiquitous metal chelator (Herbik et al, 1999;Mari et al, 2006). In graminaceous plants (strategy II plants), NA is the precursor of mugineic acid family phytosiderophores and, consequently, plays a key role in iron uptake.…”

Crystallographic snapshots of iterative substrate translocations during nicotianamine synthesis in archaea

“…S1) (20). In barley, NAS is a single polypeptide whose functional form has been reported to be a monomer (20) or a trimer (21). The trimer form was taken as an indication that the catalytic mechanism involves the cooperation of three monomers to achieve the triple condensation of aminopropyl moieties.…”

“…However, recent sequencing projects have revealed nas-like genes in the genome of various organisms including plants, fungi, and archaea (21,24). Apart from in plants, both NA and a functional NAS have only been detected in Neurospora crassa, a filamentous fungus (24).…”