iron ( fe ) and copper ( cu ) are essential elements for all living organisms because of their unique property of being able to catalyze oxidation / reduction reactions . conversely , an excess of fe , especially fe , is detrimental since it catalyzes the production of reactive oxygen species ( ros ) in the fenton reaction ( fenton , 1894 ; hell and stephan , 2003 ) . for this reason , free metal ions are not likely to exist in large amounts in cells . indeed , fe and other metals are mainly present in stable complexes with organic ligands or inorganic phosphates ( haydon and cobbett , 2007 ) . nicotianamine ( na ) is one of the most investigated metal chelator molecules in plants . na is a non - proteinogenic amino acid and it results from the enzymatic condensation of three s - adenosyl methionine molecules ( sam ) catalyzed by nicotianamine synthase ( nas ; herbik et al . , 1999 ; ling et al . , 1999 ; na is able to form stable complexes with mn , fe , co , zn , ni , and cu ( benes et al . , 1983 moreover , na has a high capacity to chelate fe ( von wiren et al . , 1999 ; for all the metals considered , the stability of the na  metal complexes had its maximum at ph values above 6.5 , suggesting that na is more likely a symplastic chelator of metals and therefore would bind metals predominately within cells and the phloem ( von wiren et al . , 1999 ) . cu is an exception among the essential metals , since the cu  na complex is very stable in mild acidic conditions , which is a strong argument in favor of the possible occurrence of cu  na complexes in the apoplastic environment as prevailing in the xylem sap ( von wiren et al . , 1999 ) . nicotianamine can be transported to the various organs and tissues via oligopeptide transporters , such as yellowstripe1-like ( ysl ) proteins ( curie et al . , 2001 , 2009 ) . rice ena1 and ena2 transporters were just recently described to mediate na export from cells ( nozoye et al . , 2011 ) . studies of solanaceous and graminaceous plants as well as of hyperaccumulators showed that na functions in long - distance transport of cu ( pich and scholz , 1996 ) , short - distance and intracellular transport of fe ( becker et al . , 1995 ; curie and briat , 2003 ) , plant reproduction ( takahashi et al . , 2006 ) , detoxification of heavy metals like ni ( douchkov et al . callahan et al . , 2007 ) and zn ( becher et al . , 2004 ; weber et al . , 2004 ; talke et al . , 2006 ; van de mortel et al . , 2006 ) , and in grasses as a precursor in the biosynthesis of phytosiderophores ( mori and nishizawa , 1987 ) . several studies suggested that na could be involved in iron mobilization and accumulation in plant roots and seeds ( douchkov et al . , 2001 , 2005 ; cheng et al . , 2007 ; lee et al . , na is beneficial for increased bioavailability of fe in foods ( maurer et al . , 2010 ; taken together , na is a key compound of metal homeostasis in plants contributing to mobilization , uptake , transport , storage , and detoxification of metals . since na was found to be an important biofortification factor for essential nutrients like fe and zn in edible portions of crop plants ( zheng et al . , 2010 ) , further knowledge about the functions of na and the characterization of the essential genes for its production are of high relevance . the arabidopsis system provides all tools that allow combining genetic studies with physiological analyzes and global gene expression experiments . while not all plant species have multiple nas genes , the arabidopsis genome harbors a nas gene family comprising four members ( bauer et al . , 2004 ) . nas1 and nas2 are located on chromosome v , while nas3 and nas4 are located on chromosome i ( figure 1a ) . multiple alignment ( clustalw ) showed a close relation between nas genes located on the same chromosome with more than 80% identity while alignment of genes belonging to separate chromosomes showed an identity of about 70% ( bauer et al . , 2004 ) . gene mapping between solanum esculentum and a. thaliana suggested that the four arabidopsis genes , as well as the single tomato nas gene originated from a common ancestor nas gene . this finding is in agreement with a first genome duplication event in the evolution of arabidopsis , followed later by two independent duplication events ( bauer et al . , 2004 ) . ( a ) scheme representing the five chromosomes of arabidopsis , indicating the location of the four nas genes and their gene identity numbers ( bauer et al . , 2004 ) . ( b ) scheme representing the location of t - dna insertions , indicating the allele names . it is shown which alleles are combined in the quadruple mutants nas4x-1 and nas4x-2 ( klatte et al . , 2009 ) . ( c ) table listing the primers used for genotyping as described in ( klatte et al . , 2009 ) and the primers used for gene expression analysis by reverse transcription - qpcr according to ( klatte and bauer , 2009 ) . t - dna insertion lines of all four nas genes were identified and crossed to each other ( figure 1b ; primer sequences for genotyping are found in figure 1c ) . under regular growth conditions single , double , and triple mutants did not show any obvious phenotypes suggesting functional redundancy ( klatte et al . , 2009 ) . triple nas mutants had na levels that were reduced to 3040% of wild type levels ( klatte et al . , 2009 ) . since na can be transported short and long - distance in plants , severe metal homeostasis phenotypes are not expected in the presence of a functional nas gene . interestingly , however , upon exposure to modestly toxic ni supply , nas4 - 1 had a more chlorotic phenotype than nas3 - 1 , while nas1 - 1 and nas2 - 1 had mild phenotypes like the wild type ( klatte et al . , 2009 ) . with increasing number of nas knockout alleles , the na contents decreased in the mutants while the severity of the leaf chlorosis was enhanced ( klatte et al . , 2009 ) . , we show the seedling growth responses of single and multiple mutants in response to fe deficiency . we found that all single nas mutants tested had a stronger leaf chlorosis than wild type plants upon fe deficiency ( figure 2a ) . nas4 - 1 mutants had the strongest leaf chlorosis among the tested single mutants ( figure 2a ) . it can therefore be concluded that the nas gene functions are partially non - overlapping . alternatively , the nas isoforms might have different enzyme activities , perhaps under specific conditions like fe deficiency and ni supply . ( a ) percentage of leaf chlorosis phenotypes of multiple nas mutants , combining the alleles nas1 - 1 , nas2 - 1 , nas3 - 1 , and nas4 - 1 , germinated for 2  weeks on hoagland agar medium devoid of fe ; the medium is described in ( jakoby et al . , 2004 ) . the colors indicate the percentage of plants with light green leaves ( weak leaf chlorosis , black ) , light green intercostal areas ( intermediate degree of leaf chlorosis , light gray ) , yellow intercostal areas ( strong leaf chlorosis , dark gray ) . the numbers on the right side indicate the number of seedlings examined ; * indicates a strong phenotype . ( b ) table summarizing the gene expression results from ( bauer et al . , 2004 ; partial non - redundancy is further confirmed by the fact that the nas genes are differentially regulated in plants ( bauer et al . , 2004 ; klatte et al . , 2009 ; nas1 , nas2 , and nas4 were found expressed in roots , where they were induced by ni supply . nas1 and nas4 were expressed in leaves , and nas4 could be induced by fe deficiency and ni in leaves . nas3 was expressed in leaves where it was repressed by fe deficiency but strongly induced by ni supply . in flowers , nas3 was expressed in sepals and petals while nas1 , nas2 , and nas4 were not expressed ( schuler et al . , in preparation ) . taken together , nas genes evolved as a gene family in arabidopsis where they acquired overlapping and specific functions in metal homeostasis as well as differential gene regulation in response to metals . nas3 seems important for leaf and flower nicotianamine production upon fe supply as well as ni tolerance , while nas4 was more important for fe deficiency in leaves and perhaps in roots . mutant analysis showed that all four nas genes are functional , so that quadruple mutant analysis was needed to study na function . the leaf chlorosis phenotypes of quadruple nas mutants were more severe than those of single mutants . quadruple nas1 - 1 nas2 - 1 nas3 - 1 nas4 - 1 mutants ( termed nas4x-1 ) were found to have a stronger reduction of na levels than all triple mutant combinations analyzed , namely to approximately 15% in vegetative leaves and 30% in seeds compared to wild type ( klatte et al . , 2009 ) . while nas4x-1 plants had a residual na level in leaves at the vegetative stage , this was not the case in the reproductive stage in leaves . full loss of function nas1 - 1 nas2 - 2 nas3 - 1 nas4 - 1 mutants ( termed nas4x-2 ) did not contain any na ( klatte et al . the morphological phenotypes of nas4x-1 and nas4x-2 were compared ( summarized in table 1 ) . comparison of nas4x-1 and nas4x-2 . * a more detailed investigation of nas4x-2 phenotypes will be presented in schuler et al . nas4x-1 plants appeared nearly normal during the vegetative stage , unless they were grown under fe deficiency or ni supply . however , nas4x-1 plants showed an interveinal leaf chlorosis upon transition to the reproductive growth stage , and fe contents were increased in leaves at this stage compared to wild type ( klatte et al . , 2009 ) . mobilization of fe by nas4x-1 roots was up - regulated at this stage which accounts for the increased fe contents ( klatte et al . presumably , intercostal leaf areas with mesophyll cells did not acquire fe in sufficient amounts and may have emitted a long - distance fe deficiency signal that stimulated root fe uptake . nas4x-1 plants are still fertile , yet flowers and seeds were found to contain less fe than in the wild type ( klatte et al . , 2009 ) . nas4x-1 mutant plants are valuable models to study na function in late phases of plant development . these plants also served to perform a transcriptome analysis of roots and leaves upon fe supply and fe deficiency ( schuler et al . , 2011 ) . a comparison with the wild type transcriptomes confirmed that nas4x-1 was affected in metal homeostasis since a high number of genes of this category was hit by differential expression . besides this category , the mutant was also affected in biotic stress responses , leaf photosystem organization , and root carbohydrate metabolism ( schuler et al . , 2011 ) . significantly more genes of these four biological categories were affected by differential expression between mutant and wild type compared to all genes analyzed in the microarray study . a change of expression of genes from these categories can be explained as an adaptation response to altered fe levels . leaf chlorosis started during the vegetative phase ( klatte et al . , 2009 ) . closer inspection of this mutant showed that na was involved in the long - distance transport of fe to young leaves presumably using the phloem , while older leaves received fe from citrate - mediated transport in the xylem ( schuler et al . the leaf chlorosis was due to fe accumulation in the vascular system suggesting that na is involved in lateral transport of fe from vascular tissues to mesophyll ( schuler et al . , in preparation ) . furthermore , nas4x-2 mutants were affected in pollination ( schuler et al . , in preparation ) . the split of a nas locus to four nas genes in arabidopsis resulted in a partial non - redundant specialization of nas gene functions . these are conferred at least partly by differential gene expression of the nas genes in response to developmental cues , tissue specificity , and metals . it has not been investigated yet whether the enzyme activities of the nas isoforms are differentially regulated by metal supply . using the mild and severe nas4x-1 and nas4x-2 mutants novel nicotianamine functions were uncovered , such as seed fe loading , long - distance fe transport to leaves , short - distance transport from vascular tissues to mesophyll , and in pollination . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
understanding the regulated inter- and intra - cellular metal circulation is one of the challenges in the field of metal homeostasis . 
 inside organisms metal ions are bound to organic ligands to prevent their uncontrolled reactivity and to increase their solubility . 
 nicotianamine ( na ) is one of the important ligands . 
 this non - proteinogenic amino acid is synthesized by nicotianamine synthase ( nas ) . 
 na is involved in mobilization , uptake , transport , storage , and detoxification of metals . 
 much of the progress in understanding na function has been achieved by studying mutants with altered nicotianamine levels . 
 mild and strong arabidopsis mutants impaired in nicotianamine synthesis have been identified and characterized , namely nas4x-1 and nas4x-2 . 
 arabidopsis thaliana has four nas genes . in this review , 
 we summarize the structure and evolution of the nas genes in the arabidopsis genome . 
 we summarize previous results and present novel evidence that the four nas genes have partially overlapping functions when plants are exposed to fe deficiency and nickel supply . 
 we compare the phenotypes of nas4x-1 and nas4x-2 and summarize the functions of nas genes and na as deduced from the studies of mutant phenotypes .