Genome-wide identification, classification and expression profiling of nicotianamine synthase (NAS) gene family in maize
© Zhou et al.; licensee BioMed Central Ltd. 2013
Received: 30 November 2012
Accepted: 1 April 2013
Published: 10 April 2013
Nicotianamine (NA), a ubiquitous molecule in plants, is an important metal ion chelator and the main precursor for phytosiderophores biosynthesis. Considerable progress has been achieved in cloning and characterizing the functions of nicotianamine synthase (NAS) in plants including barley, Arabidopsis and rice. Maize is not only an important cereal crop, but also a model plant for genetics and evolutionary study. The genome sequencing of maize was completed, and many gene families were identified. Although three NAS genes have been characterized in maize, there is still no systematic identification of maize NAS family by genomic mining.
In this study, nine NAS genes in maize were identified and their expression patterns in different organs including developing seeds were determined. According to the evolutionary relationship and tissue specific expression profiles of ZmNAS genes, they can be subgrouped into two classes. Moreover, the expression patterns of ZmNAS genes in response to fluctuating metal status were analysed. The class I ZmNAS genes were induced under Fe deficiency and were suppressed under Fe excessive conditions, while the expression pattern of class II genes were opposite to class I. The complementary expression patterns of class I and class II ZmNAS genes confirmed the classification of this family. Furthermore, the histochemical localization of ZmNAS1;1/1;2 and ZmNAS3 were determined using in situ hybridization. It was revealed that ZmNAS1;1/1;2, representing the class I genes, mainly expressed in cortex and stele of roots with sufficient Fe, and its expression can expanded in epidermis, as well as shoot apices under Fe deficient conditions. On the contrary, ZmNAS3, one of the class II genes, was accumulated in axillary meristems, leaf primordia and mesophyll cells. These results suggest that the two classes of ZmNAS genes may be regulated on transcriptional level when responds to various demands for iron uptake, translocation and homeostasis.
These results provide significant insights into the molecular bases of ZmNAS in balancing iron uptake, translocation and homeostasis in response to fluctuating environmental Fe status.
KeywordsMaize Nicotianamine synthase Gene family Iron uptake and homeostasis Subcellular localization Expression profiling In situ hybridization
Iron is an essential micronutrient with numerous cellular functions in animals and plants. The anemia caused by iron-deficiency is still a prevalent nutrient problem affecting more than half of the world’s population, especially in developing countries . Besides, iron is also an essential metal nutrient factor for plants, as it plays critical roles during many development processes, including photosynthesis, respiration, and other biochemical reactions that need Fe as a co-factor. Iron deficiency in plants may lead to leaf senescence, and in turn severely reduced the yield and quality. The total amount of Fe in soil is not limited; however, it can be merely soluble under aerobic conditions, especially in alkaline and calcareous soil . In order to acquire enough Fe without toxicity, plants have developmented iron uptake, utilization and storage system regulated by environmental Fe availability. The mechanism of Fe acquisition in plants can be divided into two categories: strategy I and strategy II . The strategy I was applied by nongraminaceous plants, which includes the reduction of ferric to ferrous on the root surface, and absorption of ferrous across the root plasma membrane by Fe2+ transporters. The FRO2  and IRT1  were firstly cloned from Arabidopsis and responsible for these processes. The graminaceous plants, such as rice, corn and barley, applied strategy II, which includes the synthesis and secretion of mugineic acid (MAs) family phytosiderophores (PS) from roots and the uptake of Fe3+-PS complexes by specific plasma membrane transporters. MAs can be synthesized by a conserved pathway begin with trimerization of three molecular of S-adenosyl-L-methionine into nicotianamine (NA) by nicotianamine synthase (NAS) , and then NA is converted into 2′-deoxymugineic acid (DMA), the precursor of MAs, by nicotianamine aminotransferase (NAAT)  and deoxymugineic acid synthase (DMAS) . In some graminaceous plants MAs can be obtained by hydroxylation of DMA [9, 10]. NA is known as a metal chelator, which can bind a range of metals, including Fe, Zn, Mn and Cu [11–15]. When iron was absorbed in plants, its translocation is thought to be associated with appropriate chelators, such as citrate [16, 17], NA [1, 14], and MAs [18, 19]. Citrate is essential in Fe transportation in xylem sap , while NA play a dominant role in the chelating and trafficking of Fe in phloem . In graminaceous plants, yellow strip like (YSL) family transporter, YS1, was reported facilitating the Fe3+-DMA uptake from rhizosphere , while AtYSL1 and AtYSL3 involved in long-distance translocation of Fe2+-NA in nongraminaceous plants [20, 22–24]. A tomato NA synthesis mutant, chloronerva (chln), show phenotype defects in Fe utilization and homeostasis [25, 26]. In addition, transgenic tobacco plants that continuously expressed barley NAAT exhibited disorders in internal metal transport, such as interveinal chlorosis in young leaves and abnormally shaped and sterilized flowers . In the NAAT tobacco, the endogenous NA was consumed as a result of excessive produced NAAT, suggesting that NA play critical roles in the regulation of metal transfer in plants, and maintaining sufficient amount of NA is required for inner metal homeostasis. A recent study reported that activation of OsNAS3 resulted in elevated Fe and Zn content in both vegetative tissue and seeds. Anemic mice fed with the OsNAS3 activated rice recovered more rapidly than those with wild type rice. Moreover, activated OsNAS3 expression also leads to increased tolerance to both Fe/Zn deficiencies and heavy-metal toxicity . This report suggested that NA is critical for Fe acquisition and storage, as well as detoxification of excessive intracellular Fe in plants.
Maize (Zea mays) is a major crop plant for feed industry and food, as well as a research model for monocotyledon plant. Although the iron content in corn is relatively higher than that in brown rice , it can barely meet the increasing demand for feed production. Therefore, investigating the mechanisms of iron acquisition, translocation and homeostasis in maize may support a model for understanding that in other crop plants, and provide gene resources for further breeding maize varieties with enhanced iron content. Since NA is the key for regulating Fe homeostasis in plants, considerable progress has been achieved in cloning and characterizing the functions of NAS in plants, including barley [29, 30], Arabidopsis, rice , tomato  and maize . Although it has been demonstrated that NA facilitate iron acquisition and translocation by forming Fe2+–NA complexes and serving as the precursor of MAs, the mechanism regulating these two pathways under fluctuating environmental iron status is still unclear. Systematic analyses in NAS gene families revealed that there are three NAS genes in rice and four in Arabidopsis, which suggested that NAS are encoded by a few genes instead of a gene family [31, 34]. However, nine NAS members were mapped in barley by combined approaches ; and it was also suggested that there are five genes encode NAS in maize, though only three of them were cloned due to the lack of genome information . The relatively larger numbers of NAS genes in barley and maize indicates that NAS may duplicate and evolve during the emergence of new species and breeding process.
The maize genome had been thoroughly sequenced and assembled recently, whereas there is still no systematic identification and characterization of NAS family. To better understand the roles of ZmNAS genes in iron uptake, translocation and homeostasis, the sequences encoding NAS were analyzed by searching the maize inbred line B73 genome database. Nine similar sequences encoding putative NAS family members were explored. In this study, we provided detailed information on the phylogeny, subcellular localization, expression patterns and histochemical localization of the family. In particular, the ZmNAS family was subgrouped into class I and II depending on the phylogenetic relationship between graminaceous and nongraminaceous plants. Moreover, a comparison of the expression in different tissues and under various metal status provides further evidence for the specialization of ZmNAS in iron acquisition and homeostasis.
Identification and cloning of ZmNASgenes
BLAST analysis for the maize Nicotianamine Synthase genes ( ZmNAS ) based on the genome database
Given name (previous name)
Genomic locus (bp)
cDNA length (bp)
Subcellular localization of ZmNASs
Complementary expression patterns of class I and class II ZmNASgenes
Histochemical localization of ZmNASgenes
Identification of ZmNASfamily
NAS was firstly identified in barley for catalyzing the trimerization of SAM into one molecule of NA , which is a key molecular chelating divalent metal ion and facilitating metal translocation in plants. In addition, NA is also the precursor for MA biosynthesis in graminaceous plants, suggesting its critical role in regulating iron uptake and homeostasis. There is a broad consensus that NAS is ubiquitously present in higher plants, though the number of encoding genes was limited in rice and Arabidopsis[31, 34]. However, nine NAS genes were identified in barley by a combined screening strategy, indicating that NAS proteins may be encoded by a gene family and providing a possible link between the number of NAS genes and iron uptake strength . In the previous study, due to unavailability of maize genomic sequence, only three ZmNAS were identified by screening a genomic library, though five ZmNAS proteins was predicted by western analysis . Recently, many gene families were identified in maize by genomic mining, and it was also suggested that relatively more family members existed in maize than in another cereal crop, rice [38–41]. In our study, nine ZmNAS genes were systematically identified and characterized through genome wide analysis using the current version of maize inbred line B73 genome database. It is known that cereal genome undergoes two rounds of whole genome duplications associated with genome evolution. The fist occurred in all cereals before the specification of rice, sorghum and maize, whereas the second take place specifically in the lineage leading to maize . Therefore, it is not surprising to identify more genes encoding NAS in maize than in rice. Besides, the increasing biomass and enhanced iron uptake and restoration features may be another driving force for the evolution and duplication of NAS in maize and barley. It was also interesting to find that NASs from graminaceous plants were divided into two classes by phylogenetic analysis, and relative more members were existed in class I in maize and barley than in rice. It was suggested that approximately one fourth of the genes in the maize genome possess closely related paralogs resulted from the genome duplication . We found the class I ZmNAS genes duplicated as paralogs, and localized at duplicated region of maize genome, suggesting possible functional redundancy between them. Unlike class I, ZmNAS3, ZmNAS4 and ZmNAS5 share relatively lower identity, indicating a possibility of functional divergence between them. Interestingly, the paralogs, ZmNAS2;1 and ZmNAS2;2, are consisted of two full length NAS domain in tandem repeated. It was previously reported bacterium expressed ZmNAS2 (ZmNAS2;1) exhibited no NAS activity , though expression analysis revealed that ZmNAS2;1/2;2 accumulated in roots and stems, and responded to fluctuated environmental iron status. Anyway, in vivo evidence are necessary to exclude (or confirm) the possibility they are not pseudogenes.
Cytoplasm localization of ZmNAS
It can be assumed that the subcellular localization of NAS may affect NA compartmentalization in plant cell, and thus regulate the downstream utilizing of NA as an iron chelator or a precursor of MAs. It has been reported “particular vesicles” formed in the Fe-deficient barley root cells, which was suggested as the sites secreting MAs . Pervious study showed ZmNAS1 (ZmNAS1;1) and ZmNAS2 (ZmNAS2;1) located to spot organelles in the cytoplasm, while ZmNAS3 distributed throughout the cytoplasm. The spot organelles were suggested as vesicles derived from the endoplasmic reticulum, which was thought to be the place for MAs synthesis . In our study, the subcellular localization of each ZmNAS was determined by transient expressing the GFP fusion proteins in Arabidopsis mesophyll protoplasts (Figure 3) and onion epidermal cells (Additional file 3). Unexpected, all ZmNASs were localized at cytoplasm, suggesting that the N-terminal variable domain has little effect on subcellular localization. Since it is generally considered that over accumulation of the GFP fusion protein may lead to spot-like localization, the distinct results obtained between the present and pervious study may due to different transcription strength of the GFP fusion protein. Because the spot-like organelles in cytoplasm were not characterized in detail, further study concerning the subcellular localization of NAS family proteins should be applied by alternative methods, such as immunofluorescence.
The complementary expression patterns of class I and class II ZmNASgenes links to their specific physiological functions
To date, the underlying mechanisms regulating iron uptake and translocation in plants are still not well understood, as well as the delicate transcriptional regulatory network involved in response to fluctuating environmental iron status. It has been reported the genes in strategy II Fe uptake system, such as YS1/YSL[21, 45], NAS[6, 33, 34], NAAT, DAMS and TOM1 (a MAs efflux transporter) , were strongly induced under Fe deficiency, while those associated with metal detoxification were stimulated in response excessive environmental Fe . Since the NA concentrations in tomato increase in response to Fe overload , arose the possibility that NA may play a critical role in regulating the balance between acquisition of environmental Fe and detoxification of excessive intracellular Fe. Therefore, it would be worthy to determine the response of ZmNAS genes to fluctuated environmental Fe status. It has been showed that the expression of OsNAS1 and OsNAS2 were increased in both roots and leaves under Fe deficiency, while that of OsNAS3 was decreased in leaves and induced in roots in response to Fe deficiency . Similar results were observed for ZmNAS1 and ZmNAS2, though ZmNAS3 was the first one reported to be repressed in roots under Fe deficiency .
Biofortification of maize with high level of bioavailable Fe and Zn
Micronutrient deficiencies are mainly responsible for “hidden hunger”. In particular, the anemia caused by iron-deficiency is a prevalent nutrient problem in developing countries . Maize is a major cereal crop for food supply and feed industry worldwide, though the lack of bioavailable Fe in corn can barely meet the demand. Therefore, addition essential metal elements were usually added in feeds to fulfil daily needs of animals. Alternatively, transgenic approaches can be applied to biofortificate the micronutrient content of crop plants. In the past, efforts were made in overexpressing ferritin from soybean and Phaseolus vulgaris in rice, and the Fe content was increased up to 3 and 2 fold [50, 51]. Recently, NAS was chosen as a new candidate for improving micronutrient content. It was showed that activation of OsNAS3 led to enhanced bioavailable Fe and Zn . Similar result was obtained for OsNAS1 and OsNAS2. Endosperm specific overexpression of OsNAS1 enhance the Fe and Zn content up to 1.45 and 1.55 fold in unpolished grains, respectively . Likewise, the Fe content in seeds of OsNAS2-activated rice was 3 fold higher than wild type . Moreover, it was found that endosperm specific expression of OsNAS1 could avoid negative effects on agronomic performance caused by constitutively overexpression , which suggested the original expression profile of NAS is essential for Fe homeostasis and thus affects plant growth. Therefore, the temporal and spatial RNA accumulation patterns of ZmNAS genes detected in this study may provide a delicate strategy to biofortificate maize with increased bioavailable iron.
In this study, nine NAS genes in maize were identified by genomic mining. According to the evolutionary relationship of NAS from maize, barley, rice and Arabidopsis, ZmNAS and HvNAS can be subgrouped into two classes. Moreover, the temporal and spatial RNA accumulation patterns of ZmNAS genes were investigated in various organs including developing seeds, which further support the classification of ZmNAS gene family. Histochemical localizations of the ZmNAS1;1/1;2 and ZmNAS3, which belongs to class I and class II, were determined by in situ hybridization. The complementary expression patterns of ZmNAS genes indicate maintaining sufficient NA is essential for overcoming fluctuating iron status. It was also hypothesized that the class I ZmNAS may be mainly responsible for supporting the precursor for MAs synthesis and long distance translocation of Fe, while the class II ZmNAS produce NA for local distribution of Fe and detoxification of excess intracellular Fe. These results provide significant insights into the molecular bases of ZmNAS in balancing iron uptake, translocation and homeostasis.
Maize inbred line B73 was surface-sterilized and germinated in vermiculite for 12 days in a greenhouse at 28°C. Then the seedlings were transferred into culture boxes and hydroponically grown to three-leaf stage in Hoagland nutrient solution. For metal-deficient treatment, the seedlings were transferred to Hoagland solution lacking indicated metals. For Fe and Zn excess treatment, 500 μM Fe3+-EDTA and 200 μM ZnSO4 were used. The shoots and roots from treated seedlings were sampled at indicated times and immediately frozen in liquid nitrogen and stored at −80°C until use. To detect the histochemical localization of ZmNAS, the samples were collected from 96 h treated seedlings and fixated in FAA.
Identification of maize NASgenes
The sequences encoding putative NAS family members were identified using the TBLASTN program from the MaizeSequence database (http://www.maizesequence.org), using the protein sequence of previously identified ZmNAS1 as a query. The threshold of e-value and score for TBLASTN was set at 1e-80 and 600, respectively. In order to confirm the predicted genes encode ZmNASs, the protein sequences were searched in the Pfam database (http://pfam.sanger.ac.uk). In addition, full length coding cDNA sequences of all ZmNAS genes were further confirmed by cloning and sequencing. The primers used for cloning ZmNAS genes were listed in Additional file 4.
Sequence alignment and phylogenetic tree construction
The deduced protein sequences of ZmNAS proteins were aligned with AtNAS1 and OsNAS1 using ClustalX 2.0.5 program. The phylogenetic tree was constructed with NAS proteins from Maize (Zm), Barley (Hv), Rice (Os), Arabidopsis thaliana (At) and Solanum lycopersicum (chlN) using the neighbor-joining method in MEGA 4.0 software. The proteins and their accession numbers used for alignment and phylogenetic tree construction are as follows: ZmNAS1;1 [MaizeSequence:GRMZM2G385200], ZmNAS1;2 [MaizeSequence:GRMZM2G312481], ZmNAS2;1 [MaizeSequence:GRMZM2G030036], ZmNAS2;2 [MaizeSequence:GRMZM2G124785], ZmNAS3 [MaizeSequence:GRMZM2G478568], ZmNAS4 [MaizeSequence:GRMZM2G439195], ZmNAS5 [MaizeSequence:GRMZM2G050108], ZmNAS6;1 [MaizeSequence:GRMZM2G704488], and ZmNAS6;2 [MaizeSequence:AC233955.1_FGT003] from Maize (Zea mays); NASHOR1 [GenBank:AF136941], NASHOR2 [GenBank:AF136942], HvNAS1 [GenBank:AB010086], HvNAS2 [GenBank:AB011265], HvNAS3 [GenBank:AB011264], HvNAS4 [GenBank:AB011266], HvNAS5 [GenBank:AB011268], HvNAS6 [GenBank:AB011269] and HvNAS7 [GenBank:AB019525] from barley (Hordeum vulgare), OsNAS1 [GenBank:AB021746], OsNAS2 [GenBank:AB023818] and OsNAS3 [GenBank:AB023819] from rice (Oryza sativa); AtNAS1 [GenBank:NM_120577], AtNAS2 [GenBank:NM_124990], AtNAS3 [GenBank:NM_100794] and AtNAS4 [GenBank:NM_104521] from Arabidopsis thaliana; chlN [GenBank:AJ242045] from Lycopersicon esculen-tum.
RNA isolation and real-timeRT-PCR analysis
Total RNA was isolated using TRIzol reagent according to the manufacturer’s instructions (Invitrogen) Genomic DNA contaminants were removed from RNA samples using DNaseI (NEB). The amount and quality of the total RNA was confirmed by electrophoresis in 1% formamide agarose gel. Approximately 2 μg of total RNA was reverse transcribed to cDNA in 20 μL reaction using oligo-dT and M-MLV reverse transcriptase (Fermentas). Real-time PCR primers were designed to amplify a 100–200 bp fragments in untranslated regions. All primers were designed for 60°C annealing and their sequences are as follows: ZmNAS1;1/1;2, 5'-GAGGAGATGGCGACCACGACAGAGC-3′ and 5′-AGAAGTGCATGAGAAATTCAGAAGC-3′; ZmNAS2;1/2;2, 5′-AGTGCTGCAAGATGGAGGCGAAC-3′ and 5′-AGTTACACGAGAGATTGAAACAG-3′; ZmNAS3, 5′-GGCTCACCAGAAGATGGAGGAG-3′ and 5′-TCACGCATGTGGTGTAGACACG-3′; ZmNAS4, 5′-CACGGCACACACCACAAGCAACAAG-3′ and 5′-ATCCATGCGGTGTGGGCACATAGAC-3′; ZmNAS5, 5′-ACCGGCGTCCTCGCTTTCTTGTC-3′ and 5′-ACGATATGCGGATGCGGTCAGCCAG-3′; ZmNAS6;1/6;2 5′-CTTGCAGCACCAAGTTGTCGAAC-3′ and 5′-CATGGAAGTTGTGGTTGCTACGG-3′; ZmActin1, 5′-ATGTTTCCTGGGATTGCCGAT-3′ and 5′-CCAGTTTCGTCATACTCTCCCTTG-3′. Real-time RT-PCR was performed with an ABI7500 cycler (Applied Biosystems) using the SYBR Premix Ex-Taq master Mix (TakaRa). Reactions were performed in a total volume of 20 μL with 2 μL of 20×diluted cDNA, 0.2 mM gene-specific primers and 10 μL of 2×SYBR premix. The PCR conditions were initial denaturation at 95°C for 30 s, followed by 40 cycles composed of 5 s denaturation at 95°C and 34 s of annealing/extension at 60°C. To verify specific amplification, melting-curve analysis was performed and the PCR products were separated by electrophoresis and sequenced. Data were analyzed with the ABI7500 software (version 2.0.5) via the ΔΔCT method, and the expression levels of ZmActin1 were used as an internal control. For all real-time PCR analysis, two biological replicates were used and three technical replicates were performed for each biological replicate.
Subcellular localization of the ZmNAS-GFP fusion protein
The coding region of GFP was amplified with the following primers, 5′- CTCGAGGGATCCCCGGGAATTCCATGGAGCTCGGTACCTCTAGAATGGTGAGCAAGGGCGAG 3′ and 5′- TACTAGTTTACTTGTACAGCTCGTCCATGC -3′, and the resulting fragment was cloned into the XhoI-XbaI sites of plant expression vector pRTL2 to generate the plasmid pRTL2GFP. To examine the subcellular localization of ZmNAS proteins, the entire coding region of each gene were cloned in between the cauliflower mosaic virus 35S promoter and GFP of pRTL2GFP vector. The primers used for cloning coding regions of ZmNAS genes are listed in Additional file 5. The ZmNAS-GFP fusion constructs were transformed into Arabidopsis mesophyll protoplasts as described previously . After incubation in the dark at 26°C for 14 h, the fluorescence was examined using a confocal microscope (LSM700; Carl Zeiss).
mRNA in situhybridization
In situ hybridization was performed as described previously  with slight modifications. The shoots and roots were collected from Fe-deficient and excessive treated seedlings and fixed in FAA solution containing 50% ethanol, 5% acetic acid, and 3.7% formaldehyde. To examine the mRNA localization of ZmNAS1;1/1;2 and ZmNAS3, the specific sequences corresponding to the 3′-region of mRNA were amplified with the following primers, ZmNAS1;1/1;2, 5′- TTCCATGGATCGTCGATCCTGAGGACATTCGTC -3′ and 5′- TTACTAGTAGAAGTGCATGAGAAATTCAGAAGC -3′; ZmNAS3, 5′- TTAAGCTTACTCCGTCATCATCGCCCGCAAGC -3′ and 5′- TTACTAGTAAATTAGGCCAGCCTGTTCGCTC -3′; The PCR products were cloned into the vector pEasy-T3 to generate pEasy-NAS1ISH and pEasy-NAS3ISH, then the resulting plasmids were sequenced and linerized. The Digoxigenin-labeled sense and antisense RNA probes were in vitro transcripted by T7 and SP6 RNA polymerase (Roche) using SpeI and NcoI digested pEasy-NAS1ISH, and SpeI and HindIII digested pEasy-NAS3ISH, respectively. The hybridization was performed with a probe concentration of 0.4 ng μL-1 at 55°C in a wet chamber. The enzyme-catalyzed insoluble purple signal was visualized with a Zeiss Axioscop 4.0 microscope and photographed (Zeiss Mrc5, Germany).
Deoxymugineic acid synthase
Yellow strip like transporter
Green fluorescent protein.
This work was supported by the National Natural Science Foundation of China (grant number 31101095) and by the National Special Program for GMO Development of China (grant number 2008ZX08003-002).
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