Genome-wide analysis of NAAT, DMAS, TOM, and ENA gene families in maize reveals their roles in regulating iron homeostasis

Genome-wide analysis of NAAT, DMAS, TOM, and ENA gene families in maize reveals their roles in regulating iron homeostasis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Genome-wide analysis of NAAT, DMAS, TOM, and ENA gene families in maize reveals their roles in regulating iron homeostasis Xin Zhang, Xiaojin Zhou, Suzhen Li, Jiaxing Huang, Sen Pang, Rumei Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-19256/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Nicotianamine (NA) serves as not only the major chelator for iron transport but also the intermediate for synthesizing mugineic acid family phytosiderophores (MAs) which are secreted by graminaceous plants for Fe uptake. Therefore, the production and secretion of MAs are key steps for maintaining iron homeostasis in plants. Nicotianamine aminotransferase (NAAT), 2’-deoxymugineic acid synthase (DMAS), MAs efflux transporter (TOM), and efflux transporter of NA (ENA) were identified to be involved in these processes in rice and barley, whereas little systematic study has been performed in maize (Zea mays.L). Results: Here, we identified five ZmNAAT, nine ZmDMAS, eleven ZmTOM, and two ZmENA genes in maize by genome mining. RNA-sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of these genes exhibited diverse tissue specificity and different responses to environmental iron conditions. Moreover, the expression patterns were related to their evolution relationships. In particular, the ZmNAAT family can be classified into two subgroups, with one group showed inhibited expression in root under iron excess status and another subclass were repressed in shoot under both iron deficiency and excess. Likewise, the expression of ZmDMAS1 was stimulated under iron deficiency, while the remaining genes fell into two sub-clades with different expression patterns. Significant up-regulation of ZmTOM1, ZmTOM3 and ZmENA1 were observed under iron starvation, while ZmTOM2 was induced under both iron-excess and deficiency. These results reflect changing demands for the synthesis and secretion of NA/MAs to balance iron homeostasis under fluctuating conditions. All the examined ZmNAAT and ZmDMAS proteins localized in cytoplasm, while plasma and tonoplast membrane, endomembrane, and vesicle localization were observed for ZmTOM and ZmENA proteins. These results indicate that ZmTOM and ZmENA proteins may contribute to not only intercellular export but also intracellular sequestration of NA and MAs to facilitate iron homeostasis. Conclusions: Our results suggest that different gene expression profiles and subcellular localization of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA members may enable dedicate regulation of NA and phytosiderophores (PS) metabolism, shedding light on the understanding of iron-homeostasis in maize. Additionally, we also provided candidate genes for breeding iron-rich maize varieties. Epigenetics & Genomics Maize iron homeostasis nicotianamine aminotransferase 2’-deoxymugineic acid synthase MAs efflux transporter efflux transporter of NA expression profiles subcellular localization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Iron (Fe) is an essential micronutrient for all organisms and plays an important role in numerous cellular functions in plants, including respiration, photosynthesis, and chlorophyll biosynthesis, as well as cofactors of many enzymes. Deficiency of iron in diet leads to health problems and more than a billion people suffer from different levels of anemia resulting from insufficient iron intake [ 1 ]. In plants, the deficiency of Fe causes leaf senescence, and therewith limits plant growth and reduces the quality and yield [ 2 ]. However, excessive amounts of iron generates toxic effects, because free Fe gives rise to reactive oxygen species which can damage the membrane lipids [ 3 ]. Correspondingly, plants have evolved a well-balanced iron uptake, chelation, transport, and storage mechanism to maintain sufficient Fe level while avoid over accumulation of Fe in cells [ 4 , 5 ]. Despite of rich content of iron in the soil, it is not readily bioavailable for plant uptake as the majority of iron is insoluble in ferric (Fe 3+ ) form under aerobic conditions [ 6 ]. Therefore, plants have developed two distinct strategies for iron acquisition [ 7 ]. Non-graminaceous plants use a reduction strategy (strategy I), which involves the secretion of protons and phenolic acids from root cells to the rhizosphere to acidify the soil and increase the solubility of ferric. Ferric is then reduced to ferrous (Fe 2+ ), which is transported into root cells by iron-regulated transporter 1 (IRT1) [ 8 , 9 ]. In contrast, strategy II is predominantly characterized in graminaceous plants, which relies upon biosynthesis of MAs which are secreted to rhizosphere by MAs efflux transporter 1 (TOM1) [ 10 , 11 ]. Fe 3+ -MAs chelators are formed in the rhizosphere and transported into plant root cells by YELLOW STRIPE 1/ YELLOW STRIPE 1–LIKE (YS1/YSL) iron transporters [ 12 ]. The synthetic pathway of MAs is conserved in graminaceous plants, which uses NA as a precursor. First, NA is synthesized from three molecules of S-adenosyl-L-methionine by nicotianamine synthase (NAS), which step is shared by both strategy I and II as NA chelates to ferrous and assists iron trafficking in both graminaceous and nongraminaceous plants [ 13 – 15 ]. NA is then converted to deoxymugineic acid (DMA) by nicotianamine aminotransferase and 2’-deoxymugineic acid synthase [ 16 , 17 ]. Although DMA is the final product of PS synthesized in rice, it can be further converted into MA by a hydroxylation reaction in barley and other graminaceous plants [ 18 ]. Iron is transported in a chelated form in plants, due to its low solubility and high reactivity. DMA, NA, and citrate, are three major Fe-chelators which assist long distance trafficking of iron in various species [ 19 – 21 ]. Since DMA-Fe 3+ and NA-Fe 2+ have been detected in phloem sap of rice, they are proposed to be chelators for phloem Fe transport [ 22 – 24 ]. In accordance with these evidences, OsYSL2, OsYSL9, and OsYSL15 were characterized as iron transporters in phloem with different selectivity for NA-Fe 2+ or DMA-Fe 3+ , indicating possible switching of chelator between NA and DMA in phloem [ 25 – 27 ]. Citrate is considered to play a dominant role in xylem Fe transport, because citrate-Fe 3+ and citrate-Fe 2+ were detected in xylem sap of Arabidopsis and rice ( Oryza sativa .L), meanwhile knockout of a citrate efflux transporter OsFRDL1 leads to decreased citrate and Fe contents in the xylem sap [ 19 , 28 ]. Since concentrations of DMA and NA in xylem are significantly lower than citrate, they are considered to play minor roles in xylem Fe transport. However, in response to Fe deficiency, DMA accumulates in rice xylem sap and the expression of DMA efflux transporters, OsTOM1 and OsTOM2 were induced in root stele, indicating that DMA serves as a compensatory chelator in xylem Fe transport in response to fluctuating Fe status [ 11 , 29 , 30 ]. Interference of PS synthesis and release generates strong Fe deficient phenotypes and growth defects. Two classical maize chlorosis mutants, ys1 and ys3 , are characterized by yellow stripe phenotype (yellow interveinal regions and green veins), which is result from impaired MA-Fe 3+ uptake and disrupted PS secretion, respectively [ 31 , 32 ]. Moreover, typical Fe deficiency has also been observed as a result of disturbed DMA/NA metabolism. Constitutive expression of HvNAAT in transgenic tobacco plants leads to over consumption of NA and thereby causes interveinal chlorosis in young leaves [ 33 ]. In summary, these observations imply that regulation of NA-PS metabolism is crucial for Fe-homeostasis in graminaceous plants. In line with these speculation, recent studies revealed that different DMA/NA ratio impacted the content of Zn and Fe in embryo and endosperm, and altered the local distribution patterns of Fe in embryo [ 34 ], shedding light on the balance between DMA and NA affects Fe-homeostasis. In addition to providing precursors for DMA synthesis, NA in the cytoplasm is also consumed by efflux transporter of NA 1 (ENA1), which possibly exports NA out of cells and transports NA between intracellular compartments [ 35 ]. Therefore, NA seems to act as a hub for PS metabolism, because it not only serves as the biosynthetic intermediate of PS, but also regulates metal transport. However, in spite of progresses in functional characterizations of individual NAAT, DMAS, TOM, and ENA members in rice and barley, there are still many gaps in the understanding of their regulation mechanisms in response to fluctuating environmental Fe status. It has been reported that the expression of NAAT and DMAS genes are induced in roots under Fe deficiency, indicating accumulation of DMA is necessary for dealing with Fe deficient conditions [ 16 , 36 ]. Besides, the release of NA and DMA/MAs are also regulated by different members of efflux transporter, as OsTOM1 expressed in root cells is involved in the secretion of MAs to the rhizosphere, while OsTOM2 and OsTOM3 with specific expression patterns associate with iron transport in a narrow arrange of tissues [ 30 ]. These observations suggest different expression specificity, subcellular localization, and possible enzyme activity of NAAT, DMAS, TOM, and ENA provides various levels of regulation in the production and secretion of DMA/MAs. Maize is not only a major crop worldwide, but also a monocotyledon model plant. Although the iron content in corn is higher than that in brown rice [ 37 ], it still hardly meet the growing demand of food and feed industry. Therefore, breeding maize varieties with enriched iron content is of essential, and the understanding of NA-MAs metabolism pathway may provide key information and gene resources. We previously reported the duplication of NAS family genes in maize, which suggests that dedicated regulation of NA synthesis is required for balancing DMA synthesis and NA secretion. This result arises the question that whether NAAT, DMAS, TOM, and ENA were also encoded by mutli-gene families, thereby regulate DMA synthesis and NA/DMA export in response to changing demands of Fe uptake, transport, detoxication, and storage. It has been reported that NAAT and DMAS genes are duplicated in bread wheat, but there is no systematic study of NAAT , DMAS , TOM , and ENA genes in maize in spite of functional identification of ZmDMAS1 and ZmTOM1. In this study, genes encoding NAAT, DMAS, TOM, and ENA were explored in maize genome. Additionally, we provided detailed information on subcellular localization, and expression patterns in different tissues as well as in response to fluctuating environmental Fe conditions. Our results provide a better understanding of the regulation of NA-MAs metabolism and give gene resources for biofortification in Fe-enriched maize varieties. Results Genome-wide identification of genes associated with the synthesis and secretion of PS in maize In order to identify enzymes for the biosynthesis of PS, as well as transporters for NA and MAs, the maize B73 genome (v4) was mined using identified NAAT, DMAS, TOM and ENA proteins as quires (Table S1). Consistent with the observation that ZmNAS genes have duplicated in maize, we found 5, 9, 11, and 2 genes encoded putative ZmNAAT, ZmDMAS, ZmTOM, and ZmENA proteins, respectively. Table 1 Detailed information of ZmNAAT, ZmDMAS, ZmTOM , and ZmENA genes in maize Gene name Gene ID Chromosome NO. Genomic locus(bp) Protein length (AA) CDS length (bp) cDNA length (bp) ZmNAAT1 a Zm00001d053281 4 223,892,837 − 223,896,295 434 1305 1543 ZmNAAT-L1 Zm00001d007462 2 232,247,283 − 232,255,516 474 1425 1685 ZmNAAT-L2 Zm00001d048736 4 4,607,651-4,610,357 455 1368 1771 ZmNAAT-L3 Zm00001d053107 4 214,129,873 − 214,132,827 438 1317 1694 ZmNAAT-L4 Zm00001d016441 5 162,429,370 − 162,433,900 440 1323 1792 ZmDMAS1a Zm00001d028360 1 32,049,647 − 32,052,463 314 945 1209 ZmDMAS-L1 Zm00001d003524 2 46,658,256 − 46,660,165 329 990 1310 ZmDMAS-L2 Zm00001d003525 2 46,712,428 − 46,719,096 360 1083 4502 ZmDMAS-L3 Zm00001d005932 2 193,258,782 − 193,262,494 343 1032 1393 ZmDMAS-L4 Zm00001d042869 3 182,508,943 − 182,514,234 310 933 1470 ZmDMAS-L5 Zm00001d000060 10 292,300 − 293,833 358 1077 1339 ZmDMAS-L6 Zm00001d025057 10 102,026,193 − 102,028,561 313 942 1465 ZmDMAS-L7 Zm00001d025528 10 121,509,129 − 121,526,342 344 1035 3967 ZmDMAS-L8 Zm00001d025533 10 121,567,456 − 121,569,106 331 996 1350 ZmTOM1 a Zm00001d041111 3 97,974,955 − 97,982,185 476 1431 1780 ZmTOM2 a Zm00001d052435 4 189,981,671 − 189,987,040 589 1770 2560 ZmTOM3 a Zm00001d005001 2 153,144,332 − 153,148,743 492 1479 2001 ZmTOM-L1 Zm00001d031789 1 201,868,285 − 201,873,117 503 1512 1903 ZmTOM-L2 Zm00001d005002 2 153,238,644 − 153,242,689 502 1509 1917 ZmTOM-L3 Zm00001d040422 3 42,672,281 − 42,680,006 541 1626 2344 ZmTOM-L4 Zm00001d040468 3 45,120,037–45,134,869 473 1422 1708 ZmTOM-L5 Zm00001d040947 3 81,710,564 − 81,722,446 317 954 1900 ZmTOM-L6 Zm00001d044640 3 233,816,809 − 233,822,007 494 1485 1796 ZmTOM-L7 Zm00001d052434 4 189,873,712 − 189,877,696 508 1527 1988 ZmTOM-L8 Zm00001d008227 8 1,969,000–1,975,048 480 1443 1761 ZmENA1 Zm00001d052532 4 192,393,400 − 192,396,824 452 1359 1615 ZmENA2 Zm00001d014611 5 55,313,125 − 55,314,896 188 567 714 a Previous identified genes, Gene ID is obtained from Gramene ( http://ensembl.gramene.org/Zea_mays/ ), No : number, bp : base pair, CDS : coding sequence, AA : amino acid The gene ID, chromosome location, and deductive opening reading frame (ORF) length are listed in Table 1 . Previous identified genes were annotated, and newly-identified genes were named as ZmNAAT-like1-4 , ZmDMAS-like1-8 , ZmTOM-like1-8 , and ZmENA1-2 according to their chromosome locations. The amino acid length for ZmNAAT and ZmDMAS proteins were ranging between 434–474 AA and 310–360 AA respectively, suggesting conserved catalytic activity within these families. Both TOM and ENA proteins belong to the major facilitator superfamily (MFS), but they were varied significantly in protein lengths, as 317–589 AA and 188–452 AA, respectively. This result indicates that TOM and ENA proteins putatively vary in substrate affinity or subcellular localization. It was proposed that duplication of genes usually derived from chromosome rearrangements [ 38 ]. Therefore, the chromosome location of ZmNAAT , ZmDMAS , ZmTOM , and ZmENA genes were mapped on the maize genome (Fig. 1 ). We found that 22 out of 27 genes were located on chromosome 2, 3, 4, 10, while the rest of 5 genes were mapped on chromosome 1, 5, 8. Noteworthy, we found several closely localized gene pairs, including ZmDMAS-L1/ZmDMAS-L2 , ZmDMAS-L7/ZmDMAS-L8 , ZmTOM3/ZmTOM-L2 , ZmTOM-L3/ZmTOM-L4 , and ZmTOM-L7/ZmTOM2/ZmENA1 . Phylogenetic Analysis And Conserved Domains The deduced amino acid sequences of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA proteins were aligned with functional characterized isoforms (Fig. 2 ). High identities were observed within NAAT and DMAS proteins (Fig. 3 A and 3 B), while TOM and ENA members exhibited modest similarities (Fig. 3 C and 3 D). Besides, a variable N-terminal region was found in NAAT proteins, which may relate with potential functional divergence. To gain an insight into the phylogenetic relationships of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA proteins with their homologs in other species, a phylogenetic tree was constructed using Neighbor-joining (NJ) method (Fig. 2 ). ZmNAAT proteins are closely related to their rice homologs, as ZmNAAT1 and ZmNAAT-L4 form a sub-cluster with previously characterized OsNAAT1, while ZmNAAT-L1, ZmNAAT-L2, and ZmNAAT-L3 form another sub-cluster with OsNAAT2 and OsNAAT3, suggesting similar functions within the same sub-cluster (Fig. 2 A). In addition, we found a close relationship between HvNAATs and TaNAATs (Fig. 2 A), indicating that these genes may arose prior than the emergence of barley and wheat. The in vitro DMA synthesis activity of OsDMAS1and ZmDMAS1 were reported, and they are highly orthologous to each other [ 17 ]. The amino acid sequences of ZmDMAS-L1-L8 were highly conserved with ZmDMAS1 and OsDMAS1 (Fig. 3 B), and they both had three conserved motifs for Aldo/keto reductase activity, indicating these newly identified ZmDMAS genes may have the synthesis activity. However, the phylogenetic distance between ZmDMAS1 and ZmDMAS-like proteins are further than that between ZmDMAS1 and OsDMAS1 (Fig. 2 B), suggesting that ZmDMAS-L1-L8 may evolved after the divergence of maize and rice. Although TOM and ENA belong to MFS family, they fall into different clusters, suggesting functional divergence (Fig. 2 C). Expression profiles of ZmNAAT , ZmDMAS , ZmTOM , and ZmENA genes under Fe-excessive and Fe-deficient conditions The synthesis and secretion pathway of PS are known to be essential for Fe acquisition in roots, while increasing evidences indicate that NA, DMA and MAs are important for chelating Fe in phloem tissues and thereby they are involved in Fe transport and detoxification in vegetative tissues. Therefore, we examined the responses of ZmNAAT , ZmDMAS , ZmTOM , and ZmENA genes under different environmental Fe conditions using qRT-PCR. Serving as enzymes regulating the first step of DMA synthesis, the expression of ZmNAAT genes exhibited two types of patterns. The transcript accumulation of ZmNAAT1 and ZmNAAT-L4 were significantly reduced in Fe-excessive roots, whereas ZmNAAT-L2 and ZmNAAT-L3 showed reduction trends in both Fe-deficient and Fe excessive conditions in shoots (Fig. 4 ). Interestingly, the distinct expression profile of ZmNAAT genes was consistent with their phylogenetic classification, as ZmNAAT-L2/ZmNAAT-L3 and ZmNAAT1/ZmNAAT-L4 belongs to different sub-clades, suggesting potential function divergence. ZmDMAS genes also showed different response to fluctuating environmental Fe status. The function of ZmDMAS1 was characterized in mediating NA/DMA synthesis for Fe uptake in roots [ 17 ]. In Fe-starved root tissues, ZmDMAS1 was stimulated as expected, while ZmDMAS-L4 and ZmDMAS-L 6 (Fig. 4 ) repressed. In Fe-excessive shoot samples, the expression of ZmDMAS-L2 , ZmDMAS-L3 , ZmDMAS-L7 , and ZmDMAS-L8 were induced (Fig. 4 ). Besides, the different expression patterns of ZmDMAS genes also match their phylogenetic divergence. The up-regulation of ZmDMAS genes in shoots under high environmental Fe status may reflect the demands for DMA synthesis, which facilities Fe transport and detoxification (Fig. 4 ). ZmTOM1 was characterized as the causal gene for ys3 mutant [ 32 ]. We found the expression of ZmTOM1 and ZmTOM3 were significantly induced in Fe-deficient root, suggesting their functions of MAs secretion in root. In Fe-starved shoots, the transcript accumulation of ZmTOM-L7 was increased, while that of ZmTOM-L5 was reduced. Moreover, ZmTOM2 was stimulated in both Fe-deficient and Fe-excess shoots. The up-regulation of ZmTOM genes observed in shoots, indicating they may be involved in Fe transport (Fig. 4 ). Unlike ZmTOM1, the physiological function of ZmENA1 was not reported. We found the expression of ZmENA1 was increased in response to Fe-starvation, while that was repressed by Fe-excess (Fig. 4 ). Gene expression profiles in different tissues We analyzed the expression of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA genes in different tissues using RNA-seq data (Figure 5) and qRT-PCR (Figure 6). The heatmap was generated using normalized expression values of 79 samples covering the whole lifespan of maize. Accordingly, the expression patterns of genes associated with PS synthesis and secretion can be classified into 5 groups, including leaf preferred (ZmDMAS1-L1/L2/L3/L4/L5/L8, ZmNAAT-L2/L4, ZmTOM2/L3), embryo preferred (ZmTOM-L7, ZmDMAS1-L7), root preferred (ZmTOM-1/3/L2/L6, ZmDMAS1/L6, ZmNAAT1/L1/L3, ZmENA2), endosperm specific (ZmTOM-L1), and anther specific (ZmTOM-L8). Interestingly, previously identified ZmNAAT1, ZmDMAS1, and ZmTOM1 exhibited root preferential expression pattern, suggesting they may work together in PS synthesis and secretion in root cells. Besides, newly identified genes expressed in almost all tissues with different specificities, implying essential roles of PS in Fe trafficking and storage. Since expression value of some genes were not available in the RNA-sequencing data set, qRT-PCR was used to determine and further verify expression of these genes in different tissues (root, crow root, stem, leaf, ear and tassel before pollination and developing embryo and endosperm). Interestingly, we found some genes exhibited abundant accumulation in ear and/or tassel, including ZmNAAT1/L2/L4, ZmDMAS1/L2/L3/L4/L7, and ZmTOM1/3/L5/L7, suggesting the involvement of PS-mediated Fe trafficking in maize reproductive development. Reduced specificities were also observed by qRT-PCR, as some leaf- and root- preferential genes showed expression in stem, tassel, ear and developing seeds, which is possibly due to different set of tissues and/or developmental stages were applied. Additionally, the expression patterns of ZmTOM-L4/L5 and ZmENA1 were revealed by qRT-PCR. ZmTOM-L4 was expressed preferentially in both leaf and root, while the transcript of ZmTOM-L5 was mainly detected in embryo at 12 days after pollination (DAP). ZmENA1 expressed in almost all tissues with relatively high expression in ear and embryo on 12 DAP. Subcellular Localization The localization of enzymes and transporters may determine the subcellular compartments or organelles where synthesis and secretion of PS take place. Therefore, to study the subcellular localization of ZmNAAT, ZmDMAS, ZmTOM and ZmENA proteins, green fluorescent protein (GFP) -fusion proteins of randomly selected isoforms were transiently expressed in maize mesophyll protoplasts. As showed in Figure 7, we found that all the selected ZmNAAT and ZmDMAS proteins (ZmNAAT1, ZmNAAT-L4, ZmDMAS1, and ZmDMAS-L1) localized in cytoplasm and nucleus, which pattern was also revealed for GFP control. In contrast, ZmTOM and ZmENA proteins were determined to localize at membrane systems. Since both the plasma membrane and complex endomembrane localization were observed for ZmTOM1, ZmENA1, and ZmENA2, an endoplasmic reticulum (ER) marker was co-transformed to determine the inner membrane. Unexpectedly, we could not find perfect match between mcherry fluorescence of ER and GFP signals in both single and z-stacked images, indicating the inner membrane localizations are not ER. Besides, we found spot-like signals for ZmTOM1 and ZmENA2, suggesting they are also targeted to small vesicles. Noteworthy, ZmTOM2 was found localized to both tonoplast and spot-like vesicles, indicating it may mediate import-flux of PS into vacuolar. Discussion The graminaceous plants use a chelation strategy to uptake enough iron and prevent overaccumulation of Fe, in regulating these processes the balance of NA-MAs metabolism plays a crucial role. DMA and MAs are secreted into the rhizosphere for acquisition of Fe 3+ , while NA and DMA are also essential for intercellular, phloem, and possible xylem Fe transport. Since NA also serves as the intermediate for DMA/MAs biosynthesis, the export of NA which mediated by ENA and conversion of DMA which mediated by NAAT and DMAS are thus key steps affecting Fe homeostasis in plants. Over-consumption of NA by constitutively expressing HvNAAT in transgenic tobacco plants leads to interveinal chlorosis in young leaves [ 33 ]. Similarly, in spite of the high content of iron in leaves and roots, lack of NA result in chlorosis leaf in the tomato mutant chloronerva [ 39 ]. These results indicate that NA not only plays role in long-distance metal transfer but also in the regulation of metal transport among cells. Besides, disrupted PS secretion also generates yellow stripe phenotypes. The maize ys3 mutant was identified in the defect of MA release, and the expression of ZmTOM1 was also notably decreased [ 31 , 40 ]. Therefore, it can be concluded that disturbed metabolism and transport of NA/DMA may lead to inadequate Fe uptake and distribution. Many studies revealed that NAS genes are encoded by multi-gene families in a broad range of graminaceous plants, including maize, rice, wheat and barley [ 15 , 41 – 43 ]. ZmNAS genes were firstly grouped into two classes, class I and class II, and then this classification was further applied in other species [ 41 , 43 ]. Moreover, it was found that class I NAS genes were preferentially expression in root and stem, and their expressions were up-regulated in response to iron deficiency; while class II genes were mainly expressed in leaf and they were induced under excess iron conditions [ 41 , 44 , 45 ]. In line with these observations, recent expression and function study of OsNAS3 suggests that NA synthesized by OsNAS3 under excess Fe conditions is associated with Fe detoxification, redistribution, and storage, while NA produced by OsNAS1 and OsNAS2 under normal Fe conditions may play roles in assisting Fe transport [ 46 ]. These results indicate that two classes of NAS genes may be deferentially regulated in mRNA level under fluctuating Fe status, by which mechanism they may associated with different transporters or enzymes, and thereby participate in either NA secretion or DMA synthesis to balancing Fe uptake, transport and storage. Meanwhile, it raises a question of whether enzymes associated with NA-DMA metabolism (NAAT and DMAS) and transports corresponding for NA/DMA secretion (ENA and TOM) are also encoded by multi-gene families, which provides chances for dedicate regulation of these pathways. Previous studies reported that NAAT and DMAS genes were duplicated in wheat. In addition, six NAATs were identified in rice, while only OsNAAT1 responded to iron fluctuations [ 47 ]. Here, in spite of previously reported ZmNAAT1, ZmDMAS1 , and ZmTOM1/2/3 , we newly identified 4 ZmNAATs , 8 ZmDMASs , 8 ZmTOMs and 2 ZmENAs in maize. The alignment shows that ZmNAAT and ZmDMAS are highly conserved in amino acid sequence (Fig. 3 A and 3 B). However, their expressions were differently regulated in response to environmental Fe conditions, and the expression patterns were correlated with phylogenetic classifications. ZmNAAT1 and ZmNAAT-L4 belonged to the same sub-class and they were repressed in Fe-excessive roots, whereas ZmNAAT-L2 and ZmNAAT-L3 were grouped into another sub-class and they exhibited reduced expression in both Fe-deficient and Fe excessive conditions in shoots (Fig. 4 ). Likewise, three different expression patterns were observed for ZmDMAS genes. In response to Fe deficiency, ZmDMAS1 showed similar expression trend with its orthologs, OsDMAS1 , HvDMAS1 , OsDMAS1 , and TaDMAS1 , as they were induced in root [ 17 ]. The closely phylogenetic relationship and similar expression trends under changing environmental Fe conditions indicate that ZmDMAS1 plays similar function as OsDMAS1. Indeed, both ZmDMAS1 and OsDMAS1 showed DMA synthesis activities in vitro . Moreover, the physiological function of OsDMAS1 and its involvement in mediating Fe homeostasis was further confirmed by knock-down plants [ 48 ]. In contrast to the increased expression of ZmDMAS1 under Fe deficiency, the accumulation of ZmDMAS-L2 , ZmDMAS-L3 , ZmDMAS-L7 , and ZmDMAS-L8 were stimulated in Fe-excessive shoots, while the expression of ZmDMAS-L4 and ZmDMAS-L 6 were repressed in roots under Fe-deficient conditions. These newly identified expression trends of DMAS family genes correlated with their classifications, indicating potential roles of these genes in balancing Fe uptake and homeostasis. Although TOM and ENA belong to MFS family, they were less conserved in amino acid sequences which may lead to different substrate specificity. ENA was characterized as an efflux transporter of NA and the expression of OsENA1 was strongly up-regulated under Fe-deficient conditions [ 11 , 35 ]. Similarly, induced expression in response to Fe-starvation was also observed for ZmENA1 . As another sub-class of MFS family, TOM was firstly identified as a MA efflux transporter in rice and barley, and the expression of both OsTOM1/2 and HvTOM1 were induced in Fe deficient roots [ 11 , 30 ]. Interestingly, only three TOM and two ENA encoding genes were identified in rice, though they had a broad spectrum of expression patterns. OsTOM1 expressed in both shoot and root, while OsTOM2 accumulates transcript in the epithelium, scutellum, and dorsal vascular bundles of seeds [ 11 , 30 ]. The expression of OsENA1 was stimulated under Fe deficiency [ 35 ]. Here, we found that the expression of ZmENA1 was induced and repressed in shoot under Fe deficiency and excess, respectively. Besides, it was observed that the TOM gene family expanded in maize, and the expression of these ZmTOM genes exhibited diverse tissue specificity and different responses to environmental conditions. Even though most of ZmTOM genes were leaf and root-preferentially expressed, embryo ( ZmTOM-L5/L7 ), endosperm ( ZmTOM-L1 ) and anther ( ZmTOM-L8 ) preferred expression pattern were also identified. In addition, the expression of ZmTOM1 and ZmTOM3 were both induce by Fe-deficiency, while that of ZmTOM2 was stimulated under both Fe-excess and deficiency. Therefore, it can be speculated that duplication of ZmTOM genes may result from the need for dedicate regulation of DMA/MAs secretion in maize. The subcellular compartmentalization of enzymes and transporters provides another layer of functional regulation. It was proposed that NA and DMA are synthesized in specific vesicles, which may derive from ER [ 49 , 50 ]. In line with this model, OsNAS2 was found to localize in moving vesicles, depending on its tyrosine (YXXφ) and di-leucine (LL) motifs [ 51 ]. These vesicles were considered to be the place for NA and MAs synthesis, and provided a compartment sequestrating MA from cytosol to prevent disturbance of Fe homeostasis. However, since these vesicles could not fuse directly with the cell membrane, unidentified transporters might be needed to transport NA/MAs form vesicles into cytoplasm [ 51 ]. Different from OsNAS, we previous revealed ZmNAS proteins were distributed uniformly in the cytoplasm of mesophyll protoplasts [ 41 ]. In line with this observation, all the examined ZmNAAT and ZmDMAS proteins showed cytoplasm localization, suggesting the synthesis of NA and DMA takes place in the cytoplasm in maize. Interestingly, ZmTOM1, ZmENA1, and ZmENA2 localized at plasma membrane and endomembrane. Moreover, spot-like signals were also observed for ZmTOM1 and ZmENA2. Likewise, OsTOM1 and OsTOM2 were determined to locate on the cell membrane [ 11 , 30 ], while OsENA1 localized mainly to the plasma membrane and partially to vesicular structures in rice roots [ 35 ]. This result indicates that OsENA1 is responsible for NA trafficking between cell membrane and cellular compartments by vesicular transport. Therefore, we speculate that, in maize, the cytosolic NA and MAs might be exported into vesicles and out of cell membrane by ZmENA and ZmTOM family proteins. Since the vesicles were proposed to be derived from ER [ 52 ], we applied an ER-marker to determine the nature of endomembrane. Unexpectedly, it seems that the endomembrane localization of ZmTOM1, ZmENA1, and ZmENA2 is not ER, though it may interact or related to ER as similar but not identical pattern were found for GFP and mcherry signals. Vacuole, chloroplast, and mitochondria are considered to act as cellular Fe pools, and the mobilization of Fe between cytoplasm and these organelles play essential roles in maintaining Fe hemostasis [ 53 – 55 ]. ZINC-INDUCED FACILITATOR 1 (ZIF1) is a vacuolar membrane MFS family protein which was hypothesized to transport NA from cytoplasm into vacuoles [ 56 ]. Here, ZmTOM2 was found to target to tonoplast and vesicles, suggesting that ZmTOM2 may act in both vacuolar and vesicular transport of MAs. In summary, our results indicate that ZmTOM and ZmENA proteins may contribute to not only the export of NA/MAs into intercellular space, but also the sequestration of NA/MAs into vacuolar and vesicles to detoxify excess Fe. It is crucial to improve micro-essential nutrients in human diet by enhancing the iron content in cereal grains. To date, several approaches were taken to enhance iron content in seeds, including increasing the transcript accumulation of genes associated with Fe uptake and transport, as well as modifying expression of endosperm specific genes [ 54 , 57 , 58 ]. It is noteworthy that excessive production of NA lead to subsequently increasing of DMA, which may increase Fe content in seed[ 34 ]. Consistently, different NA to DMA ratio regulated by NAS and NAAT had different effects on Fe content in grains [ 34 , 59 ]. Therefore, exploring enzymes involved in the synthesis and transport of NA and DMA may provide a theoretical basis to optimize iron biofortification in cereals. Conclusions In this study, we identified five ZmNAAT , nine ZmDMA , eleven ZmTOM , and two ZmENA genes in maize. In addition, the phylogenetic relationship, subcellular localization, and gene expression patterns in different tissue and under fluctuating environmental conditions were also determined. We found NAAT and DMAS members are highly conserved in protein sequence, and they can be classified into different subgroups. Moreover, genes within these subgroups showed distinct expression patterns in response to deficient and excess Fe conditions, reflecting functional divergence between different sub-classes. Likewise, various expression profiles were also observed for ZmTOM and ZmENA genes. These results suggest a changing balance of the metabolism and secretion of NA/PS is needed for Fe homeostasis under fluctuating Fe conditions. All the examined ZmNAAT and ZmDMAS proteins localized in cytoplasm, whereas plasma and tonoplast membrane, endomembrane, and vesicle localization were observed for ZmTOM and ZmENA proteins. This result indicates that ZmTOM and ZmENA proteins may contribute to not only intercellular export but also intracellular sequestration of NA and PS. In spite of expression in root, stem and leaf, we found abundant transcript accumulation of these genes in tassel, ear, and developing seeds, indicating essential roles of NA and PS in facilitating Fe transport during reproductive developments. In summary, expansion of genes associated with the biosynthesis and secretion of PS may provide various levels of regulation in substrate affinity, enzymatic activity, expression, and subcellular compartmentalization, which suggests that dedicate regulation of NA-PS metabolism contributes to the Fe-homeostasis in maize. Methods Plant material and growth The maize inbred line Z58 was provided by Chinese Academy of Agricultural Sciences, and planted in a greenhouse. For expression analysis in different tissues, we collected root, crown, stem, leaf, ear, and tassel before pollination. In addition, embryo and endosperm at 12 DAP, 21 DAP, and 28 DAP were also collected for determine expression patterns in developing seeds. For different environmental Fe treatments, Z58 seedlings were cultured at 28 °C with 16-h-light/8-h-dark photoperiod. The seeds were germinated and the seedlings were incubated in normal Hoagland nutrient solution with standard Fe content (c(Fe 3+ ) = 100 µm/L ) until trefoil stage. Then, the seedlings were transferred to the Hoagland nutrient solution without Fe or with excessive Fe content (c(Fe 3+ ) = 500 µm/L) for deficient and excess Fe treatment, respectively. Shoots and roots were harvested at 24 h, 48 h and 96 h after different Fe-treatments. Seedlings sampled prior to Fe-treatments were used as controls (0 h). All samples were frozen by liquid nitrogen immediately and quickly stored at -80 °C. Bioinformatics Analysis In order to identify the NAAT , DMAS , TOM , and ENA family genes in maize, the previously characterized members of these families in rice ( Oryza sativa .L) and maize were used as queries (Table S1). The TBLASTN program at Gramene ( http://http://ensembl.gramene.org/Zea_mays/Tools/Blast ) was used to obtain the full length cDNAs. All acquired genes were mapped to maize chromosomes according to their locations. The amino acid sequences of selected genes were compared with known genes by the MegAlign software of Lasergene using Clustal W method, and the alignments were visualized by GeneDoc software. The phylogenetic tree was constructed for NAAT, DMAS, TOM and ENA proteins from different species using the Neighbor-Joining method in MEGA version 4.0. Expression Analysis Using RNA-Seq Data The expression values in various tissues of all selected genes were retrieved from previous RNA-Seq data of 79 tissues covering a whole life-span of maize [ 60 ]. The heatmap was construed using Cluster 3.0 software, and the result was visualized using Treeview software. Quantitative Real-time PCR Total RNA was extracted using a Plant RNA Kit (Transgen, Beijing, China) following the manufacturer’s instruction. The first strand cDNA was obtained by reverse transcription using cDNA TranScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, Beijing, China). Primers for qRT-PCR assays (Table S2) were designed using the Primer 3.0 website ( http://bioinfo.ut.ee/primer3-0.4.0/ ). qRT-PCR were performed in 20 µL volume containing 10 µL SYBR Green (Takara, Japan), 0.4 µL ROX II (Takara, Japan), 0.2 µM gene-specific primers, and 5 µL 5 × diluted cDNA. The reaction was performed using the ABI 7500 Real Time Thermal Cycler. The expression of ZmActin1 was used as an internal control (Table S2). For qRT-PCR assays, three biological replicates were applied, with three technical replicates per biological replicate. Subcellular localization To construct plant transient expression vectors, the amplified ORFs were cloned into the XhoI-XbaI site of plasmid pRTL-2NGFP [41], which express C-terminal GFP fusion proteins. Gene-specific primers were designed to amplify the full length ORFs without stop codons (Table S2). To determine the subcellular localization of randomly selected NAAT, DMAS, ENA, and TOM1 family proteins, the GFP-fusion proteins were co-expressed in maize mesophyll protoplasts with a mcherry-fused ER marker [61] . For mesophyll protoplast transformation, plasmids were extracted using the Wizard Plus Miniprep DNA Purification System kit (Promega, Beijing, China). Maize seedlings were grown in a green house in dark. Protoplasts were extracted and transformed using polyethylene glycol 4000 as described previously [62]. After co-transformation, the protoplasts were incubated in dark at 26°C for 12-16 h. Then, a confocal microscope (LSM700; Carl Zeiss) was used to visualize the fluorescence. GFP and mcherry signal were excitated at 488 nm and 555 nm, while they were collected under 500-530 nm and 610 nm emission, respectively. The autofluorescence of chlorophyll were observed using the 630 emission filter. ZEN light edition 2009 software was used for picture processing. Abbreviations NA: Nicotianamine; MAs: mugineic acid family phytosiderophores; NAAT: Nicotianamine aminotransferase; DMAS: 2’-Deoxymugineic acid synthase; TOM: MAs efflux transporter; ENA: efflux transporter of NA; RNA-seq: RNA sequencing; Fe: qRT-PCR: Quantitative reverse transcription polymerase chain reaction; PS: Phytosiderophores; Iron; Fe 3+ : Ferric; Fe 2+ : Ferrous; DMA: Deoxymugineic acid; MFS: Major facilitator superfamily; NJ: Neighbor-joining; IRT1: Iron-regulated transporter 1; PS: Phytosiderophores; YS1: YELLOW STRIPE 1; YSL: YELLOW STRIPE 1-LIKE; NAS: Nicotianamine synthase; DAP: Days after pollination; ER: endoplasmic reticulum; GFP: Green fluorescent protein. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this published article [and its supplementary information files]. Competing interests The authors declare that they have no competing interests. Funding This study was supported by the National Special Program for GMO Development of China (grant number 2016ZX08003-002). The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Authors' contributions XJZ conceived and designed the study. XZ and XJZ conducted the bioinformatics analysis. XZ contributed to perform the experiments and drafted the manuscript. SZL provided ER maker. XJZ, SZL, JXH, SP, and RMC revised the manuscript. All authors read and approved the final manuscript. Acknowledgments We are grateful to anonymous reviewers for comments on this manuscript. 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A multi-color set of in vivo organelle markers for colocalization studies in Arabidopsis and other plants[J]. 2007, 51(6): 1126-1136. 62 Yoo SD, Cho YH, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis[J]. Nat Protoc, 2007, 2(7): 1565-1572. Supplementary Files additionalfile2.docx additionalfile1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-19256","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":460141,"identity":"3856731d-599b-4730-9448-de9b6ec6e310","order_by":1,"name":"Xin Zhang","email":"","orcid":"","institution":"Biotechnology Research Institute，Chinese Academy of Agricultural Sciences \u0026 Department of Applied Chemistry, College of Science, China Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zhang","suffix":""},{"id":460142,"identity":"6584400b-9fed-4bda-bff7-0a9f4af2986f","order_by":2,"name":"Xiaojin Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYDACCRBhwMDA2MzA+IBkLcwGJGiBADYJ3MqQgPzs5mPSPAV2csztvMcqftTY2TOwnz2AVwvjnGNpkjMMko0Zm/nSbvYcS05s4MlLwKuFWSLHTOKDAXNiYzOP2W0GNuYEBgke/H5iA2lJMKgHaylm+FdvT1ALD8SWw2AtzIxthxkbCGmRkEhLtpxhcBzoFx5jyd6+44ltPDn4tcjPSD54m+dPtZxh/xnDDz++Vdvzs58hMn4MG2C+I049yDqiVY6CUTAKRsGIAwCmyzhb2pHjggAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-3046-5808","institution":"Biotechnology Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiaojin","middleName":"","lastName":"Zhou","suffix":""},{"id":460143,"identity":"a6cee330-dc56-4bbd-b394-dd0ceeb5f03e","order_by":3,"name":"Suzhen Li","email":"","orcid":"","institution":"Biotechnology Research Institute,Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Suzhen","middleName":"","lastName":"Li","suffix":""},{"id":460144,"identity":"0362bc4c-7fd4-4f2b-87b9-8b24cb2896a1","order_by":4,"name":"Jiaxing Huang","email":"","orcid":"","institution":"Department of Applied Chemistry, College of Science, China Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiaxing","middleName":"","lastName":"Huang","suffix":""},{"id":460145,"identity":"4429a752-659c-4b52-92bd-9f43e671f87f","order_by":5,"name":"Sen Pang","email":"","orcid":"","institution":"Department of Applied Chemistry, College of Science, China Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sen","middleName":"","lastName":"Pang","suffix":""},{"id":460146,"identity":"c6532cd6-7bf2-4abf-8bcb-afd14425c7bc","order_by":6,"name":"Rumei Chen","email":"","orcid":"","institution":"Biotechnology Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rumei","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2020-03-24 12:18:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-19256/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-19256/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":833336,"identity":"22e1883c-5498-4ad2-8c58-279fe82d5bdd","added_by":"auto","created_at":"2020-04-03 15:29:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":72305,"visible":true,"origin":"","legend":"The chromosomal locations of NAAT, DMAS, TOM, and ENA genes in maize. The positions of identified genes were mapped on the genome of maize. The length (bp) of the chromosome and the position (bp) of each gene are indicated at the bottom and left side of each chromosome, respectively. The green, blue, purple, and red lines indicate ZmNAAT, ZmDMAS, ZmTOM, and ZmENA genes, respectively.","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-19256/v1/fig1.png"},{"id":833338,"identity":"9d0a8f10-db3b-409c-8f06-5d1121693405","added_by":"auto","created_at":"2020-04-03 15:29:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":117263,"visible":true,"origin":"","legend":"Neighbor-joining phylogenetic trees of NAAT, DMAS, TOM, and ENA members from various species. a Phylogenetic tree of NAAT proteins; b Phylogenetic tree of DMAS proteins; c Phylogenetic tree of TOM and ENA proteins. The phylogenetic trees were built with proteins from maize (Zm), wheat (Ta), rice (Os), and barley (Hv), using the neighbor-joining method in MEGA 4.0 software. The proteins and accession numbers used in phylogenetic trees can be referenced in the methods. The scale bar corresponds to a distance of 10 changes per 100 amino acid positions.","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-19256/v1/fig2.png"},{"id":833339,"identity":"a670911f-7b74-452b-9b1c-4d6a7f0255ae","added_by":"auto","created_at":"2020-04-03 15:29:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":408196,"visible":true,"origin":"","legend":"The multiple sequence alignments of NAAT, DMAS, TOM, and ENA proteins. Amino acid alignment of NAAT (a), DMAS (b), TOM (c), and ENA (d) proteins were performed using Clustal W method. N-Ter: N-terminus; AKR1: Aldo/Keto reductase family signature 1; AKR2: Aldo/Keto reductase family signature 2; AKR3: Aldo/Keto reductase family putative active site signature; TMD: Transmembrane region. The light or dark shaded backgrounds suggest segmental or entire conservative residues.","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-19256/v1/fig3.png"},{"id":833340,"identity":"429cf783-ab54-44e5-a53b-45cd29aec83e","added_by":"auto","created_at":"2020-04-03 15:29:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":156697,"visible":true,"origin":"","legend":"Expression profiles of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA genes in response to different Fe treatments. The maize seedlings were cultured to three-leaf stage in standard Hoagland solution, and then they were transferred to Hoagland solution with 0 μmol/L and 500 μmol/L Fe for deficiency (Fe--) and Fe excess (Fe++) treatments, respectively. The shoots (sh) and roots (Root) were harvested at 0 h, 24 h, 48 h, and 96 h after treatments. Maize Actin1 was used to normalize relative gene expressions. The error bars indicate standard deviations.","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-19256/v1/fig4.png"},{"id":833341,"identity":"6d0f5193-bf6f-459b-84cb-9c44965c9eb0","added_by":"auto","created_at":"2020-04-03 15:29:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":126266,"visible":true,"origin":"","legend":"Heatmap showing the expression patterns of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA genes in different tissues and development stages. The heatmap was generated using RNA-Seq data of 79 samples covering the whole lifespan of maize. Color scale represents expression intensity as log2-fold change.","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-19256/v1/fig5.png"},{"id":833342,"identity":"8851456f-db5d-4ae9-8b21-300e17b2555e","added_by":"auto","created_at":"2020-04-03 15:29:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":222981,"visible":true,"origin":"","legend":"Expression profiles of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA genes in developing seeds and different organs. Total RNA was extracted from endosperm (En) and embryo (Em) at indicated days after pollination (12, 21, and 28 DAP), as well as different organs including root (Root), crown (Crow), stem (Stem), leaf (Leaf) and tassel (Tass). Maize Actin1 was used for normalizing relative expressions of each gene. The error bars indicate standard deviations.","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-19256/v1/fig6.png"},{"id":833343,"identity":"a9059221-adf3-43c6-a974-27dc133aea05","added_by":"auto","created_at":"2020-04-03 15:29:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":382956,"visible":true,"origin":"","legend":"Subcellular localization of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA proteins in Maize mesophyll protoplasts. GFP was fused with the C-terminal of each gene and the fusion proteins were co-expressed with a mcherry-fused ER marker in maize mesophyll protoplasts. The GFP signal is indicated in green, the ER marker is indicated in red and chlorophyll autofluorescence (Chl) is shown in blue. The images were obtained by a confocal microscope, and both z-tacked and single optical slide of the merged channels are shown. The cytoplasm localization of GFP was used as a control. The scale bar represents 10 μm.","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-19256/v1/fig7.png"},{"id":13496904,"identity":"d42a3b89-a8ed-40d9-9c44-0515c2cac33b","added_by":"auto","created_at":"2021-09-16 22:51:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1548981,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-19256/v1/d7d3a8d0-70b5-46ee-b4a8-4bef4b73cba0.pdf"},{"id":833335,"identity":"ace58b81-5148-4ac1-a3eb-2241f135db33","added_by":"auto","created_at":"2020-04-03 15:29:21","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":22885,"visible":true,"origin":"","legend":"","description":"","filename":"additionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-19256/v1/additional file 2.docx"},{"id":833337,"identity":"1f72859b-6e3d-48d2-92e0-f8c600d8fd2c","added_by":"auto","created_at":"2020-04-03 15:29:21","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16511,"visible":true,"origin":"","legend":"","description":"","filename":"additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-19256/v1/additional file 1.docx"}],"financialInterests":"","formattedTitle":"Genome-wide analysis of NAAT, DMAS, TOM, and ENA gene families in maize reveals their roles in regulating iron homeostasis","fulltext":[{"header":"Background","content":" \u003cp\u003eIron (Fe) is an essential micronutrient for all organisms and plays an important role in numerous cellular functions in plants, including respiration, photosynthesis, and chlorophyll biosynthesis, as well as cofactors of many enzymes. Deficiency of iron in diet leads to health problems and more than a billion people suffer from different levels of anemia resulting from insufficient iron intake [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In plants, the deficiency of Fe causes leaf senescence, and therewith limits plant growth and reduces the quality and yield [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, excessive amounts of iron generates toxic effects, because free Fe gives rise to reactive oxygen species which can damage the membrane lipids [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Correspondingly, plants have evolved a well-balanced iron uptake, chelation, transport, and storage mechanism to maintain sufficient Fe level while avoid over accumulation of Fe in cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite of rich content of iron in the soil, it is not readily bioavailable for plant uptake as the majority of iron is insoluble in ferric (Fe\u003csup\u003e3+\u003c/sup\u003e) form under aerobic conditions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, plants have developed two distinct strategies for iron acquisition [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Non-graminaceous plants use a reduction strategy (strategy I), which involves the secretion of protons and phenolic acids from root cells to the rhizosphere to acidify the soil and increase the solubility of ferric. Ferric is then reduced to ferrous (Fe\u003csup\u003e2+\u003c/sup\u003e), which is transported into root cells by iron-regulated transporter 1 (IRT1) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In contrast, strategy II is predominantly characterized in graminaceous plants, which relies upon biosynthesis of MAs which are secreted to rhizosphere by MAs efflux transporter 1 (TOM1) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Fe\u003csup\u003e3+\u003c/sup\u003e-MAs chelators are formed in the rhizosphere and transported into plant root cells by YELLOW STRIPE 1/ YELLOW STRIPE 1\u0026ndash;LIKE (YS1/YSL) iron transporters [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The synthetic pathway of MAs is conserved in graminaceous plants, which uses NA as a precursor. First, NA is synthesized from three molecules of S-adenosyl-L-methionine by nicotianamine synthase (NAS), which step is shared by both strategy I and II as NA chelates to ferrous and assists iron trafficking in both graminaceous and nongraminaceous plants [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. NA is then converted to deoxymugineic acid (DMA) by nicotianamine aminotransferase and 2\u0026rsquo;-deoxymugineic acid synthase [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although DMA is the final product of PS synthesized in rice, it can be further converted into MA by a hydroxylation reaction in barley and other graminaceous plants [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIron is transported in a chelated form in plants, due to its low solubility and high reactivity. DMA, NA, and citrate, are three major Fe-chelators which assist long distance trafficking of iron in various species [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Since DMA-Fe\u003csup\u003e3+\u003c/sup\u003e and NA-Fe\u003csup\u003e2+\u003c/sup\u003e have been detected in phloem sap of rice, they are proposed to be chelators for phloem Fe transport [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In accordance with these evidences, OsYSL2, OsYSL9, and OsYSL15 were characterized as iron transporters in phloem with different selectivity for NA-Fe\u003csup\u003e2+\u003c/sup\u003e or DMA-Fe\u003csup\u003e3+\u003c/sup\u003e, indicating possible switching of chelator between NA and DMA in phloem [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Citrate is considered to play a dominant role in xylem Fe transport, because citrate-Fe\u003csup\u003e3+\u003c/sup\u003e and citrate-Fe\u003csup\u003e2+\u003c/sup\u003e were detected in xylem sap of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eArabidopsis\u003c/span\u003e and rice (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOryza sativa\u003c/span\u003e.L), meanwhile knockout of a citrate efflux transporter OsFRDL1 leads to decreased citrate and Fe contents in the xylem sap [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Since concentrations of DMA and NA in xylem are significantly lower than citrate, they are considered to play minor roles in xylem Fe transport. However, in response to Fe deficiency, DMA accumulates in rice xylem sap and the expression of DMA efflux transporters, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOsTOM1\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOsTOM2\u003c/span\u003e were induced in root stele, indicating that DMA serves as a compensatory chelator in xylem Fe transport in response to fluctuating Fe status [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterference of PS synthesis and release generates strong Fe deficient phenotypes and growth defects. Two classical maize chlorosis mutants, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eys1\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eys3\u003c/span\u003e, are characterized by yellow stripe phenotype (yellow interveinal regions and green veins), which is result from impaired MA-Fe\u003csup\u003e3+\u003c/sup\u003e uptake and disrupted PS secretion, respectively [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Moreover, typical Fe deficiency has also been observed as a result of disturbed DMA/NA metabolism. Constitutive expression of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eHvNAAT\u003c/span\u003e in transgenic tobacco plants leads to over consumption of NA and thereby causes interveinal chlorosis in young leaves [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In summary, these observations imply that regulation of NA-PS metabolism is crucial for Fe-homeostasis in graminaceous plants. In line with these speculation, recent studies revealed that different DMA/NA ratio impacted the content of Zn and Fe in embryo and endosperm, and altered the local distribution patterns of Fe in embryo [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], shedding light on the balance between DMA and NA affects Fe-homeostasis.\u003c/p\u003e \u003cp\u003eIn addition to providing precursors for DMA synthesis, NA in the cytoplasm is also consumed by efflux transporter of NA 1 (ENA1), which possibly exports NA out of cells and transports NA between intracellular compartments [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, NA seems to act as a hub for PS metabolism, because it not only serves as the biosynthetic intermediate of PS, but also regulates metal transport. However, in spite of progresses in functional characterizations of individual NAAT, DMAS, TOM, and ENA members in rice and barley, there are still many gaps in the understanding of their regulation mechanisms in response to fluctuating environmental Fe status. It has been reported that the expression of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eNAAT\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eDMAS\u003c/span\u003e genes are induced in roots under Fe deficiency, indicating accumulation of DMA is necessary for dealing with Fe deficient conditions [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Besides, the release of NA and DMA/MAs are also regulated by different members of efflux transporter, as \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOsTOM1\u003c/span\u003e expressed in root cells is involved in the secretion of MAs to the rhizosphere, while \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOsTOM2\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOsTOM3\u003c/span\u003e with specific expression patterns associate with iron transport in a narrow arrange of tissues [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These observations suggest different expression specificity, subcellular localization, and possible enzyme activity of NAAT, DMAS, TOM, and ENA provides various levels of regulation in the production and secretion of DMA/MAs.\u003c/p\u003e \u003cp\u003eMaize is not only a major crop worldwide, but also a monocotyledon model plant. Although the iron content in corn is higher than that in brown rice [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], it still hardly meet the growing demand of food and feed industry. Therefore, breeding maize varieties with enriched iron content is of essential, and the understanding of NA-MAs metabolism pathway may provide key information and gene resources. We previously reported the duplication of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eNAS\u003c/span\u003e family genes in maize, which suggests that dedicated regulation of NA synthesis is required for balancing DMA synthesis and NA secretion. This result arises the question that whether NAAT, DMAS, TOM, and ENA were also encoded by mutli-gene families, thereby regulate DMA synthesis and NA/DMA export in response to changing demands of Fe uptake, transport, detoxication, and storage. It has been reported that \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eNAAT\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eDMAS\u003c/span\u003e genes are duplicated in bread wheat, but there is no systematic study of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eNAAT\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eDMAS\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eTOM\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eENA\u003c/span\u003e genes in maize in spite of functional identification of ZmDMAS1 and ZmTOM1. In this study, genes encoding NAAT, DMAS, TOM, and ENA were explored in maize genome. Additionally, we provided detailed information on subcellular localization, and expression patterns in different tissues as well as in response to fluctuating environmental Fe conditions. Our results provide a better understanding of the regulation of NA-MAs metabolism and give gene resources for biofortification in Fe-enriched maize varieties.\u003c/p\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003cp\u003eGenome-wide identification of genes associated with the synthesis and secretion of PS in maize\u003c/p\u003e \u003cp\u003eIn order to identify enzymes for the biosynthesis of PS, as well as transporters for NA and MAs, the maize B73 genome (v4) was mined using identified NAAT, DMAS, TOM and ENA proteins as quires (Table S1). Consistent with the observation that \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAS\u003c/span\u003e genes have duplicated in maize, we found 5, 9, 11, and 2 genes encoded putative ZmNAAT, ZmDMAS, ZmTOM, and ZmENA proteins, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003eDetailed information of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT, ZmDMAS, ZmTOM\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmENA\u003c/span\u003e genes in maize\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eGene name\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eGene ID\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003eChromosome NO.\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003eGenomic locus(bp)\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003eProtein length (AA)\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003eCDS length (bp)\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003ecDNA length (bp)\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT1\u003c/span\u003e\u003csup\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ea\u003c/span\u003e\u003c/sup\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d053281\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e223,892,837\u0026thinsp;\u0026minus;\u0026thinsp;223,896,295\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e434\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1305\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1543\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT-L1\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d007462\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e232,247,283\u0026thinsp;\u0026minus;\u0026thinsp;232,255,516\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e474\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1425\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1685\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT-L2\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d048736\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e4,607,651-4,610,357\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e455\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1368\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1771\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT-L3\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d053107\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e214,129,873\u0026thinsp;\u0026minus;\u0026thinsp;214,132,827\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e438\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1317\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1694\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT-L4\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d016441\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e5\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e162,429,370\u0026thinsp;\u0026minus;\u0026thinsp;162,433,900\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e440\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1323\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1792\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS1a\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d028360\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e1\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e32,049,647\u0026thinsp;\u0026minus;\u0026thinsp;32,052,463\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e314\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e945\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1209\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L1\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d003524\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e46,658,256\u0026thinsp;\u0026minus;\u0026thinsp;46,660,165\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e329\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e990\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1310\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L2\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d003525\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e46,712,428\u0026thinsp;\u0026minus;\u0026thinsp;46,719,096\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e360\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1083\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e4502\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L3\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d005932\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e193,258,782\u0026thinsp;\u0026minus;\u0026thinsp;193,262,494\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e343\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1032\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1393\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L4\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d042869\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e182,508,943\u0026thinsp;\u0026minus;\u0026thinsp;182,514,234\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e310\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e933\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1470\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L5\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d000060\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e10\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e292,300\u0026thinsp;\u0026minus;\u0026thinsp;293,833\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e358\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1077\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1339\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L6\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d025057\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e10\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e102,026,193\u0026thinsp;\u0026minus;\u0026thinsp;102,028,561\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e313\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e942\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1465\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L7\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d025528\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e10\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e121,509,129\u0026thinsp;\u0026minus;\u0026thinsp;121,526,342\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e344\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1035\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e3967\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L8\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d025533\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e10\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e121,567,456\u0026thinsp;\u0026minus;\u0026thinsp;121,569,106\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e331\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e996\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1350\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM1\u003c/span\u003e\u003csup\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ea\u003c/span\u003e\u003c/sup\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d041111\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e97,974,955\u0026thinsp;\u0026minus;\u0026thinsp;97,982,185\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e476\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1431\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1780\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM2\u003c/span\u003e\u003csup\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ea\u003c/span\u003e\u003c/sup\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d052435\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e189,981,671\u0026thinsp;\u0026minus;\u0026thinsp;189,987,040\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e589\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1770\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e2560\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM3\u003c/span\u003e\u003csup\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ea\u003c/span\u003e\u003c/sup\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d005001\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e153,144,332\u0026thinsp;\u0026minus;\u0026thinsp;153,148,743\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e492\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1479\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e2001\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L1\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d031789\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e1\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e201,868,285\u0026thinsp;\u0026minus;\u0026thinsp;201,873,117\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e503\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1512\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1903\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L2\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d005002\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e153,238,644\u0026thinsp;\u0026minus;\u0026thinsp;153,242,689\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e502\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1509\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1917\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L3\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d040422\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e42,672,281\u0026thinsp;\u0026minus;\u0026thinsp;42,680,006\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e541\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1626\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e2344\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L4\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d040468\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e45,120,037\u0026ndash;45,134,869\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e473\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1422\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1708\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L5\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d040947\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e81,710,564\u0026thinsp;\u0026minus;\u0026thinsp;81,722,446\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e317\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e954\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1900\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L6\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d044640\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e233,816,809\u0026thinsp;\u0026minus;\u0026thinsp;233,822,007\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e494\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1485\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1796\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L7\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d052434\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e189,873,712\u0026thinsp;\u0026minus;\u0026thinsp;189,877,696\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e508\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1527\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1988\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L8\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d008227\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e8\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e1,969,000\u0026ndash;1,975,048\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e480\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1443\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1761\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmENA1\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d052532\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e192,393,400\u0026thinsp;\u0026minus;\u0026thinsp;192,396,824\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e452\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1359\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e1615\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmENA2\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZm00001d014611\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e5\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e55,313,125\u0026thinsp;\u0026minus;\u0026thinsp;55,314,896\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e188\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e567\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e714\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u003c/sup\u003e Previous identified genes, Gene ID is obtained from Gramene (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ensembl.gramene.org/Zea_mays/\u003c/span\u003e\u003c/span\u003e), No : number, bp : base pair, CDS : coding sequence, AA : amino acid\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe gene ID, chromosome location, and deductive opening reading frame (ORF) length are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Previous identified genes were annotated, and newly-identified genes were named as \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT-like1-4\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-like1-8\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-like1-8\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmENA1-2\u003c/span\u003e according to their chromosome locations. The amino acid length for ZmNAAT and ZmDMAS proteins were ranging between 434\u0026ndash;474 AA and 310\u0026ndash;360 AA respectively, suggesting conserved catalytic activity within these families. Both TOM and ENA proteins belong to the major facilitator superfamily (MFS), but they were varied significantly in protein lengths, as 317\u0026ndash;589 AA and 188\u0026ndash;452 AA, respectively. This result indicates that TOM and ENA proteins putatively vary in substrate affinity or subcellular localization. It was proposed that duplication of genes usually derived from chromosome rearrangements [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, the chromosome location of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmENA\u003c/span\u003e genes were mapped on the maize genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We found that 22 out of 27 genes were located on chromosome 2, 3, 4, 10, while the rest of 5 genes were mapped on chromosome 1, 5, 8. Noteworthy, we found several closely localized gene pairs, including \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L1/ZmDMAS-L2\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L7/ZmDMAS-L8\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM3/ZmTOM-L2\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L3/ZmTOM-L4\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L7/ZmTOM2/ZmENA1\u003c/span\u003e. \u003c/p\u003e \u003c/div\u003e \n\u003cp\u003ePhylogenetic Analysis And Conserved Domains\u003c/p\u003e\n \u003cp\u003eThe deduced amino acid sequences of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA proteins were aligned with functional characterized isoforms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). High identities were observed within NAAT and DMAS proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), while TOM and ENA members exhibited modest similarities (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Besides, a variable N-terminal region was found in NAAT proteins, which may relate with potential functional divergence. To gain an insight into the phylogenetic relationships of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA proteins with their homologs in other species, a phylogenetic tree was constructed using Neighbor-joining (NJ) method (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). ZmNAAT proteins are closely related to their rice homologs, as ZmNAAT1 and ZmNAAT-L4 form a sub-cluster with previously characterized OsNAAT1, while ZmNAAT-L1, ZmNAAT-L2, and ZmNAAT-L3 form another sub-cluster with OsNAAT2 and OsNAAT3, suggesting similar functions within the same sub-cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In addition, we found a close relationship between HvNAATs and TaNAATs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), indicating that these genes may arose prior than the emergence of barley and wheat. The \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e DMA synthesis activity of OsDMAS1and ZmDMAS1 were reported, and they are highly orthologous to each other [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The amino acid sequences of ZmDMAS-L1-L8 were highly conserved with ZmDMAS1 and OsDMAS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and they both had three conserved motifs for Aldo/keto reductase activity, indicating these newly identified ZmDMAS genes may have the synthesis activity. However, the phylogenetic distance between ZmDMAS1 and ZmDMAS-like proteins are further than that between ZmDMAS1 and OsDMAS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), suggesting that ZmDMAS-L1-L8 may evolved after the divergence of maize and rice. Although TOM and ENA belong to MFS family, they fall into different clusters, suggesting functional divergence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eExpression profiles of\u003c/span\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eZmNAAT\u003c/span\u003e, \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eZmDMAS\u003c/span\u003e, \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eZmTOM\u003c/span\u003e, \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eand\u003c/span\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eZmENA\u003c/span\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003egenes under Fe-excessive and Fe-deficient conditions\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe synthesis and secretion pathway of PS are known to be essential for Fe acquisition in roots, while increasing evidences indicate that NA, DMA and MAs are important for chelating Fe in phloem tissues and thereby they are involved in Fe transport and detoxification in vegetative tissues. Therefore, we examined the responses of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmENA\u003c/span\u003e genes under different environmental Fe conditions using qRT-PCR. Serving as enzymes regulating the first step of DMA synthesis, the expression of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT\u003c/span\u003e genes exhibited two types of patterns. The transcript accumulation of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT1\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT-L4\u003c/span\u003e were significantly reduced in Fe-excessive roots, whereas \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT-L2\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT-L3\u003c/span\u003e showed reduction trends in both Fe-deficient and Fe excessive conditions in shoots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Interestingly, the distinct expression profile of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT\u003c/span\u003e genes was consistent with their phylogenetic classification, as ZmNAAT-L2/ZmNAAT-L3 and ZmNAAT1/ZmNAAT-L4 belongs to different sub-clades, suggesting potential function divergence. \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS\u003c/span\u003e genes also showed different response to fluctuating environmental Fe status. The function of ZmDMAS1 was characterized in mediating NA/DMA synthesis for Fe uptake in roots [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In Fe-starved root tissues, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS1\u003c/span\u003e was stimulated as expected, while \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L4\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L\u003c/span\u003e6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) repressed. In Fe-excessive shoot samples, the expression of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L2\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L3\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L7\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L8\u003c/span\u003e were induced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Besides, the different expression patterns of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS\u003c/span\u003e genes also match their phylogenetic divergence. The up-regulation of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS\u003c/span\u003e genes in shoots under high environmental Fe status may reflect the demands for DMA synthesis, which facilities Fe transport and detoxification (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM1\u003c/span\u003e was characterized as the causal gene for \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eys3\u003c/span\u003e mutant [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We found the expression of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM1\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM3\u003c/span\u003e were significantly induced in Fe-deficient root, suggesting their functions of MAs secretion in root. In Fe-starved shoots, the transcript accumulation of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L7\u003c/span\u003e was increased, while that of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L5\u003c/span\u003e was reduced. Moreover, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM2\u003c/span\u003e was stimulated in both Fe-deficient and Fe-excess shoots. The up-regulation of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM\u003c/span\u003e genes observed in shoots, indicating they may be involved in Fe transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Unlike ZmTOM1, the physiological function of ZmENA1 was not reported. We found the expression of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmENA1\u003c/span\u003e was increased in response to Fe-starvation, while that was repressed by Fe-excess (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \n\u003cp\u003eGene expression profiles in different tissues\u003c/p\u003e\n\u003cp\u003eWe analyzed the expression of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA genes in different tissues using RNA-seq data (Figure 5) and qRT-PCR (Figure 6). The heatmap was generated using normalized expression values of 79 samples covering the whole lifespan of maize. Accordingly, the expression patterns of genes associated with PS synthesis and secretion can be classified into 5 groups, including leaf preferred (ZmDMAS1-L1/L2/L3/L4/L5/L8, ZmNAAT-L2/L4, ZmTOM2/L3), embryo preferred (ZmTOM-L7, ZmDMAS1-L7), root preferred (ZmTOM-1/3/L2/L6, ZmDMAS1/L6, ZmNAAT1/L1/L3, ZmENA2), endosperm specific (ZmTOM-L1), and anther specific (ZmTOM-L8). Interestingly, previously identified ZmNAAT1, ZmDMAS1, and ZmTOM1 exhibited root preferential expression pattern, suggesting they may work together in PS synthesis and secretion in root cells. Besides, newly identified genes expressed in almost all tissues with different specificities, implying essential roles of PS in Fe trafficking and storage.\u003c/p\u003e\n\u003cP\u003eSince expression value of some genes were not available in the RNA-sequencing data set, qRT-PCR was used to determine and further verify expression of these genes in different tissues (root, crow root, stem, leaf, ear and tassel before pollination and developing embryo and endosperm). Interestingly, we found some genes exhibited abundant accumulation in ear and/or tassel, including ZmNAAT1/L2/L4, ZmDMAS1/L2/L3/L4/L7, and ZmTOM1/3/L5/L7, suggesting the involvement of PS-mediated Fe trafficking in maize reproductive development. Reduced specificities were also observed by qRT-PCR, as some leaf- and root- preferential genes showed expression in stem, tassel, ear and developing seeds, which is possibly due to different set of tissues and/or developmental stages were applied. Additionally, the expression patterns of ZmTOM-L4/L5 and ZmENA1 were revealed by qRT-PCR. ZmTOM-L4 was expressed preferentially in both leaf and root, while the transcript of ZmTOM-L5 was mainly detected in embryo at 12 days after pollination (DAP). ZmENA1 expressed in almost all tissues with relatively high expression in ear and embryo on 12 DAP.\u003c/p\u003e\n\u003cp\u003eSubcellular Localization\u003c/p\u003e \u003cp\u003eThe localization of enzymes and transporters may determine the subcellular compartments or organelles where synthesis and secretion of PS take place. Therefore, to study the subcellular localization of ZmNAAT, ZmDMAS, ZmTOM and ZmENA proteins, green fluorescent protein (GFP) -fusion proteins of randomly selected isoforms were transiently expressed in maize mesophyll protoplasts. As showed in Figure 7, we found that all the selected ZmNAAT and ZmDMAS proteins (ZmNAAT1, ZmNAAT-L4, ZmDMAS1, and ZmDMAS-L1) localized in cytoplasm and nucleus, which pattern was also revealed for GFP control. In contrast, ZmTOM and ZmENA proteins were determined to localize at membrane systems. Since both the plasma membrane and complex endomembrane localization were observed for ZmTOM1, ZmENA1, and ZmENA2, an endoplasmic reticulum (ER) marker was co-transformed to determine the inner membrane. Unexpectedly, we could not find perfect match between mcherry fluorescence of ER and GFP signals in both single and z-stacked images, indicating the inner membrane localizations are not ER. Besides, we found spot-like signals for ZmTOM1 and ZmENA2, suggesting they are also targeted to small vesicles. Noteworthy, ZmTOM2 was found localized to both tonoplast and spot-like vesicles, indicating it may mediate import-flux of PS into vacuolar.\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eThe graminaceous plants use a chelation strategy to uptake enough iron and prevent overaccumulation of Fe, in regulating these processes the balance of NA-MAs metabolism plays a crucial role. DMA and MAs are secreted into the rhizosphere for acquisition of Fe\u003csup\u003e3+\u003c/sup\u003e, while NA and DMA are also essential for intercellular, phloem, and possible xylem Fe transport. Since NA also serves as the intermediate for DMA/MAs biosynthesis, the export of NA which mediated by ENA and conversion of DMA which mediated by NAAT and DMAS are thus key steps affecting Fe homeostasis in plants. Over-consumption of NA by constitutively expressing \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eHvNAAT\u003c/span\u003e in transgenic tobacco plants leads to interveinal chlorosis in young leaves [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Similarly, in spite of the high content of iron in leaves and roots, lack of NA result in chlorosis leaf in the tomato mutant \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003echloronerva\u003c/span\u003e [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. These results indicate that NA not only plays role in long-distance metal transfer but also in the regulation of metal transport among cells. Besides, disrupted PS secretion also generates yellow stripe phenotypes. The maize \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eys3\u003c/span\u003e mutant was identified in the defect of MA release, and the expression of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM1\u003c/span\u003e was also notably decreased [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Therefore, it can be concluded that disturbed metabolism and transport of NA/DMA may lead to inadequate Fe uptake and distribution.\u003c/p\u003e \u003cp\u003eMany studies revealed that \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eNAS\u003c/span\u003e genes are encoded by multi-gene families in a broad range of graminaceous plants, including maize, rice, wheat and barley [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAS\u003c/span\u003e genes were firstly grouped into two classes, class I and class II, and then this classification was further applied in other species [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Moreover, it was found that class I \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eNAS\u003c/span\u003e genes were preferentially expression in root and stem, and their expressions were up-regulated in response to iron deficiency; while class II genes were mainly expressed in leaf and they were induced under excess iron conditions [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In line with these observations, recent expression and function study of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOsNAS3\u003c/span\u003e suggests that NA synthesized by OsNAS3 under excess Fe conditions is associated with Fe detoxification, redistribution, and storage, while NA produced by OsNAS1 and OsNAS2 under normal Fe conditions may play roles in assisting Fe transport [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. These results indicate that two classes of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eNAS\u003c/span\u003e genes may be deferentially regulated in mRNA level under fluctuating Fe status, by which mechanism they may associated with different transporters or enzymes, and thereby participate in either NA secretion or DMA synthesis to balancing Fe uptake, transport and storage. Meanwhile, it raises a question of whether enzymes associated with NA-DMA metabolism (NAAT and DMAS) and transports corresponding for NA/DMA secretion (ENA and TOM) are also encoded by multi-gene families, which provides chances for dedicate regulation of these pathways.\u003c/p\u003e \u003cp\u003ePrevious studies reported that \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eNAAT\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eDMAS\u003c/span\u003e genes were duplicated in wheat. In addition, six \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eNAATs\u003c/span\u003e were identified in rice, while only \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOsNAAT1\u003c/span\u003e responded to iron fluctuations [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Here, in spite of previously reported \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT1, ZmDMAS1\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM1/2/3\u003c/span\u003e, we newly identified 4 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAATs\u003c/span\u003e, 8 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMASs\u003c/span\u003e, 8 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOMs\u003c/span\u003e and 2 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmENAs\u003c/span\u003e in maize. The alignment shows that ZmNAAT and ZmDMAS are highly conserved in amino acid sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). However, their expressions were differently regulated in response to environmental Fe conditions, and the expression patterns were correlated with phylogenetic classifications. \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT1\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT-L4\u003c/span\u003e belonged to the same sub-class and they were repressed in Fe-excessive roots, whereas \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT-L2\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT-L3\u003c/span\u003e were grouped into another sub-class and they exhibited reduced expression in both Fe-deficient and Fe excessive conditions in shoots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Likewise, three different expression patterns were observed for \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS\u003c/span\u003e genes. In response to Fe deficiency, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS1\u003c/span\u003e showed similar expression trend with its orthologs, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOsDMAS1\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eHvDMAS1\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOsDMAS1\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eTaDMAS1\u003c/span\u003e, as they were induced in root [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The closely phylogenetic relationship and similar expression trends under changing environmental Fe conditions indicate that ZmDMAS1 plays similar function as OsDMAS1. Indeed, both ZmDMAS1 and OsDMAS1 showed DMA synthesis activities \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e. Moreover, the physiological function of OsDMAS1 and its involvement in mediating Fe homeostasis was further confirmed by knock-down plants [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In contrast to the increased expression of ZmDMAS1 under Fe deficiency, the accumulation of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L2\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L3\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L7\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L8\u003c/span\u003e were stimulated in Fe-excessive shoots, while the expression of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L4\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMAS-L\u003c/span\u003e6 were repressed in roots under Fe-deficient conditions. These newly identified expression trends of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eDMAS\u003c/span\u003e family genes correlated with their classifications, indicating potential roles of these genes in balancing Fe uptake and homeostasis.\u003c/p\u003e \u003cp\u003eAlthough TOM and ENA belong to MFS family, they were less conserved in amino acid sequences which may lead to different substrate specificity. ENA was characterized as an efflux transporter of NA and the expression of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOsENA1\u003c/span\u003e was strongly up-regulated under Fe-deficient conditions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Similarly, induced expression in response to Fe-starvation was also observed for \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmENA1\u003c/span\u003e. As another sub-class of MFS family, TOM was firstly identified as a MA efflux transporter in rice and barley, and the expression of both \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOsTOM1/2\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eHvTOM1\u003c/span\u003e were induced in Fe deficient roots [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Interestingly, only three \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eTOM\u003c/span\u003e and two \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eENA\u003c/span\u003e encoding genes were identified in rice, though they had a broad spectrum of expression patterns. \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOsTOM1\u003c/span\u003e expressed in both shoot and root, while \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOsTOM2\u003c/span\u003e accumulates transcript in the epithelium, scutellum, and dorsal vascular bundles of seeds [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The expression of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOsENA1\u003c/span\u003e was stimulated under Fe deficiency [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Here, we found that the expression of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmENA1\u003c/span\u003e was induced and repressed in shoot under Fe deficiency and excess, respectively. Besides, it was observed that the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eTOM\u003c/span\u003e gene family expanded in maize, and the expression of these \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM\u003c/span\u003e genes exhibited diverse tissue specificity and different responses to environmental conditions. Even though most of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM\u003c/span\u003e genes were leaf and root-preferentially expressed, embryo (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L5/L7\u003c/span\u003e), endosperm (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L1\u003c/span\u003e) and anther (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM-L8\u003c/span\u003e) preferred expression pattern were also identified. In addition, the expression of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM1\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM3\u003c/span\u003e were both induce by Fe-deficiency, while that of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM2\u003c/span\u003e was stimulated under both Fe-excess and deficiency. Therefore, it can be speculated that duplication of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM\u003c/span\u003e genes may result from the need for dedicate regulation of DMA/MAs secretion in maize.\u003c/p\u003e \u003cp\u003eThe subcellular compartmentalization of enzymes and transporters provides another layer of functional regulation. It was proposed that NA and DMA are synthesized in specific vesicles, which may derive from ER [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In line with this model, OsNAS2 was found to localize in moving vesicles, depending on its tyrosine (YXXφ) and di-leucine (LL) motifs [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. These vesicles were considered to be the place for NA and MAs synthesis, and provided a compartment sequestrating MA from cytosol to prevent disturbance of Fe homeostasis. However, since these vesicles could not fuse directly with the cell membrane, unidentified transporters might be needed to transport NA/MAs form vesicles into cytoplasm [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Different from OsNAS, we previous revealed ZmNAS proteins were distributed uniformly in the cytoplasm of mesophyll protoplasts [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In line with this observation, all the examined ZmNAAT and ZmDMAS proteins showed cytoplasm localization, suggesting the synthesis of NA and DMA takes place in the cytoplasm in maize. Interestingly, ZmTOM1, ZmENA1, and ZmENA2 localized at plasma membrane and endomembrane. Moreover, spot-like signals were also observed for ZmTOM1 and ZmENA2. Likewise, OsTOM1 and OsTOM2 were determined to locate on the cell membrane [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], while OsENA1 localized mainly to the plasma membrane and partially to vesicular structures in rice roots [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This result indicates that OsENA1 is responsible for NA trafficking between cell membrane and cellular compartments by vesicular transport. Therefore, we speculate that, in maize, the cytosolic NA and MAs might be exported into vesicles and out of cell membrane by ZmENA and ZmTOM family proteins. Since the vesicles were proposed to be derived from ER [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], we applied an ER-marker to determine the nature of endomembrane. Unexpectedly, it seems that the endomembrane localization of ZmTOM1, ZmENA1, and ZmENA2 is not ER, though it may interact or related to ER as similar but not identical pattern were found for GFP and mcherry signals. Vacuole, chloroplast, and mitochondria are considered to act as cellular Fe pools, and the mobilization of Fe between cytoplasm and these organelles play essential roles in maintaining Fe hemostasis [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. ZINC-INDUCED FACILITATOR 1 (ZIF1) is a vacuolar membrane MFS family protein which was hypothesized to transport NA from cytoplasm into vacuoles [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Here, ZmTOM2 was found to target to tonoplast and vesicles, suggesting that ZmTOM2 may act in both vacuolar and vesicular transport of MAs. In summary, our results indicate that ZmTOM and ZmENA proteins may contribute to not only the export of NA/MAs into intercellular space, but also the sequestration of NA/MAs into vacuolar and vesicles to detoxify excess Fe.\u003c/p\u003e \u003cp\u003eIt is crucial to improve micro-essential nutrients in human diet by enhancing the iron content in cereal grains. To date, several approaches were taken to enhance iron content in seeds, including increasing the transcript accumulation of genes associated with Fe uptake and transport, as well as modifying expression of endosperm specific genes [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. It is noteworthy that excessive production of NA lead to subsequently increasing of DMA, which may increase Fe content in seed[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Consistently, different NA to DMA ratio regulated by NAS and NAAT had different effects on Fe content in grains [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Therefore, exploring enzymes involved in the synthesis and transport of NA and DMA may provide a theoretical basis to optimize iron biofortification in cereals.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eIn this study, we identified five \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmNAAT\u003c/span\u003e, nine \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmDMA\u003c/span\u003e, eleven \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM\u003c/span\u003e, and two \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmENA\u003c/span\u003e genes in maize. In addition, the phylogenetic relationship, subcellular localization, and gene expression patterns in different tissue and under fluctuating environmental conditions were also determined. We found NAAT and DMAS members are highly conserved in protein sequence, and they can be classified into different subgroups. Moreover, genes within these subgroups showed distinct expression patterns in response to deficient and excess Fe conditions, reflecting functional divergence between different sub-classes. Likewise, various expression profiles were also observed for \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmTOM\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmENA\u003c/span\u003e genes. These results suggest a changing balance of the metabolism and secretion of NA/PS is needed for Fe homeostasis under fluctuating Fe conditions. All the examined ZmNAAT and ZmDMAS proteins localized in cytoplasm, whereas plasma and tonoplast membrane, endomembrane, and vesicle localization were observed for ZmTOM and ZmENA proteins. This result indicates that ZmTOM and ZmENA proteins may contribute to not only intercellular export but also intracellular sequestration of NA and PS. In spite of expression in root, stem and leaf, we found abundant transcript accumulation of these genes in tassel, ear, and developing seeds, indicating essential roles of NA and PS in facilitating Fe transport during reproductive developments. In summary, expansion of genes associated with the biosynthesis and secretion of PS may provide various levels of regulation in substrate affinity, enzymatic activity, expression, and subcellular compartmentalization, which suggests that dedicate regulation of NA-PS metabolism contributes to the Fe-homeostasis in maize.\u003c/p\u003e "},{"header":"Methods","content":" \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003cp\u003ePlant material and growth\u003c/p\u003e \u003cp\u003eThe maize inbred line Z58 was provided by Chinese Academy of Agricultural Sciences, and planted in a greenhouse. For expression analysis in different tissues, we collected root, crown, stem, leaf, ear, and tassel before pollination. In addition, embryo and endosperm at 12 DAP, 21 DAP, and 28 DAP were also collected for determine expression patterns in developing seeds. For different environmental Fe treatments, Z58 seedlings were cultured at 28\u0026nbsp;\u0026deg;C with 16-h-light/8-h-dark photoperiod. The seeds were germinated and the seedlings were incubated in normal Hoagland nutrient solution with standard Fe content (c(Fe\u003csup\u003e3+\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;100\u0026nbsp;\u0026micro;m/L ) until trefoil stage. Then, the seedlings were transferred to the Hoagland nutrient solution without Fe or with excessive Fe content (c(Fe\u003csup\u003e3+\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;500\u0026nbsp;\u0026micro;m/L) for deficient and excess Fe treatment, respectively. Shoots and roots were harvested at 24\u0026nbsp;h, 48\u0026nbsp;h and 96\u0026nbsp;h after different Fe-treatments. Seedlings sampled prior to Fe-treatments were used as controls (0\u0026nbsp;h). All samples were frozen by liquid nitrogen immediately and quickly stored at -80\u0026nbsp;\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \n\u003cp\u003eBioinformatics Analysis\u003c/p\u003e\n \u003cp\u003eIn order to identify the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eNAAT\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eDMAS\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eTOM\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eENA\u003c/span\u003e family genes in maize, the previously characterized members of these families in rice (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eOryza sativa\u003c/span\u003e.L) and maize were used as queries (Table S1). The TBLASTN program at Gramene (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://http://ensembl.gramene.org/Zea_mays/Tools/Blast\u003c/span\u003e\u003c/span\u003e) was used to obtain the full length cDNAs. All acquired genes were mapped to maize chromosomes according to their locations. The amino acid sequences of selected genes were compared with known genes by the MegAlign software of Lasergene using Clustal W method, and the alignments were visualized by GeneDoc software. The phylogenetic tree was constructed for NAAT, DMAS, TOM and ENA proteins from different species using the Neighbor-Joining method in MEGA version 4.0.\u003c/p\u003e \n\u003cp\u003eExpression Analysis Using RNA-Seq Data\u003c/p\u003e\n \u003cp\u003eThe expression values in various tissues of all selected genes were retrieved from previous RNA-Seq data of 79 tissues covering a whole life-span of maize [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The heatmap was construed using Cluster 3.0 software, and the result was visualized using Treeview software.\u003c/p\u003e\n\u003cp\u003eQuantitative Real-time PCR\u003c/p\u003e \n \u003cp\u003eTotal RNA was extracted using a Plant RNA Kit (Transgen, Beijing, China) following the manufacturer\u0026rsquo;s instruction. The first strand cDNA was obtained by reverse transcription using cDNA TranScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, Beijing, China). Primers for qRT-PCR assays (Table S2) were designed using the Primer 3.0 website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinfo.ut.ee/primer3-0.4.0/\u003c/span\u003e\u003c/span\u003e). qRT-PCR were performed in 20 \u0026micro;L volume containing 10 \u0026micro;L SYBR Green (Takara, Japan), 0.4 \u0026micro;L ROX II (Takara, Japan), 0.2\u0026nbsp;\u0026micro;M gene-specific primers, and 5 \u0026micro;L 5\u0026thinsp;\u0026times;\u0026thinsp;diluted cDNA. The reaction was performed using the ABI 7500 Real Time Thermal Cycler. The expression of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eZmActin1\u003c/span\u003e was used as an internal control (Table S2). For qRT-PCR assays, three biological replicates were applied, with three technical replicates per biological replicate.\u003c/p\u003e \n\u003cp\u003eSubcellular localization\u003c/p\u003e\n\u003cp\u003eTo construct plant transient expression vectors, the amplified ORFs were cloned into the XhoI-XbaI site of plasmid pRTL-2NGFP [41], which express C-terminal GFP fusion proteins. Gene-specific primers were designed to amplify the full length ORFs without stop codons (Table S2). To determine the subcellular localization of randomly selected NAAT, DMAS, ENA, and TOM1 family proteins, the GFP-fusion proteins were co-expressed in maize mesophyll protoplasts with a mcherry-fused ER marker [61] . For mesophyll protoplast transformation, plasmids were extracted using the Wizard Plus Miniprep DNA Purification System kit (Promega, Beijing, China). Maize seedlings were grown in a green house in dark. Protoplasts were extracted and transformed using polyethylene glycol 4000 as described previously [62]. After co-transformation, the protoplasts were incubated in dark at 26°C for 12-16 h. Then, a confocal microscope (LSM700; Carl Zeiss) was used to visualize the fluorescence. GFP and mcherry signal were excitated at 488 nm and 555 nm, while they were collected under 500-530 nm and 610 nm emission, respectively. The autofluorescence of chlorophyll were observed using the 630 emission filter. ZEN light edition 2009 software was used for picture processing.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNA: Nicotianamine; MAs: mugineic acid family phytosiderophores; NAAT: Nicotianamine aminotransferase; DMAS: 2\u0026rsquo;-Deoxymugineic acid synthase; TOM: MAs efflux transporter; ENA: efflux transporter of NA; RNA-seq: RNA sequencing; Fe: qRT-PCR: Quantitative reverse transcription polymerase chain reaction; PS: Phytosiderophores; Iron; Fe\u003csup\u003e3+\u003c/sup\u003e: Ferric; Fe\u003csup\u003e2+\u003c/sup\u003e: Ferrous; DMA: Deoxymugineic acid; MFS: Major facilitator superfamily; NJ: Neighbor-joining; IRT1: Iron-regulated transporter 1; PS: Phytosiderophores; YS1: YELLOW STRIPE 1; YSL: YELLOW STRIPE 1-LIKE; NAS: Nicotianamine synthase; DAP: Days after pollination; ER: endoplasmic reticulum; GFP: Green fluorescent protein.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Special Program for GMO Development of China (grant number 2016ZX08003-002). The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXJZ conceived and designed the study. XZ and XJZ conducted the bioinformatics analysis. XZ contributed to perform the experiments and drafted the manuscript. SZL provided ER maker. XJZ, SZL, JXH, SP, and RMC revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to anonymous reviewers for comments on this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1 \u003c/sup\u003eBiotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People\u0026rsquo;s Republic of China.\u003csup\u003e 2 \u003c/sup\u003eDepartment of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, People\u0026rsquo;s Republic of China.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e1\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Zimmermann MB, Lancet RFHJ. 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Nat Protoc, 2007, 2(7): 1565-1572.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Maize, iron homeostasis, nicotianamine aminotransferase, 2’-deoxymugineic acid synthase, MAs efflux transporter, efflux transporter of NA, expression profiles, subcellular localization","lastPublishedDoi":"10.21203/rs.3.rs-19256/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-19256/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Nicotianamine (NA) serves as not only the major chelator for iron transport but also the intermediate for synthesizing mugineic acid family phytosiderophores (MAs) which are secreted by graminaceous plants for Fe uptake. Therefore, the production and secretion of MAs are key steps for maintaining iron homeostasis in plants. Nicotianamine aminotransferase (NAAT), 2’-deoxymugineic acid synthase (DMAS), MAs efflux transporter (TOM), and efflux transporter of NA (ENA) were identified to be involved in these processes in rice and barley, whereas little systematic study has been performed in maize (Zea mays.L). \u003c/p\u003e\u003cp\u003eResults: Here, we identified five ZmNAAT, nine ZmDMAS, eleven ZmTOM, and two ZmENA genes in maize by genome mining. RNA-sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of these genes exhibited diverse tissue specificity and different responses to environmental iron conditions. Moreover, the expression patterns were related to their evolution relationships. In particular, the ZmNAAT family can be classified into two subgroups, with one group showed inhibited expression in root under iron excess status and another subclass were repressed in shoot under both iron deficiency and excess. Likewise, the expression of ZmDMAS1 was stimulated under iron deficiency, while the remaining genes fell into two sub-clades with different expression patterns. Significant up-regulation of ZmTOM1, ZmTOM3 and ZmENA1 were observed under iron starvation, while ZmTOM2 was induced under both iron-excess and deficiency. These results reflect changing demands for the synthesis and secretion of NA/MAs to balance iron homeostasis under fluctuating conditions. All the examined ZmNAAT and ZmDMAS proteins localized in cytoplasm, while plasma and tonoplast membrane, endomembrane, and vesicle localization were observed for ZmTOM and ZmENA proteins. These results indicate that ZmTOM and ZmENA proteins may contribute to not only intercellular export but also intracellular sequestration of NA and MAs to facilitate iron homeostasis. \u003c/p\u003e\u003cp\u003eConclusions: Our results suggest that different gene expression profiles and subcellular localization of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA members may enable dedicate regulation of NA and phytosiderophores (PS) metabolism, shedding light on the understanding of iron-homeostasis in maize. Additionally, we also provided candidate genes for breeding iron-rich maize varieties.\u003c/p\u003e","manuscriptTitle":"Genome-wide analysis of NAAT, DMAS, TOM, and ENA gene families in maize reveals their roles in regulating iron homeostasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-04-03 15:28:46","doi":"10.21203/rs.3.rs-19256/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a7c5d274-4207-40d8-8571-0c421b3a6c71","owner":[],"postedDate":"April 3rd, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":78844,"name":"Epigenetics \u0026 Genomics"}],"tags":[],"updatedAt":"2020-06-27T12:40:42+00:00","versionOfRecord":[],"versionCreatedAt":"2020-04-03 15:28:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-19256","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-19256","identity":"rs-19256","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}