Nicotianamine is a major player in plant Zn homeostasis

Nicotianamine (NA) is among the most studied plant metal chelators. A large body of evidence supports its crucial role for Fe distribution in plants and as a precursor of phytosiderophore synthesis in grasses. NA forms stable complexes in vitro not only with Fe(II) and Fe(III) but also with various other divalent metal cations including Zn(II). Early observations indicated a possible contribution of NA to Zn trafficking in plants. Numerous studies on transgenic monocot and dicot plants with modulated NA levels have since then reported Zn accumulation phenotypes. NAS genes were shown to represent promising targets for biofortification efforts. For instance, NA was found to bind Zn in rice grains in a form bioavailable for humans. Recently, additional strong support for the existence of Zn–NA complexes in planta has been obtained in rice, Arabidopsis thaliana and the Zn hyperaccumulating plant A. halleri. We review the evidence for a role of NA in the intercellular and long-distance transport of Zn in plants and discuss open questions.     

Three nicotianamine synthase genes isolated from maize are differentially regulated by iron nutritional status

Nicotianamine synthase (NAS) is an enzyme that is critical for the biosynthesis of the mugineic acid family of phytosiderophores in graminaceous plants, and for the homeostasis of metal ions in nongraminaceous plants. We isolated one genomic NAS clone, ZmNAS3, and two cDNA NAS clones, ZmNAS1 and ZmNAS2, from maize (Zea mays cv Alice). In agreement with the increased secretion of phytosiderophores with Fe deficiency, ZmNAS1 and ZmNAS2 were positively expressed only in Fe-deficient roots. In contrast, ZmNAS3 was expressed under Fe-sufficient conditions, and was negatively regulated by Fe deficiency. This is the first report describing down-regulation of NAS gene expression in response to Fe deficiency in plants, shedding light on the role of nicotianamine in graminaceous plants, other than as a precursor in phytosiderophore production. ZmNAS1-green fluorescent protein (sGFP) and ZmNAS2-sGFP were localized at spots in the cytoplasm of onion (Allium cepa) epidermal cells, whereas ZmNAS3-sGFP was distributed throughout the cytoplasm of these cells. ZmNAS1 and ZmNAS3 showed NAS activity in vitro, whereas ZmNAS2 showed none. Due to its duplicated structure, ZmNAS2 was much larger (65.8 kD) than ZmNAS1, ZmNAS3, and previously characterized NAS proteins (30-38 kD) from other plant species. We reveal that maize has two types of NAS proteins based on their expression pattern and subcellular localization.     

The Analysis of Arabidopsis Nicotianamine Synthase Mutants Reveals Functions for Nicotianamine in Seed Iron Loading and Iron Deficiency Responses

Nicotianamine chelates and transports micronutrient metal ions in plants. It has been speculated that nicotianamine is involved in seed loading with micronutrients. A tomato (Solanum lycopersicum) mutant (chloronerva) and a tobacco (Nicotiana tabacum) transgenic line have been utilized to analyze the effects of nicotianamine loss. These mutants showed early leaf chlorosis and had sterile flowers. Arabidopsis (Arabidopsis thaliana) has four NICOTIANAMINE SYNTHASE (NAS) genes. We constructed two quadruple nas mutants: one had full loss of NAS function, was sterile, and showed a chloronerva-like phenotype (nas4x-2); another mutant, with intermediate phenotype (nas4x-1), developed chlorotic leaves, which became severe upon transition from the vegetative to the reproductive phase and upon iron (Fe) deficiency. Residual nicotianamine levels were sufficient to sustain the life cycle. Therefore, the nas4x-1 mutant enabled us to study late nicotianamine functions. This mutant had no detectable nicotianamine in rosette leaves of the reproductive stage but low nicotianamine levels in vegetative rosette leaves and seeds. Fe accumulated in the rosette leaves, while less Fe was present in flowers and seeds. Leaves, roots, and flowers showed symptoms of Fe deficiency, whereas leaves also showed signs of sufficient Fe supply, as revealed by molecular-physiological analysis. The mutant was not able to fully mobilize Fe to sustain Fe supply of flowers and seeds in the normal way. Thus, nicotianamine is needed for correct supply of seeds with Fe. These results are fundamental for plant manipulation approaches to modify Fe homeostasis regulation through alterations of NAS genes.     

Trehalose Metabolism-Related Genes in Maize

Maize is a cereal crop that is grown widely throughout the world in a range of agro-ecological environments. Trehalose is a nonreducing disaccharide of glucose that has been associated with tolerance to different stress conditions, including salt and drought. Bioinformatic analysis of genes involved in trehalose biosynthesis and degradation in maize has not been reported to date. Through systematic analysis, 1 degradation-related and 36 trehalose biosynthesis-related genes were identified. The conserved domains and phylogenetic relationships among the deduced maize proteins and their homologs, isolated from other plant species such as Arabidopsis and rice, were revealed. Using a comprehensive approach, the intron/exon structures and expression patterns of all identified genes and their responses to salt stress, jasmonic acid, and abscisic acid treatment were analyzed. Microarray data demonstrated that some of the genes show differential, organ-specific expression patterns in the 60 different developmental stages of maize. It was discovered that some of the key enzymes such as hexokinase, trehalose-6-phosphate synthase, and trehalose-6-phosphate phosphatase are encoded by multiple gene members with different expression patterns. The results highlight the complexity of trehalose metabolism and provide useful information for improving maize stress tolerance through genetic engineering.     

玉米中Class Ⅰ和Class ⅡNAS蛋白之间的BiFC互作研究

烟草胺合成酶（nicotianamine synthase,NAS）能够催化合成植物体内铁运输所需的螯合物烟草胺（nicotianamine,NA）,在植物维持铁稳态方面发挥重要的作用。玉米、小麦和大麦等禾本科植物的NAS蛋白进化为ClassⅠ和ClassⅡ两个亚家族,可能分别参与调节铁的吸收和运输,其家族成员之间蛋白序列同源性较高,ClassⅡNAS具有特异的N端可变结构域。通过进化分析分析玉米NAS的两个亚家族,以及建模预测两类亚家族代表基因ZmNAS1(ClassⅠ)和ZmNAS3(ClassⅡ)的蛋白结构,结果表明ZmNAS1和ZmNAS3的三维结构高度相似,推测可能通过同源或异源二聚体化发挥功能;进一步通过双分子荧光互补（bimolecular fluorescence complementation,BiFC）分析ZmNAS1和ZmNAS3的相互作用,结果表明,ZmNAS1和ZmNAS3蛋白可以互作,删除N端可变结构域的ZmNAS3?N只能与ZmNAS3蛋白互作而不与ZmNAS1互作,推测ZmNAS可以形成同源二聚体,而形成异源二聚体需要ClassⅡ家族蛋白的N端可变结构域...     

Genome-wide analysis of primary auxin-responsive Aux/IAA gene family in maize (Zea mays. L.)

The phytohormone auxin is important in various aspects of organism growth and development. Aux/IAA genes encoding short-lived nuclear proteins are responsive primarily to auxin induction. Despite their physiological importance, systematic analysis of Aux/IAA genes in maize have not yet been reported. In this paper, we presented the isolation and characterization of maize Aux/IAA genes in whole-genome scale. A total of 31 maize Aux/IAA genes (ZmIAA1 to ZmIAA31) were identified. ZmIAA genes are distributed in all the maize chromosomes except chromosome 2. Aux/IAA genes expand in the maize genome partly due to tandem and segmental duplication events. Multiple alignment and motif display results revealed major maize Aux/IAA proteins share all the four conserved domains. Phylogenetic analysis indicated Aux/IAA family can be divided into seven subfamilies. Putative cis-acting regulatory DNA elements involved in auxin response, light signaling transduction and abiotic stress adaption were observed in the promoters of ZmIAA genes. Expression data mining suggested maize Aux/IAA genes have temporal and spatial expression pattern. Collectively, these results will provide molecular insights into the auxin metabolism, transport and signaling research.     

Induction of annexin by heavy metals and jasmonic acid in Zea mays

Plant annexins are Ca²⁺- and phospholipid-binding proteins forming an evolutionary conserved multi-gene family. They are implicated in the regulation of plant growth, development, and stress responses. With the availability of the maize genome sequence information, we identified 12 members of the maize annexin genes. Analysis of protein sequence and gene structure of maize annexins led to their classification into five different orthologous groups. Expression analysis by RT-PCR revealed that these genes are responsive to heavy metals (Ni, Zn, and Cd). The maize annexin genes were also found to be regulated by Ustilago maydis and jasmonic acid. Additionally, the promoter of the maize annexin gene was analyzed for the presence of different stress-responsive cis-elements, such as ABRE, W-box, GCC-box, and G-box. RT-PCR and microarray data show that all 12 maize annexin genes present differential, organ-specific expression patterns in the maize developmental steps. These results indicate that maize annexin genes may play important roles in the adaptation of plants to various environmental stresses.     

Effect of colchicine-induced polyploidy on morphological characteristics and essential oil composition of ajowan (<Emphasis Type="Italic">Trachyspermum ammi</Emphasis> L.)

Cyclophilins (CYPs) belong to the immunophilin superfamily, having the peptidyl prolyl cis/trans isomerase activity that can catalyze the cis/trans isomerisation process of proline residues. Previous studies have shown their importance in plants, but no comprehensive analysis of maize CYP family has been reported. In the present study, a whole-genome-wide analysis of maize CYP family was performed and 39 ZmCYP genes (ZmCYP1 to ZmCYP39) were identified from maize genome, which were unequally distributed on maize ten chromosomes. Phylogenetic analysis revealed a weak relationship among these ZmCYP genes. Furthermore, their gene structure and motif patterns also displayed variant within the gene family. Four segmental and one tandem duplicated gene pairs were found from 39 ZmCYP genes, respectively, indicating their roles in the expansion of maize CYP family. Expression analysis of 39 ZmCYP genes in maize tissues showed their differential tissue specific expression patterns. Quantitative real-time PCR analysis of 19 selected ZmCYP genes under salinity stress indicated their stress-inducible expression profile. Heterologous expression of ZmCYP15 in E. coli enhanced tolerance against abiotic stress. Subcellular localization analysis indicated ZmCYP15 was located in nucleus and cytoplasm. Our study describes the importance of the maize CYP gene family in stress response, and provides a reference for future study and application for maize genetic improvement.     

Isolation and characterization of three polyamine oxidase genes from Zea mays

Isolation and sequencing of three genes, MPAO1, MPAO2 and MPAO3, coding for polyamine oxidase (PAO) from maize (Zea mays) are reported here. Gene organization is extremely conserved among these copies, being composed of eight exons and seven introns. Furthermore, these genes encode for a protein of an almost identical amino acid sequence. These data suggest that the three MPAO copies have been derived from gene duplication of a common ancestor gene. Long inverted repeat sequences, also present in other maize genes, have been found within the second intron. Promoter sequences of MPAO1 and MPAO2 genes have been analysed for putative cis-acting elements. According to genomic Southern blot analysis, the MPAO gene family in maize and other monocots is represented by a small number of copies. Northern and western blot analysis have revealed a tissue-specific accumulation of both MPAO mRNA and protein.     

In silico analysis of PHB gene family in maize

Prohibitins (PHBs) are highly conserved proteins in species ranging from prokaryotes to eukaryotes. Plant PHBs have been implicated in various cellular processes including development, senescence and stress responses. Although PHBs have been investigated in several plant species including Arabidopsis and tobacco, no systematic gene family analysis has been carried in maize. In the present study, 16 putative PHB genes have been identified. Analysis of the conserved protein motifs and gene structures has revealed high levels of conservation within the phylogenetic subgroups. Published microarray database showed that most maize PHB genes exhibited different expression levels in different tissues and developmental stages. Cis-elements analysis showed that ZmPHB2 and ZmPHB12 may play important roles in plant development. Taken together, we provide a comprehensive bioinformatics analysis of the PHB gene family in maize genome and our data provide an important foundation for further functional study of this gene family in maize.     

Genome-wide identification and expression profiling of ankyrin-repeat gene family in maize

Members of the ankyrin repeats (ANK) gene family encode ANK domain that are common in diverse organisms and play important roles in cell growth and development, such as cell-cell signal transduction and cell cycle regulation. Recently, genome-wide identification and evolutionary analyses of the ANK gene family have been carried out in Arabidopsis and rice. However, little is known regarding the ANK genes in the entire maize genome. In this study, we described the identification and structural characterization of 71 ANK genes in maize (ZmANK). Then, comprehensive bioinformatics analyses of ZmANK genes family were performed including phylogenetic, domain and motif analysis, chromosomal localization, intron/exon structural patterns, gene duplications and expression profiling. Domain composition analyses showed that ZmANK genes formed ten subfamilies. Five tandem duplications and 14 segmental duplications were identified in ZmANK genes. Furthermore, we took comparative analysis of the total ANK gene family in Arabidopsis, rice and maize, ZmANKs were more closely paired with OsANKs than with AtANKs. At last, expression profile analyses were performed. Forty-one members of ZmANK genes held EST sequences records. Semi-quantitative expression and microarray data analysis of these 41 ZmANK genes demonstrated that ZmANK genes exhibit a various expression pattern, suggesting that functional diversification of ZmANK genes family. The results will present significant insights to explore ANK genes expression and function in future studies in maize.     

Characterization of thermostable deblocking aminopeptidases of archaeon Thermococcus onnurineus NA1 by proteomic and biochemical approaches

Thermococcus onnurineus NA1 is a hyperthermophilic archaeon that grows optimally at >80°C. The deblocking aminopeptidase (DAP) (TNA1-DAP1) encoded in Ton_1032 of T. onnurineus NA1 is considered a major DAP. However, four genes encoding putative DAP have been identified from a genomic analysis of T. onnurineus NA1. A proteomic analysis revealed that all four DAPs were differentially induced in YPS culture medium and, particularly, two DAPs (TNA1-DAP1 and TNA1-DAP2) were dominantly expressed in T. onnurineus NA1. The biochemical properties and enzyme activity of DAPs induced in an E. coli expression system suggested that the two major DAPs play complementary roles in T. onnurineus NA1.