Metabolic engineering of astaxanthin-rich maize and its use in the production of biofortified eggs.

Production of the high-value carotenoid astaxanthin, which is widely used in food and feed due to its strong antioxidant activity and colour, is less efficient in cereals than in model plants. Here, we report a new strategy for expressing ß-carotene ketolase and hydroxylase genes from algae, yeasts and flowering plants in the whole seed using a seed-specific bidirectional promoter. Engineered maize events were backcrossed to inbred maize lines with yellow endosperm to generate progenies that accumulate astaxanthin from 47.76 to 111.82 mg/kg DW in seeds, and the maximum level is approximately sixfold higher than those in previous reports (16.2-16.8 mg/kg DW) in cereals. A feeding trial with laying hens indicated that they could take up astaxanthin from the maize and accumulate it in egg yolks (12.10-14.15 mg/kg) without affecting egg production and quality, as observed using astaxanthin from Haematococcus pluvialis. Storage stability evaluation analysis showed that the optimal conditions for long-term storage of astaxanthin-rich maize are at 4 °C in the dark. This study shows that co-expressing of functional genes driven by seed-specific bidirectional promoter could dramatically boost astaxanthin biosynthesis in every parts of kernel including embryo, aleurone layer and starch endosperm other than previous reports in the starch endosperm only. And the staple crop maize could serve as a cost-effective plant factory for reliably producing astaxanthin.

Kernel size-related genes revealed by an integrated eQTL analysis during early maize kernel development.

In maize, kernel traits strongly impact overall grain yields, and it is known that sophisticated spatiotemporal programs of gene expression coordinate kernel development, so advancing our knowledge of kernel development can help efforts to improve grain yields. Here, using phenotype, genotype and transcriptomics data of maize kernels at 5 and 15 days after pollination (DAP) for a large association mapping panel, we employed multiple quantitative genetics approaches-genome-wide association studies (GWAS) as well as expression quantitative trait loci (eQTL) and quantitative trait transcript (QTT) analyses-to gain insights about molecular genetic basis of kernel development in maize. This resulted in the identification of 137 putative kernel length-related genes at 5 DAP, of which 43 are located in previously reported QTL regions. Strikingly, we identified an eQTL that overlaps the locus encoding a maize homolog of the recently described m6 A methylation reader protein ECT2 from Arabidopsis; this putative epi eQTL is associated with 53 genes and may represent a master epi-transcriptomic regulator of kernel development. Notably, among the genes associated with this epi eQTL, 10 are for the main storage proteins in the maize endosperm (zeins) and two are known regulators of zein expression or endosperm development (Opaque2 and ZmICE1). Collectively, beyond cataloging and characterizing genomic attributes of a large number of eQTL associated with kernel development in maize, our study highlights how an eQTL approach can bolster the impact of both GWAS and QTT studies and can drive insights about the basic biology of plants.

Maize NCP1 negatively regulates drought and ABA responses through interacting with and inhibiting the activity of transcription factor ABP9.

KEY MESSAGE: NCP1, a NINJA family protein lacking EAR motif, acts as a negative regulator of ABA signaling by interacting with and inhibiting the activity of transcriptional activator ABP9. The phytohormone abscisic acid plays a pivotal role in regulating plant responses to a variety of abiotic stresses including drought and salinity. Maize ABP9 is an ABRE-binding bZIP transcription activator that enhances plant tolerance to multiple stresses by positively regulating ABA signaling, but the molecular mechanism by which ABP9 is regulated in mediating ABA responses remains unknown. Here, we report the identification of an ABP9-interacting protein, named ABP Nine Complex Protein 1 (NCP1) and its functional characterization. NCP1 belongs to the recently identified NINJA family proteins, but lacks the conserved EAR motif, which is a hallmark of this class of transcriptional repressors. In vitro and in vivo assays confirmed that NCP1 physically interacts with ABP9 and that they are co-localized in the nucleus. In addition, NCP1 and ABP9 are similarly induced with similar patterns by ABA treatment and osmotic stress. Interestingly, NCP1 over-expressing Arabidopsis plants exhibited a reduced sensitivity to ABA and decreased drought tolerance. Transient assay in maize protoplasts showed that NCP1 inhibits the activity of ABP9 in activating ABRE-mediated reporter gene expression, a notion further supported by genetic analysis of drought and ABA responses in the transgenic plants over-expressing both ABP9 and NCP1. These data together suggest that NCP1 is a novel negative regulator of ABA signaling via interacting with and inhibiting the activity of ABP9.

An operon consisting of a P-type ATPase gene and a transcriptional regulator gene responsible for cadmium resistances in Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25.

BACKGROUND: Cadmium (Cd) is a severely toxic heavy metal to most microorganisms. Many bacteria have developed Cd2+ resistance. RESULTS: In this study, we isolated two different Cd2+ resistance Bacillus sp. strains, Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25, which could be grown in the presence of Cd2+ at concentration up to 0.3 mM and 0.8 mM, respectively. According to the genomic sequencing, transcriptome analysis under cadmium stress, and other related experiments, a gene cluster in plasmid p25 was found to be a major contributor to Cd2+ resistance in B. marisflavi 151-25. The cluster in p25 contained orf4802 and orf4803 which encodes an ATPase transporter and a transcriptional regulator protein, respectively. Although 151-6 has much lower Cd2+ resistance than 151-25, they contained similar gene cluster, but in different locations. A gene cluster on the chromosome containing orf4111, orf4112 and orf4113, which encodes an ATPase transporter, a cadmium efflux system accessory protein and a cadmium resistance protein, respectively, was found to play a major role on the Cd2+ resistance for B. vietamensis 151-6. CONCLUSIONS: This work described cadmium resistance mechanisms in newly isolated Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25. Based on homologies to the cad system (CadA-CadC) in Staphylococcus aureus and analysis of transcriptome under Cd2+ induction, we inferred that the mechanisms of cadmium resistance in B. marisflavi 151-25 was as same as the cad system in S. aureus. Although Bacillus vietamensis 151-6 also had the similar gene cluster to B. marisflavi 151-25 and S. aureus, its transcriptional regulatory mechanism of cadmium resistance was not same. This study explored the cadmium resistance mechanism for B. vietamensis 151-6 and B. marisflavi 151-25 and has expanded our understanding of the biological effects of cadmium.

Genome-scale mining of root-preferential genes from maize and characterization of their promoter activity.

BACKGROUND: Modification of root architecture and improvement of root resistance to stresses can increase crop productivity. Functional analyses of root-specific genes are necessary for root system improvement, and root-specific promoters enable research into the regulation of root development and genetic manipulation of root traits. Maize is an important crop species; however, little systematic mining of root-specific genes and promoters has been performed to date. RESULTS: Genomic-scale mining based on microarray data sets followed by transcript detection resulted in the identification of 222 root-specific genes. Gene Ontology enrichment analyses revealed that these 222 root-specific genes were mainly involved in responses to chemical, biotic, and abiotic stresses. Of the 222 genes, 33 were verified by quantitative reverse transcription polymerase chain reaction, and 31 showed root-preferential activity. About 2 kb upstream 5 of the 31 identified root-preferential genes were cloned from the maize genome as putative promoters and named p8463, p5023, p1534, p8531 and p6629. GUS staining of transgenic maize-derived promoter-GUS constructs revealed that the five promoters drove GUS expression in a root-preferential manner. CONCLUSIONS: We mined root-preferential genes and their promoters in maize and verified p8463, p5023, p1534, p8531 and p6629 as root-preferential promoters. Our research enables the identification of other tissue-specific genes and promoters in maize and other species. In addition, the five promoters may enable enhancement of target gene(s) of maize in a root-preferential manner to generate novel maize cultivars with resistance to water, fertilizer constraints, or biotic stresses.

Synthesis of Seed-Specific Bidirectional Promoters for Metabolic Engineering of Anthocyanin-Rich Maize.

Tissue-specific promoters play an important role in plant molecular farming. Here, we describe a strategy to modify the tissue specificity of a maize embryo-specific bidirectional promoter PZmBD1. Six types of cis-elements, i.e. RY repeats (R), GCN4 (G), the prolamin box (P), Skn-1 (S), and the ACGT and AACA (A) motifs, were collected and fused to PZmBD1 to generate eight chimeric putative bidirectional promoters. Qualitative and quantitative analysis of reporter genes driven by the promoters showed that two promoters exhibited high seed-specific bidirectional activity in maize transient and stable transformed systems. The stronger one was chosen and fused to the intergenic region of two gene clusters consisting of four anthocyanin biosynthesis-related genes (ZmBz1, ZmBz2, ZmC1 and ZmR2) and seven reporter genes, resulting in the first embryo and endosperm anthocyanin-rich purple maize. Anthocyanin analysis showed that the total anthocyanin content reaches 2,910 mg kg-1 DW in transgenic maize and cyanidin is the major anthocyanin in transgenic maize, as in natural varieties. The expression profile analysis of endogenous genes showed that the anthocyanin biosynthesis pathway was activated by two transgenic transcription factor genes ZmC1 and ZmR2. Our results indicate that both the modification strategy and these functionally characterized tissue-specific bidirectional promoters generated could be used for genetic research and development of plant biotechnology products. The anthocyanin-rich purple maize could provide economic natural colorants for the food and beverage industry, and valuable germplasm for developing anthocyanin-rich fresh corn.

Six new soil-inhabiting Cladosporium species from plateaus in China.

Cladosporium species are ubiquitous in various environments but are hitherto rarely isolated from soil. In the present study, six new Cladosporium species inhabiting the plateau soils of China are described as C. neopsychrotolerans, C. paralimoniforme, C. prolongatum, C. sinuatum, C. tianshanense, and C. verruculosum. These species are phylogenetically distinct and morphologically different from known species. This study increased the number of species classified in the C. cladosporioides and C. herbarum complexes and revealed Chinese plateau soil as a rich niche of Cladosporium species diversity.

Analysis of weighted co-regulatory networks in maize provides insights into new genes and regulatory mechanisms related to inositol phosphate metabolism.

BACKGROUND: D-myo-inositol phosphates (IPs) are a series of phosphate esters. Myo-inositol hexakisphosphate (phytic acid, IP6) is the most abundant IP and has negative effects on animal and human nutrition. IPs play important roles in plant development, stress responses, and signal transduction. However, the metabolic pathways and possible regulatory mechanisms of IPs in maize are unclear. In this study, the B73 (high in phytic acid) and Qi319 (low in phytic acid) lines were selected for RNA-Seq analysis from 427 inbred lines based on a screening of IP levels. By integrating the metabolite data with the RNA-Seq data at three different kernel developmental stages (12, 21 and 30 days after pollination), co-regulatory networks were constructed to explore IP metabolism and its interactions with other pathways. RESULTS: Differentially expressed gene analyses showed that the expression of MIPS and ITPK was related to differences in IP metabolism in Qi319 and B73. Moreover, WRKY and ethylene-responsive transcription factors (TFs) were common among the differentially expressed TFs, and are likely to be involved in the regulation of IP metabolism. Six co-regulatory networks were constructed, and three were chosen for further analysis. Based on network analyses, we proposed that the GA pathway interacts with the IP pathway through the ubiquitination pathway, and that Ca(2+) signaling functions as a bridge between IPs and other pathways. IP pools were found to be transported by specific ATP-binding cassette (ABC) transporters. Finally, three candidate genes (Mf3, DH2 and CB5) were identified and validated using Arabidopsis lines with mutations in orthologous genes or RNA interference (RNAi)-transgenic maize lines. Some mutant or RNAi lines exhibited seeds with a low-phytic-acid phenotype, indicating perturbation of IP metabolism. Mf3 likely encodes an enzyme involved in IP synthesis, DH2 encodes a transporter responsible for IP transport across organs and CB5 encodes a transporter involved in IP co-transport into vesicles. CONCLUSIONS: This study provides new insights into IP metabolism and regulation, and facilitates our development of a better understanding of the functions of IPs and how they interact with other pathways involved in plant development and stress responses. Three new genes were discovered and preliminarily validated, thereby increasing our knowledge of IP metabolism.

A novel protein, Rsf1/Pxd1, is critical for the single-strand annealing pathway of double-strand break repair in Schizosaccharomyces pombe.

The process of single-strand annealing (SSA) repairs DNA double-strand breaks that are flanked by direct repeat sequences through the coordinated actions of a series of proteins implicated in recombination, mismatch repair and nucleotide excision repair (NER). Many of the molecular and mechanistic insights gained in SSA repair have principally come from studies in the budding yeast Saccharomyces cerevisiae. However, there is little molecular understanding of the SSA pathway in the fission yeast Schizosaccharomyces pombe. To further our understanding of this important process, we established a new chromosome-based SSA assay in fission yeast. Our genetic analyses showed that, although many homologous components participate in SSA repair in these species indicating that some evolutionary conservation, Saw1 and Slx4 are not principal agents in the SSA repair pathway in fission yeast. This is in marked contrast to the function of Saw1 and Slx4 in budding yeast. Additionally, a novel genus-specific protein, Rsf1/Pxd1, physically interacts with Rad16, Swi10 and Saw1 in vitro and in vivo. We find that Rsf1/Pxd1 is not required for NER and demonstrate that, in fission yeast, Rsf1/Pxd1, but not Saw1, plays a critical role in SSA recombination.

Interactions of Oryza sativa OsCONTINUOUS VASCULAR RING-LIKE 1 (OsCOLE1) and OsCOLE1-INTERACTING PROTEIN reveal a novel intracellular auxin transport mechanism.

Little is known about the transport mechanism of intracellular auxin. Here, we report two vacuole-localized proteins, Oryza sativa OsCONTINUOUS VASCULAR RING-LIKE 1 (OsCOLE1) and OsCOLE1-INTERACTING PROTEIN (OsCLIP), that regulate intracellular auxin transport and homoeostasis. Overexpression of OsCOLE1 markedly increased the internode length and auxin content of the stem base, whereas these parameters were decreased in RNA interference (RNAi) plants. OsCOLE1 was localized on the tonoplast and preferentially expressed in mature tissues. We further identified its interacting protein OsCLIP, which was co-localized on the tonoplast. Protein-protein binding assays demonstrated that the N-terminus of OsCOLE1 directly interacted with OsCLIP in yeast cells and the rice protoplast. Furthermore, (3) H-indole-3-acetic acid ((3) H-IAA) transport assays revealed that OsCLIP transported IAA into yeast cells, which was promoted by OsCOLE1. The results indicate that OsCOLE1 affects rice development by regulating intracellular auxin transport through interaction with OsCLIP, which provides a new insight into the regulatory mechanism of intracellular transport of auxin and the roles of vacuoles in plant development.

The intergenic region of the maize defensin-like protein genes Def1 and Def2 functions as an embryo-specific asymmetric bidirectional promoter.

Bidirectional promoters are identified in diverse organisms with widely varied genome sizes, including bacteria, yeast, mammals, and plants. However, little research has been done on any individual endogenous bidirectional promoter from plants. Here, we describe a promoter positioned in the intergenic region of two defensin-like protein genes, Def1 and Def2 in maize (Zea mays). We examined the expression profiles of Def1 and Def2 in 14 maize tissues by qRT-PCR, and the results showed that this gene pair was expressed abundantly and specifically in seeds. When fused to either green fluorescent protein (GFP) or ß-glucuronidase (GUS) reporter genes, P ZmBD1 , P ZmDef1 , and P ZmDef2 were active and reproduced the expression patterns of both Def1 and Def2 genes in transformed immature maize embryos, as well as in developing seeds of transgenic maize. Comparative analysis revealed that PZmBD1 shared most of the expression characteristics of the two polar promoters, but displayed more stringent embryo specificity, delayed expression initiation, and asymmetric promoter activity. Moreover, a truncated promoter study revealed that the core promoters only exhibit basic bidirectional activity, while interacting with necessary cis-elements, which leads to polarity and different strengths. The sophisticated interaction or counteraction between the core promoter and cis-elements may potentially regulate bidirectional promoters.

ZmGRF, a GA regulatory factor from maize, promotes flowering and plant growth in Arabidopsis.

Transcription factors that act as positive regulators of gibberellin (GA) biosynthetic genes in plants are not well understood. A nuclear-localized basic leucine zipper transcription factor, ZmGRF, was isolated from maize. The core DNA sequence motif recognized for binding by ZmGRF was CCANNTGGC. ZmGRF overexpression in transgenic Arabidopsis plants promoted flowering, stem elongation, and cell expansion. Chromatin immunoprecipitation assays revealed that ZmGRF bound directly to the cis-element CCANNTGGC in the promoter of the Arabidopsis ent-kaurene oxidase (AtKO1) gene and promoted AtKO1 expression. GA4 content increased by 372-567% in transgenic Arabidopsis plants overexpressing ZmGRF compared to wild-type control plants. The GIBBERELLIN-INSENSITIVE DWARF1 gene, which encodes a GA receptor, was also upregulated and the growth-repressing DELLA protein gene GA INSENSITIVE was downregulated. Our results showed ZmGRF functioned through the GA-signaling pathway.

Transcriptome analysis of nitrogen-starvation-responsive genes in rice.

BACKGROUND: Nitrogen (N), a critical macronutrient for plant growth and development, is a major limiting factor in most agricultural systems. Microarray analyses have been conducted to investigate genome-wide gene expression in response to changes in N concentrations. Although RNA-Seq analysis can provide a more precise determination of transcript levels, it has not previously been employed to investigate the expression of N-starvation-induced genes. RESULTS: We constructed cDNA libraries from leaf sheaths and roots of rice plants grown under N-deficient or -sufficient conditions for 12 h. Sequencing the libraries resulted in identification of 33,782 annotated genes. A comparison of abundances revealed 1,650 transcripts that were differentially expressed (fold-change ≥ 2) due to an N-deficiency. Among them, 1,158 were differentially expressed in the leaf sheaths (548 up-regulated and 610 down-regulated) and 492 in the roots (276 up, 216 down). Among the 36 deficiency-induced genes first identified via RNA-Seq analyses, 34 were subsequently confirmed by qRT-PCR. Our RNA-Seq data identified 8,509 multi-exonic genes with 19,628 alternative splicing events. However, we saw no significant difference in alternative splicing between N-sufficient and -deficient conditions. We found 2,986 novel transcripts, of which 192 were regulated under the N-deficiency. CONCLUSION: We identified 1,650 genes that were differentially expressed after 12 h of N-starvation. Responses by those genes to a limited supply of N were confirmed by RT-PCR and GUS assays. Our results provide valuable information about N-starvation-responsive genes and will be useful when investigating the signal transduction pathway of N-utilization.

Insights into the substrate specificity and synergy with mannanase of family 27 α-galactosidases from Neosartorya fischeri P1.

Thermophilic Neosartorya fischeri P1 is an excellent carbohydrate-active enzyme (CAZyme) producer. Two α-galactosidases of GH (glycoside hydrolase ) family 27 with a very low sequence identity (28.7%), Gal27A and Gal27B, were identified in strain P1 and functionally expressed in Pichia pastoris. In comparison to other characterized GH27 fungal counterparts, rGal27B has a higher temperature optimum (75 °C) and better thermostability (>50% activity at 70 °C for 15 min), and rGal27A shows stability over the broadest pH range (pH 2.0-12.0). Moreover, great distinctions lie in the two enzymes. When using pNPG as the substrate, rGal27B had a higher turnover number (1621.4 vs. 368.3 s(-1)) but lower affinity (2.84 vs. 0.8 mM) and catalytic efficiency (460.8 vs. 580.3 s(-1) mM(-1)) than rGal27A. rGal27B acted on galacto-oligosaccharides, whereas rGal27A was active on polymeric substrates. Although both enzymes showed synergy in galactomannan degradation when combined with a ß-mannanase of the same strain, enzyme combinations including rGal27A released more reducing sugars (up to 11.67-fold). Homology modeling predicts different loops in N. fischeri α-galactosidases, highlighting the larger tunnel structure in Gal27A to accommodate/bind branched galactomannan with high galactose contents. Phylogenetic analysis reveals the far relationship of Gal27A and Gal27B that they may evolve in different action modes, and their coexistence widens the substrate spectrum for nutrient utilization. This study illustrates the substrate profiles and synergistic mechanism of GH27 α-galactosidases of different structures.

Identification and functional characterization of bidirectional gene pairs and their intergenic regions in maize.

BACKGROUND: Bidirectional gene pairs exist as a specific form of gene organization in microorganisms and mammals as well as in model plant species, such as Arabidopsis and rice. Little is known about bidirectional gene pairs in maize, which has a large genome and is one of the most important grain crops. RESULTS: We conducted a genome-wide search in maize using genome sequencing results from the inbred line B73. In total, 1696 bidirectional transcript pairs were identified using a modified search model. We functionally characterized the promoter activity of the intergenic regions of most of the bidirectional transcript pairs that were expressed in embryos using a maize embryo transient expression system. A comparative study of bidirectional gene pairs performed for three monocot (Zea mays, Sorghum bicolor and Oryza sativa) and two dicot (Arabidopsis thaliana and Glycine max) plant genomes showed that bidirectional gene pairs were abundant in the five plant species. Orthologous bidirectional gene pairs were clearly distinguishable between the monocot and dicot species although the total numbers of orthologous bidirectional genes were similar. Analysis of the gene pairs using the Blast2GO software suite showed that the molecular functions (MF), cellular components (CC), and biological processes (BP) associated with the bidirectional transcripts were similar among the five plant species. CONCLUSIONS: The evolutionary analysis of the function and structure of orthologous bidirectional gene pairs in various plant species revealed a potential pathway of their origin, which may be required for the evolution of a new species.

Identification and characterization of promoters specifically and strongly expressed in maize embryos.

The use of maize seeds as bioreactors has several advantages for the production of recombinant proteins in plant biotechnology, but available embryo-specific and strong promoters are limited. Here, we describe a genome-scale microarray-based approach to identify embryo-specifically and strongly expressed genes and their promoters in maize. We identified 28 embryo-preferred and abundantly expressed genes based on our microarray data. These embryo-preferred genes were further analysed using the UniGene database and by quantitative reverse transcriptase-PCR leading to the identification of seven genes (Zm.2098, Zm.13387, Zm.66589, Zm.85502, Zm.68129, Zm.3896 and Zm.2941) as embryo-specific genes with higher expression levels relative to maize globulin-1. The putative promoters of five embryo-specific genes (Zm.13387, Zm.66589, Zm.85502, Zm.3896 and Zm.2941) were isolated and all exhibited strong promoter activities when transiently expressed in maize embryos of 20 DAP. The embryo specificity and expression levels of the promoters of four genes (Zm.13387, Zm.85502, Zm.3896 and Zm.2941) were further examined in transgenic maize plants, revealing that they are strong promoters in embryos of all four developmental stages tested compared with reference globulin-1 promoter. Moreover, Zm.2941 and Zm.3896 promoters are stringently embryo-specific promoters, while Zm.85502 promoter is basically embryo specific yet wounding inducible in non-seed tissues, and Zm.13387 promoter is developmentally expressed in both embryo and aleurone with wounding-induced activity in non-seed tissues. Our study provides novel embryo-specific and strong promoters that are suitable for production of high-level recombinant proteins in maize embryos.

The differential transcription network between embryo and endosperm in the early developing maize seed.

Transcriptome analysis of early-developing maize (Zea mays) seed was conducted using Illumina sequencing. We mapped 11,074,508 and 11,495,788 paired-end reads from endosperm and embryo, respectively, at 9 d after pollination to define gene structure and alternative splicing events as well as transcriptional regulators of gene expression to quantify transcript abundance in both embryo and endosperm. We identified a large number of novel transcribed regions that did not fall within maize annotated regions, and many of the novel transcribed regions were tissue-specifically expressed. We found that 50.7% (8,556 of 16,878) of multiexonic genes were alternatively spliced, and some transcript isoforms were specifically expressed either in endosperm or in embryo. In addition, a total of 46 trans-splicing events, with nine intrachromosomal events and 37 interchromosomal events, were found in our data set. Many metabolic activities were specifically assigned to endosperm and embryo, such as starch biosynthesis in endosperm and lipid biosynthesis in embryo. Finally, a number of transcription factors and imprinting genes were found to be specifically expressed in embryo or endosperm. This data set will aid in understanding how embryo/endosperm development in maize is differentially regulated.

The mitochondrial folylpolyglutamate synthetase gene is required for nitrogen utilization during early seedling development in arabidopsis.

Investigations into the biochemical processes and regulatory mechanisms of nitrogen (N) utilization can aid in understanding how N is used efficiently in plants. This report describes a deficiency in N utilization in an Arabidopsis (Arabidopsis thaliana) transfer DNA insertion mutant of the mitochondrial folylpolyglutamate synthetase gene DFC, which catalyzes the conjugation of glutamate residues to the tetrahydrofolate during folate synthesis. The mutant seedlings displayed several metabolic changes that are typical of plant responses to low-N stress, including increased levels of starch and anthocyanin synthesis as well as decreased levels of soluble protein and free amino acid, as compared with those in wild-type seedlings when external N was sufficient. More striking changes were observed when dfc seedlings were grown under N-limited conditions, including shorter primary roots, fewer lateral roots, higher levels of glycine and carbon-N ratios, and lower N content than those in wild-type seedlings. Gene expression studies in mutant seedlings revealed altered transcript levels of several genes involved in folate biosynthesis and N metabolism. The biochemical and metabolic changes also suggested that N assimilation is drastically perturbed due to a loss of DFC function. The observation that elevated CO(2) partly rescued the dfc phenotypes suggests that the alterations in N metabolism in dfc may be mainly due to a defect in photorespiration. These results indicate that DFC is required for N utilization in Arabidopsis and provide new insight into a potential interaction between folate and N metabolism.

ZmSOC1, a MADS-box transcription factor from Zea mays, promotes flowering in Arabidopsis.

Zea mays is an economically important crop, but its molecular mechanism of flowering remains largely uncharacterized. The gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), integrates multiple flowering signals to regulate floral transition in Arabidopsis. In this study, ZmSOC1 was isolated from Zea mays. Sequence alignment and phylogenetic analysis demonstrated that the ZmSOC1 protein contained a highly conserved MADS domain and a typical SOC1 motif. ZmSOC1 protein was localized in the nucleus in protoplasts and showed no transcriptional activation activity in yeast cells. ZmSOC1 was highly expressed in maize reproductive organs, including filaments, ear and endosperm, but expression was very low in embryos; on the other hand, the abiotic stresses could repress ZmSOC1 expression. Overexpression of ZmSOC1 resulted in early flowering in Arabidopsis through increasing the expression of AtLFY and AtAP1. Overall, these results suggest that ZmSOC1 is a flowering promoter in Arabidopsis.

Genome-wide identification, classification and expression profiling of nicotianamine synthase (NAS) gene family in maize.

BACKGROUND: Nicotianamine (NA), a ubiquitous molecule in plants, is an important metal ion chelator and the main precursor for phytosiderophores biosynthesis. Considerable progress has been achieved in cloning and characterizing the functions of nicotianamine synthase (NAS) in plants including barley, Arabidopsis and rice. Maize is not only an important cereal crop, but also a model plant for genetics and evolutionary study. The genome sequencing of maize was completed, and many gene families were identified. Although three NAS genes have been characterized in maize, there is still no systematic identification of maize NAS family by genomic mining. RESULTS: In this study, nine NAS genes in maize were identified and their expression patterns in different organs including developing seeds were determined. According to the evolutionary relationship and tissue specific expression profiles of ZmNAS genes, they can be subgrouped into two classes. Moreover, the expression patterns of ZmNAS genes in response to fluctuating metal status were analysed. The class I ZmNAS genes were induced under Fe deficiency and were suppressed under Fe excessive conditions, while the expression pattern of class II genes were opposite to class I. The complementary expression patterns of class I and class II ZmNAS genes confirmed the classification of this family. Furthermore, the histochemical localization of ZmNAS1;1/1;2 and ZmNAS3 were determined using in situ hybridization. It was revealed that ZmNAS1;1/1;2, representing the class I genes, mainly expressed in cortex and stele of roots with sufficient Fe, and its expression can expanded in epidermis, as well as shoot apices under Fe deficient conditions. On the contrary, ZmNAS3, one of the class II genes, was accumulated in axillary meristems, leaf primordia and mesophyll cells. These results suggest that the two classes of ZmNAS genes may be regulated on transcriptional level when responds to various demands for iron uptake, translocation and homeostasis. CONCLUSION: These results provide significant insights into the molecular bases of ZmNAS in balancing iron uptake, translocation and homeostasis in response to fluctuating environmental Fe status.