Rumei Chen

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

BackgroundNicotianamine (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.ResultsIn 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.ConclusionThese results provide significant insights into the molecular bases of ZmNAS in balancing iron uptake, translocation and homeostasis in response to fluctuating environmental Fe status.

A Maize Jasmonate Zim-Domain Protein, ZmJAZ14, Associates with the JA, ABA, and GA Signaling Pathways in Transgenic Arabidopsis

Jasmonate (JA) is an important signaling molecule involved in the regulation of many physiological and stress-related processes in plants. Jasmonate ZIM-domain (JAZ) proteins have been implicated in regulating JA signaling pathways and the cross talk between various phytohormones. Maize is not only an important cereal crop, but also a model plant for monocotyledon studies. Although many JAZ proteins have been characterized in Arabidopsis and rice, few reports have examined the function of JAZ proteins in maize. In this report, we examined the phylogenetic relationship and expression pattern of JAZ family genes in maize. In addition, a tassel and endosperm-specific JAZ gene, ZmJAZ14, was identified using microarray data analysis and real-time RT-PCR, and its expression was induced by polyethylene glycol (PEG), jasmonate (JA), abscisic acid (ABA), and gibberellins (GAs). ZmJAZ14 was shown to be localized in the nucleus and possessed no transcriptional activating activity, suggesting that it functions as a transcriptional regulator. We found that overexpression of ZmJAZ14 in Arabidopsis enhanced plant tolerance to JA and ABA treatment, as well as PEG stress, while it promoted growth under GA stimulus. Moreover, ZmJAZ14 interacted with a subset of transcription factors in Arabidopsis, and the accumulation of several marker genes involved in JA, ABA, and GA signaling pathways were altered in the overexpression lines. These results suggest that ZmJAZ14 may serve as a hub for the cross talk among the JA, ABA, and GA signaling pathways. Our results can be used to further characterize the function of JAZ family proteins in maize, and the gene cloned in this study may serve as a candidate for drought tolerance and growth promotion regulation in maize.

Overexpression of ZmIRT1 and ZmZIP3 Enhances Iron and Zinc Accumulation in Transgenic Arabidopsis

Iron and zinc are important micronutrients for both the growth and nutrient availability of crop plants, and their absorption is tightly controlled by a metal uptake system. Zinc-regulated transporters, iron-regulated transporter-like proteins (ZIP), is considered an essential metal transporter for the acquisition of Fe and Zn in graminaceous plants. Several ZIPs have been identified in maize, although their physiological function remains unclear. In this report, ZmIRT1 was shown to be specifically expressed in silk and embryo, whereas ZmZIP3 was a leaf-specific gene. Both ZmIRT1 and ZmZIP3 were shown to be localized to the plasma membrane and endoplasmic reticulum. In addition, transgenic Arabidopsis plants overexpressing ZmIRT1 or ZmZIP3 were generated, and the metal contents in various tissues of transgenic and wild-type plants were examined based on ICP-OES and Zinpyr-1 staining. The Fe and Zn concentration increased in roots and seeds of ZmIRT1-overexpressing plants, while the Fe content in shoots decreased. Overexpressing ZmZIP3 enhanced Zn accumulation in the roots of transgenic plants, while that in shoots was repressed. In addition, the transgenic plants showed altered tolerance to various Fe and Zn conditions compared with wild-type plants. Furthermore, the genes associated with metal uptake were stimulated in ZmIRT1 transgenic plants, while those involved in intra- and inter- cellular translocation were suppressed. In conclusion, ZmIRT1 and ZmZIP3 are functional metal transporters with different ion selectivities. Ectopic overexpression of ZmIRT1 may stimulate endogenous Fe uptake mechanisms, which may facilitate metal uptake and homeostasis. Our results increase our understanding of the functions of ZIP family transporters in maize.

Genome-Wide Epigenetic Regulation of Gene Transcription in Maize Seeds.

Background Epigenetic regulation is well recognized for its importance in gene expression in organisms. DNA methylation, an important epigenetic mark, has received enormous attention in recent years as it’s a key player in many biological processes. It remains unclear how DNA methylation contributes to gene transcription regulation in maize seeds. Here, we take advantage of recent technologies to examine the genome-wide association of DNA methylation with transcription of four types of DNA sequences, including protein-coding genes, pseudogenes, transposable elements, and repeats in maize embryo and endosperm, respectively. Results The methylation in CG, CHG and CHH contexts plays different roles in the control of gene expression. Methylation around the transcription start sites and transcription stop regions of protein-coding genes is negatively correlated, but in gene bodies positively correlated, to gene expression level. The upstream regions of protein-coding genes are enriched with 24-nt siRNAs and contain high levels of CHH methylation, which is correlated to gene expression level. The analysis of sequence content within CG, CHG, or CHH contexts reveals that only CHH methylation is affected by its local sequences, which is different from Arabidopsis. Conclusions In summary, we conclude that methylation-regulated transcription varies with the types of DNA sequences, sequence contexts or parts of a specific gene in maize seeds and differs from that in other plant species. Our study helps people better understand from a genome-wide viewpoint that how transcriptional expression is controlled by DNA methylation, one of the important factors influencing transcription, and how the methylation is associated with small RNAs.

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

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

Highlight Here we cloned and intensively characterized the first natural embryo-specific bidirectional promoter which could facilitate multi-gene expression in maize, and proposed a model of regulation of bidirectional transcription initiation.

Engineering of ‘Purple Embryo Maize’ with a multigene expression system derived from a bidirectional promoter and self‐cleaving 2A peptides

Anthocyanins are polyhydroxy and polymethoxy 2-phenylbenzopyrylium glucosides that belong to a large group of plant secondary metabolites termed flavonoids. The most common anthocyanidins (anthocyanin aglycones) in higher plants are cyanidin, pelargonidin, peonidin, delphinidin, petunidin and malvidin. Flavonoid biosynthesis has been suggested to occur on the cytoplasmic face of the endoplasmic reticulum (ER), where sequential enzymes of the pathway loosely form a metabolon (Winkel-Shirley, 1999). Known structural genes along the anthocyanin biosynthesis pathway in maize include chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 30-hydroxylase (F30H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), flavonoid 3-O-glucosyltransferase (3UFGT) and glutathione Stransferase (GST). Known regulators of anthocyanin biosynthesis contain MYC-like bHLH (basic helix-loop-helix) proteins and R2R3MYB proteins. MYC-like bHLH proteins are encoded by the R locus (S, Lc and Sn) and B locus (b1, booster1), whereas MYB regulators are encoded by c1 (colorless1), pl1 (purple plant1) and p1 (pericarp color1). C1 or PL1 along with R1 or B1 and WD40 Pale Aleurone Color1 (PAC1) form a ternary MYB-bHLH-WD40 (MBW) transcription factor complex to regulate the synthesis of anthocyanins at the transcriptional level in maize kernels or vegetative plant parts (Sharma et al., 2011). In addition, other regulatory genes include viviparous1 (vp1), anthocyaninless lethal1 (anl1) and intensifier1 (in1). VP1 controls the anthocyanin pathway in maize seed development primarily through activation of the C1 gene in the aleurone layer (McCarty et al., 1989). The IN1 gene product is a repressor that shares homology with R1/B1 and functions as a competitive inhibitor of R1 by binding to C1 (Burr et al., 1996). Crop performance depends on multiple traits and metabolic pathways, and multiple traits must simultaneously be addressed during crop improvement. To meet these broad demands, diverse multigene stacking strategies have been developed to introduce multiple genes or complex metabolic pathways into plants. Sexual crossing, a simple but time-consuming and labour-intensive strategy, has been successfully applied in traditional cross-breeding (hybrid rice) but is powerless in introducing genes from species that are not sexually compatible with the crop of interest. Cotransformation mediated by biolistic gene transfer can simultaneously deliver multiple transgenes but is coupled with the drawbacks of complex genome integration of transgenes and unstable linkage inheritance between generations (Chen et al., 1998). Serial retransformation is another approach for stacking multiple transgenes but requires different selection marker genes for each transformation (Qi et al., 2004). Assembly of multiple individual cassettes into a T-DNA vector is a common multigene stacking strategy when using Agrobacterium-mediated plant genetic transformation (Zhu et al., 2017), but the challenges of potential gene silencing caused by repetitious use of the same promoter, large size of the T-DNA construct and complex recombination steps of multiple genes stacking in a vector may limit its application. 2A peptides found in various viruses and Impatiens balsamina are selfcleaving linker peptides varying from16 to 20 amino acids in length (Szymczak et al., 2004). No matter in animal or plant cells, individual proteins can be released from translated, 2A-linked, multicistrons by self-cleaving (Ha et al., 2010; Szymczak et al., 2004). In this study, we combine the advantages of a bidirectional promoter and self-cleaving 2A peptides to produce a simple and efficient multigene expression system (MES) that can be used for polyprotein expression and engineering complex metabolic pathways, as we demonstrate by rebuilding the anthocyanin biosynthesis pathway in maize embryos. Four synthesized basic multicistrons were linked by 2A peptides from different origins: (i) contained four fluorescent protein genes (GFP, YFP, CFP and mCherry), (ii) b-glucuronidase gene (GUS), (iii) two phytase genes (AO and CP53) and (iv) four anthocyanin biosynthesis-related genes (ZmBz1, ZmBz2, ZmC1 and ZmR2). Each was then fused to both ends of PZmBD1, which obtained frommaize inbred line B73 (Liu et al., 2016) to generate multigene expression constructs. Before stable transformation, these constructs were

Constitutive expression of the ZmZIP7 in Arabidopsis alters metal homeostasis and increases Fe and Zn content.

Iron (Fe) and zinc (Zn) are important micronutrients for plant growth and development. Zinc-regulated transporters and the iron-regulated transporter-like protein (ZIP) are necessary for the homeostatic regulation of these metal micronutrients. In this study, the physiological function of ZmZIP7 which encodes a ZIP family transporter was characterized. We detected the expression profiles of ZmZIP7 in maize, and found that the accumulation of ZmZIP7 in root, stem, leaf, and seed was relatively higher than tassel and young ear. ZmZIP7 overexpression transgenic Arabidopsis lines were generated and the metal contents in transgenic and wild-type (WT) plants were examined using inductively coupled plasma atomic emission spectroscopy (ICP-OES) and Zinpyr-1 staining. Fe and Zn concentrations were elevated in the roots and shoots of ZmZIP7-overexpressing plants, while only Fe content was elevated in the seeds. We also analyzed the expression profiles of endogenous genes associated with metal homeostasis. Both endogenic Fe-deficiency inducible genes and the genes responsible for Zn and Fe transport and storage were stimulated in ZmZIP7 transgenic plants. In conclusion, ZmZIP7 encodes a functional Zn and Fe transporter, and ectopic overexpression of ZmZIP7 in Arabidopsis stimulate endogenous Fe and Zn uptake mechanisms, thereby facilitating both metal uptake and homeostasis. Our results contribute to improved understanding of ZIP family transporter functions and suggest that ZmZIP7 could be used to enhance Fe levels in grains.

Isolation of a maize ZmCI - 1B promoter and characterization of its activity in transgenic maize and tobacco

Key messageThe 2-kbZmCI-1Bpromoter is active in the root and embryo and induced by wounding in maize and the 220-bp 5′-deleted segment maybe the minimal promoter.AbstractThe subtilisin–chymotrypsin inhibitor gene, CI-1B of Zea mays (ZmCI-1B), has been suggested to induce the maize defense system to resist insect attack. Real-time RT-PCR showed that ZmCI-1B gene exhibited especially high expression in roots and embryos. The 2-kb full-length promoter of ZmCI-1B gene was isolated from the maize genome and used to drive expression of a beta-glucuronidase (GUS) reporter gene for transient expression and stable expression analysis in maize. The results of GUS histochemical staining in transgenic maize plants revealed that the ZmCI-1B promoter induced GUS expression preferentially in roots and embryos and in response to wounding. A series of 5′-deleted segments of the ZmCI-1B promoter were cloned individually to drive GUS expression for further analysis. Deletion analysis combined with the histochemical staining of transgenic tobacco plants revealed 220-bp segment could drive GUS in a tissue-specific and wounding-induced expression in tobacco; thus, it maybe the minimally active promoter of ZmCI-1B gene. Furthermore, it revealed that the ZmCI-1B promoter contained tissue-specific and wounding-induced elements.