Xiaoqing Liu

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.

Identification of a highly efficient stationary phase promoter in Bacillus subtilis

A promoter that enabled high-level expression of the target gene during the stationary phase in the absence of an inducer would facilitate the efficient production of heterogeneous proteins at a low cost. In this study, a genome-scale microarray-based approach was employed to identify promoters that induced high-level expression of the target genes in Bacillus subtilis from the late log phase to the stationary phase without an inducer. Eleven candidate promoters were selected based on B. subtilis microarray data and the quantitative PCR analysis. Among the selected promoters, Pylb exhibited the highest activity with the reporter bgaB during the stationary phase. Compared with P43 (a commonly used constitutive promoter), promoter Pylb could express two reporter genes (egfp and mApple), and the expression levels of EGFP and RFP were 7.8- and 11.3-fold higher than that of P43, respectively. This finding was verified by overexpression of the genes encoding pullulanase and organophosphorus hydrolase, the activities of which were 7.4- and 2.3-fold higher, respectively, when driven by Pylb compared with P43. Therefore, our results suggest that the Pylb promoter could be used to overexpress target genes without an inducer; this method could facilitate the identification and evaluation of attractive promoters in the genome.

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

BackgroundD-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 30xa0days after pollination), co-regulatory networks were constructed to explore IP metabolism and its interactions with other pathways.ResultsDifferentially 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 Ca2+ 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.ConclusionsThis 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 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.

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

Predicting synonymous codon usage and optimizing the heterologous gene for expression in E. coli

Of the 20 common amino acids, 18 are encoded by multiple synonymous codons. These synonymous codons are not redundant; in fact, all of codons contribute substantially to protein expression, structure and function. In this study, the codon usage pattern of genes in the E. coli was learned from the sequenced genomes of E. coli. A machine learning based method, Presyncodon was proposed to predict synonymous codon selection in E. coli based on the learned codon usage patterns of the residue in the context of the specific fragment. The predicting results indicate that Presycoden could be used to predict synonymous codon selection of the gene in the E. coli with the high accuracy. Two reporter genes (egfp and mApple) were designed with a combination of low- and high-frequency-usage codons by the method. The fluorescence intensity of eGFP and mApple expressed by the (egfp and mApple) designed by this method was about 2.3- or 1.7- folds greater than that from the genes with only high-frequency-usage codons in E. coli. Therefore, both low- and high-frequency-usage codons make positive contributions to the functional expression of the heterologous proteins. This method could be used to design synthetic genes for heterologous gene expression in biotechnology.