Suzhi Zhang

Validation of Potential Reference Genes for qPCR in Maize across Abiotic Stresses, Hormone Treatments, and Tissue Types

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful and widely used technique for the measurement of gene expression. Reference genes, which serve as endogenous controls ensure that the results are accurate and reproducible, are vital for data normalization. To bolster the literature on reference gene selection in maize, ten candidate reference genes, including eight traditionally used internal control genes and two potential candidate genes from our microarray datasets, were evaluated for expression level in maize across abiotic stresses (cold, heat, salinity, and PEG), phytohormone treatments (abscisic acid, salicylic acid, jasmonic acid, ethylene, and gibberellins), and different tissue types. Three analytical software packages, geNorm, NormFinder, and Bestkeeper, were used to assess the stability of reference gene expression. The results revealed that elongation factor 1 alpha (EF1α), tubulin beta (β-TUB), cyclophilin (CYP), and eukaryotic initiation factor 4A (EIF4A) were the most reliable reference genes for overall gene expression normalization in maize, while GRP (Glycine-rich RNA-binding protein), GLU1(beta-glucosidase), and UBQ9 (ubiquitin 9) were the least stable and most unsuitable genes. In addition, the suitability of EF1α, β-TUB, and their combination as reference genes was confirmed by validating the expression of WRKY50 in various samples. The current study indicates the appropriate reference genes for the urgent requirement of gene expression normalization in maize across certain abiotic stresses, hormones, and tissue types.

Genome-scale identification of resistance gene analogs and the development of their intron length polymorphism markers in maize

As introns are vulnerable to changes such as insertions and deletions when exposed to various evolutionary forces, they constitute a repository for developing genetic markers based on intron length polymorphisms (ILP). This study developed a set of genetic markers that use the potential intron length polymorphism in resistance gene analogs (RGAs) in Zea mays. By searching the genome of Zea mays B73 for the homologs of 73 R genes which have already been identified in plants, we found 861 RGAs, 632 of which have at least one intron that can serve as putative markers targeting the intron length polymorphism in RGAs (RGA-ILP). We developed 1972 candidate markers via electronic PCR (e-PCR) with primer pairs designed in each pair of exonic regions that flank an intron. Furthermore, the performance of RGA-ILP among four maize inbred lines (Huangzao4, B73, Mo17, and Dan340) was evaluated with 69 pairs of randomly selected primers. Of them, 46.4% showed bands that had discriminating length polymorphism, and between any two of the inbred lines the proportion of polymorphism ranged from 23.2 to 31.9%. To make it convenient to use these markers for those interested in molecular breeding of disease-resistant maize, we provide all related information in a web-based database named MaizeRGA, which is available at http://www.sicau.edu.cn/web/yms/rga/maizeRGA.html.

Identification, and Functional and Expression Analyses of the CorA/MRS2/MGT-Type Magnesium Transporter Family in Maize.

Magnesium (Mg(2+)) is an essential macronutrient for plant growth and development, and the CorA/MRS2/MGT-type Mg(2+) transporters play important roles in maintaining Mg(2+) homeostasis in plants. Although the MRS2/MGT genes have been identified in two model plant species, Arabidopsis and rice, a comprehensive analysis of the MRS2/MGT gene family in other plants is lacking. In this work, 12 putative MRS2/MGT genes (ZmMGT1- ZmMGT12) were identified in maize and all of them were classified into five distinct subfamilies by phylogenetic analysis. A complementation assay in the Salmonella typhimurium MM281 strain showed that five representatives of the 12 members possess Mg(2+) transport abilities. Inhibition of ZmMGT protein activity using the hexaamminecobalt (III) (Co-Hex) inhibitor indicated that the ZmMGT protein mediated both low-affinity and high-affinity Mg(2+) transport in maize. A semi-quantitative reverse transcription-PCR (RT-PCR) analysis revealed that eight genes were constitutively expressed in all of the detected tissues, with one being specifically expressed in roots and three having no detectable expression signals. A quantitative RT-PCR analysis showed that some ZmMGT members displayed differential responses to Mg(2+) deficiency and aluminum (Al) stress. Furthermore, root growth inhibition and Mg(2+) accumulation analyses in two maize inbred lines, which conferred different levels of Al tolerance, revealed that ZmMGT proteins contributed to the Al resistance of the Al tolerance genotype. We hypothesize that ZmMGT family members function as Mg(2+) transporters and may play a role in linking Mg(2+) deficiency and Al stress responses. Our results will be valuable in a further analysis of the important biological functions of ZmMGT members in maize.

Genome-wide identification of microRNAs responding to early stages of phosphate deficiency in maize.

Phosphorus (P) is an essential element involved in numerous biochemical reactions. In plants, stress responses, such as the expression of microRNAs (miRNAs), are induced to help them adapt to low phosphate (Pi) concentrations. In this study, deep sequencing was performed using the roots and leaves of maize seedlings grown under low Pi concentrations to identify miRNAs that are differentially expressed during the early stages of Pi deficiency. Eight small RNA libraries were constructed, and 159 known miRNAs representing 32 miRNA families and 10 novel miRNAs. Members of the miR396 family were extremely abundant. Further, 28 Pi-responsive miRNAs were identified (27 known and 1 novel) of which 8 and 7 were significantly expressed exclusively in leaf and root tissues, respectively. The analysis of Pi-responsive miRNAs target genes suggested that most target genes functioning as transcription factors were involved in root and leaf development. The expression profiles of selected Pi-responsive miRNAs and target genes were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, we discuss the significance of the differences in expression patterns of these miRNAs during the early and later stages of Pi starvation. This study provides useful information concerning the role of miRNAs in response to Pi starvation and will further our understanding of the mechanisms governing Pi homeostasis in maize.

Genome-wide analysis of Gro/Tup1 family corepressors and their responses to hormones and abiotic stresses in maize

Gro/Tup1 proteins act as negative transcriptional regulators and play crucial roles in many growth and developmental processes in a wide range of organisms. However, our understanding of Gro/Tup1 protein functions in plants is confined to the model plant Arabidopsis. Here, 11 Gro/Tup1 genes, which were characterized by the typical LisH and WD40 repeat domains, were identified in maize through a genome-wide survey. A phylogenetic analysis revealed that maize Gro/Tup1 proteins could be divided into three subfamilies, in which members shared similar protein and gene structures. The predicted maize Gro/Tup1 genes were distributed on seven chromosomes and segmental duplication contributed to their expansion. Many predicted cis-elements associated with hormones, biotic- or abioticstress responses, meristem and seed development, and circadian rhythms, were found in their putative promoter regions. A potential associated protein analysis identified a large number of candidates, including transcription factors, chromatin-modifying enzymes, protein kinases, and ubiquitinconjugating enzymes. An expression profile derived from the RNA-seq data indicated that Gro/Tup1 genes in maize were widely expressed in various organs and tissues. Quantitative real-time PCR revealed that these genes responded to at least one hormone or abiotic stress, either in roots or in shoots. Our study provides useful information on the Gro/Tup1 genes in maize and will facilitate the further functional validation of these genes in growth and development, hormone responses, and biotic- or abiotic-stress resistance.

Erratum to: Mining for low-nitrogen tolerance genes by integrating meta-analysis and large-scale gene expression data from maize

Abstract Nitrogen (N) is the most important macronutrient for plant growth and development. Hence, understanding genetic architectures and functional genes involved in the response to N deficiency can greatly facilitate the development of low-N-tolerant cultivars. In this study, we collected 212 quantitative trait loci (QTL) of agronomically important traits under low-N stress conditions in maize. We then identified 21 consensus QTL (cQTL) strongly induced for low-N tolerance after excluding overlapping cQTL containing QTL simultaneously identified in meta-analyses of studies performed under other environmental conditions. Among the 21 cQTL, 30 candidate maize genes were identified from maize large-scale differential expression data derived from analyses of low-N stress, and the 12 most important maize orthologs were identified using homologous BLAST analyses of genes with known functions in N use efficiency in model plants. Furthermore, maize orthologs associated with low-N tolerance and metabolism were also predicted using large-scale expression data from other model plants. The present genetic loci and candidate genes indicate the molecular mechanisms of low-N tolerance in maize and may provide information for QTL fine mapping and molecular marker-assisted selection.

Cloning, molecular evolution and functional characterization of ZmbHLH16, the maize ortholog of OsTIP2 (OsbHLH142)

ABSTRACT The transcription factor ZmbHLH16, the maize ortholog of OsTIP2 (OsbHLH142), was isolated in the present study. Tissue expression analysis showed that ZmbHLH16 is preferentially expressed in male reproductive organs. Subcellular location analysis of ZmbHLH16 via rice protoplast indicated that it is located in the nucleus. Through nucleotide variation analysis, 36 polymorphic sites in ZmbHLH16, including 23 single nucleotide polymorphisms and 13 InDels, were detected among 78 maize inbred lines. Neutrality tests and linkage disequilibrium analysis showed that ZmbHLH16 experienced no significant evolutionary pressure. Yeast one-hybrid experiment showed that the first 80 residues in the N-terminus of ZmbHLH16 had transactivation activity, whereas the full length did not. Genome-wide coexpression analysis showed that 395 genes were coexpressed with ZmbHLH16. Among these genes, the transcription factor ZmbHLH51 had similar expression pattern and identical subcellular localization to those of ZmbHLH16. Subsequently, the interaction between ZmbHLH51 and ZmbHLH16 was verified by yeast two-hybrid experiment. Through yeast two-hybrid analysis of series truncated ZmbHLH16 fragments, we found not only the typical bHLH domain [175-221 amino acids (a.a.)], but also that the 81-160 a.a. and 241-365 a.a. of ZmbHLH16 could interact with ZmbHLH51. All these results lay the foundation for further understanding the functions of ZmbHLH16. Summary: ZmbHLH16, the maize ortholog of OsTIP2 (OsbHLH142), its subcellular location, molecular evolution, transcriptional activity domains and heterodimerization domains with ZmbHLH51 are analyzed.

DNA methylation analysis of sterile and fertile CMS-C hybrids and their parents in maize

C-type cytoplasmic male sterile (CMS-C) line plays an important role in hybrid seed production in maize. However, mechanisms of pollen abortion and fertility restoration remain unclear. This study aimed to investigate the mechanisms of CMS-C pollen abortion and fertility restoration, particularly based on epigenetics, and to understand the relationship between male fertility performance and DNA methylation status. Methylation-sensitive amplification polymorphism (MSAP) technique was conducted to analyze DNA methylation levels and patterns of tassels at pollen-mother cell, tetrad, mononuclear, and binuclear stages among four half-sib hybrids and their parents with different fertility. Results showed that DNA methylation levels in fertility restored hybrids were higher than those in sterility maintained hybrids. Nine CCGG sites, which displayed methylation polymorphism between male-fertile and male-sterile hybrids, were screened. To validate MSAP results, we performed methylation-sensitive PCR (MS-PCR) and found consistent observations in cloned methylation sites. Interestingly, a specific site named 16–1 was discovered in the region of Rf5 gene, which is one of the restorer genes of CMS-C. Thus, DNA methylation may participate in regulating the expression of fertility restorer genes of maize CMS-C.

Study on the expression of specific chimeric-orfs of C type cytoplasmic male sterile in maize

In the present study, a mutated chimeric orf364, named orf366-c, was identified in a maize C type cytoplasmic male sterile line (CMS-C) by PCR and RT-PCR. Four base pair adjacent base substitution mutations (CAAA to TTTT) and eight bp insertion mutations were found in orf366-c compared with orf364. ORF366-C was predicted to contain one membrane-spanning domain using TMHMM online software. Real-time quantitative PCR analysis showed that orf366-c was upregulated in the CMS-C line in comparison to its maintainer line at the uninucleate stage. The protokaryotic protein expression of orf366-c in Escherichia coli showed that ORF366-C could be a cytotoxic protein. All the results indicated that orf366-c may be associated with maize CMS-C.

Molecular and functional characterization of the magnesium transporter gene ZmMGT12 in maize

Magnesium (Mg) is an essential mineral element for normal plant growth and development, and the CorA/MRS2/MGT-type Mg transporters play a significant role in maintaining Mg homeostasis in plants. In total, 12 maize CorA-like Mg2+ transporters have been identified, but none of them had been functionally characterized. Accordingly, we cloned and functionally characterized ZmMGT12 from the maize CorA-like gene family. ZmMGT12 exhibited the structure typical of Mg2+ transporters, i.e., two conserved TM domains and a GMN tripeptide motif. ZmMGT12, Arabidopsis AtMGT6, and rice OsMRS2-6 shared high protein sequence identity and thus clustered in the same phylogenetic branch, suggesting that they could be homologs. A functional complementation assay in the Salmonella typhimurium MM281 mutant indicated that ZmMGT12 possessed Mg2+ transport ability. ZmMGT12 was expressed in roots, stems, and leaves, with the highest expression in leaves. Moreover, ZmMGT12 expression was induced by light and exhibited a circadian expression pattern. In addition, the expression level of ZmMGT12 in leaf tissue was related to chlorophyll synthesis. Overexpression of ZmMGT12 in Arabidopsis caused no phenotypic change in transgenic plants, including in fresh shoot weight, chlorophyll content, shoot Mg2+ content, and chloroplast Mg2+ content. Together, these results suggest that ZmMGT12 is a Mg2+ transporter and may play a role in Mg transport into chloroplasts.