Beijiu Cheng

CCCH-Type Zinc Finger Family in Maize: Genome-Wide Identification, Classification and Expression Profiling under Abscisic Acid and Drought Treatments

Background CCCH-type zinc finger proteins comprise a large protein family. Increasing evidence suggests that members of this family are RNA-binding proteins with regulatory functions in mRNA processing. Compared with those in animals, functions of CCCH-type zinc finger proteins involved in plant growth and development are poorly understood. Methodology/Principal Findings Here, we performed a genome-wide survey of CCCH-type zinc finger genes in maize (Zea mays L.) by describing the gene structure, phylogenetic relationships and chromosomal location of each family member. Promoter sequences and expression profiles of putative stress-responsive members were also investigated. A total of 68 CCCH genes (ZmC3H1-68) were identified in maize and divided into seven groups by phylogenetic analysis. These 68 genes were found to be unevenly distributed on 10 chromosomes with 15 segmental duplication events, suggesting that segmental duplication played a major role in expansion of the maize CCCH family. The Ka/Ks ratios suggested that the duplicated genes of the CCCH family mainly experienced purifying selection with limited functional divergence after duplication events. Twelve maize CCCH genes grouped with other known stress-responsive genes from Arabidopsis were found to contain putative stress-responsive cis-elements in their promoter regions. Seven of these genes chosen for further quantitative real-time PCR analysis showed differential expression patterns among five representative maize tissues and over time in response to abscisic acid and drought treatments. Conclusions The results presented in this study provide basic information on maize CCCH proteins and form the foundation for future functional studies of these proteins, especially for those members of which may play important roles in response to abiotic stresses.

Genome-wide identification, classification and analysis of heat shock transcription factor family in maize

BackgroundHeat shock response in eukaryotes is transcriptionally regulated by conserved heat shock transcription factors (Hsfs). Hsf genes are represented by a large multigene family in plants and investigation of the Hsf gene family will serve to elucidate the mechanisms by which plants respond to stress. In recent years, reports of genome-wide structural and evolutionary analysis of the entire Hsf gene family have been generated in two model plant systems, Arabidopsis and rice. Maize, an important cereal crop, has represented a model plant for genetics and evolutionary research. Although some Hsf genes have been characterized in maize, analysis of the entire Hsf gene family were not completed following Maize (B73) Genome Sequencing Project.ResultsA genome-wide analysis was carried out in the present study to identify all Hsfs maize genes. Due to the availability of complete maize genome sequences, 25 nonredundant Hsf genes, named ZmHsfs were identified. Chromosomal location, protein domain and motif organization of ZmHsfs were analyzed in maize genome. The phylogenetic relationships, gene duplications and expression profiles of ZmHsf genes were also presented in this study. Twenty-five ZmHsfs were classified into three major classes (class A, B, and C) according to their structural characteristics and phylogenetic comparisons, and class A was further subdivided into 10 subclasses. Moreover, phylogenetic analysis indicated that the orthologs from the three species (maize, Arabidopsis and rice) were distributed in all three classes, it also revealed diverse Hsf gene family expression patterns in classes and subclasses. Chromosomal/segmental duplications played a key role in Hsf gene family expansion in maize by investigation of gene duplication events. Furthermore, the transcripts of 25 ZmHsf genes were detected in the leaves by heat shock using quantitative real-time PCR. The result demonstrated that ZmHsf genes exhibit different expression levels in heat stress treatment.ConclusionsOverall, data obtained from our investigation contributes to a better understanding of the complexity of the maize Hsf gene family and provides the first step towards directing future experimentation designed to perform systematic analysis of the functions of the Hsf gene family.

Systematic Analysis of Sequences and Expression Patterns of Drought-Responsive Members of the HD-Zip Gene Family in Maize

Background Members of the homeodomain-leucine zipper (HD-Zip) gene family encode transcription factors that are unique to plants and have diverse functions in plant growth and development such as various stress responses, organ formation and vascular development. Although systematic characterization of this family has been carried out in Arabidopsis and rice, little is known about HD-Zip genes in maize (Zea mays L.). Methods and Findings In this study, we described the identification and structural characterization of HD-Zip genes in the maize genome. A complete set of 55 HD-Zip genes (Zmhdz1-55) were identified in the maize genome using Blast search tools and categorized into four classes (HD-Zip I-IV) based on phylogeny. Chromosomal location of these genes revealed that they are distributed unevenly across all 10 chromosomes. Segmental duplication contributed largely to the expansion of the maize HD-ZIP gene family, while tandem duplication was only responsible for the amplification of the HD-Zip II genes. Furthermore, most of the maize HD-Zip I genes were found to contain an overabundance of stress-related cis-elements in their promoter sequences. The expression levels of the 17 HD-Zip I genes under drought stress were also investigated by quantitative real-time PCR (qRT-PCR). All of the 17 maize HD-ZIP I genes were found to be regulated by drought stress, and the duplicated genes within a sister pair exhibited the similar expression patterns, suggesting their conserved functions during the process of evolution. Conclusions Our results reveal a comprehensive overview of the maize HD-Zip gene family and provide the first step towards the selection of Zmhdz genes for cloning and functional research to uncover their roles in maize growth and development.

Identification and characterization of Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families in maize

Eukaryotic gene expression is regulated at least by two processes, RNA interference at the post-transcriptional level and chromatin modification at the transcriptional level. Distinct small RNAs (approximately 21–24 nucleotides; sRNAs) were demonstrated to play vital roles in facilitating gene silencing. In plants, the generation of these sRNAs mainly depends on some proteins encoded by respective Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerases (RDR) gene families. Here, we analyzed the DCL, AGO and RDR gene families in maize, including gene structure, phylogenetic relationships, protein conserved motifs and genomic localization among gene family members. A total of 5 Zmdcl, 18 Zmago and 5 Zmrdr genes were identified in maize. Phylogenetic analyses clustered each of these genes families into four subfamilies. In addition, gene chromosomal localization revealed that five pairs of Zmago genes resulted from tandem or segmental duplication, respectively. EST expression data mining revealed that these newly identified genes had temporal and spatial expression pattern. Furthermore, the transcripts of these genes were detected in the leaves by two different abiotic stress treatments using semi-quantitative RT-PCR. The data demonstrated that these genes exhibited different expression levels in stress treatments. The results of this study provided basic genomic information for these gene families and insights into the probable roles of these genes in plant growth and development. This will further provide a solid foundation for future functional genomics studies of Dicer-like, Argonaute and RDR gene families in maize.

Bacterially expressed dsRNA protects maize against SCMV infection.

RNA interference (RNAi) is a sequence-specific, posttranscriptional gene silencing (PTGS) process in plants that is mediated by dsRNA homologous to the silenced gene(s). In this study, we report an efficient method to produce dsRNA using a bacterial expression system. Two fragments of the Sugarcane Mosaic Virus (SCMV) CP (coat protein) gene were amplified by RT-PCR, and cloned into the inverted-repeat cloning vector pUCCRNAi. The two recombinant plasmids were transformed individually into E. coli HT115, an RNase-III deficient strain, and dsRNA was induced by isopropyl-β-d-thiogalactopyranoside (IPTG). The crude extracts of E.coli HT115 containing large amounts of dsRNA were applied to plants as a spray and the experiment confirmed a preventative efficacy. Our findings demonstrated that spraying crude dsRNA-containing extracts inhibited SCMV infection, and the dsRNA derived from an upstream region (CP1) was more effective than was dsRNA derived from a downstream region (CP2) of the SCMV CP gene. The results provide a valuable tool for plant viral control using dsRNA and the PTGS approach.

Genome-wide analysis of soybean HD-Zip gene family and expression profiling under salinity and drought treatments.

Background Homeodomain-leucine zipper (HD-Zip) proteins, a group of homeobox transcription factors, participate in various aspects of normal plant growth and developmental processes as well as environmental responses. To date, no overall analysis or expression profiling of the HD-Zip gene family in soybean (Glycine max) has been reported. Methods and Findings An investigation of the soybean genome revealed 88 putative HD-Zip genes. These genes were classified into four subfamilies, I to IV, based on phylogenetic analysis. In each subfamily, the constituent parts of gene structure and motif were relatively conserved. A total of 87 out of 88 genes were distributed unequally on 20 chromosomes with 36 segmental duplication events, indicating that segmental duplication is important for the expansion of the HD-Zip family. Analysis of the Ka/Ks ratios showed that the duplicated genes of the HD-Zip family basically underwent purifying selection with restrictive functional divergence after the duplication events. Analysis of expression profiles showed that 80 genes differentially expressed across 14 tissues, and 59 HD-Zip genes are differentially expressed under salinity and drought stress, with 20 paralogous pairs showing nearly identical expression patterns and three paralogous pairs diversifying significantly under drought stress. Quantitative real-time RT-PCR (qRT-PCR) analysis of six paralogous pairs of 12 selected soybean HD-Zip genes under both drought and salinity stress confirmed their stress-inducible expression patterns. Conclusions This study presents a thorough overview of the soybean HD-Zip gene family and provides a new perspective on the evolution of this gene family. The results indicate that HD-Zip family genes may be involved in many plant responses to stress conditions. Additionally, this study provides a solid foundation for uncovering the biological roles of HD-Zip genes in soybean growth and development.

A novel maize homeodomain-leucine zipper (HD-Zip) I gene, Zmhdz10, positively regulates drought and salt tolerance in both rice and Arabidopsis.

Increasing evidence suggests that homeodomain-leucine zipper I (HD-Zip) I transcription factors play important roles in abiotic stress responses, but no HD-Zip I proteins have been reported in maize. Here, a drought-induced HD-Zip I gene, Zmhdz10, was isolated from maize and characterized for its role in stress responses. Real-time quantitative PCR showed that expression of Zmhdz10 was also induced by salt stress and ABA. Transient expression of Zmhdz10-green fluorescent protein (GFP) fusion proteins in onion cells showed a nuclear localization of Zmhdz10. Yeast hybrid assays demonstrated that Zmhdz10 has transactivation and DNA-binding activity in yeast cells. Overexpression of Zmhdz10 in rice led to enhanced tolerance to drought and salt stresses and increased sensitivity to ABA. Moreover, Zmhdz10 transgenic plants had lower relative electrolyte leakage (REL), lower malondialdehyde (MDA) and increased proline content relative to wild-type plants under stress conditions, which may contribute to enhanced stress tolerance. Zmhdz10 transgenic Arabidopsis plants also exhibited enhanced tolerance to drought and salt stresses that was concomitant with altered expression of stress/ABA-responsive genes, including Δ1-Pyrroline-5-carboxylate synthetase 1 (P5CS1), Responsive to dehydration 22 (RD22), Responsive to dehydration 29B (RD29B) and ABA-insensitive 1 (ABI1). Taken together, these results suggest that Zmhdz10 functions as a transcriptional regulator that can positively regulate drought and salt tolerance in plants through an ABA-dependent signaling pathway.

Whole-genome survey and characterization of MADS-box gene family in maize and sorghum

MADS-box genes comprise a large gene family, which codes for transcription factors, and play important functions in various aspects of flowering plant growth and development. However, little is known about the MADS-box genes in maize (Zea mays) and sorghum (Sorghum bicolor). Here, we performed a comprehensive bioinformatics analysis of the MADS-box gene family in the maize and sorghum genomes and identified 75 maize and 65 sorghum MADS-box genes. We subsequently carried out a comparative analysis of these genes, including the gene structure, phylogenetic relationship, conserved protein motifs, gene duplications, chromosomal locations and expression pattern between the two plants. According to these analyses, the MADS-box genes in both maize and sorghum were categorized into five (MIKCC, MIKC*, Mα, Mβ and Mγ) groups, and the MIKCC groups were further divided into 11 subfamilies. In addition, gene duplications of MADS-box genes were also investigated in the maize, sorghum, rice and Arabidopsis genomes. We found a higher percentage of MADS-box gene duplications in the maize and sorghum genomes, which contributed to the expansion of the MADS-box gene family. Furthermore, both tandem and segmental duplications played a major role in the MADS-box gene expansion in maize and sorghum. A survey of maize and sorghum EST sequences indicated that MADS-box genes exhibit a various expression pattern, suggesting diverse and novel functions of MADS-box gene families in the two plants. These results provided a useful reference for selection of candidate MADS-box genes for cloning and further functional analysis in both maize and sorghum.

Genome-wide analysis of the auxin response factor (ARF) gene family in maize (Zea mays)

Auxin response factors (ARFs) are an important family involved in auxin-mediated response through specific binding to auxin response elements (AuxREs). A few members of the ARF family have been functionally characterized in Arabidopsis, rice (Oryza sativa), Poplar (Populous trichocarpa). However, little is known about ARF genes in maize (Zea mays). We performed a comprehensive bioinformatics analysis of the maize ARF gene family including analysis of the genome sequence, conserved domains, chromosomal locations, phylogenetic relationships, gene duplication, and expression profiles. 35 ZmARF genes were identified and categorized into four groups (Class I, II, III, and IV). In addition, a segmental ZmARF duplication event was shown to play an important role in maize ARF gene expansion. 7 ZmARF genes had no expression in specific tissues we obtained, but presented in mixed tissues according to the NCBI EST database, respectively. These studies have laid the theoretical foundation for further functional verification of these ZmARF genes.

Overexpression of a maize WRKY58 gene enhances drought and salt tolerance in transgenic rice

WRKY transcription factors (TFs) are reported to play crucial roles in the processes of plant growth and development, defense regulation and stress responses. In this study, a WRKY group IId TF, designated ZmWRKY58, was isolated from maize (Zea mays L.). Expression pattern analysis revealed that ZmWRKY58 was induced by drought, salt and abscisic acid treatments. Subcellular localization experiments in onion epidermal cells showed the presence of ZmWRKY58 in the nucleus. Overexpression of ZmWRKY58 in rice resulted in delayed germination and inhibited post-germination development. Further investigation showed that ZmWRKY58 overexpressing transgenic plants had higher survival rates and relative water contents, but lower malonaldehyde contents and relative electrical leakage compared with wild-type plants, following drought and salt stress treatments, suggesting that overexpression of ZmWRKY58 leads to enhanced tolerance to drought and salt stresses in transgenic rice. Additionally, yeast two-hybrid assay showed that ZmWRKY58 could interact with ZmCaM2, suggesting that ZmWRKY58 may function as a calmodulin binding protein. Taken together, these results suggest that ZmWRKY58 may act as a positive regulator involved in the drought and salt stress responses.