Transcription factor ZmWRKY20 interacts with ZmWRKY115 to repress expression of ZmbZIP111 for salt tolerance in maize.

Maize (Zea mays) is an important cereal crop worldwide. However, its yield and quality are adversely affected by salt stress resulting from soil hypersalinity. Exploring the regulatory mechanisms of stress responses is of vital importance to increase maize seed production. In the present study, we screened ethyl methanesulfonate-induced maize mutants and identified a salt-tolerant mutant. A single base was mutated in ZmWRKY20, leading to the formation of a truncated protein variant. A detailed phenotypic analysis revealed that this mutant had significantly higher resistance to wilting and lower reactive oxygen species levels than the inbred line B73. ZmWRKY20 showed transcriptional activity in yeast and specifically bound W-boxes according to the results of our yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays. Overexpression of ZmWRKY20 decreased salt tolerance in maize. Transcriptome profiling revealed that ZmWRKY20 overexpression extensively reprogrammed genes involved in regulating defense and oxidation-reduction responses. The results substantiate that ZmWRKY20 is directly targeted to the basic leucine zipper (bZIP) motif in the transcription factor ZmbZIP111. It was also verified that ZmWRKY20 interacts with ZmWRKY115 and both proteins act jointly to enhance ZmbZIP111 repression. The results indicate that the ZmWRKY20 and ZmWRKY115 transcription factors interact in the nucleus, leading to repression of ZmbZIP111 expression by directly binding its promoter, and increase the sensitivity of maize seedlings to salt stress. The current study improves our understanding of the complicated responses of maize to salt stress.

Genome-wide association study leads to novel genetic insights into resistance to Aspergillus flavus in maize kernels.

BACKGROUND: Fungus infection in staple grains affects the food storage and threatens food security. The Aspergillus flavus is known to infect multiple grains and produce mycotoxin Aflatoxin B1, which is mutagenic, teratogenic and causes immunosuppression in animals. However, the molecular mechanism of maize resistance to A. flavus is largely unknown. RESULTS: Here we used corn kernels to investigate resistance genes to A. flavus using genome-wide association study (GWAS) of 313 inbred lines. We characterized the resistance levels of kernels after inoculating with A. flavus. The GWAS with 558,529 SNPs identified four associated loci involving 29 candidate genes that were linked to seed development, resistance or infection, and involved in signal pathways, seed development, germination, dormancy, epigenetic modification, and antimicrobial activity. In addition, a few candidate genes were also associated with several G-protein signaling and phytohormones that might involve in synergistic work conferring different resistance during seed development. Expression of 16 genes out of 29 during kernel development was also associated with resistance levels. CONCLUSIONS: We characterized the resistance levels of 313 maize kernels after inoculating with A. flavus, and found four associated loci and 16 candidate maize genes. The expressed 16 genes involved in kernel structure and kernel composition most likely contribute to mature maize kernels' resistance to A. flavus, and in particular, in the development of pericarp. The linked candidate genes could be experimentally transformed to validate and manipulate fungal resistance. Thus this result adds value to maize kernels in breeding programs.

Maize WRKY114 gene negatively regulates salt-stress tolerance in transgenic rice.

KEY MESSAGE: Overexpression in rice of the isolated salt-responsive WRKY114 gene from maize resulted in decreases in both salt-stress tolerance and abscisic acid sensitivity by regulating stress- and abscisic acid-related gene expression. WRKYs are an important family of transcription factors that widely participate in plant development, defense regulation and stress responses. In this research, WRKY114 encoding a WRKY transcription factor was cloned from maize (Zea mays L.). ZmWRKY114 expression was down-regulated by salt stress but up-regulated by abscisic acid (ABA) treatments. ZmWRKY114 is a nuclear protein with no transcriptional activation ability in yeast. A yeast one-hybrid experiment confirmed that ZmWRKY114 possesses an ability to specifically bind to W-boxes. The heterologous overexpression of ZmWRKY114 in rice enhanced the salt-stress sensitivity as indicated by the transgenic plants having reduced heights, root lengths and survival rates under salt-stress conditions. In addition, transgenic plants also retained lower proline contents, but greater malondialdehyde contents and relative electrical leakage levels. Additionally, ZmWRKY114-overexpressing plants showed less sensitivity to ABA during the early seedling growth stage. Further analyses indicated that transgenic rice accumulated higher levels of ABA than wild-type plants under salt-stress conditions. Transcriptome and quantitative real-time PCR analyses indicated that a few regulatory genes, which play vital roles in controlling plant stress responses and/or the ABA signaling pathway, were affected by ZmWRKY114 overexpression when rice was treated with NaCl. Thus, ZmWRKY114 may function as a negative factor that participates in salt-stress responses through an ABA-mediated pathway.

Genome-wide association study of maize plant architecture using F1 populations.

KEY MESSAGE: Genome-wide association study of maize plant architecture using F1 populations can better dissect various genetic effects that can provide precise guidance for genetic improvement in maize breeding. Maize grain yield has increased at least eightfold during the past decades. Plant architecture, including plant height, leaf angle, leaf length, and leaf width, has been changed significantly to adapt to higher planting density. Although the genetic architecture of these traits has been dissected using different populations, the genetic basis remains unclear in the F1 population. In this work, we perform a genome-wide association study of the four traits using 573 F1 hybrids with a mixed linear model approach and QTXNetwork mapping software. A total of 36 highly significant associated quantitative trait SNPs were identified for these traits, which explained 51.86-79.92% of the phenotypic variation and were contributed mainly by additive, dominance, and environment-specific effects. Heritability as a result of environmental interaction was more important for leaf angle and leaf length, while major effects (a, aa, and d) were more important for leaf width and plant height. The potential breeding values of the superior lines and superior hybrids were also predicted, and these values can be applied in maize breeding by direct selection of superior genotypes for the associated quantitative trait SNPs. A total of 108 candidate genes were identified for the four traits, and further analysis was performed to screen the potential genes involved in the development of maize plant architecture. Our results provide new insights into the genetic architecture of the four traits, and will be helpful in marker-assisted breeding for maize plant architecture.

A Moso Bamboo Drought-Induced 19 Protein, PeDi19-4, Enhanced Drought and Salt Tolerance in Plants via the ABA-Dependent Signaling Pathway.

Here, 10 drought-induced 19 (Di19) proteins from Phyllostachys edulis were analyzed and an important stress-related candidate gene (PeDi19-4) was isolated based on analysis of phylogenetic relationships and expression profiles. PeDi19-4 is a nuclear localization protein that can bind the conserved TACA(A/G)T sequence, as determined using enzyme-linked immunosorbent assay (EMSA). PeDi19-4 has no transcriptional activity in yeast but functions as a transcription activator in plants. Overexpression of PeDi19-4 in rice and Arabidopsis thaliana enhanced drought and salt tolerance as determined through phenotypic analysis and the use of stress-associated physiological indicators. PeDi19-4 transgenic plants showed increased sensitivity to ABA during seed germination and early seedling growth. Additionally, transgenic rice accumulated more ABA than wild-type plants under drought and salt stress conditions. Moreover, the stomata of PeDi19-4-overexpressing plants changed significantly with ABA treatment. RNA sequencing revealed that PeDi19-4 regulated the expression of a wide spectrum of stress-/ABA-responsive differentially expressed genes. The stress-responsive genes (OsZFP252 and OsNAC6) and ABA-responsive genes (OsBZ8 and OsbZIP23) were direct targets of PeDi19-4. Our research indicated that PeDi19-4 enhanced drought and salt tolerance in plants via the ABA-dependent signaling pathway.

Global transcriptome and weighted gene co-expression network analyses reveal hybrid-specific modules and candidate genes related to plant height development in maize.

KEY MESSAGE: Weighted gene co-expression network analysis was explored to find key hub genes involved in plant height regulation. Plant height, an important trait for maize breeding because of its close relatedness to lodging resistance and yield, has been reported to be determined by multiple qualitative and quantitative genes. However, few genes related to plant height have been characterized in maize. Herein, three different maize hybrids, with extremely distinct plant height, which were further classified into low (L), middle (M) and high (H) group, were selected for RNA sequencing at three key developmental stages, namely, jointing stage (I), big flare period (II) and tasseling stage (III). Intriguingly, transcriptome profiles for hybrids ranging from low to high group exhibited significantly similarity in both jointing stage and big flare period. However, remarkably larger differentially expressed genes could be detected between hybrid from low to either middle or high group in tasseling stage. These results were repeatedly observed in both phenotyping and gene ontology enrichment analysis, indicating that transition from big flare period to tasseling stage plays a critical role in determination of plant height. Furthermore, weighted gene co-expression network analysis was explored to find key hub genes involved in plant height regulation. Hundreds of candidate genes, encoding various transcription factors, and regulators involved in internode cell regulation and cell wall synthesis were identified in our network. More importantly, great majority of candidates were correlated to either metabolism or signaling pathway of several plant phytohormones. Particularly, numerous functionally characterized genes in gibberellic acid as well as brassinosteroids signaling transduction pathways were also discovered, suggesting their critical roles in plant height regulation. The present study could provide a modestly comprehensive insight into networks for regulation of plant height in maize.

The maize WRKY transcription factor ZmWRKY17 negatively regulates salt stress tolerance in transgenic Arabidopsis plants.

MAIN CONCLUSION: We cloned and characterized the ZmWRKY17 gene from maize. Overexpression of ZmWRKY17 in Arabidopsis led to increased sensitivity to salt stress and decreased ABA sensitivity through regulating the expression of some ABA- and stress-responsive genes. The WRKY transcription factors have been reported to function as positive or negative regulators in many different biological processes including plant development, defense regulation and stress response. This study isolated a maize WRKY gene, ZmWRKY17, and characterized its role in tolerance to salt stress by generating transgenic Arabidopsis plants. Expression of the ZmWRKY17 was up-regulated by drought, salt and abscisic acid (ABA) treatments. ZmWRKY17 was localized in the nucleus with no transcriptional activation in yeast. Yeast one-hybrid assay showed that ZmWRKY17 can specifically bind to W-box, and it can activate W-box-dependent transcription in planta. Heterologous overexpression of ZmWRKY17 in Arabidopsis remarkably reduced plant tolerance to salt stress, as determined through physiological analyses of the cotyledons greening rate, root growth, relative electrical leakage and malondialdehyde content. Additionally, ZmWRKY17 transgenic plants showed decreased sensitivity to ABA during seed germination and early seedling growth. Transgenic plants accumulated higher content of ABA than wild-type (WT) plants under NaCl condition. Transcriptome and quantitative real-time PCR analyses revealed that some stress-related genes in transgenic seedlings showed lower expression level than that in the WT when treated with NaCl. Taken together, these results suggest that ZmWRKY17 may act as a negative regulator involved in the salt stress responses through ABA signalling.

A novel GRAS transcription factor, ZmGRAS20, regulates starch biosynthesis in rice endosperm.

Starch occupies the maximal component of cereal grains and is pivotal for maize yield and quality. However, the regulatory mechanism of starch synthesis is still poorly understand. In this study, a GRAS transcription factor, ZmGRAS20, was isolated from maize inbred line B73 based on transcriptome sequencing. Quantitative real-time PCR indicated that ZmGRAS20 is specifically expressed in maize endosperm. Transient expression of ZmGRAS20-green fluorescent protein fusion protein in tobacco cells showed a nucleus and membrane localization of the protein. Transactivation assay of ZmGRAS20 demonstrated that it has no transactivation activity in yeast cells. Overexpression of ZmGRAS20 led to a chalky region of ventral endosperm with decreased starch content and defective agronomic characters in transgenic seeds. Moreover, ZmGRAS20-overexpression plants had fewer fractions of long-branched starch chains. Further scanning electron microscopy observation of ZmGRAS20 transgenic seeds exhibited altered starch granules morphology compared with wide type plants. Taken together, these results suggested that ZmGRAS20 may function as a starch synthesis regulatory factor in rice endosperm.

Genome-wide analysis of the IQD gene family in maize.

IQD gene family plays important roles in plant developmental processes and stress responses. To date, no systematic characterization of this gene family has been carried out in maize. In this study, 26 IQD genes, from ZmIQD1 to ZmIQD26, were identified using Blast search tools. The phylogenetic analysis showed these genes were divided into four subfamilies (IQD I-IV) and members within the same subfamily shared conserved exon/intron distribution and motif composition. The 26 ZmIQD genes are distributed unevenly on 8 of the 10 chromosomes, with 9 segmental duplication events, suggesting that the expansion of IQDs in maize was due to the segmental duplication. The analysis of Ka/Ks ratios showed that the duplicated ZmIQDs had primarily undergone strong purifying selection. In addition, the 26 ZmIQDs displayed different expression patterns at different developmental stages of maize based on transcriptome analysis. Further, quantitative real-time PCR analysis showed that all 26 ZmIQD genes were responsive to drought treatment, suggesting their crucial roles in drought stress response. Yeast two-hybrid assay proved that ZmIQD2 and ZmIQD15 can interact with ZmCaM2 and IQ or I in IQ motif is required for ZmIQD15 to combine with CaM2. Our results present a comprehensive overview of the maize IQD gene family and lay an important foundation for further analysis aimed at uncovering the biological functions of ZmIQDs in growth and development.

The WRKY transcription factor ZmWRKY92 binds to GA synthesis-related genes to regulate maize plant height.

The plant height is a crucial agronomic trait in contemporary maize breeding. Appropriate plant height can improve crop lodging resistance, increase the planting density and harvest index of crops, and thus contribute to stable and increased yields. In this study, molecular characterization showed that ZmWRKY92 is a nuclear protein and has transcriptional activation in yeast. ZmWRKY92 can specifically bind to the W-box (TTGACC), which was confirmed by double LUC experiments and Yeast one-hybrid assays. Subsequently we screened wrky92 mutants from a library of ethyl methanesulfonate (EMS)-induced mutants. The mutation of a base in ZmWRKY92 leading to the formation of a truncated protein variant is responsible for the dwarfing phenotype of the mutant, which was further verified by allelic testing. Detailed phenotypic analysis revealed that wrky92 mutants have shorter internodes due to reduced internode cell size and lower levels of GA3 and IAA. Transcriptome analysis revealed that the ZmWRKY92 mutation caused significant changes in the expression of genes related to plant height in maize. Additionally, ZmWRKY92 was found to interact with the promoters of ZmGA20ox7 and ZmGID1L2, which are associated with GA synthesis. This study shows that ZmWRKY92 significantly affects the plants height in maize and is crucial in identifying new varieties suitable for growing in high-density conditions.

Mutation of ZmWRKY86 confers enhanced salt stress tolerance in maize.

As one of the largest families of transcription factors in plants, the WRKY proteins play crucial roles in plant growth and development, defense regulation and stress responses. In this research, ZmWRKY86 encoding a WRKY transcription factor was cloned from maize (Zea mays L.). ZmWRKY86 expression was up-regulated by salt stress. ZmWRKY86 is a nuclear protein and has no transcriptional activation ability in yeast. ZmWRKY86 can specifically bind to W-box (TTGACC), which was confirmed by electrophoretic mobility shift assay (EMSA) and dual-LUC experiments. As compared with control, the wrky86 mutants showed enhanced plant tolerance to salt stress with higher survival rate, catalase activity and K+ content, but lower malondialdehyde accumulation, relative electrical leakage level and Na+ content under salt-stress condition. Transcriptome and quantitative real-time PCR analyses indicated that the mutation of ZmWRKY86 led to significant changes in the expression of stress-related genes in maize. Further assays showed that ZmWRKY86 directly interacted with the promoter of two salt stress-related genes Zm00001d020840 and Zm00001d046813. In summary, we identified a WRKY transcription factor ZmWRKY86 that participates in salt stress regulation through controlling the expression of stress-related genes.

Overexpression of the maize WRKY114 gene in transgenic rice reduce plant height by regulating the biosynthesis of GA.

WRKYs represent an important family of transcription factors that are widely involved in plant development, defense regulation and stress response. Transgenic rice that constitutively expressed ZmWRKY114 had shorter plant height and showed less sensitivity to gibberellic acid (GA3). Further investigation proved that transgenic rice accumulated lower levels of bioactive GAs than that in wild-type plants. Application of exogenous GA3 fully rescued the semi-dwarf phenotype of ZmWRKY114 transgenic plants. Transcriptome and qRT-PCR analyses indicated that the expression of OsGA2ox4, encoding the repressor of GA biosynthesis, was markedly increased. Electrophoretic mobility shift assay and dual-luciferase reporter assay indicated that ZmWRKY114 directly binds to a W-box motif in the OsGA2ox4 promoter. Taken together, these results confirm that ZmWRKY114 is a GA-responsive gene and is participated in the regulation of plant height in rice.

A moso bamboo WRKY gene PeWRKY83 confers salinity tolerance in transgenic Arabidopsis plants.

The WRKY family are transcription factors, involved in plant development, and response to biotic and abiotic stresses. Moso bamboo is an important bamboo that has high ecological, economic and cultural value and is widely distributed in the south of China. In this study, we performed a genome-wide identification of WRKY members in moso bamboo and identified 89 members. By comparative analysis in six grass genomes, we found the WRKY gene family may have experienced or be experiencing purifying selection. Based on relative expression levels among WRKY IIc members under three abiotic stresses, PeWRKY83 functioned as a transcription factor and was selected for detailed analysis. The transgenic Arabidopsis of PeWRKY83 showed superior physiological properties compared with the WT under salt stress. Overexpression plants were less sensitive to ABA at both germination and postgermination stages and accumulated more endogenous ABA under salt stress conditions. Further studies demonstrated that overexpression of PeWRKY83 could regulate the expression of some ABA biosynthesis genes (AtAAO3, AtNCED2, AtNCED3), signaling genes (AtABI1, AtPP2CA) and responsive genes (AtRD29A, AtRD29B, AtABF1) under salt stress. Together, these results suggested that PeWRKY83 functions as a novel WRKY-related TF which plays a positive role in salt tolerance by regulating stress-induced ABA synthesis.

Identification and Expression Analysis of BURP Domain-Containing Genes in Medicago truncatula.

BURP domain-containing proteins belong to a newly identified protein class that is unique to plants and plays an important role in plant development and metabolism. Although systematic characterization of BURP domain-containing proteins have been carried out in many species, such as rice, poplar and maize, little is known about BURP domain-containing proteins in Medicago. In this study, multiple bioinformatics approaches were employed to identify all the members of BURP family genes in Medicago. A complete set of 39 BURP family genes were identified. These genes have diverse structures and were distributed on chromosome 1-8 except 7. According to phylogenetic analysis, these BURP family genes could be classified into eight classes. Motif and exon-intron organization, stress-related cis-elements in promoter regions and microarray analysis of MtBURPs were also performed. Furthermore, transcript level analysis of MtBURP genes in response to drought stress revealed that all of the 39 BURP genes were regulated by drought stress. The results of this study reveal a comprehensive overview of the Medicago BURP gene family and provide the first step toward the selection of MtBURP genes for cloning and functional analysis of the BURP gene family in Medicago truncatula.

Genome-wide identification, classification and expression analysis of the PHD-finger protein family in Populus trichocarpa.

The plant homeobox domain (PHD) proteins are widespread in eukaryotes, and play important roles in regulating chromatin and transcription. Comprehensive analyses of PHD-finger proteins have been performed in animals, but few plant PHD-finger proteins involved in growth and development have been characterized functionally. In this study, we conducted a genome-wide survey of PHD-finger proteins in Populus trichocarpa by describing the phylogenetic relationship, gene structure, and chromosomal location and microarray analyses of each predicted PHD-finger family member. We identified 73 PHD-finger genes (PtPHD1-73) and classified them into eleven subfamilies (A-K) by phylogenetic analysis. Seventy-two of the 73 genes were unevenly distributed on all 19 chromosomes, with seven segmental duplication events. Analysis of the Ka (non-synonymous substitution rate)/Ks (synonymous substitution rate) ratios suggested that the duplicated genes of the PHD-finger family mainly underwent purifying selection with restrictive functional divergence after the duplication events. Expression profiles analysis indicated that 67 PHD-finger genes were differentially expressed in various tissues. Quantitative real-time RT-PCR (qRT-PCR) analyses of nine selected PtPHD genes under high salinity, drought and cold stresses were also performed to explore their stress-related expression patterns. The results of this study provide a thorough overview of poplar PHD-finger proteins and will be valuable for further functional research of poplar PHD-finger genes to unravel their biological roles.

The Lipoxygenase Gene Family in Poplar: Identification, Classification, and Expression in Response to MeJA Treatment.

BACKGROUND: Lipoxygenases (LOXs) are important dioxygenases in cellular organisms. LOXs contribute to plant developmental processes and environmental responses. However, a systematic and comprehensive analysis has not been focused on the LOX gene family in poplar. Therefore, in the present study, we performed a comprehensive analysis of the LOX gene family in poplar. RESULTS: Using bioinformatics methods, we identified a total of 20 LOX genes. These LOX genes were clustered into two subfamilies. The gene structure and motif composition of each subfamily were relatively conserved. These genes are distributed unevenly across nine chromosomes. The PtLOX gene family appears to have expanded due to high tandem and low segmental duplication events. Microarray analysis showed that a number of PtLOX genes have different expression pattern across disparate tissues and under various stress treatments. Quantitative real-time PCR (qRT-PCR) analysis was further performed to confirm the responses to MeJA treatment of the 20 poplar LOX genes. The results show that the PtLOX genes are regulated by MeJA (Methyl jasmonate) treatment. CONCLUSIONS: This study provides a systematic analysis of LOX genes in poplar. The gene family analysis reported here will be useful for conducting future functional genomics studies to uncover the roles of LOX genes in poplar growth and development.

Identification and Characterization of Novel Maize Mirnas Involved in Different Genetic Background.

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition in plants and animals. At present there is relatively little information regarding the role of miRNAs in the response to drought stress in maize. In this study, two small RNA libraries were sequenced, and a total of 11,973,711 and 14,326,010 raw sequences were generated from growing leaves of drought-tolerant and drought-sensitive maize seedlings, respectively. Further analysis identified 192 mature miRNAs, which include 124 known maize (zma) miRNAs and 68 potential novel miRNA candidates. Additionally, 167 target genes (259 transcripts) of known and novel miRNAs were predicted to be differentially expressed between two maize inbred lines. Of these, three novel miRNAs were up-regulated and two were down-regulated under drought stress. The expression of these five miRNAs and nine target genes was confirmed using quantitative reverse transcription PCR. The expression of three of the miRNAs and their putative target genes exhibited an inverse correlation, and expression analysis suggested that all five may play important roles in maize leaves. Finally, GO annotations of the target genes indicated a potential role in photosynthesis, may therefore contribute to the drought stress response. This study describes the identification and characterization of novel miRNAs that are the differentially expressed in drought-tolerant and drought-sensitive inbred maize lines. This provides the foundation for further investigation into the mechanism of miRNA function in response to drought stress in maize.

Identification and characterization of the RCI2 gene family in maize (Zea mays).

Rare-cold-inducible (RCI2) genes are structurally conserved members that encode small, highly hydrophobic proteins involved in response to various abiotic stresses. Phylogenetic and functional analyses of these genes have been conducted in Arabidopsis, but an extensive investigation of the RCI2 gene family has not yet been carried out in maize. In the present study, 10 RCI2 genes were identified in a fully sequenced maize genome. Structural characterization and expression pattern analysis of 10 ZmRCI2s (Zea mays RCI2 genes) were subsequently determined. Sequence and phylogenetic analyses indicated that ZmRCI2s are highly conserved, and most of them could be grouped with their orthologues from other organisms. Chromosomal location analysis indicated that ZmRCI2s were distributed unevenly on seven chromosomes with two segmental duplication events, suggesting that maize RCI2 gene family is an evolutionarily conserved family. Putative stress-responsive cis-elements were detected in the 2-kb promoter regions of the 10 ZmRCI2s. In addition, the 10 ZmRCI2s showed different expression patterns in maize development based on transcriptome analysis. Further, microarray and quantitative real-time PCR (qRT-PCR) analysis showed that each maize RCI2 genes were responsive to drought stress, suggesting their important roles in drought stress response. The results of this work provide a basis for future cloning and application studies of maize RCI2 genes.