Membrane-Bound Transcription Factor ZmNAC074 Positively Regulates Abiotic Stress Tolerance in Transgenic Arabidopsis.

Maize (Zea mays L.) is one of the most widely cultivated crops for humans, making a vital contribution to human nutrition and health. However, in recent years, due to the influence of external adverse environments, the yield and quality of maize have been seriously affected. NAC (NAM, ATAF1/2 and CUC2) transcription factors (TFs) are important plant-unique TFs, which are crucial for regulating the abiotic stress response of plants. Therefore, it is of great biological significance to explore the underlying regulatory function of plant NAC TFs under various abiotic stresses. In this study, wild-type and ZmNAC074-overexpressed transgenic Arabidopsis were used as experimental materials to dissect the stress-resistant function of ZmNAC074 in transgenic Arabidopsis at phenotypic, physiological and molecular levels. The analyses of seed germination rate, survival rate, phenotype, the content of chlorophyll, carotenoids, malondialdehyde (MDA), proline and other physiological indexes induced by distinct abiotic stress conditions showed that overexpression of ZmNAC074 could confer the enhanced resistance of salt, drought, and endoplasmic reticulum (ER) stress in transgenic Arabidopsis, indicating that ZmNAC074 plays an important regulatory role in plant response to abiotic stress, which provides an important theoretical foundation for further uncovering the molecular regulation mechanism of ZmNAC074 under abiotic stresses.

Genome-Wide Identification of Maize Protein Arginine Methyltransferase Genes and Functional Analysis of ZmPRMT1 Reveal Essential Roles in Arabidopsis Flowering Regulation and Abiotic Stress Tolerance.

Histone methylation, as one of the important epigenetic regulatory mechanisms, plays a significant role in growth and developmental processes and stress responses of plants, via altering the methylation status or ratio of arginine and lysine residues of histone tails, which can affect the regulation of gene expression. Protein arginine methyltransferases (PRMTs) have been revealed to be responsible for histone methylation of specific arginine residues in plants, which is important for maintaining pleiotropic development and adaptation to abiotic stresses in plants. Here, for the first time, a total of eight PRMT genes in maize have been identified and characterized in this study, named as ZmPRMT1-8. According to comparative analyses of phylogenetic relationship and structural characteristics among PRMT gene family members from several representative species, all maize 8 PRMT proteins were categorized into three distinct subfamilies. Further, schematic structure and chromosome location analyses displayed evolutionarily conserved structure features and an unevenly distribution on maize chromosomes of ZmPRMT genes, respectively. The expression patterns of ZmPRMT genes in different tissues and under various abiotic stresses (heat, drought, and salt) were determined. The expression patterns of ZmPRMT genes indicated that they play a role in regulating growth and development and responses to abiotic stress. Eventually, to verify the biological roles of ZmPRMT genes, the transgenic Arabidopsis plants overexpressing ZmPRMT1 gene was constructed as a typical representative. The results demonstrated that overexpression of ZmPRMT1 can promote earlier flowering time and confer enhanced heat tolerance in transgenic Arabidopsis. Taken together, our results are the first to report the roles of ZmPRMT1 gene in regulating flowering time and resisting heat stress response in plants and will provide a vital theoretical basis for further unraveling the functional roles and epigenetic regulatory mechanism of ZmPRMT genes in maize growth, development and responses to abiotic stresses.

ZmNAC074, a maize stress-responsive NAC transcription factor, confers heat stress tolerance in transgenic Arabidopsis.

The harsh environment such as high temperature greatly limits the growth, development and production of crops worldwide. NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) play key regulatory roles in abiotic stress responses of plants. However, the functional roles of NAC TFs in heat stress response of maize remain elusive. In our present study, we identified and isolated a stress-responsive NAC transcription factor gene in maize, designated as ZmNAC074 and orthologous with rice OsNTL3. Further studies revealed that ZmNAC074 may encode a membrane-bound transcription factor (MTF) of NAC family in maize, which is comprised of 517 amino acid residues with a transmembrane domain at the C-terminus. Moreover, ZmNAC074 was highly expressed and induced by various abiotic stresses in maize seedlings, especially in leaf tissues under heat stress. Through generating ZmNAC074 transgenic plants, phenotypic and physiological analyses further displayed that overexpression of ZmNAC074 in transgenic Arabidopsis confers enhanced heat stress tolerance significantly through modulating the accumulation of a variety of stress metabolites, including reactive oxygen species (ROS), antioxidants, malondialdehyde (MDA), proline, soluble protein, chlorophyll and carotenoid. Further, quantitative real-time PCR analysis showed that the expression levels of most ROS scavenging and HSR- and UPR-associated genes in transgenic Arabidopsis were significantly up-regulated under heat stress treatments, suggesting that ZmNAC074 may encode a positive regulator that activates the expression of ROS-scavenging genes and HSR- and UPR-associated genes to enhance plant thermotolerance under heat stress conditions. Overall, our present study suggests that ZmNAC074 may play a crucial role in conferring heat stress tolerance in plants, providing a key candidate regulatory gene for heat stress tolerance regulation and genetic improvement in maize as well as in other crops.

Genome-Wide Identification and Functional Analysis of Polyamine Oxidase Genes in Maize Reveal Essential Roles in Abiotic Stress Tolerance.

Polyamines (PAs) play a critical role in growth and developmental processes and stress responses in plants. Polyamine oxidase (PAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme that plays a major role in PA catabolism. Here, for the first time, PAO genes in maize were screened for the whole genome-wide and nine ZmPAO genes were identified in this study, named as ZmPAO1-9. Based on structural characteristics and a comparison of phylogenetic relationships of PAO gene families from seven representative species, all nine PAO proteins in maize were categorized into three distinct subfamilies. Further, chromosome location and schematic structure revealed an unevenly distribution on chromosomes and evolutionarily conserved structure features of ZmPAO genes in maize, respectively. Furthermore, transcriptome analysis demonstrated that ZmPAO genes showed differential expression patterns at diverse developmental stages of maize, suggesting that these genes may play functional developmental roles in multiple tissues. Further, through qRT-PCR validation, these genes were confirmed to be responsive to heat, drought and salinity stress treatments in three various tissues, indicating their potential roles in abiotic stress responses. Eventually, to verify the biological function of ZmPAO genes, the transgenic Arabidopsis plants overexpressing ZmPAO6 gene were constructed as a typical representative to explore functional roles in plants. The results demonstrated that overexpression of ZmPAO6 can confer enhanced heat tolerance through mediating polyamine catabolism in transgenic Arabidopsis, which might result in reduced H2O2 and MDA accumulation and alleviated chlorophyll degradation under heat stress treatment, indicating that ZmPAO6 may play a crucial role in enhancing heat tolerance of transgenic Arabidopsis through the involvement in various physiological processes. Further, the expression analysis of related genes of antioxidant enzymes including glutathione peroxidase (GPX) and ascorbate peroxidase (APX) demonstrated that ZmPAO6 can enhance heat resistance in transgenic Arabidopsis through modulating heat-induced H2O2 accumulation in polyamine catabolism. Taken together, our results are the first to report the ZmPAO6 gene response to heat stress in plants and will serve to present an important theoretical basis for further unraveling the function and regulatory mechanism of ZmPAO genes in growth, development and adaptation to abiotic stresses in maize.

Genome-wide identification and expression profiling analysis of maize AP2/ERF superfamily genes reveal essential roles in abiotic stress tolerance.

BACKGROUND: As one of the largest transcription factor families in plants, the APETALA2/Ethylene-Responsive Factor (AP2/ERF) superfamily is involved in various biological processes and plays significant roles in plant growth, development and responses to various stresses. Although identification and characterization of AP2/ERF superfamily genes have been accomplished in many plant species, very little is known regarding the structure and function of AP2/ERF genes in maize. RESULTS: In this study, a total of 214 genes encoding ZmAP2/ERF proteins with complete AP2/ERF domain were eventually identified according to the AGPv4 version of the maize B73 genome. Based on the number of AP2/ERF domain and similarities of amino acid sequences among AP2/ERF proteins from Arabidopsis, rice and maize, all 214 putative ZmAP2/ERF proteins were categorized into three distinct families, including the AP2 family (44), the ERF family (166) and the RAV family (4), respectively. Among them, the ERF family was further subdivided into two diverse subfamilies, including the DREB and ERF subfamilies with 61 and 105 members, respectively. Further, based on phylogenetic analysis, the members of DREB and ERF subfamilies were subdivided into four (Group I-IV) and eight (Group V-XII) groups, respectively. The characteristics of exon-intron structure of these putative ZmAP2/ERF genes and conserved protein motifs of their encoded ZmAP2/ERF proteins were also presented respectively, which was in accordance with the results of group classification. Promoter analysis suggested that ZmAP2/ERF genes shared many stress- and hormone-related cis-regulatory elements. Gene duplication and synteny analysis revealed that tandem or segmental duplication and purifying selection might play significant roles in evolution and functional differentiation of AP2/ERF superfamily genes among three various gramineous species (maize, rice and sorghum). Using RNA-seq data, transcriptome analysis indicated that the majority of ZmAP2/ERF genes displayed differential expression patterns at different developmental stages of maize. In addition, the following analyses of co-expression network among ZmAP2/ERF genes and protein protein interaction between ZmAP2 and ZmERF proteins further enabled us to understand the regulatory relationship among members of the AP2/ERF superfamily in maize. Furthermore, by quantitative real-time PCR analysis, twenty-seven selected ZmAP2/ERF genes were further confirmed to respond to three different abiotic stresses, suggesting their potential roles in various abiotic stress responses. Collectively, these results revealed that these ZmAP2/ERF genes play essential roles in abiotic stress tolerance. CONCLUSIONS: Taken together, the present study will serve to present an important theoretical basis for further exploring the function and regulatory mechanism of ZmAP2/ERF genes in the growth, development, and adaptation to abiotic stresses in maize.

Genome-wide identification and analysis of WRKY gene family in maize provide insights into regulatory network in response to abiotic stresses.

BACKGROUND: The WRKY transcription factor family plays significant roles in biotic and abiotic stress responses, which has been associated with various biological processes in higher plants. However, very little is known regarding the structure and function of WRKY genes in maize. RESULTS: In this study, a total of 140 ZmWRKY proteins encoded by 125 ZmWRKY genes were eventually identified in maize. On the basis of features of molecular structure and a comparison of phylogenetic relationships of WRKY transcription factor families from Arabidopsis, rice and maize, all 140 ZmWRKY proteins in maize were divided into three main groups (Groups I, II and III) and the Group II was further classified into five subgroups. The characteristics of exon-intron structure of these putative ZmWRKY genes and conserved protein motifs of their encoded ZmWRKY proteins were also presented respectively, which was in accordance with the group classification results. Promoter analysis suggested that ZmWRKY genes shared many abiotic stress-related elements and hormone-related elements. Gene duplication analysis revealed that the segmental duplication and purifying selection might play a significant role during the evolution of the WRKY gene family in maize. Using RNA-seq data, transcriptome analysis indicated that most of ZmWRKY genes displayed differential expression patterns at different developmental stages of maize. Further, by quantitative real-time PCR analysis, twenty-one ZmWRKY genes were confirmed to respond to two different abiotic stress treatments, suggesting their potential roles in various abiotic stress responses. In addition, RNA-seq dataset was used to conduct weighted gene co-expression network analysis (WGCNA) in order to recognize gene subsets possessing similar expression patterns and highly correlated with each other within different metabolic networks. Further, subcellular localization prediction, functional annotation and interaction analysis of ZmWRKY proteins were also performed to predict their interactions and associations involved in potential regulatory network. CONCLUSIONS: Taken together, the present study will serve to present an important theoretical basis for further exploring function and regulatory mechanism of ZmWRKY genes in the growth, development, and adaptation to abiotic stresses in maize.

Genome-wide identification, classification and expression analysis of the Hsf and Hsp70 gene families in maize.

Heat shock factors (Hsfs) and heat shock proteins (Hsps) play a critical role in the molecular mechanisms such as plant development and defense against abiotic. As an important food crop, maize is vulnerable to adverse environment such as heat stress and water logging, which leads to a decline in yield and quality. To date, very little is known regarding the structure and function of Hsf and Hsp genes in maize. Although some Hsf and Hsp genes have been characterized in maize, analysis of the entire Hsf and Hsp70 gene families were not completed following Maize (B73) Genome Sequencing Project. Therefore, studying their molecular mechanism and revealing their biological function in plant stress resistance process will contribute to reveal important theoretical significance and application value for improving corn yield and quality. In this study, we have identified 25 ZmHsf and 22 ZmHsp70 genes in maize. The structural characteristics and phylogenetic relationships of the Hsf and Hsp70 gene families of Arabidopsis thaliana, rice and maize were compared. The final 25 ZmHsf proteins and 22 ZmHsp70 proteins were divided into three and four subfamilies, respectively. In addition, chromosomal localization indicated that the ZmHsf and ZmHsp70 genes were unevenly distributed on the chromosome, and the gene structure map revealed the characteristics of their structures. Finally, transcriptome analysis indicated that most of the ZmHsf and ZmHsp70 genes showed different expression patterns at different developmental stages of maize. Further, by semi-quantitative RT-PCR and quantitative real-time PCR analysis, all 25 ZmHsf and 22 ZmHsp70 genes were confirmed to respond to heat stress treatment, indicating that they have potential effects in heat stress response. The analyses performed by combining co-expression network with protein-protein interaction network among the members of the Hsf and Hsp70 gene families in maize further enabled us to recognize components involved in the regulatory network associated with hsfs and hsp70s complex. The predicted subcellular location revealed that maize Hsp70 proteins exhibited a various subcellular distribution, which may be associated with functional diversification in heat stress response. Taken together, our study provides comprehensive information on the members of Hsf and Hsp70 gene families and will help in elucidating their exact function in maize.

Genome-wide identification, classification and expression analysis of the JmjC domain-containing histone demethylase gene family in maize.

BACKGROUND: Histone methylation mainly occurs on the lysine residues and plays a crucial role during flowering and stress responses of plants, through changing the methylation status or ratio of lysine residues. Histone lysine residues of plants can arise in three forms of methylation (single, double and triple) and the corresponding demethylation can also ensue on certain occasions, by which the plants can accommodate the homeostasis of histone methylation by means of lysine methyltransferase and demethylase. The JmjC domain-containing proteins, an important family of histone lysine demethylases, play a vital role in maintaining homeostasis of histone methylation in vivo. RESULTS: In this study, we have identified 19 JmjC domain-containing histone demethylase (JHDM) proteins in maize. Based on structural characteristics and a comparison of phylogenetic relationships of JHDM gene families from Arabidopsis, rice and maize, all 19 JHDM proteins in maize were categorized into three different subfamilies. Furthermore, chromosome location and schematic structure revealed an unevenly distribution on chromosomes and structure features of ZmJMJ genes in maize, respectively. Eventually, the 19 ZmJMJ genes displayed different expression patterns at diverse developmental stages of maize based on transcriptome analysis. Further, quantitative real-time PCR analysis showed that all 19 ZmJMJ genes were responsive to heat stress treatment, suggesting their potential roles in heat stress response. CONCLUSIONS: Overall, our study will serve to present an important theoretical basis for future functional verification of JHDM genes to further unravel the mechanisms of epigenetic regulation in plants.

The Dynamics of DNA methylation in the maize (Zea mays L.) inbred line B73 response to heat stress at the seedling stage.

High temperature stress has become a major concern for crop production worldwide because it greatly affects the growth, development, and productivity of plants. The mechanisms underlying the development of heat-tolerance need to be better understood for important agricultural crops. Recent research shows that DNA methylation is dynamic during plant development. However, the molecular mechanism regulating these dynamic DNA methylation patterns remains to be elucidated. In this study, six MethylRAD libraries were constructed using DNA isolated from leaves of maize. A total of 42,561,144 and 48,157,284 clean reads were generated from CK (Control condition) and HTP (Heat stress condition) treatments, respectively. The results showed that a total of 25,470 methylated genes were found in six tested samples, including 325 differentially methylated genes (200 in CCGG sites and 125 in CCWGG sites) between the CK and HTP samples. KEGG pathway enrichment analysis for DMGs indicated that Spliceosome, Homologous recombination, RNA transport, Ubiquitin mediated proteolysis and Carbon metabolism pathways play a central role in maize response to heat stress. Taken together, this research revealed the genome-wide DNA methylation pattern of maize leaves in response to heat exposure and identified candidate genes potentially involved in response to heat stress at the methylation level, which will facilitate future studies to elucidate the epigenetic mechanisms underlying the responses of maize to heat stress.

Transcriptomic analysis of the maize (Zea mays L.) inbred line B73 response to heat stress at the seedling stage.

High temperature is a common stress, which influences the growth and reproduction of plants. Maize is one of the most important crops all over the world. However, heat stress reduces significantly the yield and quality of maize. Therefore, it is important to illuminate molecular mechanism of maize response to heat stress. To estimate genes related to heat stress, we analyzed the transcriptome of maize in response to heat stress. In this study, six cDNA libraries were constructed form total RNA isolated from leaves of maize. A total of 35,209,446 and 35,205,472 clean reads were generated from CK (Control condition) and HTP (Heat stress condition) treatments, respectively. The results showed that 1857 DEGs were identified in maize after heat stress (1029 up-regulated and 828 down-regulated). KEGG pathway enrichment analysis for DEGs indicated that protein processing in endoplasmic reticulum pathways play a central role in maize response to heat stress. In addition, in the present study, 167 putative TFs were identified, which belong to various TF families (e.g., MYB, AP2-EREBP, b-ZIP, bHLH, NAC and WRKY), and may be associated with heat stress response of maize. This research may contribute to understand the molecular mechanism of maize inbred line B73 response to heat stress, which is beneficial for developing maize cultivars to improve yield and quality.

Genome-wide identification and expression profiling of DNA methyltransferase gene family in maize.

KEY MESSAGE: In this study, we identified eight DNA MTase genes in maize and the diversity of expression patterns of them was presented by EST mining, microarray and semi-quantitative expression profile analyses. DNA methylation plays a pivotal role in promoting genomic stability through diverse biological processes including regulation of gene expression during development and chromatin organization. Although this important biological process is mainly regulated by several conserved Cytosine-5 DNA methyltransferases encoded by a smaller multigene family in plants, investigation of the plant C5-MTase-encoding gene family will serve to elucidate the epigenetic mechanism diversity in plants. Recently, genome-wide identification and evolutionary analyses of the C5-MTase-encoding gene family have been characterized in multiple plant species including Arabidopsis, rice, carrot and wheat. However, little is known regarding the C5-MTase-encoding genes in the entire maize genome. Here, genome-wide identification and expression profile analyses of maize C5-MTase-encoding genes (ZmMETs) were performed from the latest version of the maize (B73) genome. Phylogenetic analysis indicated that the orthologs from the three species (maize, Arabidopsis and rice) were categorized into four classes. Chromosomal location of these genes revealed that they are unevenly distributed on 6 of all 10 chromosomes with three chromosomal/segmental duplication events, suggesting that gene duplication played a key role in expansion of the maize C5-MTase-encoding gene family. Furthermore, EST expression data mining, microarray data and semi-quantitative expression profile analyses detected in the leaves by two different abiotic stress treatments have demonstrated that these genes had temporal and spatial expression pattern and exhibited different expression levels in stress treatments, suggesting that functional diversification of ZmMET genes family. Overall, our study will serve to present signification insights to explore the plant C5-MTase-encoding gene expression and function and also be beneficial for future experimental research to further unravel the mechanisms of epigenetic regulation in plants.

Identification and characterization of the SET domain gene family in maize.

Histone lysine methylation plays a pivotal role in a variety of developmental and physiological processes through modifying chromatin structure and thereby regulating eukaryotic gene transcription. The SET domain proteins represent putative candidates for lysine methyltransferases containing the evolutionarily-conserved SET domain, and important epigenetic regulators present in eukaryotes. In recent years, increasing evidence reveals that SET domain proteins are encoded by a large multigene family in plants and investigation of the SET domain gene family will serve to elucidate the epigenetic mechanism diversity in plants. Although the SET domain gene family has been thoroughly characterized in multiple plant species including two model plant systems, Arabidopsis and rice, through their sequenced genomes, analysis of the entire SET domain gene family in maize was not completed following maize (B73) genome sequencing project. Here, we performed a genome-wide structural and evolutionary analysis of maize SET domain genes from the latest version of the maize (B73) genome. A complete set of 43 SET domain genes (Zmset1-43) were identified in the maize genome using Blast search tools and categorized into seven classes (Class I-VII) based on phylogeny. Chromosomal location of these genes revealed that they are unevenly distributed on all ten chromosomes with seven segmental duplication events, suggesting that segmental duplication played a key role in expansion of the maize SET domain gene family. EST expression data mining revealed that these newly identified genes had temporal and spatial expression pattern and suggested that many maize SET domain genes play functional developmental roles in multiple tissues. Furthermore, the transcripts of the 18 genes (the Class V subfamily) 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. Overall, our study will serve to better understand the complexity of the maize SET domain gene family and also be beneficial for future experimental research to further unravel the mechanisms of epigenetic regulation in plants.

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.

Reactivation of a silenced minimal Mutator transposable element system following low-energy nitrogen ion implantation in maize.

In maize, Mutator transposable elements are either active or silenced within the genome. In response to environmental stress, silenced Mutator elements could be reactivated, leading to changes in genome structure and gene function. However, there is no direct experimental evidence linking environmental stress and Mutator transposon reactivation. Using a maize line that contains a single inactive MuDR and a lone nonautonomous Mutator element, a Mu1 insertion in the recessive reporter allele a1-mum2 in an inactive Mutator background, we directly assessed Mutator reactivation following low-energy nitrogen ion implantation. We observed that N(+) implantation decreased cytosine methylation in MuDR terminal inverted repeats and increased expression of mudrA and mudrB. Both changes were associated with increased transpositional activity of MuDR through reactivation of the inactive minimal Mutator transposable element system. This study provides direct evidence linking environmental stress agents and Mutator transposon mobilization in maize. In addition, the observed changes to DNA methylation suggest a new mechanism for mutations by low-energy ion implantation.

A genomic analysis of disease-resistance genes encoding nucleotide binding sites in Sorghum bicolor.

A large set of candidate nucleotide-binding site (NBS)-encoding genes related to disease resistance was identified in the sorghum (Sorghum bicolor) genome. These resistance (R) genes were characterized based on their structural diversity, physical chromosomal location and phylogenetic relationships. Based on their N-terminal motifs and leucine-rich repeats (LRR), 50 non-regular NBS genes and 224 regular NBS genes were identified in 274 candidate NBS genes. The regular NBS genes were classified into ten types: CNL, CN, CNLX, CNX, CNXL, CXN, NX, N, NL and NLX. The vast majority (97%) of NBS genes occurred in gene clusters, indicating extensive gene duplication in the evolution of S. bicolor NBS genes. Analysis of the S. bicolor NBS phylogenetic tree revealed two major clades. Most NBS genes were located at the distal tip of the long arms of the ten sorghum chromosomes, a pattern significantly different from rice and Arabidopsis, the NBS genes of which have a random chromosomal distribution.

A genomic analysis of disease-resistance genes encoding nucleotide binding sites in Sorghum bicolor

A large set of candidate nucleotide-binding site (NBS)-encoding genes related to disease resistance was identified in the sorghum (Sorghum bicolor) genome. These resistance (R) genes were characterized based on their structural diversity, physical chromosomal location and phylogenetic relationships. Based on their N-terminal motifs and leucine-rich repeats (LRR), 50 non-regular NBS genes and 224 regular NBS genes were identified in 274 candidate NBS genes. The regular NBS genes were classified into ten types: CNL, CN, CNLX, CNX, CNXL, CXN, NX, N, NL and NLX. The vast majority (97 percent) of NBS genes occurred in gene clusters, indicating extensive gene duplication in the evolution of S. bicolor NBS genes. Analysis of the S. bicolor NBS phylogenetic tree revealed two major clades. Most NBS genes were located at the distal tip of the long arms of the ten sorghum chromosomes, a pattern significantly different from rice and Arabidopsis, the NBS genes of which have a random chromosomal distribution.