Identification and Functional Characterization of ZmSCYL2 Involved in Phytosterol Accumulation in Plants.

Phytosterols are natural active substances widely found in plants and play an important role in hypolipidemia, antioxidants, antitumor, immunomodulation, plant growth, and development. In this study, phytosterols were extracted and identified from the seed embryos of 244 maize inbred lines. Based on this, a genome-wide association study (GWAS) was used to predict the possible candidate genes responsible for phytosterol content; 9 SNPs and 32 candidate genes were detected, and ZmSCYL2 was identified to be associated with phytosterol accumulation. We initially confirmed its functions in transgenic Arabidopsis and found that mutation of ZmSCYL2 resulted in slow plant growth and a significant reduction in sterol content, while overexpression of ZmSCYL2 accelerated plant growth and significantly increased sterol content. These results were further confirmed in transgenic tobacco and suggest that ZmSCYL2 was closely related to plant growth; overexpression of ZmSCYL2 not only facilitated plant growth and development but also promoted the accumulation of phytosterols.

Transcriptomic and weighted gene co-expression network analysis of tropic and temperate maize inbred lines recovering from heat stress.

Heat stress (HS) causes imbalance of cellular homeostasis, growth impairment and extensively yield loss in crop production. In the present study, the tropic maize inbred CIMBL55 showed more thermotolerance than the maize temperate inbred B73, with less leaf damage rate and ROS accumulation. Transcriptome profiling of CIMBL55 and B73 upon (exposing at 45 â„ƒ for 0, 1, and 6 h) and post (recovering at 28 â„ƒ for 1 and 6 h) HS were further assessed and a total of 20204 DEGs were identified. Functional annotation revealed that HS activated unfolded protein response in endoplasmic reticulum in both two inbreds. Moreover, in CIMBL55, far more primary and secondary metabolism pathways were transcriptional altered. Afterwards, weighted gene co-expression analysis grouped all expressed genes into eighteen co-expressed modules. Four HS responsive and four CIMBL55 recovery-related modules were subsequently identified. Highly connected genes (hub genes) in these modules were characterized as transcription factors, heat shock proteins, Ca2+ signaling related genes and various enzymes. Moreover, one hub gene, ZmHsftf13 was verified to positively regulate thermotolerance by heterologous expressing in Arabidopsis and its Mu insertion mutant. The present research provides promising genes related to HS response in maize and is of great significance for breeding.

Whole-Genome and Transposed Duplication Contributes to the Expansion and Diversification of TLC Genes in Maize.

TLC (TRAM/LAG/CRN8) proteins play important roles in ceramide metabolism and mycotoxin resistance. Herein a comparative genomics analysis of TLCs was performed in 31 plant and 3 species from other kingdoms, with an emphasis mainly on maize. TLCs were conserved across kingdoms and expanded in angiosperms, largely due to whole-genome/segmental duplication (WGD/SD) under purifying selection. Phylogeny reconstruction by maximum-likelihood method uncovered five TLC clades, subsequently named as TRAM/LAG, CLN8, PS-TLC, TM136 and TLCD clades. Each clade of TLCs shared specific transmembrane regions and motif composition. Divisions of conserved motifs to subunits may have occurred in TM136-type TLCs. Focusing on maize, five WGD and two DNA-mediated transposed duplication (TD) pairs were discovered, accounting for 61.11% ZmTLCs. Combined with further expression analysis, significant divergence was found in expression patterns between most maize WGD pairs, indicating subfunctionalization or/and neofunctionalization. Moreover, ZmTLC5, a deduced parental copy in a TD pair, was highly induced under FB1 and fungus pathogen injection and exhibited potential capacity to respond to environmental stimuli. Additionally, population genetics analysis showed that ZmTLC10 in the CLN8-clade may have experienced significant positive selection and differentiated between wild and inbred maize populations. Overall, our results help to decipher the evolutionary history of TLCs in maize and plants, facilitating further functional analysis of them.

Transcriptome analysis reveals comprehensive responses to cadmium stress in maize inoculated with arbuscular mycorrhizal fungi.

Biological strategy of utilization of plants-microbe's interactions to remediate cadmium (Cd) contaminated soils is effective and practical. However, limited evidence at transcriptome level is available about how microbes work with host plants to alleviate Cd stress. In the present study, comparative transcriptomic analysis was performed between maize seedlings inoculated with arbuscular mycorrhizal (AM) fungi and non-AM fungi inoculation under distinct concentrations of CdCl2 (0, 25, and 50 mg per kg soil). Significantly higher levels of Cd were found in root tissues of maize colonized by AM fungi, whereas, Cd content was reduced as much as 50% in leaf tissues when compared to non-AM seedlings, indicating that symbiosis between AM fungi and maize seedlings can significantly block translocation of Cd from roots to leaf tissues. Moreover, a total of 5827 differentially expressed genes (DEG) were determined and approximately 68.54% DEGs were downregulated when roots were exposed to high Cd stress. In contrast, 67.16% (595) DEGs were significantly up-regulated when seedlings were colonized by AM fungi under 0 mg CdCl2. Based on hierarchical clustering analysis, global expression profiles were split into eight distinct clusters. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that hundreds of genes functioning in plant hormone signal transduction, mitogen-activated protein kinase (MAPK) signaling pathway and glutathione metabolism were enriched. Furthermore, MapMan pathway analysis indicated a more comprehensive overview response, including hormone metabolism, especially in JA, glutathione metabolism, transcription factors and secondary metabolites, to Cd stress in mycorrhizal maize seedlings. These results provide an overview, at the transcriptome level, of how inoculation of maize seedlings by AM fungi could facilitate the relief of Cd stress.

Maize factors ZmUBP15, ZmUBP16 and ZmUBP19 play important roles for plants to tolerance the cadmium stress and salt stress.

Ubiquitin-Specific Protease16 (UBP16) has been described involved in cadmium stress and salt stress in Arabidopsis, however nothing is known about the functions of its homologs in maize. In this study, we investigate the functions of ZmUBP15, ZmUBP16 and ZmUBP19, three Arabidopsis UBP16 homologs in maize. Our results indicate that ZmUBP15, ZmUBP16 and ZmUBP19 are ubiquitously expressed throughout plant development, and ZmUBP15, ZmUBP16 and ZmUBP19 proteins are mainly localized in plasma membrane. Complementation analyses show that over-expression of ZmUBP15 or ZmUBP16 can rescue the defective phenotype of ubp16-1 in cadmium stress. In addition, over-expression of ZmUBP15, ZmUBP16 or ZmUBP19 can increase the plant tolerance to cadmium stress. These results indicate that ZmUBP15, ZmUBP16 and ZmUBP19 are required for plant to tolerance the cadmium stress. Consistent with this point, cadmium-related genes are markedly up-regulated in seedlings over-expressing ZmUBP15, ZmUBP16 or ZmUBP19. Furthermore, our data indicate that ZmUBP15, ZmUBP16 and ZmUBP19 partially rescue the salt-stress phenotype of ubp16-1. Thus, our research uncover the functions of three novel maize proteins, ZmUBP15, ZmUBP16 and ZmUBP19, which are required for plants in response to cadmium stress and salt stress.

Expression of Maize MADS Transcription Factor ZmES22 Negatively Modulates Starch Accumulation in Rice Endosperm.

As major component in cereals grains, starch has been one of the most important carbohydrate consumed by a majority of world's population. However, the molecular mechanism for regulation of biosynthesis of starch remains elusive. In the present study, ZmES22, encoding a MADS-type transcription factor, was modestly characterized from maize inbred line B73. ZmES22 exhibited high expression level in endosperm at 10 days after pollination (DAP) and peaked in endosperm at 20 DAP, indicating that ZmES22 was preferentially expressed in maize endosperm during active starch synthesis. Transient expression of ZmES22 in tobacco leaf revealed that ZmES22 protein located in nucleus. No transactivation activity could be detected for ZmES22 protein via yeast one-hybrid assay. Transformation of overexpressing plasmid 35S::ZmES22 into rice remarkedly reduced 1000-grain weight as well as the total starch content, while the soluble sugar was significantly higher in transgenic rice lines. Moreover, overexpressing ZmES22 reduced fractions of long branched starch. Scanning electron microscopy images of transverse sections of rice grains revealed that altered expression of ZmES22 also changed the morphology of starch granule from densely packed, polyhedral starch granules into loosely packed, spherical granules with larger spaces. Furthermore, RNA-seq results indicated that overexpressing ZmES22 could significantly influence mRNA expression levels of numerous key regulatory genes in starch synthesis pathway. Y1H assay illustrated that ZmES22 protein could bind to the promoter region of OsGIF1 and downregulate its mRNA expression during rice grain filling stages. These findings suggest that ZmES22 was a novel regulator during starch synthesis process in rice endosperm.

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.

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.

Selective sweep with significant positive selection serves as the driving force for the differentiation of japonica and indica rice cultivars.

BACKGROUND: Asian cultivated rice (Oryza sativa L.), including japonica and indica, is unarguable the most important crop in Asia as well as worldwide. However, a decisive conclusion of its origination and domestication processes are still lacking. Nowadays, the ever-increasing high-throughput sequencing data of numerous rice samples have provided us new opportunities to get close to the answer of these questions. RESULTS: By compiling 296 whole-genome sequenced rice cultivars and 39 diverse wild rice, two types of domesticated regions (DR-I and DR-II) with strong selective sweep signals between different groups were detected. DR-I regions included 28 blocks which significantly differentiated between japonica and indica subspecies, while DR-II regions were consisted of another 28 blocks which significantly differentiated between wild and cultivated rice, each covered 890 kb and 640 kb, respectively. In-depth analysis suggested that both DR-Is and DR-IIs could have originated from Indo-China Peninsula to southern China, and DR-IIs might be introgressed from indica to japonica. Functional bias with significant positive selection has also been detected in the genes of DR-I, suggesting important role of the selective sweep in differentiation of japonica and indica. CONCLUSIONS: This research promoted a new possible model of the origin of the cultivated rice that DR-Is in japonica and indica maybe independently originated from the divergent wild rice in the Indo-China Peninsula to southern China, and then followed by frequent introgression. Genes with significant positive selection and biased functions were also detected which could play important roles in rice domestication and differentiation processes.

Dissimilar Manifestation of Heterosis in Superhybrid Rice at Early-Tillering Stage under Nutrient-Deficient and Nutrient-Sufficient Condition.

Heterosis has long been exploited for crop breeding; however, the genetic mechanisms, particularly the initial establishment of heterosis during the early vegetative growth phase, remain elusive. The biggest challenge for that is to exclude noise genes from the identified heterosis-related candidates. Herein, we use nutrient-deficient hybrid with no measurable growth heterosis as control. After filtering these irrelevant genes, only 336 differentially expressed genes (DEGs), which is significantly lower than in previous reports, were identified as heterosis-related genes in a superhybrid rice of Liang-You-Pei-Jiu (LYP9) at early-tillering stage. Among the DEGs that could be mapped to quantitative trait loci (QTL), approximately 72.8% could be covered by yield or growth vigor-related QTL, thereby suggesting that our DEGs were reliable and may have potential value to rice breeding. Among the 336 DEGs identified, a majority showed intermediate expression relative to that of its parental lines (i.e. additive effects), particularly, expression was frequently more similar to the paternal line rather than the maternal line (44.1% versus 32.7%); the remaining 27.1% were exclusively up- or down-regulated between the hybrid and either parent. Interestingly, up-regulated genes encoded various enzymes, whereas down-regulated genes were enriched in responses to stress, indicating that hybrids may benefit from both activating metabolism-related pathways and alleviating fitness cost through allelic interactions. Furthermore, a significantly larger proportion of divergent genes and higher nonsynonymous substitutions rates were detected in these DEGs, suggesting a potential contribution to the establishment of heterosis in superhybrid rice LYP9.

Genomic variations and distinct evolutionary rate of rare alleles in Arabidopsis thaliana.

BACKGROUND: The variation rate in genomic regions associated with different alleles, impacts to distinct evolutionary patterns involving rare alleles. The rare alleles bias towards genome-wide association studies (GWASs), aim to detect different variants at genomic loci associated with single-nucleotide polymorphisms (SNPs) inclined to produce different haplotypes. Here, we sequenced Arabidopsis thaliana and compared its coding and non-coding genomic regions with its closest outgroup relative, Arabidopsis lyrta, which accounted for the ancestral misinference. The use of genome-wide SNPs interpret the genetic architecture of rare alleles in Arabidopsis thaliana, elucidating a significant departure from a neutral evolutionary model and the pattern of polymorphisms around a selected locus will exclusively influence natural selection. RESULTS: We found 23.4% of the rare alleles existing randomly in the genome. Notably, in our results significant differences (P < 0.01) were estimated in the relative rates between rare versus intermediate alleles, between fixed versus non-fixed mutations, and between type I versus type II rare-mutations by using the χ (2)-test. However, the rare alleles generating negative values of Tajima's D suggest that they generated under selective sweeps. Relative to polymorphic sites including SNPs, 67.5% of the fixed mutations were attributed, indicating major contributors to speciation. Substantially, an evolution occurred in the rare allele that was 1.42-times faster than that in a major haplotype. CONCLUSION: Our results interpret that rare alleles fits a random occurrence model, indicating that rare alleles occur at any locus in a genome and in any accession in a species. Based on the higher relative rate of derived to ancient mutations and higher average D xy, we conclude that rare alleles evolve faster than the higher frequency alleles. The rapid evolution of rare alleles indicates that they must have been newly generated with fixed mutations, compared with the other alleles. Eventually, PCR and sequencing results, in the flanking regions of rare allele loci confirm that they are of short extension, indicating the absence of a genome-wide pattern for a rare haplotype. The indel-associated model for rare alleles assumes that indel-associated mutations only occur in an indel heterozygote.

A genome-wide survey reveals abundant rice blast R genes in resistant cultivars.

Plant resistance genes (R genes) harbor tremendous allelic diversity, constituting a robust immune system effective against microbial pathogens. Nevertheless, few functional R genes have been identified for even the best-studied pathosystems. Does this limited repertoire reflect specificity, with most R genes having been defeated by former pests, or do plants harbor a rich diversity of functional R genes, the composite behavior of which is yet to be characterized? Here, we survey 332 NBS-LRR genes cloned from five resistant Oryza sativa (rice) cultivars for their ability to confer recognition of 12 rice blast isolates when transformed into susceptible cultivars. Our survey reveals that 48.5% of the 132 NBS-LRR loci tested contain functional rice blast R genes, with most R genes deriving from multi-copy clades containing especially diversified loci. Each R gene recognized, on average, 2.42 of the 12 isolates screened. The abundant R genes identified in resistant genomes provide extraordinary redundancy in the ability of host genotypes to recognize particular isolates. If the same is true for other pathogens, many extant NBS-LRR genes retain functionality. Our success at identifying rice blast R genes also validates a highly efficient cloning and screening strategy.

Dynamic evolution of NBS-LRR genes in bread wheat and its progenitors.

Extensive studies have focused on the largest class of disease resistance genes (nucleotide binding site-leucine-rich repeat, NBS-LRR) in various plants. However, no research on the dynamic evolution of these genes in domesticated species and their progenitors has been reported. Recently published genome sequences of bread wheat and its two ancestors provide a good opportunity for comparing NBS-encoding genes between ancestors and their progeny. Over 2000 NBS-encoding genes have been identified in bread wheat, which is the largest number having been reported so far. Compared with other grass species, its two progenitors also contained more NBS-encoding genes, indicating that there was an expansion of these genes in their common ancestor. Interestingly, the inherited relationships of NBS-LRR genes among the bread wheat and its two progenitors were ambiguous and only 3 % single-copy orthologues retained gene order in three-way genome comparisons of the three genomes. Lots of NBS-encoding genes present in the either ancestor could not be found in the bread wheat. These results indicated that NBS-LRR genes in bread wheat might have evolved rapidly through a rapid loss of ancestor genes.

Comparative study of the marR genes within the family Enterobacteriaceae.

marR genes are members of an ancient family originally identified in Escherichia coli. This family is widely distributed in archaea and bacteria. Homologues of this family have a conserved winged helix fold. MarR proteins are involved in non-specific resistance systems conferring resistance to multiple antibiotics. Extensive studies have shown the importance of MarR proteins in physiology and pathogenicity in Enterobacteria, but little is known about their origin or evolution. In this study, all the marR genes in 43 enterobacterial genomes representing 14 genera were identified, and the phylogenetic relationships and genetic parameters were analyzed. Several major findings were made. Three conserved marR genes originated earlier than Enterobacteriaceae and a geneloss event was found to have taken place in Yersinia pestis Antiqua. Three functional genes, rovA, hor, and slyA, were found to be clear orthologs among Enterobacteriaceae. The copy number of marR genes in Enterobacteriaceae was found to vary from 2 to 11. These marR genes exhibited a faster rate of nucleotide substitution than housekeeping genes did. Specifically, the regions of marR domain were found to be subject to strong purifying selection. The phylogenetic relationship and genetic parameter analyses were consistent with conservation and specificity of marR genes. These dual characters helped MarR to maintain a conserved binding motif and variable C-terminus, which are important to adaptive responses to a number of external stimuli in Enterobacteriaceae.