Gene editing of ZmGA20ox3 improves plant architecture and drought tolerance in maize.

KEY MESSAGE: Editing ZmGA20ox3 can achieve the effect similar to applying Cycocel, which can reduce maize plant height and enhance stress resistance. Drought stress, a major plant abiotic stress, is capable of suppressing crop yield performance severely. However, the trade-off between crop drought tolerance and yield performance turns out to be significantly challenging in drought-resistant crop breeding. Several phytohormones [e.g., gibberellin (GA)] have been reported to play a certain role in plant drought response, which also take on critical significance in plant growth and development. In this study, the loss-of-function mutations of GA biosynthesis enzyme ZmGA20ox3 were produced using the CRISPR-Cas9 system in maize. As indicated by the result of 2-year field trials, the above-mentioned mutants displayed semi-dwarfing phenotype with the decrease of GA1, and almost no yield loss was generated compared with wild-type (WT) plants. Interestingly, as revealed by the transcriptome analysis, differential expressed genes (DEGs) were notably enriched in abiotic stress progresses, and biochemical tests indicated the significantly increased ABA, JA, and DIMBOA levels in mutants, suggesting that ZmGA20ox3 may take on vital significance in stress response in maize. The in-depth analysis suggested that the loss function of ZmGA20ox3 can enhance drought tolerance in maize seedling, reduce Anthesis-Silking Interval (ASI) delay while decreasing the yield loss significantly in the field under drought conditions. The results of this study supported that regulating ZmGA20ox3 can improve plant height while enhancing drought resistance in maize, thus serving as a novel method for drought-resistant genetic improvement in maize.

Exploring the roles of ZmARM gene family in maize development and abiotic stress response.

Armadillo (ARM) was a gene family important to plants, with crucial roles in regulating plant growth, development, and stress responses. However, the properties and functions of ARM family members in maize had received limited attention. Therefore, this study employed bioinformatics methods to analyze the structure and evolution of ARM-repeat protein family members in maize. The maize (Zea mays L.) genome contains 56 ARM genes distributed over 10 chromosomes, and collinearity analysis indicated 12 pairs of linkage between them. Analysis of the physicochemical properties of ARM proteins showed that most of these proteins were acidic and hydrophilic. According to the number and evolutionary analysis of the ARM genes, the ARM genes in maize can be divided into eight subgroups, and the gene structure and conserved motifs showed similar compositions in each group. The findings shed light on the significant roles of 56 ZmARM domain genes in development and abiotic stress, particularly drought stress. RNA-Seq and qRT-PCR analysis revealed that drought stress exerts an influence on specific members of the ZmARM family, such as ZmARM4, ZmARM12, ZmARM34 and ZmARM36. The comprehensive profiling of these genes in the whole genome, combined with expression analysis, establishes a foundation for further exploration of plant gene function in the context of abiotic stress and reproductive development.

Enhanced charge transfer and photocatalytic carbon dioxide reduction of copper sulphide@cerium dioxide p-n heterojunction hollow cubes.

Hollow structure hybrids have gained considerable attention for their ability to reduce CO2 owing to their rich active sites, high gas adsorption ability, and excellent light utilization capacity. Herein, a template-engaged strategy was provided to fabricate copper sulphide@cerium dioxide (CuS@CeO2) p-n heterojunction hollow cube photocatalysts using Cu2O cubes as a sacrificial template. The sequential steps of loading of CeO2 nanolayer, sulfidation, and etching reaction facilitate the formation of CuS@CeO2 p-n heterojunction hollow cubes. Compared with the single CuS, CeO2, and their physical mixture, the CuS@CeO2 p-n heterojunction hollow cube photocatalyst expresses a higher performance toward photocatalytic CO2 reduction under solid-gas reaction conditions due to the faster separation of photogenerated charges. The further enhanced performance of CuS@CeO2 p-n heterojunction hollow cubes was achieved by decorating pt nanoparticles due to the fact that Pt nanoparticles had a high electron affinity and CO2 adsorption capacity, and the highest CO and CH4 yields of the optimized hybrid reached 195.8 µmol g-1 h-1 and 19.96 µmol g-1 h-1, respectively. This work might provide a strategy for designing and synthesizing efficient hollow heterostructured photocatalysts for solar energy conversion and utilization.

Complex genetic architecture underlying the plasticity of maize agronomic traits.

Phenotypic plasticity is the ability of a given genotype to produce multiple phenotypes in response to changing environmental conditions. Understanding the genetic basis of phenotypic plasticity and establishing a predictive model is highly relevant to future agriculture under a changing climate. Here we report findings on the genetic basis of phenotypic plasticity for 23 complex traits using a diverse maize population planted at five sites with distinct environmental conditions. We found that latitude-related environmental factors were the main drivers of across-site variation in flowering time traits but not in plant architecture or yield traits. For the 23 traits, we detected 109 quantitative trait loci (QTLs), 29 for mean values, 66 for plasticity, and 14 for both parameters, and 80% of the QTLs interacted with latitude. The effects of several QTLs changed in magnitude or sign, driving variation in phenotypic plasticity. We experimentally validated one plastic gene, ZmTPS14.1, whose effect was likely mediated by the compensation effect of ZmSPL6 from a downstream pathway. By integrating genetic diversity, environmental variation, and their interaction into a joint model, we could provide site-specific predictions with increased accuracy by as much as 9.9%, 2.2%, and 2.6% for days to tassel, plant height, and ear weight, respectively. This study revealed a complex genetic architecture involving multiple alleles, pleiotropy, and genotype-by-environment interaction that underlies variation in the mean and plasticity of maize complex traits. It provides novel insights into the dynamic genetic architecture of agronomic traits in response to changing environments, paving a practical way toward precision agriculture.

A multi-omics integrative network map of maize.

Networks are powerful tools to uncover functional roles of genes in phenotypic variation at a system-wide scale. Here, we constructed a maize network map that contains the genomic, transcriptomic, translatomic and proteomic networks across maize development. This map comprises over 2.8 million edges in more than 1,400 functional subnetworks, demonstrating an extensive network divergence of duplicated genes. We applied this map to identify factors regulating flowering time and identified 2,651 genes enriched in eight subnetworks. We validated the functions of 20 genes, including 18 with previously unknown connections to flowering time in maize. Furthermore, we uncovered a flowering pathway involving histone modification. The multi-omics integrative network map illustrates the principles of how molecular networks connect different types of genes and potential pathways to map a genome-wide functional landscape in maize, which should be applicable in a wide range of species.

Genome sequencing reveals evidence of adaptive variation in the genus Zea.

Maize is a globally valuable commodity and one of the most extensively studied genetic model organisms. However, we know surprisingly little about the extent and potential utility of the genetic variation found in wild relatives of maize. Here, we characterize a high-density genomic variation map from 744 genomes encompassing maize and all wild taxa of the genus Zea, identifying over 70 million single-nucleotide polymorphisms. The variation map reveals evidence of selection within taxa displaying novel adaptations. We focus on adaptive alleles in highland teosinte and temperate maize, highlighting the key role of flowering-time-related pathways in their adaptation. To show the utility of variants in these data, we generate mutant alleles for two flowering-time candidate genes. This work provides an extensive sampling of the genetic diversity of Zea, resolving questions on evolution and identifying adaptive variants for direct use in modern breeding.

Engineered chimeric insecticidal crystalline protein improves resistance to lepidopteran insects in rice (Oryza sativa L.) and maize (Zea mays L.).

The insecticidal crystalline proteins (Crys) are a family of insect endotoxin functioning in crop protection. As insects keep evolving into tolerance to the existing Crys, it is necessary to discover new Cry proteins to overcome potential threatens. Crys possess three functional domains at their N-termini, and the most active region throughout evolution was found at the domain-III. We swapped domain-IIIs from various Cry proteins and generated seven chimeric proteins. All recombinants were expressed in Escherichia coli and their toxicity was assessed by dietary exposure assays. Three of the seven Crys exhibited a high toxicity to Asian corn borer over the controls. One of them, Cry1Ab-Gc, a chimeric Cry1Ab being replaced with the domain-III of Cry1Gc, showed the highest toxicity to rice stem borer when it was over-expressed in Oryza sativa. Furthermore, it was also transformed into maize, backcrossed into commercial maize inbred lines and then produced hybrid to evaluate their commercial value. Transgenic maize performed significant resistance to the Asian corn borer without affecting the yield. We further showed that this new protein did not have adverse effects on the environment. Our results indicated that domain III swapped of Crys could be used as an efficient method for developing new engineered insecticidal protein.

A Na2CO3-Responsive Chitinase Gene From Leymus chinensis Improve Pathogen Resistance and Saline-Alkali Stress Tolerance in Transgenic Tobacco and Maize.

Salinity and microbial pathogens are the major limiting factors for crop production. Although the manipulation of many genes could improve plant performance under either of these stresses, few genes have reported that could improve both pathogen resistance and saline-alkali stress tolerance. In this study, we identified a new chitinase gene CHITINASE 2 (LcCHI2) that encodes a class II chitinase from Leymus chinensis, which grows naturally on alkaline-sodic soil. Overexpression of LcCHI2 increased chitinase activity in transgenic plants. The transgenic tobacco and maize exhibited improved pathogen resistance and enhanced both neutral salt and alkaline salt stress tolerance. Overexpression of LcCHI2 reduced sodium (Na+) accumulation, malondialdehyde content and relative electrical conductivity in transgenic tobacco under salt stress. In addition, the transgenic tobacco showed diminished lesion against bacterial and fungal pathogen challenge, suggesting an improved disease resistance. Similar improved performance was also observed in LcCHI2-overexpressed maize under both pathogen and salt stresses. It is worth noting that this genetic manipulation does not impair the growth and yield of transgenic tobacco and maize under normal cultivation condition. Apparently, application of LcCHI2 provides a new train of thought for genetically engineering saline-alkali and pathogen resistant crops of both dicots and monocots.

Development of dwarfish and yield-effective GM maize through passivation of bioactive gibberellin.

During the Green Revolution in the 1960s, breeding dwarf cultivars turned out to be a landmark, leading to a significant increase in the global production of wheat and rice. The most direct and effective strategy for breeding dwarf crops, among others, would be to control endogenous gibberellin (GA) levels of the crops. GA 2-oxidases are a group of 2-oxoglutarate-dependent dioxygenases that catalyze the deactivation of bioactive GAs. The ArabidopsisAtGA2ox1 gene was transformed into maize with the aim of obtaining a height-reduced GM maize. The characterization of the GM maize revealed that the highest plant height reduction was accomplished by a 74% decline in GA1 level, and by approximately twofold increases in both chlorophyll content and root/shoot ratio over the wild-type (WT). Interestingly, the stem cells of the GM maize were condensed, and the typical vascular bundle structure was found to be deformed. Based on a 2-season field trial, the GM maize exhibited a higher harvest index (9-17%) and grain yield (10-14%) than the WT. The current results suggest that a modulation of the endogenous GA level would be a sensible approach for improving the crop architecture and grain yield in maize.

ZmCOL3, a CCT gene represses flowering in maize by interfering with the circadian clock and activating expression of ZmCCT.

Flowering time is a trait vital to the adaptation of flowering plants to different environments. Here, we report that CCT domain genes play an important role in flowering in maize (Zea mays L.). Among the 53 CCT family genes we identified in maize, 28 were located in flowering time quantitative trait locus regions and 15 were significantly associated with flowering time, based on candidate-gene association mapping analysis. Furthermore, a CCT gene named ZmCOL3 was shown to be a repressor of flowering. Overexpressing ZmCOL3 delayed flowering time by approximately 4 d, in either long-day or short-day conditions. The absence of one cytosine in the ZmCOL3 3'UTR and the presence of a 551 bp fragment in the promoter region are likely the causal polymorphisms contributing to the maize adaptation from tropical to temperate regions. We propose a modified model of the maize photoperiod pathway, wherein ZmCOL3 acts as an inhibitor of flowering either by transactivating transcription of ZmCCT, one of the key genes regulating maize flowering, or by interfering with the circadian clock.

Affinity chromatography revealed insights into unique functionality of two 14-3-3 protein species in developing maize kernels.

The 14-3-3 proteins are a group of regulatory proteins of divergent functions in plants. However, little is known about their roles in maize kernel development. Using publically available gene expression profiling data, we found that two 14-3-3 species genes, zmgf14-4 and zmgf14-6, exhibited prominent expression profiles over other 14-3-3 protein genes during maize kernel development. More than 5000 transcripts of these two genes were identified accounting for about 1/10 of the total transcripts of genes correlating to maize kernel development. We constructed a proteomics pipeline based on the affinity chromatography, in combination with 2-DE and LC-MS/MS technologies to identify the specific client proteins of the two proteins for their functional characterization. Consequently, we identified 77 specific client proteins from the developing kernels of the inbred maize B73. More than 60% of the client proteins were commonly affinity-identified by the two 14-3-3 species and are predicted to be implicated in the fundamental functions of metabolism, protein destination and storage. In addition, we found ZmGF14-4 specifically bound to the disease- or defense-relating proteins, whilst ZmGF14-6 tended to interact with the proteins involving metabolism and cell structure. Our findings provide primary insights into the functional roles of 14-3-3 proteins in maize kernel development. BIOLOGICAL SIGNIFICANCE: Maize kernel development is a complicated physiological process for its importance in both genetics and cereal breeding. 14-3-3 proteins form a multi-gene family participating in regulations of developmental processes in plants. However, the correlation between this protein family and maize kernel development has hardly been studied. We have for the first time found 12 14-3-3 protein genes from maize genome and studied in silico the gene transcription profiling of these genes. Comparative studies revealed that maize kernel development aroused a great number of gene expression, among which 14-3-3 protein genes took a significant proportion. We applied affinity chromatographic approach, in combination with 2-DE and LC-MS/MS, to explore the specific client proteins of two crucial 14-3-3 protein species that exhibit prominent gene expression over other members in the family during the kernel development. Assessments of the identified client proteins resulted in important information toward understanding the functional mechanism of 14-3-3 protein family in maize kernel development.

Molecular dynamics analysis reveals structural insights into mechanism of nicotine N-demethylation catalyzed by tobacco cytochrome P450 mono-oxygenase.

CYP82E4, a cytochrome P450 monooxygenase, has nicotine N-demethylase (NND) activity, which mediates the bioconversion of nicotine into nornicotine in senescing tobacco leaves. Nornicotine is a precursor of the carcinogen, tobacco-specific nitrosamine. CYP82E3 is an ortholog of CYP82E4 with 95% sequence identity, but it lacks NND activity. A recent site-directed mutagenesis study revealed that a single amino acid substitution, i.e., cysteine to tryptophan at the 330 position in the middle of protein, restores the NND activity of CYP82E3 entirely. However, the same amino acid change caused the loss of the NND activity of CYP82E4. To determine the mechanism of the functional turnover of the two molecules, four 3D structures, i.e., the two molecules and their corresponding cys-trp mutants were modeled. The resulting structures exhibited that the mutation site is far from the active site, which suggests that no direct interaction occurs between the two sites. Simulation studies in different biological scenarios revealed that the mutation introduces a conformation drift with the largest change at the F-G loop. The dynamics trajectories analysis using principal component analysis and covariance analysis suggests that the single amino acid change causes the opening and closing of the transfer channels of the substrates, products, and water by altering the motion of the F-G and B-C loops. The motion of helix I is also correlated with the motion of both the F-G loop and the B-C loop and; the single amino acid mutation resulted in the curvature of helix I. These results suggest that the single amino acid mutation outside the active site region may have indirectly mediated the flexibility of the F-G and B-C loops through helix I, causing a functional turnover of the P450 monooxygenase.

Molecular dynamics simulations reveal the disparity in specific recognition of GCC-box by AtERFs transcription factors super family in Arabidopsis.

Arabidopsis ethylene responsive element binding factors (AtERFs) form a transcription factor super family. While the functions of most AtERFs are unknown, a number of AtERFs appear to be involved in regulation of stress-related genes through their DNA binding domains (DBD), namely ERF domains, which recognize a consensus motif GCC-box at the regulatory region. In this study, molecular dynamics simulations were performed on the four ERF domain-GCC-box complexes, AtERF1, AtERF4, AtEBP and CFBF1, to determine disparity in specific binding to the GCC-box by the AtERFs. Our results suggested that three amino acid residues Arg29, Glu39 and Arg41, played a vital role in direct readout of DNA. The position of the consensus sequence GCCGCC has an intrinsic disparity on binding with ERF domains. The third C, fourth G and the last C in the GCC motif was compulsory for recognition by ERF domains. Our results provide structural evidence for a sequence-dependent recognition mechanism for AtERFs.

An in silico strategy identified the target gene candidates regulated by dehydration responsive element binding proteins (DREBs) in Arabidopsis genome.

Identification of downstream target genes of stress-relating transcription factors (TFs) is desirable in understanding cellular responses to various environmental stimuli. However, this has long been a difficult work for both experimental and computational practices. In this research, we presented a novel computational strategy which combined the analysis of the transcription factor binding site (TFBS) contexts and machine learning approach. Using this strategy, we conducted a genome-wide investigation into novel direct target genes of dehydration responsive element binding proteins (DREBs), the members of AP2-EREBPs transcription factor super family which is reported to be responsive to various abiotic stresses in Arabidopsis. The genome-wide searching yielded in total 474 target gene candidates. With reference to the microarray data for abiotic stresses-inducible gene expression profile, 268 target gene candidates out of the total 474 genes predicted, were induced during the 24-h exposure to abiotic stresses. This takes about 57% of total predicted targets. Furthermore, GO annotations revealed that these target genes are likely involved in protein amino acid phosphorylation, protein binding and Endomembrane sorting system. The results suggested that the predicted target gene candidates were adequate to meet the essential biological principle of stress-resistance in plants.