Comprehensive identification of maize ZmE2F transcription factors and the positive role of ZmE2F6 in response to drought stress.

BACKGROUND: The early 2 factor (E2F) family is characterized as a kind of transcription factor that plays an important role in cell division, DNA damage repair, and cell size regulation. However, its stress response has not been well revealed. RESULTS: In this study, ZmE2F members were comprehensively identified in the maize genome, and 21 ZmE2F genes were identified, including eight E2F subclade members, seven DEL subfamily genes, and six DP genes. All ZmE2F proteins possessed the DNA-binding domain (DBD) characterized by conserved motif 1 with the RRIYD sequence. The ZmE2F genes were unevenly distributed on eight maize chromosomes, showed diversity in gene structure, expanded by gene duplication, and contained abundant stress-responsive elements in their promoter regions. Subsequently, the ZmE2F6 gene was cloned and functionally verified in drought response. The results showed that the ZmE2F6 protein interacted with ZmPP2C26, localized in the nucleus, and responded to drought treatment. The overexpression of ZmE2F6 enhanced drought tolerance in transgenic Arabidopsis with longer root length, higher survival rate, and biomass by upregulating stress-related gene transcription. CONCLUSIONS: This study provides novel insights into a greater understanding and functional study of the E2F family in the stress response.

Maize ZmLAZ1-3 gene negatively regulates drought tolerance in transgenic Arabidopsis.

BACKGROUND: Molecular mechanisms in response to drought stress are important for the genetic improvement of maize. In our previous study, nine ZmLAZ1 members were identified in the maize genome, but the function of ZmLAZ1 was largely unknown. RESULTS: The ZmLAZ1-3 gene was cloned from B73, and its drought-tolerant function was elucidated by expression analysis in transgenic Arabidopsis. The expression of ZmLAZ1-3 was upregulated by drought stress in different maize inbred lines. The driving activity of the ZmLAZ1-3 promoter was induced by drought stress and related to the abiotic stress-responsive elements such as MYB, MBS, and MYC. The results of subcellular localization indicated that the ZmLAZ1-3 protein localized on the plasma membrane and chloroplast. The ectopic expression of the ZmLAZ1-3 gene in Arabidopsis significantly reduced germination ratio and root length, decreased biomass, and relative water content, but increased relative electrical conductivity and malondialdehyde content under drought stress. Moreover, transcriptomics analysis showed that the differentially expressed genes between the transgenic lines and wild-type were mainly associated with response to abiotic stress and biotic stimulus, and related to pathways of hormone signal transduction, phenylpropanoid biosynthesis, mitogen-activated protein kinase signaling, and plant-pathogen interaction. CONCLUSION: The study suggests that the ZmLAZ1-3 gene is a negative regulator in regulating drought tolerance and can be used to improve maize drought tolerance via its silencing or knockout.

Maize ZmBES1/BZR1-1 transcription factor negatively regulates drought tolerance.

Drought stress is a common abiotic factor and restricts plant growth and development. Exploring maize stress-related genes and their regulatory mechanisms is crucial for ensuring agricultural productivity and food security. The BRI1-EMS1 suppressor (BES1)/brassinazole-resistant 1 (BZR1) transcription factors (TFs) play important roles in plant growth, development, and stress response. However, maize ZmBES1/BZR1s are rarely reported. In the present study, the ZmBES1/BZR1-1 gene was cloned from maize B73 and functionally characterized in transgenic Arabidopsis and rice in drought stress response. The ZmBES1/BZR1-1 protein possessed a conserved bHLH domain characterized by BES1/BZR1 TFs, localized in the nucleus, and showed transcription activation activity. The expression of ZmBES1/BZR1-1 exhibited no tissue specificity but drought-inhibitory expression in maize. Under drought stress, overexpression of ZmBES1/BZR1-1 resulted in the enhancement of drought sensitivity of transgenic Arabidopsis and rice with a lower survival rate, reactive oxygen species (ROS) level and relative water content (RWC) and a higher stomatal aperture and relative electrolyte leakage (REL). The RNA-seq results showed that 56 differentially expressed genes (DEGs) were regulated by ZmBES1/BZR1-1 by binding to E-box elements in their promoters. The GO analysis showed that the DEGs were significantly annotated with response to oxidative stress and oxygen level. The study suggests that the ZmBES1/BZR1-1 gene negatively regulates drought stress, which provides insights into further underlying molecular mechanisms in the drought stress response mediated by BZR1/BES1s.

Comprehensive Identification of the Pum Gene Family and Its Involvement in Kernel Development in Maize.

The Pumilio (Pum) RNA-binding protein family regulates post-transcription and plays crucial roles in stress response and growth. However, little is known about Pum in plants. In this study, a total of 19 ZmPum genes were identified and classified into two groups in maize. Although each ZmPum contains the conserved Pum domain, the ZmPum members show diversity in the gene and protein architectures, physicochemical properties, chromosomal location, collinearity, cis-elements, and expression patterns. The typical ZmPum proteins have eight α-helices repeats, except for ZmPum2, 3, 5, 7, and 14, which have fewer α-helices. Moreover, we examined the expression profiles of ZmPum genes and found their involvement in kernel development. Except for ZmPum2, ZmPum genes are expressed in maize embryos, endosperms, or whole seeds. Notably, ZmPum4, 7, and 13 exhibited dramatically high expression levels during seed development. The study not only contributes valuable information for further validating the functions of ZmPum genes but also provides insights for improvement and enhancing maize yield.

The Maize ZmBES1/BZR1-9 Transcription Factor Accelerates Flowering in Transgenic Arabidopsis and Rice.

In model plants, the BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1 (BZR1) transcription factors play vital roles in regulating growth, development, and stimuli response. However, the roles of maize ZmBES1/BZR1 members are largely unknown. In this research, the ZmBES1/BZR1-9 gene was ectopically expressed in Arabidopsis and rice for the phenotyping of flowering. We found that the complementation and overexpression of ZmBES1/BZR1-9 in bes1-D mutant and wild type Arabidopsis both resulted in early flowering that was about 10 days shorter than in the untransformed control under long-day conditions. In addition, there was no difference in the rosette leaf number between all transgenic lines and the control. Subsequently, the ZmBES1/BZR1-9 gene was overexpressed in rice. It was found that overexpression lines of rice exhibited early flowering with heading dates that were 8 days shorter compared with untransformed plants. Moreover, the results of RNA-seq and qRT-PCR showed that five flowering-regulated genes, namely At2-MMP, AtPCC1, AtMYB56, AtPELPK1, and AtPRP10, were significantly up-regulated in all complementary and overexpressing lines of Arabidopsis. Meanwhile, the results of RNA-seq showed that 69 and 33 differentially expressed genes (DEGs) were up- and down-regulated in transgenic rice, respectively. Four flowering-related genes, namely OsGA20OX1, OsCCR19, OsBTBN19, and OsRNS4 were significantly up-regulated in transgenic lines. To sum up, our findings demonstrate that ZmBES1/BZR1-9 is involved in controlling flowering and provide insights into further underlying roles of BES1/BZR1s in regulating growth and development in crops.

Genome-Wide Characterization and Function Analysis of ZmERD15 Genes' Response to Saline Stress in Zea mays L.

Early responsive dehydration (ERD) genes can be rapidly induced by dehydration. ERD15 genes have been confirmed to regulate various stress responses in plants. However, the maize ERD15 members have not been characterized. In the present study, a total of five ZmERD15 genes were identified from the maize genome and named ZmERD15a, ZmERD15b, ZmERD15c, ZmERD15d, and ZmERD15e. Subsequently, their protein properties, gene structure and duplication, chromosomal location, cis-acting elements, subcellular localization, expression pattern, and over-expression in yeast were analyzed. The results showed that the ZmERD15 proteins were characterized by a similar size (113-159 aa) and contained a common domain structure, with PAM2 and adjacent PAE1 motifs followed by an acidic region. The ZmERD15 proteins exhibited a close phylogenetic relationship with OsERD15s from rice. Five ZmERD15 genes were distributed on maize chromosomes 2, 6, 7, and 9 and showed a different exon-intron organization and were expanded by duplication. Besides, the promoter region of the ZmERD15s contained abundant cis-acting elements that are known to be responsive to stress and hormones. Subcellular localization showed that ZmERD15b and ZmERD15c were localized in the nucleus. ZmERD15a and ZmERD15e were localized in the nucleus and cytoplasm. ZmERD15d was localized in the nucleus and cell membrane. The results of the quantitative real-time PCR (qRT-PCR) showed that the expression of the ZmERD15 genes was regulated by PEG, salinity, and ABA. The heterologous expression of ZmERD15a, ZmERD15b, ZmERD15c, and ZmERD15d significantly enhanced salt tolerance in yeast. In summary, a comprehensive analysis of ZmERD15s was conducted in the study. The results will provide insights into further dissecting the biological function and molecular mechanism of ZmERD15s regulating of the stress response in maize.

Genomic Characteristics of Elite Maize Inbred Line 18-599 and Its Transcriptional Response to Drought and Low-Temperature Stresses.

Elite inbred line 18-599 was developed via triple test cross from introduced hybrid P78599 and used as parents of dozens of maize hybrids adapting to the diverse ecological conditions of the maize ecological region in Southwest China. In this study, its genomic DNA was resequenced and aligned with the B73 genome sequence to identify single nucleotide polymorphism (SNP), and insertion (In) and deletion (Del) loci. These loci were aligned with those between B73 and 1020 inbred lines in the HapMap database to identify specific variation loci of 18-599. The results showed that there were 930,439 specific SNPs and 358,750 InDels between 18-599 and the 1020 lines. In total, 21,961 of them showed significant impacts on the functions of 12,297 genes, such as frameshift, change of splicing site, stop gain, change of start site, and stop loss. Phylogenetic analysis showed that 18-599 was closely related to inbred lines ZEAxujRAUDIAAPE and 2005-4, but far from some inbred lines directly isolated from P78599. This result indicated that 18-599 not only pyramided the elite genes of P78599, but also acquired genetic divergence during the repetitive backcrosses of triple test cross to confer its elite agronomic characteristics. Subsequently, the RNA of 18-599 was sequenced. The aligned 9713 and 37,528 of the 165,098 unigenes were screened and aligned with annotated transcripts of the B73 genome differentially expressed under drought and low-temperature stress, respectively, and their functions were involved in the responses to these stresses. The quantitative PCR results of fourteen random genes verified the RNA sequencing results. These findings suggest that the transcriptional responses of many resistance-related genes were an important mechanism for 18-599 to adapt to diverse ecological conditions.

Comprehensive Identification and Functional Analysis of Stress-Associated Protein (SAP) Genes in Osmotic Stress in Maize.

Stress-associated proteins (SAPs) are a kind of zinc finger protein with an A20/AN1 domain and contribute to plants' adaption to various abiotic and biological stimuli. However, little is known about the SAP genes in maize (Zea mays L.). In the present study, the SAP genes were identified from the maize genome. Subsequently, the protein properties, gene structure and duplication, chromosomal location, and cis-acting elements were analyzed by bioinformatic methods. Finally, their expression profiles under osmotic stresses, including drought and salinity, as well as ABA, and overexpression in Saccharomyces cerevisiae W303a cells, were performed to uncover the potential function. The results showed that a total of 10 SAP genes were identified and named ZmSAP1 to ZmSAP10 in maize, which was unevenly distributed on six of the ten maize chromosomes. The ZmSAP1, ZmSAP4, ZmSAP5, ZmSAP6, ZmSAP7, ZmSAP8 and ZmSAP10 had an A20 domain at N terminus and AN1 domain at C terminus, respectively. Only ZmSAP2 possessed a single AN1 domain at the N terminus. ZmSAP3 and ZmSAP9 both contained two AN1 domains without an A20 domain. Most ZmSAP genes lost introns and had abundant stress- and hormone-responsive cis-elements in their promoter region. The results of quantitative real-time PCR showed that all ZmSAP genes were regulated by drought and saline stresses, as well as ABA induction. Moreover, heterologous expression of ZmSAP2 and ZmSAP7 significantly improved the saline tolerance of yeast cells. The study provides insights into further underlying the function of ZmSAPs in regulating stress response in maize.

Transgenic Improvement for Biotic Resistance of Crops.

Biotic constraints, including pathogenic fungi, viruses and bacteria, herbivory insects, as well as parasitic nematodes, cause significant yield loss and quality deterioration of crops. The effect of conventional management of these biotic constraints is limited. The advances in transgenic technologies provide a direct and directional approach to improve crops for biotic resistance. More than a hundred transgenic events and hundreds of cultivars resistant to herbivory insects, pathogenic viruses, and fungi have been developed by the heterologous expression of exogenous genes and RNAi, authorized for cultivation and market, and resulted in a significant reduction in yield loss and quality deterioration. However, the exploration of transgenic improvement for resistance to bacteria and nematodes by overexpression of endogenous genes and RNAi remains at the testing stage. Recent advances in RNAi and CRISPR/Cas technologies open up possibilities to improve the resistance of crops to pathogenic bacteria and plant parasitic nematodes, as well as other biotic constraints.

Low-Density Reference Fingerprinting SNP Dataset of CIMMYT Maize Lines for Quality Control and Genetic Diversity Analyses.

CIMMYT maize lines (CMLs), which represent the tropical maize germplasm, are freely available worldwide. All currently released 615 CMLs and fourteen temperate maize inbred lines were genotyped with 180 kompetitive allele-specific PCR single nucleotide polymorphisms to develop a reference fingerprinting SNP dataset that can be used to perform quality control (QC) and genetic diversity analyses. The QC analysis identified 25 CMLs with purity, identity, or mislabeling issues. Further field observation, purification, and re-genotyping of these CMLs are required. The reference fingerprinting SNP dataset was developed for all of the currently released CMLs with 152 high-quality SNPs. The results of principal component analysis and average genetic distances between subgroups showed a clear genetic divergence between temperate and tropical maize, whereas the three tropical subgroups partially overlapped with one another. More than 99% of the pairs of CMLs had genetic distances greater than 0.30, showing their high genetic diversity, and most CMLs are distantly related. The heterotic patterns, estimated with the molecular markers, are consistent with those estimated using pedigree information in two major maize breeding programs at CIMMYT. These research findings are helpful for ensuring the regeneration and distribution of the true CMLs, via QC analysis, and for facilitating the effective utilization of the CMLs, globally.

Three strategies of transgenic manipulation for crop improvement.

Heterologous expression of exogenous genes, overexpression of endogenous genes, and suppressed expression of undesirable genes are the three strategies of transgenic manipulation for crop improvement. Up to 2020, most (227) of the singular transgenic events (265) of crops approved for commercial release worldwide have been developed by the first strategy. Thirty-eight of them have been transformed by synthetic sequences transcribing antisense or double-stranded RNAs and three by mutated copies for suppressed expression of undesirable genes (the third strategy). By the first and the third strategies, hundreds of transgenic events and thousands of varieties with significant improvement of resistance to herbicides and pesticides, as well as nutritional quality, have been developed and approved for commercial release. Their application has significantly decreased the use of synthetic pesticides and the cost of crop production and increased the yield of crops and the benefits to farmers. However, almost all the events overexpressing endogenous genes remain at the testing stage, except one for fertility restoration and another for pyramiding herbicide tolerance. The novel functions conferred by the heterologously expressing exogenous genes under the control of constitutive promoters are usually absent in the recipient crops themselves or perform in different pathways. However, the endogenous proteins encoded by the overexpressing endogenous genes are regulated in complex networks with functionally redundant and replaceable pathways and are difficult to confer the desirable phenotypes significantly. It is concluded that heterologous expression of exogenous genes and suppressed expression by RNA interference and clustered regularly interspaced short palindromic repeats-cas (CRISPR/Cas) of undesirable genes are superior to the overexpression of endogenous genes for transgenic improvement of crops.

Characterization of phenylalanine ammonia-lyase genes facilitating flavonoid biosynthesis from two species of medicinal plant Anoectochilus.

Background: Anoectochilus roxburghii and Anoectochilus formosanus, belong to the Anoectochilus genus, have been used for Chinese herbal drugs as well as health food. Phenylalanine ammonia-lyase (PAL), the key enzyme in primary metabolism and phenylpropanoid metabolism, produces secondary metabolites (flavonoids) in plants, which are beneficial for the biosynthesis of phenylpropanoid metabolites. Methods: The PAL genes were cloned from A. formosanus and A. roxburghii according to our previous transcriptomic analysis. The PALs were introduced into pCAMBIA2300-35S-PAL-eGFP to generate 35S-PAL-eGFP. The constructs were further used for subcellular localization and transgenic Arabidopsis. The expression of AfPAL and ArPAL under precursor substance (L-Phe), NaCl, UV, and red-light were analyzed by real-time quantitative PCR (RT-qPCR). Results: AfPAL and ArPAL , encoding 2,148 base pairs, were cloned from A. formosanus and A. roxburghii. The subcellular localization showed that the ArPAL and AfPAL were both localized in the nucleus with GPF. Quantitative RT-PCR analysis indicated that the ArPAL and AfPAL genes function in the phenylalanine pathway as well as response to induced conditions. Overexpression of the AfPAL and ArPAL could increase flavonoids and anthocyanin content in the transgenic Arabidopsis. Discussion: The results suggest that AfPAL and ArPAL play a crucial role in the flavonoid biosynthesis in Anoectochilus. Also, our study provides new insights into the enrichment of secondary metabolites of traditional Chinese medicines A. formosanus and A. roxburghii, which can improve their medicinal active ingredients and be used for drug discovery in plants.

Maize ZmBES1/BZR1-3 and -9 Transcription Factors Negatively Regulate Drought Tolerance in Transgenic Arabidopsis.

The BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1(BZR1) transcription factors play crucial roles in plant growth, development, and stress response. However, little is known about the function of maize's BES1/BZR1s. In this study, the ZmBES1/BZR1-3 and ZmBES1/BZR1-9 genes were cloned from maize's inbred line, B73, and they were functionally evaluated by analyzing their expression pattern, subcellular localization, transcriptional activation activity, as well as their heterologous expression in Arabidopsis, respectively. The results of the qRT-PCR showed that the ZmBES1/BZR1-3 and ZmBES1/BZR1-9 genes were predominantly expressed in the root, and their expression was significantly down-regulated by drought stress. The ZmBES1/BZR1-3 and ZmBES1/BZR1-9 proteins localized in the nucleus but showed no transcriptional activation activity as a monomer. Subsequently, it was found that the heterologous expression of the ZmBES1/BZR1-3 and ZmBES1/BZR1-9 genes in Arabidopsis decreased drought tolerance, respectively. The transgenic lines showed a more serious wilting phenotype, shorter root length, lower fresh weight, and higher relative electrolyte leakage (REL) and malondialdehyde (MDA) content compared to the control under drought stress. The RNA-sequencing data showed that the 70.67% and 93.27% differentially expressed genes (DEGs) were significantly down-regulated in ZmBES1/BZR1-3 and ZmBES1/BZR1-9 transgenic Arabidopsis, respectively. The DEGs of ZmBES1/BZR1-3 gene's expressing lines were mainly associated with oxidative stress response and amino acid metabolic process and enriched in phenylpropanoid biosynthesis and protein processing in the endoplasmic reticulum. But the DEGs of the ZmBES1/BZR1-9 gene's expressing lines were predominantly annotated with water deprivation, extracellular stimuli, and jasmonic acid and enriched in phenylpropanoid biosynthesis and plant hormone signal transduction. Moreover, ZmBES1/BZR1-9 increased stomatal aperture in transgenic Arabidopsis under drought stress. This study indicates that ZmBES1/BZR1-3 and ZmBES1/BZR1-9 negatively regulate drought tolerance via different pathways in transgenic Arabidopsis, and it provides insights into the underlying the function of BES1/BZR1s in crops.

Zinc Transporter ZmLAZ1-4 Modulates Zinc Homeostasis on Plasma and Vacuolar Membrane in Maize.

Zinc is an essential micronutrient for plant growth and development, and functions as a cofactor for hundreds of transcription factors and enzymes in numerous biological processes. Zinc deficiency is common abiotic stress resulting in yield loss and quality deterioration of crops, but zinc excess causes toxicity for biological systems. In plants, zinc homeostasis is tightly modulated by zinc transporters and binding compounds that uptake/release, transport, localize, and store zinc, as well as their upstream regulators. Lazarus 1 (LAZ1), a member of DUF300 protein family, functions as transmembrane organic solute transporter in vertebrates. However, the function of LAZ1 in plants is still obscure. In the present study, the ZmLAZ1-4 protein was confirmed to bind to zinc ions by bioinformatic prediction and thermal shift assay. Heterologous expression of ZmLAZ1-4 in the zinc-sensitive yeast mutant, Arabidopsis, and maize significantly facilitated the accumulation of Zn2+ in transgenic lines, respectively. The result of subcellular localization exhibited that ZmLAZ1-4 was localized on the plasma and vacuolar membrane, as well as chloroplast. Moreover, the ZmLAZ1-4 gene was negatively co-expressed with ZmBES1/BZR1-11 gene through co-expression and real-time quantitative PCR analysis. The results of yeast one-hybrid and dual-luciferase assay suggested that ZmBES1/BZR1-11 could bind to ZmLAZ1-4 promoter to inhibit its transcription. All results indicated that ZmLAZ1-4 was a novel zinc transporter on plasma and vacuolar membrane, and transported zinc under negative regulation of the ZmBES1/BZR1-11 transcription factor. The study provides insights into further underlying the mechanism of ZmLAZ1-4 regulating zinc homeostasis.

ZmPP2C26 Alternative Splicing Variants Negatively Regulate Drought Tolerance in Maize.

Serine/threonine protein phosphatase 2C (PP2C) dephosphorylates proteins and plays crucial roles in plant growth, development, and stress response. In this study, we characterized a clade B member of maize PP2C family, i.e., ZmPP2C26, that negatively regulated drought tolerance by dephosphorylating ZmMAPK3 and ZmMAPK7 in maize. The ZmPP2C26 gene generated ZmPP2C26L and ZmPP2C26S isoforms through untypical alternative splicing. ZmPP2C26S lost 71 amino acids including an MAPK interaction motif and showed higher phosphatase activity than ZmPP2C26L. ZmPP2C26L directly interacted with, dephosphorylated ZmMAPK3 and ZmMAPK7, and localized in chloroplast and nucleus, but ZmPP2C26S only dephosphorylated ZmMAPK3 and localized in cytosol and nucleus. The expression of ZmPP2C26L and ZmPP2C26 was significantly inhibited by drought stress. Meanwhile, the maize zmpp2c26 mutant exhibited enhancement of drought tolerance with higher root length, root weight, chlorophyll content, and photosynthetic rate compared with wild type. However, overexpression of ZmPP2C26L and ZmPP2C26S significantly decreased drought tolerance in Arabidopsis and rice with lower root length, chlorophyll content, and photosynthetic rate. Phosphoproteomic analysis revealed that the ZmPP2C26 protein also altered phosphorylation level of proteins involved in photosynthesis. This study provides insights into understanding the mechanism of PP2C in response to abiotic stress.

Antifreeze protein from Ammopiptanthus nanus functions in temperature-stress through domain A.

Temperature stress restricts plant growth and development. Antifreeze protein (AFP) can improve plants antifreeze ability. In our previous study, the AnAFP gene cloned from Ammopiptanthus nanus was confirmed to be an excellent candidate enhancing plant cold resistance. But, AnAFP protein shared similar structures with KnS type dehydrins including K, N and S domains except ice crystal binding domain A. Here, we generated AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS, and transformed them into ordinary and cold sensitive strains of E. coli, and Arabidopsis KS type dehydrin mutant to evaluate their function. Expression of AnAFPΔA decreases cold and heat tolerance in E. coli, meanwhile, AnAFP enhances heat tolerance in Arabidopsis, suggesting that domain A is a thermal stable functional domain. AnAFP, AnAFPΔA and AnAFPΔS localize in whole cell, but AnAFPΔK and AnAFPΔN only localizes in nucleus and cytoplasm, respectively, exhibiting that K and N domains control localization of AnAFP. Likewise, K domain blocks interaction between AnAFP and AnICE1. The result of RT-qPCR showed that expression of AnAFP, AnICE1 and AnCBF genes was significantly induced by high-temperature, indicating that the AnAFP is likely regulated by ICE1-CBF-COR signal pathway. Taken together, the study provides insights into understanding the mechanism of AnAFP in response to temperature stress and gene resource to improve heat or cold tolerance of plants in transgenic engineering.

Genome-Wide Identification and Expression Analyses of AnSnRK2 Gene Family under Osmotic Stress in Ammopiptanthus nanus.

Sucrose non-fermenting-1 (SNF1)-related protein kinase 2's (SnRK2s) are plant-specific serine/threonine protein kinases and play crucial roles in the abscisic acid signaling pathway and abiotic stress response. Ammopiptanthus nanus is a relict xerophyte shrub and extremely tolerant of abiotic stresses. Therefore, we performed genome-wide identification of the AnSnRK2 genes and analyzed their expression profiles under osmotic stresses including drought and salinity. A total of 11 AnSnRK2 genes (AnSnRK2.1-AnSnRK2.11) were identified in the A. nanus genome and were divided into three groups according to the phylogenetic tree. The AnSnRK2.6 has seven introns and others have eight introns. All of the AnSnRK2 proteins are highly conserved at the N-terminus and contain similar motif composition. The result of cis-acting element analysis showed that there were abundant hormone- and stress-related cis-elements in the promoter regions of AnSnRK2s. Moreover, the results of quantitative real-time PCR exhibited that the expression of most AnSnRK2s was induced by NaCl and PEG-6000 treatments, but the expression of AnSnRK2.3 and AnSnRK2.6 was inhibited, suggesting that the AnSnRK2s might play key roles in stress tolerance. The study provides insights into understanding the function of AnSnRK2s.

Maize transcription factor ZmBES1/BZR1-5 positively regulates kernel size.

The BES1/BZR1 transcription factors regulate the expression of genes responsive to brassinosteroids and play pivotal roles in plant development, but their role in regulating kernel development in maize remains unclear. In this study, we found that ZmBES1/BZR1-5 positively regulates kernel size. Association analysis of candidate genes in 513 diverse maize inbred lines indicated that three SNPs related to ZmBES1/BZR1-5 were significantly associated with kernel width and whilst four SNPs were related to 100-kernel weight. Overexpression of ZmBES1/BZR1-5 in Arabidopsis and rice both significantly increased seed size and weight, and smaller kernels were produced in maize Mu transposon insertion and EMS mutants. The ZmBES1/BZR1-5 protein locates in the nucleus, contains bHLH and BAM domains, and shows no transcriptional activity as a monomer but forms a homodimer through the BAM domain. ChIP-sequencing analysis, and yeast one-hybrid and dual-luciferase assays demonstrated that the protein binds to the promoters of AP2/EREBP genes (Zm00001d010676 and Zm00001d032077) and inhibits their transcription. cDNA library screening showed that ZmBES1/BZR1-5 interacts with casein kinase II subunit ß4 (ZmCKIIß4) and ferredoxin 2 (ZmFdx2) in vitro and in vivo, respectively. Taken together, our study suggests that ZmBES1/BZR1-5 positively regulates kernel size, and provides new insights into understanding the mechanisms of kernel development in maize.

Isolation and characterization of maize ZmPP2C26 gene promoter in drought-response.

The clade A members of serine/threonine protein phosphatase 2Cs (PP2Cs) play crucial roles in plant growth, development, and stress response via the ABA signaling pathway. But little is known about other PP2C clades in plants. Our previous study showed that maize the ZmPP2C26, a clade B member of ZmPP2Cs, negatively regulated drought tolerance in transgenic Arabidopsis. However, the upstream regulatory mechanism of ZmPP2C26 remains unclear. In the present study, the expression of ZmPP2C26 gene in maize was analyzed by quantitative real time PCR (qRT-PCR). The results showed that the expression of ZmPP2C26 in shoot and root was both significantly inhibited by drought stress. Subsequently, a 2175 bp promoter of ZmPP2C26 was isolated from maize genome (P 2175). To validate whether the promoter possess some key cis-element and negatively drive ZmPP2C26 expression in drought stress, three 5´-deletion fragments of 1505, 1084 and 215 bp was amplified from P 2175 and were fused to ß-glucuronidase (GUS) and luciferase gene (LUC) to produce promoter::GUS and promoter::LUC constructs, and transformed into tobacco, respectively. Transient expression assays indicated that all promoters could drive GUS and LUC expression. The GUS and LUC activity were both significantly inhibited by PEG-6000 treatment. Notably, the - 1084 to - 215 bp promoter possess one MBS element and inhibits the expression of GUS and LUC under drought stress. Meanwhile, we found that the 215 bp length is enough to drive ZmPP2C26 expression. These findings will provide insights into understanding the transcription-regulatory mechanism of ZmPP2C26 negatively regulating drought tolerance.

Maize ZmBES1/BZR1-5 Decreases ABA Sensitivity and Confers Tolerance to Osmotic Stress in Transgenic Arabidopsis.

The BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1 (BZR1) transcription factors, key components in the brassinosteroid signaling pathway, play pivotal roles in plant growth and development. However, the function of BES1/BZR1 in crops during stress response remains poorly understood. In the present study, we characterized ZmBES1/BZR1-5 from maize, which was localized to the nucleus and was responsive to abscisic acid (ABA), salt and drought stresses. Heterologous expression of ZmBES1/BZR1-5 in transgenic Arabidopsis resulted in decreased ABA sensitivity, facilitated shoot growth and root development, and enhanced salt and drought tolerance with lower malondialdehyde (MDA) content and relative electrolyte leakage (REL) under osmotic stress. The RNA sequencing (RNA-seq) analysis revealed that 84 common differentially expressed genes (DEGs) were regulated by ZmBES1/BZR1-5 in transgenic Arabidopsis. Subsequently, gene ontology and KEGG pathway enrichment analyses showed that the DEGs were enriched in response to stress, secondary metabolism and metabolic pathways. Furthermore, 30 DEGs were assigned to stress response and possessed 2-15 E-box elements in their promoters, which could be potentially recognized and bound by ZmBES1/BZR1-5. Taken together, our results reveal that the ZmBES1/BZR1-5 transcription factor positively regulates salt and drought tolerance by binding to E-box to induce the expression of downstream stress-related genes. Therefore, our study contributes to the better understanding of BES1/BZR1 function in the stress response of plants.