ZmEREB92 plays a negative role in seed germination by regulating ethylene signaling and starch mobilization in maize.

Rapid and uniform seed germination is required for modern cropping system. Thus, it is important to optimize germination performance through breeding strategies in maize, in which identification for key regulators is needed. Here, we characterized an AP2/ERF transcription factor, ZmEREB92, as a negative regulator of seed germination in maize. Enhanced germination in ereb92 mutants is contributed by elevated ethylene signaling and starch degradation. Consistently, an ethylene signaling gene ZmEIL7 and an α-amylase gene ZmAMYa2 are identified as direct targets repressed by ZmEREB92. OsERF74, the rice ortholog of ZmEREB92, shows conserved function in negatively regulating seed germination in rice. Importantly, this orthologous gene pair is likely experienced convergently selection during maize and rice domestication. Besides, mutation of ZmEREB92 and OsERF74 both lead to enhanced germination under cold condition, suggesting their regulation on seed germination might be coupled with temperature sensitivity. Collectively, our findings uncovered the ZmEREB92-mediated regulatory mechanism of seed germination in maize and provide breeding targets for maize and rice to optimize seed germination performance towards changing climates.

OsWRKY10 extensively activates multiple rice diterpenoid phytoalexin biosynthesis genes to enhance rice blast resistance.

Phytoalexin is the main chemical weapon against pathogens in plants. Rice (Oryza sativa L.) produces a number of phytoalexins to defend against pathogens, most of which belong to the class of diterpenoid phytoalexins. Three biosynthetic gene clusters (BGCs) and a few non-BGC genes are responsible for rice diterpenoid phytoalexin biosynthesis. The corresponding regulatory mechanism of these phytoalexins in response to pathogen challenges still remains unclear. Here we identified a transcription factor, OsWRKY10, which positively regulates rice diterpenoid phytoalexin biosynthesis. Knockout mutants of OsWRKY10 obtained by CRISPR/Cas9 technology are more susceptible to Magnaporthe oryzae infection, while overexpression of OsWRKY10 enhances resistance to rice blast. Further analysis revealed that overexpression of OsWRKY10 increases accumulation of multiple rice diterpenoid phytoalexins and expression of genes in three BGCs and non-BGC genes in response to M. oryzae infection. Knockout of OsWRKY10 impairs upregulation of rice diterpenoid phytoalexin biosynthesis gene expression by blast pathogen and CuCl2 treatment. OsWRKY10 directly binds to the W-boxes or W-box-like elements (WLEs) of rice diterpenoid phytoalexin biosynthesis gene promoters to regulate gene expression. This study identified an extensive regulator (OsWRKY10) with broad transcriptional regulatory effects on rice diterpenoid phytoalexin biosynthesis genes, providing insight into the regulation of chemical defense to improve disease resistance in rice.

Genome-wide identification of ZmMYC2 binding sites and target genes in maize.

BACKGROUND: Jasmonate (JA) is the important phytohormone to regulate plant growth and adaption to stress signals. MYC2, an bHLH transcription factor, is the master regulator of JA signaling. Although MYC2 in maize has been identified, its function remains to be clarified. RESULTS: To understand the function and regulatory mechanism of MYC2 in maize, the joint analysis of DAP-seq and RNA-seq is conducted to identify the binding sites and target genes of ZmMYC2. A total of 3183 genes are detected both in DAP-seq and RNA-seq data, potentially as the directly regulating genes of ZmMYC2. These genes are involved in various biological processes including plant growth and stress response. Besides the classic cis-elements like the G-box and E-box that are bound by MYC2, some new motifs are also revealed to be recognized by ZmMYC2, such as nGCATGCAnn, AAAAAAAA, CACGTGCGTGCG. The binding sites of many ZmMYC2 regulating genes are identified by IGV-sRNA. CONCLUSIONS: All together, abundant target genes of ZmMYC2 are characterized with their binding sites, providing the basis to construct the regulatory network of ZmMYC2 and better understanding for JA signaling in maize.

ZmEREB92 interacts with ZmMYC2 to activate maize terpenoid phytoalexin biosynthesis upon Fusarium graminearum infection through jasmonic acid/ethylene signaling.

Maize (Zea mays) terpenoid phytoalexins (MTPs) induced by multiple fungi display extensive antimicrobial activities, yet how maize precisely regulates MTP accumulation upon pathogen infection remains elusive. In this study, pretreatment with jasmonic acid (JA)/ethylene (ET)-related inhibitors significantly reduced Fusarium graminearum-induced MTP accumulation and resulted in enhanced susceptibility to F. graminearum, indicating the involvement of JA/ET in MTP regulatory network. ZmEREB92 positively regulated MTP biosynthetic gene (MBG) expression by correlation analysis. Knockout of ZmEREB92 significantly compromised maize resistance to F. graminearum with delayed induction of MBGs and attenuated MTP accumulation. The activation of ZmEREB92 on MBGs is dependent on the interaction with ZmMYC2, which directly binds to MBG promoters. ZmJAZ14 interacts both with ZmEREB92 and with ZmMYC2 in a competitive manner to negatively regulate MBG expression. Altogether, our findings illustrate the regulatory mechanism for JA/ET-mediated MTP accumulation upon F. graminearum infection with the involvement of ZmEREB92, ZmMYC2, and ZmJAZ14, which provides new insights into maize disease responses.

Maize Transcription Factor ZmHsf28 Positively Regulates Plant Drought Tolerance.

Identification of central genes governing plant drought tolerance is fundamental to molecular breeding and crop improvement. Here, maize transcription factor ZmHsf28 is identified as a positive regulator of plant drought responses. ZmHsf28 exhibited inducible gene expression in response to drought and other abiotic stresses. Overexpression of ZmHsf28 diminished drought effects in Arabidopsis and maize. Gene silencing of ZmHsf28 via the technology of virus-induced gene silencing (VIGS) impaired maize drought tolerance. Overexpression of ZmHsf28 increased jasmonate (JA) and abscisic acid (ABA) production in transgenic maize and Arabidopsis by more than two times compared to wild-type plants under drought conditions, while it decreased reactive oxygen species (ROS) accumulation and elevated stomatal sensitivity significantly. Transcriptomic analysis revealed extensive gene regulation by ZmHsf28 with upregulation of JA and ABA biosynthesis genes, ROS scavenging genes, and other drought related genes. ABA treatment promoted ZmHsf28 regulation of downstream target genes. Specifically, electrophoretic mobility shift assays (EMSA) and yeast one-hybrid (Y1H) assay indicated that ZmHsf28 directly bound to the target gene promoters to regulate their gene expression. Taken together, our work provided new and solid evidence that ZmHsf28 improves drought tolerance both in the monocot maize and the dicot Arabidopsis through the implication of JA and ABA signaling and other signaling pathways, shedding light on molecular breeding for drought tolerance in maize and other crops.

Rice contains a biosynthetic gene cluster associated with production of the casbane-type diterpenoid phytoalexin ent-10-oxodepressin.

Diterpenoids play important roles in rice microbial disease resistance as phytoalexins, as well as acting in allelopathy and abiotic stress responses. Recently, the casbane-type phytoalexin ent-10-oxodepressin was identified in rice, but its biosynthesis has not yet been elucidated. Here ent-10-oxodepressin biosynthesis was investigated via co-expression analysis and biochemical characterisation, with use of the CRISPR/Cas9 technology for genetic analysis. The results identified a biosynthetic gene cluster (BGC) on rice chromosome 7 (c7BGC), containing the relevant ent-casbene synthase (OsECBS), and four cytochrome P450 (CYP) genes from the CYP71Z subfamily. Three of these CYPs were shown to act on ent-casbene, with CYP71Z2 able to produce a keto group at carbon-5 (C5), while the closely related paralogues CYP71Z21 and CYP71Z22 both readily produce a keto group at C10. Together these C5 and C10 oxidases can elaborate ent-casbene to ent-10-oxodepressin (5,10-diketo-ent-casbene). OsECBS knockout lines no longer produce casbane-type diterpenoids and exhibit impaired resistance to the rice fungal blast pathogen Magnaporthe oryzae. Elucidation of ent-10-oxodepressin biosynthesis and the associated c7BGC provides not only a potential target for molecular breeding, but also, gives the intriguing parallels to the independently assembled BGCs for casbene-derived diterpenoids in the Euphorbiaceae, further insight into plant BGC evolution, as discussed here.

Maize WRKY Transcription Factor ZmWRKY79 Positively Regulates Drought Tolerance through Elevating ABA Biosynthesis.

Drought stress causes heavy damages to crop growth and productivity under global climatic changes. Transcription factors have been extensively studied in many crops to play important roles in plant growth and defense. However, there is a scarcity of studies regarding WRKY transcription factors regulating drought responses in maize crops. Previously, ZmWRKY79 was identified as the regulator of maize phytoalexin biosynthesis with inducible expression under different elicitation. Here, we elucidated the function of ZmWRKY79 in drought stress through regulating ABA biosynthesis. The overexpression of ZmWRKY79 in Arabidopsis improved the survival rate under drought stress, which was accompanied by more lateral roots, lower stomatal aperture, and water loss. ROS scavenging was also boosted by ZmWRKY79 to result in less H2O2 and MDA accumulation and increased antioxidant enzyme activities. Further analysis detected more ABA production in ZmWRKY79 overexpression lines under drought stress, which was consistent with up-regulated ABA biosynthetic gene expression by RNA-seq analysis. ZmWRKY79 was observed to target ZmAAO3 genes in maize protoplast through acting on the specific W-boxes of the corresponding gene promoters. Virus-induced gene silencing of ZmWRKY79 in maize resulted in compromised drought tolerance with more H2O2 accumulation and weaker root system architecture. Together, this study substantiates the role of ZmWRKY79 in the drought-tolerance mechanism through regulating ABA biosynthesis, suggesting its broad functions not only as the regulator in phytoalexin biosynthesis against pathogen infection but also playing the positive role in abiotic stress response, which provides a WRKY candidate gene to improve drought tolerance for maize and other crop plants.

Probing Enzymatic Structure and Function in the Dihydroxylating Sesquiterpene Synthase ZmEDS.

Terpene synthases (TPSs) play a vital role in forming the complex hydrocarbon backbones that underlie terpenoid diversity. Notably, some TPSs can add water prior to terminating the catalyzed reaction, leading to hydroxyl groups, which are critical for biological activity. A particularly intriguing example of this is the maize (Zea mays) sesquiterpene TPS whose major product is eudesmanediol, ZmEDS. This production of dual hydroxyl groups is presumably enabled by protonation of the singly hydroxylated transient stable intermediate hedycaryol. To probe the enzymatic structure-function relationships underlying this unusual reaction, protein modeling and docking were used to direct mutagenesis of ZmEDS. Previously, an F303A mutant was shown to produce only hedycaryol, suggesting a role in protonation. Here this is shown to be dependent on the steric bulk positioning of hedycaryol, including a supporting role played by the nearby F299, rather than π-cation interaction. Among the additional residues investigated here, G411 at the conserved kink in helix G is of particular interest, as substitution of this leads to predominant production of the distinct (-)-valerianol, while substitution for the aliphatic I279 and V306 can lead to significant production of the alternative eudesmane-type diols 2,3-epi-cryptomeridiol and 3-epi-cryptomeridol, respectively. Altogether, nine residues that are important for this unusual reaction were investigated here, with the results not only emphasizing the importance of reactant positioning suggested by the stereospecificity observed among the various product types but also highlighting the potential role of the Mg2+-diphosphate complex as the general acid for the protonation-initiated (bi)cyclization of hedycaryol.

ZmMYC2 exhibits diverse functions and enhances JA signaling in transgenic Arabidopsis.

KEY MESSAGE: ZmMYC2 was identified as the key regulator of JA signaling in maize and exhibited diverse functions through binding to many gene promoters as well as enhanced JA signaling in transgenic Arabidopsis. The plant hormone jasmonate (JA) extensively coordinates plant growth, development and defensive responses. MYC2 is the master regulator of JA signaling and has been widely studied in many plant species. However, little is known about this transcription factor in maize. Here, we identified one maize transcription factor with amino acid identity of 47% to the well-studied Arabidopsis AtMYC2, named as ZmMYC2. Gene expression analysis demonstrated inducible expression patterns of ZmMYC2 in response to multiple plant hormone treatments, as well as biotic and abiotic stresses. The yeast two-hybrid assay indicated physical interaction among ZmMYC2 and JA signal repressors ZmJAZ14, ZmJAZ17, AtJAZ1 and AtJAZ9. ZmMYC2 overexpression in Arabidopsis myc2myc3myc4 restored the sensitivity to JA treatment, resulting in shorter root growth and inducible anthocyanin accumulation. Furthermore, overexpression of ZmMYC2 in Arabidopsis elevated resistance to Botrytis cinerea. Further ChIP-Seq analysis revealed diverse regulatory roles of ZmMYC2 in maize, especially in the signaling crosstalk between JA and auxin. Hence, we identified ZmMYC2 and characterized its roles in regulating JA-mediated growth, development and defense responses.

[Cloning and functional characterization of farnesyl diphosphate synthase in Senecio scandens].

As a secondary metabolite, sesquiterpenes are not only have important functions in plant defense and signaling, but also play potential roles in basic materials for pharmaceuticals, cosmetic and flavor. As a traditional Chinese herbal medicine, Senecio scandens exhibits effects of anti-inflammatory and immunosuppressive, as well as invigorating the blood and removing extravasated blood. Over 600 sesquiterpenes with diverse structures were isolated from S. scandens and related species in the same genus. To characterize sesquiterpenes synthesis, two FPS genes(SsFPS1 and SsFPS2) were identified in S. scandens through transcriptomic analysis. Bioinformatic analysis showed that both SsFPSs have conserved motifs for FPS function. Both SsFPSs exhibited constitutive gene expression in S. scandens tissues and SsFPS2 accumulated higher transcript in leaves and roots than SsFPS1. Meanwhile consistent with constitutive sesquiterpene accumulation in S.scandens tissues, most of these sesquiterpenes were detected in leaves and roots more than stems and flowers. Recombinant expression through Escherichia coli metabolic engineering, SsFPS1 or SsFPS2 was co-transformed with ZmTPS11(maize ß-macrocarpene synthase) into BL21 competent cells. The results showed that the content of ß-macrocarpene was increased by co-transformation with SsFPSs. It is demonstrated that SsFPS1 and SsFPS2 catalyzed E,E-FPP formation and provided FPP precursor for downstream sesquiterpene synthases. Characterization of SsFPSs provided the foundation for the exploration of biosynthesis of sesquiterpenoid with diverse structures and potential pharmaceutical values in S.scandens, and provide an important theoretical basis for the development of S. scandens abundant resources.

ZmWRKY79 positively regulates maize phytoalexin biosynthetic gene expression and is involved in stress response.

Maize (Zea mays) accumulates maize terpenoid phytoalexins (MTPs), kauralexins and zealexins in response to various elicitations. Although the key biosynthetic genes for these have been characterized, the regulatory mechanism remains unclear. Through co-correlation analysis, a transcription factor (TF), ZmWRKY79, was identified as highly correlated with expression of MTP biosynthetic genes. Gene expression analysis indicated that ZmWRKY79 was induced by Fusarium graminearum infection, phytohormone treatment, and multiple stresses. Overexpression of ZmWRKY79 in maize protoplasts increased expression of genes involved in MTP biosynthesis, jasmonic acid and ethylene pathways, and scavenging of reactive oxygen species (ROS). Subsequent transient RNAi in maize protoplast compromised the induction of MTP biosynthetic genes by jasmonic acid and ethylene combined treatment. Such regulation was further demonstrated to be dependent on a W-box or WLE cis-element. Transient overexpression of ZmWRKY79 in tobacco conferred resistance against Rhizoctonia solani infection through reducing ROS production. Our results indicate that MTP biosynthesis is regulated by the common transcription factor ZmWRKY79, which plays a broad role as a potential master regulator in stress response through involvement in phytohormone metabolism or signaling and ROS scavenging.

A Tandem Array of ent-Kaurene Synthases in Maize with Roles in Gibberellin and More Specialized Metabolism.

While most commonly associated with its role in gibberellin phytohormone biosynthesis, ent-kaurene also serves as an intermediate in more specialized diterpenoid metabolism, as exemplified by the more than 800 known derived natural products. Among these are the maize kauralexins. However, no ent-kaurene synthases (KSs) have been identified from maize. The maize gibberellin-deficient dwarf-5 (d5) mutant has been associated with a loss of KS activity. The relevant genetic lesion has been previously mapped, and was found here to correlate with the location of the KS-like gene ZmKSL3. Intriguingly, this forms part of a tandem array with two other terpene synthases (TPSs). Although one of these, ZmTPS1, has been previously reported to encode a sesquiterpene synthase, and both ZmTPS1 and that encoded by the third gene, ZmKSL5, have lost the N-terminal γ-domain prototypically associated with KS(L)s, all three genes fall within the KS(L) or TPS-e subfamily. Here it is reported that all three genes encode enzymes that are targeted to the plastid in planta, where diterpenoid biosynthesis is initiated, and which all readily catalyze the production of ent-kaurene. Consistent with the closer phylogenetic relationship of ZmKSL3 with previously identified KSs from cereals, only transcription of this gene is affected in d5 plants. On the other hand, the expression of all three of these genes is inducible, suggesting a role in more specialized metabolism, such as that of the kauralexins. Thus, these results clarify not only gibberellin phytohormone, but also diterpenoid phytoalexin biosynthesis in this important cereal crop plant.

MicroRNA155 in the growth and invasion of salivary adenoid cystic carcinoma.

BACKGROUND: The carcinogenesis mechanism of adenoid cystic carcinoma (ACC) of the salivary gland is poorly understood. MicroRNA155 (miRNA155) has been involved in the carcinogenesis of many malignant tumors. The present study aims to examine the role of miRNA155 in tumor growth and invasion of ACC. METHODS: MiRNA155 expression was determined in ACC specimens along with normal salivary glands by quantitative PCR. Using ACC-2 cells as a model for ACC, cell proliferation was examined by MTT assay after knocking down miRNA155 expression, and cell cycle analysis was performed. Invasive capacity of ACC-2 cells was examined by a Transwell culture assay. The effect of miRNA155 on tumor growth was also examined in vivo using mouse models. The effect of miRNA155 on epidermal growth factor receptor (EGFR)/NF-κB was studied by quantitative PCR and electrophoretic mobility shift assay. RESULTS: MiRNA155 was over-expressed in ACC. Proliferation of ACC-2 cells was markedly inhibited by knocking down miRNA155, resulting from a blockade of cell cycle in the G1 phase. Inhibition of miRNA155 significantly suppressed the invasive capacity of ACC-2 cells. In vivo growth of ACC-2 cell-derived tumors was significantly slower by inhibition of miRNA155. Inhibition of miRNA155 also resulted in decreased expression of EGFR and RelA (NF-κB). CONCLUSION: The results suggest that miRNA155 facilitates cell cycle progression and promotes invasion in ACC and that the EGFR/NF-κB pathway might participate in mediating the effects of miRNA155. This study has provided insights into the carcinogenic mechanisms of ACC and identified new targets for intervention of salivary ACC.

Maize transcription factor ZmNAC2 enhances osmotic stress tolerance in transgenic Arabidopsis.

Osmotic stress seriously limits crop yield and quality. Among plant-specific transcription factors families, the NAC family of transcription factors is extensively involved in various growth, development and stress responses. Here we identified a maize NAC family transcription factor ZmNAC2 with inducible gene expression in response to osmotic stress. The subcellular localization showed that it was localized in the nucleus and overexpression of ZmNAC2 in Arabidopsis significantly promoted seed germination and elevated cotyledon greening under osmotic stress. ZmNAC2 also enhanced stomatal closure and decreased water loss in transgenic Arabidopsis. Overexpression of ZmNAC2 activated ROS scavenging and the transgenic lines accumulated less MDA and developed more lateral roots with drought or mannitol treatment. Further RNA-seq and qRT-PCR analysis showed that ZmNAC2 up-regulated a number of genes related to osmotic stress resistance, as well as plant hormone signaling genes. All together, ZmNAC2 enhances osmotic stress tolerance by regulating multiple physiological processes and molecular mechanisms, and exhibits potential as the target gene in crop breeding to increase osmotic stress resistance.

Maize transcription factor ZmEREB20 enhanced salt tolerance in transgenic Arabidopsis.

Soil salinity severely limits agricultural crop production worldwide. As one of the biggest plant specific transcription factor families, AP2/ERF members have been extensively studied to regulate plant growth, development and stress responses. However, the role of AP2/ERF family in maize salt tolerance remains largely unknown. In this study, we identified a maize AP2-ERF family member ZmEREB20 as a positive salinity responsive gene. Overexpression of ZmEREB20in Arabidopsis enhanced ABA sensitivity and resulted in delayed seed germination under salt stress through regulating ABA and GA related genes. ZmEREB20 overexpression lines also showed higher survival rates with elevated ROS scavenging toward high salinity. Furthermore, root hair growth inhibition by salt stress was markedly rescued in ZmEREB20 overexpression lines. Auxin transport inhibitor TIBA drastically enhanced root hair growth in ZmEREB20 overexpression Arabidopsis under salt stress, together with the increased expression of auxin-related genes, ion transporter genes and root hair growth genes by RNA-seq analysis. ZmEREB20 positively regulated salt tolerance through the molecular mechanism associated with hormone signaling, ROS scavenging and root hair plasticity, proving the potential target for crop breeding to improve salt resistance.

Transcriptional Factors Regulate Plant Stress Responses through Mediating Secondary Metabolism.

Plants are adapted to sense numerous stress stimuli and mount efficient defense responses by directing intricate signaling pathways. They respond to undesirable circumstances to produce stress-inducible phytochemicals that play indispensable roles in plant immunity. Extensive studies have been made to elucidate the underpinnings of defensive molecular mechanisms in various plant species. Transcriptional factors (TFs) are involved in plant defense regulations through acting as mediators by perceiving stress signals and directing downstream defense gene expression. The cross interactions of TFs and stress signaling crosstalk are decisive in determining accumulation of defense metabolites. Here, we collected the major TFs that are efficient in stress responses through regulating secondary metabolism for the direct cessation of stress factors. We focused on six major TF families including AP2/ERF, WRKY, bHLH, bZIP, MYB, and NAC. This review is the compilation of studies where researches were conducted to explore the roles of TFs in stress responses and the contribution of secondary metabolites in combating stress influences. Modulation of these TFs at transcriptional and post-transcriptional levels can facilitate molecular breeding and genetic improvement of crop plants regarding stress sensitivity and response through production of defensive compounds.

A Wheat ß-Patchoulene Synthase Confers Resistance Against Herbivory in Transgenic Arabidopsis.

Terpenoids play important roles in plant defense. Although some terpene synthases have been characterized, terpenoids and their biosynthesis in wheat (Triticumaestivum L.) still remain largely unknown. Here, we describe the identification of a terpene synthase gene in wheat. It encodes a sesquiterpene synthase that catalyzes ß-patchoulene formation with E,E-farnesyl diphosphate (FPP) as the substrate, thus named as TaPS. TaPS exhibits inducible expression in wheat in response to various elicitations. Particularly, alamethicin treatment strongly induces TaPS gene expression and ß-patchoulene accumulation in wheat. Overexpression of TaPS in Arabidopsis successfully produces ß-patchoulene, verifying the biochemical function of TaPS in planta. Furthermore, these transgenic Arabidopsis plants exhibit resistance against herbivory by repelling beet armyworm larvae feeding, thereby indicating anti-herbivory activity of ß-patchoulene. The catalytic mechanism of TaPS is also explored by homology modeling and site-directed mutagenesis. Two key amino acids are identified to act in protonation and stability of intermediates and product formation. Taken together, one wheat sesquiterpene synthase is identified as ß-patchoulene synthase. TaPS exhibits inducible gene expression and the sesquiterpene ß-patchoulene is involved in repelling insect infestation.