Probing enzymatic structure-function in the di-hydroxylating 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, a F303A mutant was shown to produce only hedycaryol, suggesting a role in protonation. Here this is shown to be dependent on 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 for 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 important for this unusual reaction were investigated here, with the results not only emphasizing the importance of reactant positioning suggested by the stereospecificity observed between 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

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 (GA) 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 GA-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 sub-family. 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

Radiation Induced Surface Modification of Nanoparticles and Their Dispersion in the Polymer Matrix

Polymer grafted inorganic nanoparticles attract significant attention, but pose challenges because of the complexity. In this work, a facile strategy to the graft polymer onto the surface of nanoparticles have been introduced. The vinyl functionalized SiO2 nanoparticles (NPs) were first prepared by the surface modification of the unmodified SiO2 using γ-methacryloxy propyl-trimethoxylsilane. The NPs were then mixed with polyvinylidene fluoride (PVDF), which was followed by the Co-60 Gamma radiation at room temperature. PVDF molecular chains were chemically grafted onto the surface of SiO2 nanoparticles by the linking of the double bond on the NPs. The graft ratio of PVDF on SiO2 NPs surface can be precisely controlled by adjusting the absorbed dose and reactant feed ratio (maximum graft ratio was 31.3 wt%). The strategy is simple and it should be applied to the surface modification of many other nanoparticles. The prepared PVDF-grafted SiO2 NPs were then dispersed in the PVDF matrix to make the nanocomposites. It was found that the modified NPs can be precisely dispersed into the PVDF matrix, as compared with pristine silica. The filling content of modifications SiO2 NPs on the PVDF nanocomposites is almost doubled than the pristine SiO2 counterpart. Accordingly, the mechanical property of the nanocomposites is significantly improved

Transcription factor ZmNAC59 regulates plant salt resistance in Zea mays L.

[ Objective ] The NAC transcription factor family is a class of transcription factors that is widely studied in plants. It plays an important role in regulating plant growth and development and responding to abiotic stress. Maize is one of the three major food crops , which faces various adverse stresses in growth and development. Salt stress is one of the main environmental factors limiting crop growth and produc- tion. Therefore , it is of great significance to identify maize salt resistance genes and analyze their salt re- sistance mechanism for breeding stress-resistant maize varieties. [ Methods ] This study cloned the maize transcription factor ZmNAC59 and analyzed its conserved domain and phylogenetic relationship using bioinformatics methods. The expression pattern of the gene in leaves under NaCl and MeJA treatments was analyzed using the quantitative real-time PCR method. The stable transgenic system was used to het- erologously express the gene in Arabidopsis thaliana for phenotype observation. At the same time , virus- induced silencing technology was used to silence the gene in maize , followed by phenotype observation un- der salt stress and enzyme activity detection. [ Results ] The qRT-PCR results showed that ZmNAC59 was up-regulated by NaCl and MeJA treatments. After virus-induced silencing of ZmNAC59 , the silenced strains were more sensitive to salt stress and accumulated more ROS. After over expression of ZmNAC59 in A . thaliana , the over expression lines had higher survival rate , less accumulation of ROS , and lower Na + / K + ratios under salt stress , indicating that ZmNAC59 , as a positive regulator of salt stress , can im- prove plant salt resistance by regulating ion homeostasis. qRT-PCR results showed that Na + and K + transport related genes were significantly up-regulated in A . thaliana overexpression lines. After transient expression of ZmNAC59 in maize protoplasts , the expression of ZmSOS1 , ZmNHX1 , and ZmNHX7 were significantly up-regulated after salt treatment. The Dual-LUC experiment showed that ZmSOS1 was the target gene of ZmNAC59. [ Conclusion ] This study finds that ZmNAC59 activates ZmSOS1 promoter and regulates its expression to promote ion transport under salt stress , thereby improving plant salt resist- ance. This provides a basis for the screening of maize stress resistant genes and variety cultivation

Maize Transcription Factor <i>ZmHsf28</i> 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

A Wheat β-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 (Triticum aestivum L.) still remain largely unknown. Here, we describe the identification of a terpene synthase gene in wheat. It encodes a sesquiterpene synthase that catalyzes &beta;-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 &beta;-patchoulene accumulation in wheat. Overexpression of TaPS in Arabidopsis successfully produces &beta;-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 &beta;-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 &beta;-patchoulene synthase. TaPS exhibits inducible gene expression and the sesquiterpene &beta;-patchoulene is involved in repelling insect infestation

Probing enzymatic structure-function in the di-hydroxylating 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, a F303A mutant was shown to produce only hedycaryol, suggesting a role in protonation. Here this is shown to be dependent on 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 for 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 important for this unusual reaction were investigated here, with the results not only emphasizing the importance of reactant positioning suggested by the stereospecificity observed between 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.This is a manuscript of an article published as Liang, Jin, Liping Wang, Jiang Liu, Qinqin Shen, Jingye Fu, Reuben J. Peters, and Qiang Wang. "Probing enzymatic structure-function in the di-hydroxylating sesquiterpene synthase ZmEDS." Biochemistry (2020). doi: 10.1021/acs.biochem.0c00395. Posted with permission.</p

Formation of Interfacial Janus Nanomicelles by Reactive Blending and Their Compatibilization Effects on Immiscible Polymer Blends

Micellization of in situ formed graft copolymers during reactive blending is commonly observed. Numerous studies have been carried out to minimize the formation of micelles and enhance emulsification efficiency. Herein, we investigated the formation of interfacial Janus nanomicelles (JNMs) and their compatibilization effects on immiscible polymer blends when reactive graft copolymers (RGCs) are used as compatibilizers. Poly­(styrene<i>-co-</i>glycidyl methacrylate)<i>-graft-</i>poly­(methyl methacrylate) RGCs were synthesized and used as compatibilizers for immiscible poly­(l-lactide) (PLLA)/poly­(vinylidene fluoride) (PVDF) blends. Numerous nanomicelles were formed in situ during melt blending by grafting of PLLA onto the RGCs. The formation and location of JNMs depended not only on the molecular architecture of the RGCs but also on the melt processing sequence and molecular weight of the components. Interfacial JNMs can effectively improve the miscibility of polymer blends, thereby enhancing the performance of immiscible polymer blends