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The mechanism and stereoselectivity of an asymmetric cyclopropanation reaction between 3-alkenyl-oxindole and sulfoxonium ylide catalyzed by a chiral N,N'-dioxide-Mg(II) complex were explored using the B3LYP-D3(BJ) functional and the def2-TZVP basis set. The noncatalytic reaction occurred via a stepwise mechanism, with activation barriers of 21.6-23.5 kcal mol-1. The C2-Cα bond formed followed by the carbanion SN2 substitution, constructing a three-membered ring in spiro-cyclopropyl oxindoles, accompanied by the release of dimethylsulfoxide. The electron-withdrawing N-protecting t-butyloxy carbonyl (Boc) and acetyl (Ac) groups in isatin enhanced the local electrophilicity of the C2 atom and the repulsion between the two COPh groups in the reactants, contributing to high reactivity as well as good diastereoselectivity results. The N-Boc-3-phenacylideneoxindole coordinated to the chiral ligand (L-PiPr2) in a bidentate fashion, forming a hexacoordinate-Mg(II) complex as the reactive species. The origin of enantioselectivity was from the shielding effect of 2,6-diisopropylphenyl groups in the ligand toward the si-face of oxindole. The repulsion between the SO(CH3)2 and COPh groups in 3-alkenyl-oxindole and the neighboring ortho-iPr group in the ligand directed the re-face of ylide to attack the re-face of oxindole preferably, contributing to the high diastereoselectivity of the product. A metal-ion-ligand matching relationship was important for a good asymmetric induction effect of the chiral N,N'-dioxide-metal catalyst. A large chiral cavity in the Zn(II) catalyst weakened the shielding effect of 2,6-diisopropylphenyl groups in the ligand toward the prochiral face of oxindole, leading to inferior enantioselectivity observed in the experiment.

The reaction mechanism and enantioselectivity of asymmetric Michael addition reaction between alkynone (R1) with α-angelica lactone (R2) catalyzed by chiral N, N'-dioxide-Sc(III) complex were investigated at the M06/6-31G(d,p) (acetonitrile, SMD) level. The α-angelica lactone substrate could isomerize to the active enolized form in the presence of Sc(OTf)3 reagent, assisted by the counter trifluoromethanesulfonate anion OTf-. The alkynone substrate and enolized angelica lactone (or its anion) coordinated to Sc(III) center of N,N'-dioxide-Sc(III) complex catalyst simultaneously, forming a high active hexacoordinate-Sc(III) complex. The catalytic reaction occurred via a two-step mechanism, in which C2 Cγ bond formation was predicted to be the chirality-controlling step as well as the rate-determining step (RDS), affording predominant S-enantiomer. The counterion OTf- facilitated C H construction as a proton-shuttle, producing mainly E-configuration product observed in experiment. The steric repulsion from the ortho-substituent of amide moiety as well as the chiral backbone of N, N'-dioxide-Sc(III) catalyst played the key role for chiral induction in the asymmetric reaction. The less destabilizing Pauli repulsion and more stabilizing attractive interaction, especially the orbital interaction, along the si-face attack pathway enhanced the enantiodifference of the two competing pathways for high enantioselectivity. The energy barriers for E/Z isomerization of S or R-enantiomer assisted by HOTf was as high as 34.6–35.0 kcal mol−1, indicating that the product with Z-conformation was difficult to be obtained. These results were in good agreement with experimental observations.

We reported a mechanistic study on asymmetric O–H insertion reaction of α-diazoester with carboxylic acid using Rh2(OAc)4/chiral guanidine-amide as the cocatalyst by density functional theory [B3LY...

Terpene trilactones (TTLs) are the main secondary metabolites of Ginkgo biloba. As one of the rate-limiting enzymes in the mevalonic acid (MVA) pathway of TTL biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the 3-hydroxy-3-methylglutaryl coenzyme A to form MVA. In this study, two cDNA sequences of HMGR genes, namely, GbHMGR2 and GbHMGR3, were cloned from G. biloba. The protein sequences of GbHMGR2 and GbHMGR3, which contain several functional domains, were analyzed. Regulatory elements related to light, hormone, and stress response were detected in the promoter regions of GbHMGR2 and GbHMGR3. The catalytic activity of these genes was verified by a functional complement experiment in yeast. Quantitative real-time PCR (qRT-PCR) showed the distinct expression patterns of the two genes in different organs. The TTL contents in the organs were detected by high-performance liquid chromatography– evaporative light scattering detector. GbHMGR2 and GbHMGR3 were responded to cold, dark, methyl jasmonate (MJ), abscisic acid (ABA), salicylic acid (SA), and ethephon (Eth) treatments. The TTL contents were also regulated by cold, dark, MJ, ABA, SA, and Eth treatment. In conclusion, GbHMGR2 and GbHMGR3 may participate in the MVA pathway of TTL biosynthesis.

The reaction mechanism and enantioselectivity of the asymmetric [2 + 2] cycloaddition between an alkynone (R1) and a cyclic enol silyl ether (R2) were studied theoretically by the DFT method at the...

Increasing cadmium (Cd) pollution severely affects plant growth and development, posing risks to human health via food chains. The Cd toxicity could be mitigated by improving Fe nutrient in plants. IMA1 and IMA3, two novel small peptides functionally epistatic to the key transcription factor bHLH39 but independent of bHLH104, were recently identified as the newest additions to the Fe regulatory cascade, but their roles in Cd uptake and toxicity remain not addressed. Here, the functions of two IMAs and two transcription factors related to Cd tolerance were verified. Overexpression of either bHLH39 or bHLH104 in Arabidopsis showed weak roles in Cd tolerance, but overexpression of IMAs, which activates the Fe-deficient response, significantly enhanced Cd tolerance, showing greater root elongation, biomass and chlorophyll contents. The Cd contents did not show significant difference among the overexpression lines. Further investigations revealed that the tolerance of transgenic plants to Cd mainly depended on higher Fe accumulation, which decreased the MDA contents and enhanced root elongation under Cd exposure, finally contributing to attenuating Cd toxicity. Taken together, the results suggest that increasing Fe accumulation is promising for improving plant tolerance to Cd toxicity and that IMAs are potential candidates for solving Cd toxicity problem.

Farnesyl diphosphate synthase (FPPS), an isopentenyl transferase, catalyzes the condensation reaction of five carbon isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to form fifteen carbon farnesyl pyrophosphate (FPP), which is the key precursor for sesquiterpene biosynthesis. In this study, a FPPS gene ( CnFPPS ) was cloned from Chamaemelum nobile . The full-length cDNA of CnFPPS is 1239 bp and contains an open reading frame (ORF) of 1029 bp encoding 342 amino acids. The theoretical molecular weight and pI of the CnFPPS protein are 39.38 kDa and 5.59, respectively. Multiple alignment analysis showed the protein sequence of CnFPPS had a high homology with FPPS proteins from other plants. The deduced amino acid of CnFPPS contained five conservative domains such as substrate binding pocket, substrate-Mg 2+ binding site, catalytic site, aspartate-rich region 1 and 2, suggesting CnFPPS is one member of FPPS family in C. nobile . Phylogenetic analysis based on the amino acid sequences of FPPSs showed that CnFPPS was closely related to the FPPS of Matricaria chamomilla . The result of qRT-PCR revealed that CnFPPS gene was constitutively expressed in different tissues of C. nobile, with the highest expression in the root. These findings improve the understanding of the synthesis and regulation of the terpenoid compounds at the molecular level and lay a foundation for studying the regulatory functions of CnFPPS in terpenoid biosynthetic pathway in C. nobile .

The mechanism and stereoselectivity of the phenylethylketene with isatin catalyzed by a chiral N,N′-dioxide-Sc(III) complex were studied at the B3LYP-D3(BJ)/6–311G(d,p)//B3LYP-D3(BJ)/def2-SVP(SMD,CH2Cl2) theoretical level. The calculations indicated that the reaction occurred in a one-step but asynchronous manner. The four-membered product was formed by nucleophilic attack of the electron-rich O(2) atom in the N-benzylisatin toward the electron-deficient C(β) atom in ketene in the initial stage of reaction, which was responsible to the high regioselectivity of the cycloaddition. The π-π stacking interaction between two deformed reactants stabilized the corresponding transition state, affording the enantiomer with S,R-configuration as major product in background reactions. The N-benzylisatin coordinated to the Sc3+ in bidentate manner, forming a hexacoordinated scandium-complex reactive species in the catalytic process. Different from the non-catalytic reaction, the Cα-C1 bond was formed prior to the construction of Cβ-O2 bond. The ortho-iPr in the aniline of chiral N,N′-dioxide ligand shielded re-face of isatin, and induced the ketene to attack the isatin from less hindered si-face, leading to good enantioselectivity of product. Suffering from the steric repulsion from benzyl group in N-benzylisatin, the phenylethylketene substrate preferred to approach the si-face of N-benzylisatin with its re-face, affording predominant product with S,S-configuration. Variation of substituent in reactants and ligand structure would modify the shielding degree of ortho-iPr toward prochiral face of isatin, adjusting the stereoselectivity.

The MADS-box transcription factors play diverse roles in plant development, but little is known about the role of MADS-box genes in Ginkgo biloba. Here a MADS-box transcription factor gene, designated as GbMADS9, was cloned and functionally characterized from G. biloba. The open reading frame of GbMADS9 comprised 684 bp encoding a polypeptide of 227 amino acids. Phylogenetic analysis showed that GbMADS9 belonged to the GGM13 clade of Bsister-class MADS-box proteins. Real-time PCR results revealed that GbMADS9 was preferentially expressed in strobili and ovules. In addition, its expression was up-regulated with the increase in the size of ginkgo pulp, indicating that GbMADS9 had a positive function in ginkgo ovule development. The expression of GbMADS9 in ginkgo was up-regulated in response to salinity, drought, and cold stress, as well as to phytohormones gibberellic acid and abscisic acid. GbMADS9 transgenic Arabidopsis plants presented early flowering phenotypes compared with wild-type plants. The expression of the FLOWERING LOCUS T (FT), APETALA1 (AP1), LEAFY (LFY), and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) genes involved in flowering was up-regulated, whereas the expression of AGAMOUS-LIKE24 (AGL24) and SHORT VEGETATIVE PHASE (SVP) was down-regulated in the GbMADS9 transgenic lines. The GbMADS9 transgenic plants exhibited better growth than wild-type plants under high osmotic stress. Enhanced tolerance of transgenic Arabidopsis plants to osmotic stress was confirmed by changes in the chlorophyll, proline, and malondialdehyde contents. Moreover, increased tolerance to osmotic stress in GbMADS9 transgenic lines was associated with improved antioxidant enzymatic activities. These findings suggested that GbMADS9 might be a promising gene to manipulate flowering time and improve abiotic stress tolerance in G. biloba.

The reaction mechanism and origin of asymmetric induction in inverse electron demand Diels-Alder (IEDDA) reaction of ortho-quinone methide (o-QM) and fulvene mediated by chiral N,N'-dioxide–Sc(III) catalyst were rationalized using B3LYP-D3(BJ) functional with def2-TZVP basis set. The uncatalyzed IEDDA reaction was concerted but highly asynchronous with activation barriers of 29.8∼31.8 kcal mol−1. Good linear relationship between the Hammett substituent constant (σP) of o-QM and the activation barrier (ΔG≠) of DA reaction was discovered. The secondary orbital interaction (SOI) between the conjugated diene of o-QM and fulvene moiety stabilized the endo-transition state, contributing to high endo-selectivity. The catalytic asymmetric IEDDA reaction occurred via a stepwise mechanism, including the construction of Cβ−C4 bond, followed by the formation of Cα−O1 bond. The bulky substituents (i.e., adamantyl or triphenylmethyl) in amide moiety of ligand furnished sufficient steric shielding for re-face of diene, inducing the attack of fulvene from si-face in endo-pathway. The substituent at exocyclic methylene of the unsymmetrical fulvene was crucial for the adjustment of E/Z selectivity. The steric repulsion between cyclohexyl group in fulvene and aromatic ring in o-QM raised the destabilizing strain energy (ΔEstrain) at the transition state in Z-configuration, contributing to the predominant E-product.

Wheat (Triticum aestivum L.) is one of the major crops worldwide and its production is inevitably subjected to various biotic/abiotic stresses during the life cycle. Drought, salinity and flooding are among the most severe abiotic stresses restricting wheat yields and could occur at very early stages such as seed germination. How wheat seed germination responds to these different stresses remains incomplete. To fill the information gap, a label-free proteomic analysis was applied to decipher the proteomic profiling of the germinating wheat seeds subjected to PEG, NaCl and submergence treatments. In total, 4295 proteins were detected, of which 465, 397 and 732 showed significant alterations in abundance under those stresses when compared with control. A common denominator found in the response observed to all three stresses are changes related to small molecule metabolic processes, and particularly in pathways associated with phenylpropanoid biosynthesis and fatty acid degradation. It was also noticeable that pathways like cysteine and methionine metabolism in the PEG or submergence treatment and starch and sucrose metabolism in the submergence treatment are specifically pronounced. Functional analysis of putative proteins participating in these pathways revealed distinct responsive patterns across different stresses. SIGNIFICANCE: Wheat (Triticum aestivum L.) is one of the most important staple crops in the world, but its growth and productivity are frequently restrained by stresses such as drought, salinity and flooding. To date, many resources have been documented to investigate how wheat responds and adapts to these individual stresses during plant development and yield formation, but little attention was paid to the understandings of the internal link between different conditions, especially during the germination process, a critical stage that determines the optimal growth of wheat. In this study, we carried out the proteome profiling of the germinating seeds of a common wheat cultivar, Chinese Spring, subjected to PEG, NaCl and submergence stresses. We found that the phenylpropanoid biosynthesis and fatty acid degradation pathways were enriched as the ubiquitous stress responses, while some pathways were stress-specific, for instance, starch and sucrose metabolism against submergence. The changes in some of the altered processes were further validated by physiological and molecular approaches. Our results suggest that the overall pathway profiles concerned with the three stresses were similar, but the specific procedures and components in each process varied greatly. The altered proteins and processes can be taken as effective candidates in future breeding and agronomic modification researches.

Ginkgo biloba, an industrial plant of high medicinal value, is resistant to pest, disease infections, and environmental stresses. 3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) is a key mevalonic acid pathway enzyme that plays crucial roles in plant defense mechanism. In the present study, a 1407-bp HMGS gene, GbHMGS2, encoding 468 amino acid residues was cloned from G. biloba. A promoter sequence (1479-bp long) of the gene was screened from G. biloba genome data. The promoter contained the putative light-responsive (Box 4, G-Box, GT1-motif, I-box, and Sp1), cold-responsive (LTR), hormone-responsive (auxin and methyl jasmonate), and defense-related (W-box and MBS) regulatory elements. Putative GbHMGS2 protein shared high similarities to other plant HMGSs containing the conserved motif and all conserved active sites possessed by the plant HMGS protein family. Functional complementation of GbHMGS2 in an hmgs-deficient Saccharomyces cerevisiae strain confirmed the catalytic activity of GbHMGS2 protein. GbHMGS2 was preferentially expressed in the roots of G. biloba among the plant organs. The GbHMGS2 transcription was upregulated in response to cold, dark, methyl jasmonate, salicylic acid, and abscisic acid treatments, in agreement with the regulatory elements predicted in promoter region. The present work on GbHMGS2 could help ensuing research on its function, especially in the signal transduction pathways in G. biloba.

Plant cell walls primarily comprise lignin, which performs functions of mechanical support, water transport, and stress responses. Lignin biosynthesis pathway proceeds through metabolic grid featuring complexity and diversity in enzymatic reaction. Cinnamate-4-hydroxylase (C4H, EC 1.14.13.11) is the gene encoding enzyme that catalyzes the second step of phenylpropanoid pathway responsible for biosynthesis of lignin. A full-length cDNA of C4H (designated as GbC4H), which spanned 1816-bp with a 1518-bp open reading frame encoding a 505-amino-acid protein, was cloned from Ginkgo biloba. A GbC4H genomic DNA fragment, spanning 3249-bp, was cloned and found to contain two exons and one intron. GbC4H protein showed high similarities with other plant C4Hs to include conserved domains of cytochrome P450 family. GT-1, W-box, and Myb/Myc recognition sites involved in stress response were detected in a 1265-bp upstream promoter region of GbC4H. Phylogenetic analysis suggested the common evolutionary ancestor shared by plant C4Hs including the gymnosperm enzyme. pET-28a-GbC4H plasmid was constructed and expressed in Escherichia coli strain BL21. Enzymatic assay revealed that recombinant GbC4H protein catalyzes conversion of trans-cinnamic acid to p-coumaric acid. Expression analyses in different organs showed high expression of GbC4H in stems and roots, whereas low expressions was found in fruits, carpopodium, and petioles. Further analysis indicated linear correlation of lignin contents with transcript levels of GbC4H among different tissues. GbC4H transcription was increased by treatments with UV-B, cold, salicylic acid, and abscisic acid, indicating the possible role of GbC4H in response to stresses and hormonal signal. Understanding of GbC4H function could benefit molecular breeding and reinforcement of defense mechanisms in Ginkgo.

In this study, seven watermelon (Citrullus lanatus) cultivars were tested in solution culture experiment with limiting and ample phosphorus (P) supply to evaluate P uptake and utilization of watermelon under low P stress. Different genotypes showed considerable diversity in terms of biomass accumulation, P uptake, P utilization, root morphological parameters and photosynthetic parameters under low P stress. At low P supply, genotype XN8 and ZCHY were clearly superior to other genotypes in terms of total dry matter yield. The genotype ZJ has the highest P uptake ability, while the genotype XN8 has the highest P efficiency ratio and P utilization efficiency among the seven genotypes under low P stress. The P uptake ability of these genotypes was related significantly and positively to root morphological parameters and photosynthesis parameters under low P stress, the P utilization efficiency showed significant and positive correlation with total dry matter. Results showed existence of genetic differences am...

Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs), are terpenoids that form the main active substance of Ginkgo biloba. Terpenoids in the mevalonate (MVA) biosynthetic pathway include acetyl-CoA C-acetyltransferase (AACT) and mevalonate kinase (MVK) as core enzymes. In this study, two full-length (cDNAs) encoding AACT (GbAACT, GenBank Accession No. KX904942) and MVK (GbMVK, GenBank Accession No. KX904944) were cloned from G. biloba. The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF) sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA) biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is one of the rate-limiting enzymes in the mevalonate pathway as it catalyzes the condensation of acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA. In this study, A HMGS gene (designated as GbHMGS1) was cloned from Ginkgo biloba for the first time. GbHMGS1 contained a 1422-bp open-reading frame encoding 474 amino acids. Comparative and bioinformatics analysis revealed that GbHMGS1 was extensively homologous to HMGSs from other plant species. Phylogenetic analysis indicated that the GbHMGS1 belonged to the plant HMGS superfamily, sharing a common evolutionary ancestor with other HMGSs, and had a further relationship with other gymnosperm species. The yeast complement assay of GbHMGS1 in HMGS-deficient Saccharomyces cerevisiae strain YSC6274 demonstrated that GbHMGS1 gene encodes a functional HMGS enzyme. The recombinant protein of GbHMGS1 was successfully expressed in E. coli. The in vitro enzyme activity assay showed that the kcat and Km values of GbHMGS1 were 195.4 min−1 and 689 μM, respectively. GbHMGS1 was constitutively expressed in all tested tissues, including the roots, stems, leaves, female flowers, male flowers and fruits. The transcript accumulation for GbHMGS1 was highest in the leaves. Expression profiling analyses revealed that GbHMGS1 expression was induced by abiotic stresses (ultraviolet B and cold) and hormone treatments (salicylic acid, methyl jasmonate, and ethephon) in G. biloba, indicating that GbHMGS1 gene was involved in the response to environmental stresses and plant hormones.