玉米苗中DIMBOA与几种酚酸类物质抑菌活性比较

本文从室内培养的7日龄玉米幼苗中提取、分离、鉴定了抗性次生化合物丁布(2,4-d ihydroxy-7-m ethoxy-2H-1,4-benzoxazin-3(4H)-one,D IMBOA),并就该物质对玉米纹枯病病原菌立枯丝核菌(Rhizoctonia solani)的活性与三种酚酸类物质(阿魏酸、对羟基肉桂酸和咖啡酸)进行了离体比较研究。结果表明,丁布(D IMBOA),对立枯丝核菌有很强的生物活性,在浓度为50μg/mL时即可抑制立枯丝核菌菌丝的生长,抑制率为18.52%。阿魏酸、对羟基肉桂酸和咖啡酸,这三种酚酸在浓度250μg/mL时对立枯丝核菌菌丝的生长有抑制作用,抑制率分别为26.30%、8.50%和6.30%。不仅如此,丁布与对羟基肉桂酸之间、以及三种酚酸两两组合之间还存在一定的协同作用。在浓度相等的情况下,丁布与对羟基肉桂酸的等量混合液的抑菌率显著高于这两种物质单独存在时的抑菌率之和;同样,对羟基肉桂酸与阿魏酸的等量混合液的抑菌率比单一的对羟基肉桂酸溶液的抑菌率高18.89%,比单一的阿魏酸溶液的抑菌率高13.33%;对羟基肉桂酸与咖啡酸的等量混合液,抑菌率比两者单独试验时分别高9.63%和14.83%;阿魏酸与咖啡酸的混合液,抑菌率比两酸单独试验时分别高11.48%和22.23%。这一结果提示植物体内产生适当比例不同次生化合物的组合对植物抗病性的提高是至关重要的。     

Cyanogenic glucosides and plant-insect interactions

Cyanogenic glucosides are phytoanticipins known to be present in more than 2500 plant species. They are considered to have an important role in plant defense against herbivores due to bitter taste and release of toxic hydrogen cyanide upon tissue disruption. Some specialized herbivores, especially insects, preferentially feed on cyanogenic plants. Such herbivores have acquired the ability to metabolize cyanogenic glucosides or to sequester them for use in their predator defense. A few species of Arthropoda (within Diplopoda, Chilopoda, Insecta) are able to de novo synthesize cyanogenic glucosides and, in addition, some of these species are able to sequester cyanogenic glucosides from their host plant (Zygaenidae). Evolutionary aspects of these unique plant-insect interactions with focus on the enzyme systems involved in synthesis and degradation of cyanogenic glucosides are discussed.     

Diversification of an ancient theme: hydroxynitrile glucosides

Many plants produce cyanogenic glucosides as part of their chemical defense. They are alpha-hydroxynitrile glucosides, which release toxic hydrogen cyanide (HCN) upon cleavage by endogenous plant beta-glucosidases. In addition to cyanogenic glucosides, several plant species produce beta- and gamma-hydroxynitrile glucosides. These do not release HCN upon hydrolysis by beta-glucosidases and little is known about their biosynthesis and biological significance. We have isolated three beta-hydroxynitrile glucosides, namely (2Z)-2-(beta-D-glucopyranosyloxy)but-2-enenitrile and (2R,3R)- and (2R,3S)-2-methyl-3-(beta-D-glucopyranosyloxy)butanenitrile, from leaves of Ribesuva-crispa. These compounds have not been identified previously. We show that in several species of the genera Ribes, Rhodiola and Lotus, these beta-hydroxynitrile glucosides co-occur with the L-isoleucine-derived hydroxynitrile glucosides, lotaustralin (alpha-hydroxynitrile glucoside), rhodiocyanosides A (gamma-hydroxynitrile glucoside) and D (beta-hydroxynitrile glucoside) and in some cases with sarmentosin (a hydroxylated rhodiocyanoside A). Radiolabelling experiments demonstrated that the hydroxynitrile glucosides in R. uva-crispa and Hordeum vulgare are derived from L-isoleucine and L-leucine, respectively. Metabolite profiling of the natural variation in the content of cyanogenic glucosides and beta- and gamma-hydroxynitrile glucosides in wild accessions of Lotus japonicus in combination with genetic crosses and analyses of the metabolite profile of the F2 population provided evidence that a single recessive genetic trait is most likely responsible for the presence or absence of beta- and gamma-hydroxynitrile glucosides in L. japonicus. Our findings strongly support the notion that the beta- and gamma-hydroxynitrile glucosides are produced by diversification of the cyanogenic glucoside biosynthetic pathway at the level of the nitrile intermediate.     

Cyanogenesis in plants and arthropods

Cyanogenic glucosides are phytoanticipins known to be present in more than 2500 plant species. They are regarded as having an important role in plant defense against herbivores due to bitter taste and release of toxic hydrogen cyanide upon tissue disruption, but recent investigations demonstrate additional roles as storage compounds of reduced nitrogen and sugar that may be mobilized when demanded for use in primary metabolism. Some specialized herbivores, especially insects, preferentially feed on cyanogenic plants. Such herbivores have acquired the ability to metabolize cyanogenic glucosides or to sequester them for use in their own defense against predators. A few species of arthropods (within diplopods, chilopods and insects) are able to de novo biosynthesize cyanogenic glucosides and some are able to sequester cyanogenic glucosides from their food plant as well. This applies to larvae of Zygaena (Zygaenidae). The ratio and content of cyanogenic glucosides is tightly regulated in Zygaena filipendulae, and these compounds play several important roles in addition to defense in the life cycle of Zygaena. The transfer of a nuptial gift of cyanogenic glucosides during mating of Zygaena has been demonstrated as well as the involvement of hydrogen cyanide in male attraction and nitrogen metabolism. As more plant and arthropod species are examined, it is likely that cyanogenic glucosides are found to be more widespread than formerly thought and that cyanogenic glucosides are intricately involved in many key processes in the life cycle of plants and arthropods.     

The reduction of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one by thiols

2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), a naturally occurring hydroxamic acid involved in pest resistance of cereals, was reduced by thiols to the corresponding lactam. Kinetic studies showed that the reactive species are undissociated DIMBOA and thiolate anion. Possible mechanisms for the reaction are discussed in the light of relative reactivities of DIMBOA and a compound lacking the 7-methoxy substituent, and results from molecular orbital calculations.     

The mechanism of action of glycosidases.

The factors that may contribute to the rate enhancement observed with enzymatic versus non-enzymatic hydrolysis of glycosides are discussed. The nature of the active site as deduced from labelling studies with beta-glucosidases is described. A two-step mechanism involving either an enzyme stabilized glycosyl ion or a covalent glycosyl-enzyme intermediate is proposed. Experiments with a beta-glucosidase from almonds show that even with 2-deoxy glucosides with good leaving groups as aglycon which are hydrolyzed 1000 times more slowly than the corresponding glucosides, the deglucosylation step is faster than the cleavage of the glycosidic bond.     

Crystal structure of intracellular family 1 beta-glucosidase BGL1A from the basidiomycete Phanerochaete chrysosporium

The white-rot fungus Phanerochaete chrysosporium has two intracellular beta-glucosidases (BGL1A and BGL1B) belonging to glycoside hydrolase (GH) family 1. BGL1B effectively hydrolyzes cellobiose and cellobionolactone, but BGL1A does not. We have determined the crystal structure of BGL1A in substrate-free and gluconolactone complexed forms. The overall structure and the characteristic of subsite -1 (glycone site) were similar to those of other known GH1 enzymes. The loop regions covering on the (beta/alpha)(8) barrel was significantly deviated, and they form a unique subsite +1 (aglycone site) of BGL1A.     

Effect of content and distribution of hydroxamic acids in wheat on infestation by the aphid Schizaphis graminum

The content of hydroxamic acids in wheat plants shows substantial variations between different species and cultivars (1.0–6.3 mmol/kg fr. wt). It also varies with the age of the plant and the organ assayed. The maximum concentration is reached by the fourth day in epicotyls and roots. The amount in leaves at different plant ages is always higher in the younger leaves. Based on feeding and infestation experiments, it is proposed that the distribution of aphids on leaves of different ages is regulated by the hydroxamic acid content.     

Effect of cysteine on stability and toxicity to aphids of a cyclic hydroxamic acid from gramineae

Toxicity of DIMBOA, the major cyclic hydroxamic acid in maize extracts, to the aphid Schizaphis graminum, was decreased by addition of cysteine to the insect diet. The ld₅₀ for DIMBOA on aphids was, after 24 hr, 2.1 and 0.9 mM in diets with and without added cysteine, respectively. DIMBOA decomposed 1.5 times faster in diets or buffer with added cysteine. Decomposition products of DIMBOA (4 mM) in insect diets with or without added cysteine were not toxic. It is suggested that the observed variations in toxicity of DIMBOA are a consequence of differences in its rate of disappearance from the diet.     

A beta-glucosidase from lodgepole pine xylem specific for the lignin precursor coniferin.

Coniferin, the glucoside of the monolignol coniferyl alcohol, accumulates to high levels in gymnosperms during spring-cambial reactivation. A cinnamyl alcohol glucoside/beta-glucosidase system is thought to play a key role in lignification by releasing the monolignol aglycones. Investigation of such an enzyme system in the xylem of Pinus contorta var latifolia Engelm. revealed two major beta-glucosidases. One efficiently hydrolyzed the native substrate, coniferin, and the other was more active against synthetic glucosides. The coniferin beta-glucosidase was purified to apparent homogeneity using anion exchange, hydrophobic interaction, and size-exclusion chromatography. The apparent native molecular weight was estimated to be 60,000. A dominant 28-kD protein and a minor 24-kD protein were detected in the purified preparation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunological evidence from polyclonal antibodies directed against the synthetic N-terminal peptide of the 24-kD protein suggested that the native protein is a dimer of 28-kD subunit size. The N-terminal sequence showed that coniferin beta-glucosidase has high homology to known plant beta-glucosidases. Coniferin, syringin, and a synthetic coniferin analog were preferred substrates for the coniferin beta-glucosidase. In situ localization using the chromogenic coniferin analog showed the exclusive presence of beta-glucosidase activity in the differentiating xylem, similar to peroxidase activity.     

Origin of Cyanide in Cultures of a Psychrophilic Basidiomycete

An unidentified psychrophilic basidiomycete used valine and isoleucine as precursors to hydrocyanic acid (HCN). As probable intermediates in the pathway from valine and isoleucine two cyanogenic glucosides, linamarin and lotaustralin, were demonstrated in fungus cultures. The fungus contained two beta-glucosidases and an oxynitrilase which, acting together, were capable of releasing cyanide from both linamarin and lotaustralin. The two beta-glucosidases were purified and compared as to pH optimum, Michaelis constant, energy of activation, thermal stability, and substrate specificity. The products of methyl ethyl ketone cyanohydrin and acetone cyanohydrin dissociation by the oxynitrilase were demonstrated to be HCN together with methyl ethyl ketone and acetone, respectively. The oxynitrilase attacked aliphatic hydroxynitriles, but showed no activity on aromatic hydroxynitriles.     

cis-Jasmone induces accumulation of defence compounds in wheat, Triticum aestivum

Liquid phase extraction (LPE) and vapor phase extraction (VPE) methodologies were used to evaluate the impact of the plant activator, cis-jasmone, on the secondary metabolism of wheat, Triticum aestivum, var. Solstice. LPE allowed the measurement of benzoxazinoids, i.e. 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA) and 6-methoxy-benzoxazolin-2-one (MBOA), and phenolic acids such as trans-p-coumaric acid, syringic acid, p-hydroxybenzoic acid, vanillic acid and cis- and trans-ferulic acid. Using LPE, a significantly higher level of DIMBOA was found in aerial parts and roots of T. aestivum following treatment with cis-jasmone, when compared with untreated plants. Similar results were obtained for phenolic acids, such as trans-ferulic acid and vanillic acid in roots. Using VPE, it was possible to measure levels of 2-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA), benzoxazolin-2(3H)-one (BOA), ferulic acid, syringic acid and coumaric acid. The levels of HBOA in aerial parts and roots were significantly greater in cis-jasmone treated plants compared to untreated plants. cis-Jasmone is known to be a plant activator in terms of production of defence-related volatile semiochemicals that repel aphids and increase the foraging activity of aphid parasitoids. These results show, for the first time, that cis-jasmone also induces selective production of secondary metabolites that are capable of directly reducing development of pests, diseases and weeds.     

Crystal structures of Paenibacillus polymyxa beta-glucosidase B complexes reveal the molecular basis of substrate specificity and give new insights into the catalytic machinery of family I glycosidases

Bacteria species involved in degradation of cellulosic substrates produce a variety of enzymes for processing related compounds along the hydrolytic pathway. Paenibacillus polymyxa encodes two homologous beta-glucosidases, BglA and BglB, presenting different quaternary structures and substrate specificities. We previously reported the 3D-structure of BglA, which is highly specific against cellobiose. Here, we present structural analysis of BglB, a monomeric enzyme that acts as an exo-beta-glucosidase hydrolyzing cellobiose and cellodextrins of higher degree of polymerization. The crystal structure of BglB shows that several polar residues narrow the active site pocket and contour additional subsites. The structure of the BglB-cellotetraose complex confirms these subsites, revealing the substrate-binding mode, and shows the oligosaccharide-enzyme recognition pattern in detail. Comparison between BglA and BglB crystal structures suggests that oligomerization in BglA can assist in fine-tuning the specificity of the active centre by modulating the loops surrounding the cavity. We have solved the crystal structure of BglB with bound thiocellobiose, a competitive inhibitor, which together with the BglB-cellotetraose complex delineate the general features of the aglycon site. The detailed characterization of the atomic interactions at the aglycon site show a recognition pattern common to all bacterial beta-glucosidases, and presents some differences with the aglycon site in plant beta-glycosidases essentially by means of a different orientation of the basal Trp. The crystal structures of of BglB with a covalently bound inhibitor (derived from 2-fluoroglucoside) and glucose (produced by hydrolysis of the substrate in the crystal), provide additional pictures of the binding events and the intermediates formed during the reaction. Altogether, this information can assist in the understanding of subtle differences of the enzyme mechanism and substrate recognition within this family of enzymes, and consequently it can help in the development of new enzymes with improved activity or specificity.     

Transglucosylation of tertiary alcohols using cassava beta-glucosidase

We have compared the ability of beta-glucosidases from cassava, Thai rosewood, and almond to synthesize alkyl glucosides by transglucosylating alkyl alcohols of chain length C(1)-C(8). Cassava linamarase shows greater ability to transfer glucose from p-nitrophenyl-beta-glucoside to secondary alcohol acceptors than other beta-glucosidases, and is unique in being able to synthesize C(4), C(5), and C(6) tertiary alkyl beta-glucosides with high yields of 94%, 82%, and 56%, respectively. Yields of alkyl glucosides could be optimized by selecting appropriate enzyme concentrations and incubation times. Cassava linamarase required pNP-glycosides as donors and could not use mono- or di-saccharides as sugar donors in alkyl glucoside synthesis.     

High performance liquid chromatography of steroid metabolites in the pregnenolone and progesterone pathways

Rapid separation of gonadal steroids in the progesterone(Δ⁴) and pregnenolone pathway (Δ⁵) has been accomplished by the use of high performance liquid chromatography (HPLC). Two HPLC systems are utilized. The first requires the use of two separate radial compression columns (C-18 and C-8), with steroids being eluted with a methanol-water gradient. The second employs a stainless steel C-18 (reversed phase) column with a 12% octadecylsilane coating. The latter system separates seven of the eight steroids in the Δ⁴ and Δ⁵ pathways in thirty-five minutes. For the quantitation of steroids directly, integration of the peak areas, using 254 nm absorption for the Δ⁴ pathway steroids (5 ng minimum limit), and 210 nm absorption for the Δ ⁵ pathway steroids (25 ng minimum limit) is used. For the quantitation of radiolabeled metabolites resulting from incubation of gonadal tissue with radiolabeled steroid precursors, either one of two methods is used: (1) the eluent can be recovered from the HPLC using a fraction collector, and counted in liquid scintillation counter or (2) the entire eluent (or a portion of it) can be counted immediately by directing the flow through a radioactivity detector.     

Epithiospecifier protein in turnip and changes in products of autolysis during ontogeny

An epithiospecifier protein present in turnip tissue gives rise to 1-cyano-epithioalkanes during autolysis. Volatile hydrolysis products are produced from glucosinolates during autolysis of seeds, seedlings and plant tissue more than 6 weeks after sowing.     

植物抗病性物质的研究进展

植物与病原菌相互作用后在植物体内会产生抗性物质。植物的抗病性物质包括两大类 :植物固有的抗菌物质和植物保卫素。本文就近年来对植物抗病性物质的化学组成、生物合成、代谢途径方面的研究进行了综述。     

Long-distance signalling of abscisic acid (ABA): the factors regulating the intensity of the ABA signal

Abscisic acid (ABA) is a stress signal, which moves in the xylem from the roots to the aerial parts of the plant, where it regulates stomatal movement and the activity of shoot meristems. Root growth-promoting microorganisms in the rhizosphere, lateral ABA flows in the root cortex across apoplastic barriers, ABA redistribution in the stem, leaf apoplastic pH values, and the action of beta-glucosidases, both in the apoplast and the cytosol of the mesophyll, play an important role in the regulation of signal intensity. The significance of abscisic acid glucose ester as a long-distance stress signal is discussed.