植物与病原微生物互作分子基础的研究进展

植物在与病原微生物共同进化过程中形成了复杂的免疫防卫体系。植物的先天免疫系统可大致分为两个层面。第一个层面的免疫基于细胞表面的模式识别受体对病原物相关分子模式的识别, 该免疫过程被称为病原物相关分子模式触发的免疫(PAMP-triggered immunity, PTI), 能帮助植物抵抗大部分病原微生物; 第二个层面的免疫起始于细胞内部, 主要依靠抗病基因编码的蛋白产物直接或间接识别病原微生物分泌的效应子并且激发防卫反应, 来抵抗那些能够利用效应子抑制第一层面免疫的病原微生物, 这一过程被称为效应子触发的免疫(Effector-triggered immunity, ETI)。这两个层面的免疫都是基于植物对“自我”及“非我”的识别, 依靠MAPK级联等信号网络, 将识别结果传递到细胞核内, 调控相应基因的表达, 做出适当的免疫应答。本文着重阐述了植物与病原微生物互作过程中不同层面的免疫反应所发生主要事件的分子基础及研究进展。     

植物与病原微生物互作分子基础的研究进展

植物在与病原微生物共同进化过程中形成了复杂的免疫防卫体系。植物的先天免疫系统可大致分为两个层面。第一个层面的免疫基于细胞表面的模式识别受体对病原物相关分子模式的识别,该免疫过程被称为病原物相关分子模式触发的免疫(PAMP-triggered immunity,PTI),能帮助植物抵抗大部分病原微生物;第二个层面的免疫起始于细胞内部,主要依靠抗病基因编码的蛋白产物直接或间接识别病原微生物分泌的效应子并且激发防卫反应,来抵抗那些能够利用效应子抑制第一层面免疫的病原微生物,这一过程被称为效应子触发的免疫(Effector-triggered immunity,ETI)。这两个层面的免疫都是基于植物对"自我"及"非我"的识别,依靠MAPK级联等信号网络,将识别结果传递到细胞核内,调控相应基因的表达,做出适当的免疫应答。本文着重阐述了植物与病原微生物互作过程中不同层面的免疫反应所发生主要事件的分子基础及研究进展。     

Direct evidence for the presence of a rotenone-resistant NADH dehydrogenase on the inner surface of the inner membrane of plant mitochondria

Submitochondrial particles (SMP) were produced from Jerusalem artichoke (Helianthus tuberosus L.) mitochondria by sonication and differential centrifugation. The SMP were about 50% inside-out as measured by the access of reduced cytochrome c to cytochrome c oxidase. Uncoupled NADH oxidation (1 mM NADH) by the SMP was 120 nmol O₂ min^?1mg^?1, which was reduced to 98 nmol O₂ min^?1 (mg mitochondrial protein)^?1 in the presence of EGTA. In contrast, the oxidation of NADH by intact mitochondria was completely inhibited by EGTA (from 182 to 14 nmol O₂ min^?1mg^?1). The EGTA-resistant NADH oxidation by the SMP is ascribed to the NADH dehydrogenase(s) on the inside of the inner membrane and exposed to the medium in the inside-out SMP. In the presence of EGTA it could be shown that two NADH dehydrogenase activities were present in the SMP. One had an apparent K_m of 7 μM for NADH, a V_max of 80 nmol NADH min^?1mg^?1, and was rotenone-sensitive. This dehydrogenase is equivalent to the mammalian Complex I NADH dehydrogenase. The other dehydrogenase, which was rotenone-resistant, had a K_m of 80 μM and a V_max of 131 nmol NADH min^?1mg^?1; it is probably responsible for the rotenone-resistant oxidation of organic acids often observed in plant mitochondria. The redox poise of the pyridine nucleotides had only a small effect on the relative rates of the two internal dehydrogenases. Electron flow through these dehydrogenases appears, therefore, to be regulated mainly by the concentration of NADH in the matrix of the mitochondria.     

Glucosinolates in oilseed rape: secondary metabolites that influence interactions with herbivores and their natural enemies

Glucosinolates are sulphur‐containing secondary metabolites characteristic of Brassicaceous plants. Glucosinolate breakdown products, which include isothiocyanates, are released following tissue damage when hydrolytic enzymes act on them. The isothiocyanates have toxic effects on generalist herbivores when they attempt to feed on oilseed rape, Brassica napus, and also function as repellents. However, specialist herbivores such as Brevicoryne brassicae aphids, flea beetles, Psylliodes chrysocephala and the Lepidopteran pest, Pieris rapae, are adapted to the presence of glucosinolates and thrive on plants containing them. They may do this by avoiding tissue damage to prevent the formation of isothiocyanates or by metabolising or tolerating glucosinolates. For many specialist herbivores, the isothiocyanates function as attractants and glucosinolates can even be sequestered for defence against predatory insects. Thus, these herbivores have evolved resistance to host‐plant secondary metabolites and this type of evolutionary history may have given some insects an enhanced ability to adapt to xenobiotics. In an agricultural context, this may make pests better able to evolve resistance to artificially applied pesticides. The effect of increased glucosinolate content in making oilseed rape cultivars more susceptible to specialist pests was highlighted in a seminal article in the Annals of Applied Biology in 1995. This review of the literature considers developments in this area since then.     

The mechanism of lead-induced mitochondrial Ca2+ efflux

Addition of Pb²⁺ to rat kidney mitochondria is followed by induction of several reactions: inhibition of Ca²⁺ uptake, collapse of the transmembrane potential, oxidation of pyridine nucleotides, and a fast release of accumulated Ca²⁺. When the incubation media are supplemented with ruthenium red, the effect of Pb²⁺ on NAD(P)H oxidation, membrane , and Ca²⁺ release are not prevented if malate-glutamate are the oxidizing substrates; however, the latter two lead-induced reactions are prevented by ruthenium red if succinate is the electron donor. It is proposed that in mitochondria oxidizing NAD-dependent substrates, Pb²⁺ induces Ca²⁺ release by promoting NAD(P)H oxidation and a parallel drop in due to its binding to thiol groups, located in the cytosol side of the inner membrane. In addition, it is proposed that with succinate as substrate, the Ca²⁺-releasing effect of lead is due to the collapse of the transmembrane potential as a consequence of the uptake of Pb²⁺ through the calcium uniporter, since such effect is ruthenium red sensitive.     

Metabolic signalling in defence and stress: the central roles of soluble redox couples

Plant growth and development are driven by electron transfer reactions. Modifications of redox components are both monitored and induced by cells, and are integral to responses to environmental change. Key redox compounds in the soluble phase of the cell are NAD, NADP, glutathione and ascorbate--all of which interact strongly with reactive oxygen. This review takes an integrated view of the NAD(P)-glutathione-ascorbate network. These compounds are considered not as one-dimensional 'reductants' or 'antioxidants' but as redox couples that can act together to condition cellular redox tone or that can act independently to transmit specific information that tunes signalling pathways. Emphasis is placed on recent developments highlighting the complexity of redox-dependent defence reactions, and the importance of interactions between the reduction state of soluble redox couples and their concentration in mediating dynamic signalling in response to stress. Signalling roles are assessed within the context of interactions with reactive oxygen, phytohormones and calcium, and the biochemical reactions through which redox couples could be sensed are discussed.     

Degradation of NAD by Synaptosomes and Its Inhibition by Nicotinamide Mononucleotide: Implications for the Role of NAD as a Synaptic Modulator

We have found NAD to be rapidly degraded by extracellular enzymes present on intact rat brain synaptosomes. The enzyme involved had the specificity of an NADase cleaving the molecule at the nicotinamide-glycoside linkage and was inhibited by nicotinamide mononucleotide (NMN). This inhibitor did not displace specific binding of NAD to rat brain membranes or affect electrical activity in the guinea pig hippocampus. Therefore, inclusion of NMN in binding assays allowed unambiguous demonstration of two specific NAD binding sites on rat brain synaptosomal membranes (KD1, 82 nM, KD2, 1.98 microM). The depressant action of NAD on the evoked synaptic activity of the guinea pig hippocampus was not blocked after inhibition of NAD degradation with NMN. The physiological implications of these results for the function of NAD as a neurotransmitter or neuromodulator in the CNS are discussed.     

In vitro and in vivo regulation of chloroplast glyceraldehyde-3-phosphate dehydrogenase isozymes from Chenopodium rubrum. II. In vitro modulation of the isozyme pattern

Isozymes of chloroplast glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.13) from Chenopodium rubrum were separated using inverse discontinuous ammonium sulphate gradient solubilization. Leaves were extracted at the 9th h of light and the 9th h of darkness of a 12 h light/12 h dark cycle. The ratio of "NADP-GPD I" to "NADP-GPD II" varied with the light/dark cycle. However, the "light" isozyme pattern could be obtained from "dark" plants by including NADP ⁺ or NAD ⁺ kinase in the extraction buffer. Similarly, the "dark" isozyme pattern was produced in "light" plants extracted in the presence of NAD⁺. Pyridine nucleotides had no effect on the separated, purified isozymes. It is concluded that differential binding of the isozymes at the moment of extraction to pelletable material in the crude extract determines the isozyme pattern, and that this binding is regulated by the pyridine nucleotide ratio.     

Characterization and possible redox regulation of the purified calmodulin‐dependent NAD+ kinase from Lycopersicon pimpinellifolium

The soluble and calmodulin (CaM)‐dependent NAD⁺ kinase from Lycopersicon pimpinellifolium was previously shown to be largely inactivated in isolated cells exposed to a short‐term NaCl stress (Delumeau, Morère‐Le Paven, Montrichard, Laval‐Martin (2000) Plant Cell & Environment 23, 329–336). Nevertheless, the activity could be restored by adding a high dithiothreitol concentration to the protein extract, suggesting that the salt stress triggers an oxidation of the enzyme which leads to its inactivation. It was then interesting to investigate the effect of thiol‐modifying reagents and disulphide reductants on the activity of L. pimpinellifolium NAD⁺ kinase. A three‐step purification procedure was then established and allowed isolation of the enzyme which exists under two forms: a monomer and a dimer of a 56 kDa subunit, characterized, respectively, by pIs of 6·8 and 7·1. Isolated NAD⁺ kinase had a high affinity for CaM, half saturation being obtained for 7 ng mL^?1 bovine CaM. The activity of NAD⁺ kinase was strongly inhibited by thiol‐modifying reagents and oxidized glutathione. NAD⁺ kinase was also found to be air‐inactivated, the residual activity being stimulated by disulphide reductants. The most efficient of them is reduced thioredoxin from Escherichia coli which induced a five‐fold increase in activity and restored 80% of the initial activity. These results which can be related to those previously observed in vivo suggest that the activity of the L. pimpinellifolium NAD⁺ kinase, besides its dependence on CaM, is also dependent on the reduction state of the protein which could be regulated by the thioredoxin h/NADP‐thioredoxin reductase system.     

Determination of the cytosolic free NAD/NADH ratio in Saccharomyces cerevisiae under steady-state and highly dynamic conditions

The coenzyme NAD plays a major role in metabolism as a key redox carrier and signaling molecule but current measurement techniques cannot distinguish between different compartment pools, between free and protein-bound forms and/or between NAD(H) and NADP(H). Local free NAD/NADH ratios can be determined from product/substrate ratios of suitable near-equilibrium redox reactions but the application of this principle is often precluded by uncertainties regarding enzyme activity, localization and coenzyme specificity of dehydrogenases. In Saccharomyces cerevisiae, we circumvented these issues by expressing a bacterial mannitol-1-phosphate 5-dehydrogenase and determining the cytosolic free NAD/NADH ratio from the measured [fructose-6-phosphate]/[mannitol-1-phosphate] ratio. Under aerobic glucose-limited conditions we estimated a cytosolic free NAD/NADH ratio between 101(+/-14) and 320(+/-45), assuming the cytosolic pH is between 7.0 and 6.5, respectively. These values are more than 10-fold higher than the measured whole-cell total NAD/NADH ratio of 7.5(+/-2.5). Using a thermodynamic analysis of central glycolysis we demonstrate that the former are thermodynamically feasible, while the latter is not. Furthermore, we applied this novel system to study the short-term metabolic responses to perturbations. We found that the cytosolic free NAD-NADH couple became more reduced rapidly (timescale of seconds) upon a pulse of glucose (electron-donor) and that this could be reversed by the addition of acetaldehyde (electron-acceptor). In addition, these dynamics occurred without significant changes in whole-cell total NAD and NADH. This approach provides a new experimental tool for quantitative physiology and opens new possibilities in the study of energy and redox metabolism in S. cerevisiae. The same strategy should also be applicable to other microorganisms.     

Unusual conformation of nicotinamide adenine dinucleotide (NAD) bound to diphtheria toxin: a comparison with NAD bound to the oxidoreductase enzymes.

The conformation of NAD bound to diphtheria toxin (DT), an ADP-ribosylating enzyme, has been compared to the conformations of NAD(P) bound to 23 distinct NAD(P)-binding oxidoreductase enzymes, whose structures are available in the Brookhaven Protein Data Bank. For the oxidoreductase enzymes, NAD(P) functions as a cofactor in electron transfer, whereas for DT, NAD is a labile substrate in which the N-glycosidic bond between the nicotinamide ring and the N-ribose is cleaved. All NAD(P) conformations were compared by (1) visual inspection of superimposed molecules, (2) RMSD of atomic positions, (3) principal component analysis, and (4) analysis of torsion angles and other conformational parameters. Whereas the majority of oxidoreductase-bound NAD(P) conformations are found to be similar, the conformation of NAD bound to DT is found to be unusual. Distinctive features of the conformation of NAD bound to DT that may be relevant to DT''s function as an ADP-ribosylating enzyme include (1) an unusually short distance between the PN and N1N atoms, reflecting a highly folded conformation for the nicotinamide mononucleotide (NMN) portion of NAD, and (2) a torsion angle chi N approximately 0 degree about the scissile N-glycosidic bond, placing the nicotinamide ring outside of the preferred anti and syn orientations. In NAD bound to DT, the highly folded NMN conformation and torsion angle chi N approximately 0 degree could contribute to catalysis, possibly by orienting the C1''N atom of NAD for nucleophilic attack, or by placing strain on the N-glycosidic bond, which is cleaved by DT. The unusual overall conformation of NAD bound to DT is likely to reflect the structure of DT, which is unusual among NAD(P)-binding enzymes. In DT, the NAD binding site is formed at the junction of two antiparallel beta-sheets. In contrast, although the 24 oxidoreductase enzymes belong to at least six different structural classes, almost all of them bind NAD(P) at the C-terminal end of a parallel beta-sheet. The structural alignments and principal component analysis show that enzymes of the same structural class bind to particularly similar conformations of NAD(P), with few exceptions. The conformation of NAD bound to DT superimposes closely with that of an NAD analogue bound to Pseudomonas exotoxin A, an ADP-ribosylating toxin that is structurally homologous to DT. This suggests that all of the ADP-ribosylating enzymes that are structurally homologous to DT and ETA will bind a highly similar conformation of NAD.     

The oxidation of cytosolic NAD(P)H by external NAD(P)H dehydrogenases in the respiratory chain of plant mitochondria

The respiratory chain of plant mitochondria differs from that in mammalian mitochondria by containing several rotenone-insensitive NAD(P)H dehydrogenases. Two of these are located on the outer, cytosolic surface of the inner membrane. One is specific for NADH, the other for NADPH. Only the latter is inhibited by diphenyleneiodonium (DPI). Both of these enzymes are normally dependent upon Ca²⁺ for activity and this constitutes a potentially important mechanism by which the cell can regulate the oxidation of cytosolic NAD(P)H via the concentration of free Ca²⁺. This and other potential regulatory mechanisms such as the substrate concentration and polyamines are discussed.     

Regulation of intracellular levels of NAD: a novel role for CD38

Nicotinamide adenine dinucleotide (NAD) plays key roles in many cellular functions. In addition to its well-known role in energy metabolism, NAD also plays a role in signal transduction, ageing, and cellular injury. NAD is also involved in many signal transduction pathways. Therefore, it is imperative to understand the mechanisms that control intracellular NAD levels. However, to date, the mechanisms that regulate intracellular levels of NAD have not been completely elucidated. CD38 is a multifunctional enzyme ubiquitously distributed in mammalian tissues. CD38 has been implicated as the enzyme responsible for the synthesis of the second messengers. However, its major enzymatic activity is the hydrolysis of NAD, in fact, CD38 will generate one molecule of cADPR for every 100 molecules of NAD hydrolyzed. To date, the role of CD38 as a modulator of levels of NAD has not been explored. We postulated that CD38 is the major NADase in mammalian cells and that it regulates intracellular NAD levels. In the current studies we examined the NADase activities and NAD levels in a variety of tissues from both wild-type and CD38 deficient mice. In accordance with our hypothesis, we found that tissue levels of NAD in CD38 deficient mice are 10- to 20-fold higher than in wild-type animals. In addition, NADase activity in the plasma membrane, mitochondria, sarcoplasmic reticulum, and nuclei is essentially absent in most tissues from CD38 deficient mice. These data support the novel concept that CD38 is a major regulator of cellular NAD levels. These findings have implications for understanding the mechanisms that regulate intracellular NAD levels and its role in energy homeostasis, signal transduction, and ageing.     

The influence of plant age on wound induction of proteinase inhibitors in tomato

Proteinase inhibitors can be induced by wounding in shoots of tomato ( Lycopersicon esculentum [L.] Mill. cv. Moneymaker). These inhibitors are toxic to insects, but their ecological importance is not clear. Published work suggests that proteinase inhibitors may be wound-inducible in tomato only while the plants are young (less than 30 days). In the present investigation the influence of plant age on wound-inducible proteinase inhibitor was re-assessed using tomato plants grown in an outdoor polythene tunnel, with natural lighting and without supplementary heat. In contrast to previous findings, proteinase inhibitor was shown to be induced by wounding in plants of all ages. However, the systemic efficacy of wounds was much reduced in mature plants, possibly because such plants have outgrown the range of the wound-signalling system.     

Steric hindrance controls pyridine nucleotide specificity of a flavin‐dependent NADH:quinone oxidoreductase

The crystal structure of the NADH:quinone oxidoreductase PA1024 has been solved in complex with NAD⁺ to 2.2 Å resolution. The nicotinamide C4 is 3.6 Å from the FMN N5 atom, with a suitable orientation for facile hydride transfer. NAD⁺ binds in a folded conformation at the interface of the TIM‐barrel domain and the extended domain of the enzyme. Comparison of the enzyme‐NAD⁺ structure with that of the ligand‐free enzyme revealed a different conformation of a short loop (75–86) that is part of the NAD⁺‐binding pocket. P78, P82, and P84 provide internal rigidity to the loop, whereas Q80 serves as an active site latch that secures the NAD⁺ within the binding pocket. An interrupted helix consisting of two α‐helices connected by a small three‐residue loop binds the pyrophosphate moiety of NAD⁺. The adenine moiety of NAD⁺ appears to π–π stack with Y261. Steric constraints between the adenosine ribose of NAD⁺, P78, and Q80, control the strict specificity of the enzyme for NADH. Charged residues do not play a role in the specificity of PA1024 for the NADH substrate.     

Effects of short-term NaCl stress on calmodulin transcript levels and calmodulin-dependent NAD kinase activity in two species of tomato

NAD kinase is thought to play an important role in the plant cellular responses to biotic and abiotic stress as one of the isoforms of the enzyme is activated by the Ca^{2 +} –calmodulin (CaM) complex. NAD kinase activity was measured after short‐term NaCl stress applied to isolated cells from Lycopersicon esculentum, var. Volgogradskij (NaCl‐sensitive tomato) and L. pimpinellifolium, acc. PE2 (NaCl‐tolerant species). NAD kinase activity remained constant in the sensitive species, whereas a sharp decrease was observed in the tolerant one. After salt treatment, an induction of the calmodulin gene(s) was observed in the two species, together with a 30–50% decrease in ‘active’ CaM content, i.e. CaM able to activate purified NAD kinase, in L. pimpinellifolium. The decrease in NAD kinase activity could not, however, be fully explained by this decrease in active CaM content. A similar decrease in NAD kinase activity was also recorded with other ionic stresses and exposure to high temperatures, but not in the case of drought, exposure to low temperatures, hormonal (indole‐3‐acetic acid and abscisic acid) or H₂O₂ treatments. External Ca^{2 +} certainly plays a role in the biochemical mechanism(s) leading to NAD kinase inhibition, while no role could be shown for intracellular Ca^{2 +} . In addition, after salt stress, a modification of the redox state of NAD kinase seems to be responsible for the inhibition of the enzyme.     

The pyridine-nucleotide cycle in tobacco Enzyme activities for the de-novo synthesis of NAD

The enzyme activities of the pyridine-nucleotide cycle, which transform nicotinic acid mononucleotide (NaMN) into NAD, have been characterized. The investigations were based on the extraction of protein, its purification on disposable gel-filtration columns, and determination of the enzymatic activities by high-performance liquid chromatography techniques. The latter technique avoided the synthesis and use of radioactive precursors. The NaMN-adenylyltransferase which converts NaMN into NaAD (nicotinic acid adenine dinucleotide) and NAD-synthetase which converts NaAD into NAD were characterized by their kinetic parameters and their specific activities in different tobacco tissues. This is the first report on NAD-synthetase from tissue of a higher plant. It was found that NAD-synthetase accepted both glutamine and asparagine for the amide transfer. Adenylyltransfer also occured with nicotinamide mononucleotide (NMN) which was transformed to NAD, whereas the glutamine-dependent amidation was only observed with NaAD. Thus, an additional route for the synthesis of NAD (NaMNNMNNAD) obviously does not exist. A comparison of the enzyme activities in tobacco tissues with different capacities for the synthesis of nicotine showed that, in contrast to quinolinic acid phosphoribosyltransferase whose activity was strictly correlated with the nicotine content, only NaMN-adenylyltransferase showed a smooth correlation, whereas NAD-synthetase was not affected at all.Abbreviations HPLC high-performance liquid chromatography - QA quinolinic acid - NaMN nicotinic acid mononucleotide - NaAD nicotinic acid adenine dinucleotide - NMN nicotinamide mononucleotide     

Not just the sum of its parts: Geographic variation and nonadditive effects of pyrazines in the chemical defence of an aposematic moth

Chemical defences often vary within and between populations both in quantity and quality, which is puzzling if prey survival is dependent on the strength of the defence. We investigated the within- and between-population variability in chemical defence of the wood tiger moth (Arctia plantaginis). The major components of its defences, SBMP (2-sec-butyl-3-methoxypyrazine) and IBMP (2-isobutyl-3-methoxypyrazine), are volatiles that deter bird attacks. We hypothesized that (1) variation in the chemical defences of male wood tiger moths reflects the local predation pressure; (2) observed differences in quantity and quality of defence among populations have a genetic basis; and (3) increasing concentrations of SBMP and IBMP will elicit greater aversive reactions in predators, with the two pyrazines having an additive effect on predators' avoidance. We found that (1) the chemical defence of wild moths partly reflects local predator selection: high predation pressure populations (Scotland and Georgia) had stronger chemical defences, but not lower variance, than the low-predation populations (Estonia and Finland). (2) Based on the common garden results, both genetic and environmental components seem to influence the strength of chemical defence in moth populations; and (3) IBMP alone did not provide protection against bird predators but worked against bird attacks only when combined with SBMP, and while SBMP was more effective at higher concentrations, IBMP was not. Altogether this suggests that, when it comes to pyrazine concentration, more is not always better, highlighting the importance of testing the efficacy of chemical defence and its components with relevant predators, as extrapolating from chemical data may be less than straightforward.