Dynamic metabonomic responses of tobacco (Nicotiana tabacum) plants to salt stress

Metabolic responses are important for plant adaptation to osmotic stresses. To understand the dosage and duration dependence of salinity effects on plant metabolisms, we analyzed the metabonome of tobacco plants and its dynamic responses to salt treatments using NMR spectroscopy in combination with multivariate data analysis. Our results showed that the tobacco metabonome was dominated by 40 metabolites including organic acids/bases, amino acids, carbohydrates and choline, pyrimidine, and purine metabolites. A dynamic trajectory was clearly observable for the tobacco metabonomic responses to the dosage of salinity. Short-term low-dose salt stress (50 mM NaCl, 1 day) caused metabolic shifts toward gluconeogenesis with depletion of pyrimidine and purine metabolites. Prolonged salinity with high-dose salt (500 mM NaCl) induced progressive accumulation of osmolytes, such as proline and myo-inositol, and changes in GABA shunt. Such treatments also promoted the shikimate-mediated secondary metabolisms with enhanced biosynthesis of aromatic amino acids. Therefore, salinity caused systems alterations in widespread metabolic networks involving transamination, TCA cycle, gluconeogenesis/glycolysis, glutamate-mediated proline biosynthesis, shikimate-mediated secondary metabolisms, and the metabolisms of choline, pyrimidine, and purine. These findings provided new insights for the tobacco metabolic adaptation to salinity and demonstrated the NMR-based metabonomics as a powerful approach for understanding the osmotic effects on plant biochemistry.     

Comparative metabolomics of the interaction between rice and the brown planthopper

Introduction

The interactions between plants and insect herbivores are complex and multifaceted. Rice and its specialist insect pest the brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae) constitute an ideal system for studying plant–insect interactions.

Objectives

Combined metabolomics analyses of rice plant and BPH were conducted to understand the mechanism of host rice plant defense and BPH insect response.

Methods

Metabolite dynamics in rice leaf sheath and BPH honeydew was investigated using the gas chromatography–mass spectrometry (GC–MS) method. The GC–MS data were analyzed by principal component analysis and partial least squares-discriminant analysis.

Results

Twenty-six metabolites were detected in the leaf sheath extracts. Rice leaf sheath metabolomics analysis results show that BPH feeding induces distinct changes in the metabolite profiles of YHY15 and TN1 plants. These results suggest that BPH infestation enhance fatty acid oxidation, the glyoxylate cycle, gluconeogenesis and the GABA shunt in TN1 plants, and glycolysis and the shikimate pathway in YHY15. We propose that the BPH15 gene mediates a resistance reaction that increases the synthesis of secondary metabolites through the shikimate pathway. Thirty-three metabolites were identified in BPH honeydew. Honeydew metabolomics analysis results show that when BPH insects were fed on resistant YHY15 plants, most of the amino acids in honeydew were significantly decreased compared to those of BPH fed on TN1 plants. Based on metabolomics results, we propose that BPH feeding on resistant YHY15 plants would enhance amino acid absorption. At the same time, urea was significantly increased in BPH fed on YHY15.

Conclusion

Metabolomics study is valuable in understanding the complex and multifaceted interaction between plants and insect herbivores and provide essential clue for development of novel control BPH strategies.

     

Dynamic metabolic responses of brown planthoppers towards susceptible and resistant rice plants

Brown planthopper (Nilaparvata lugens Stål, BPH) causes huge economic losses in rice‐growing regions, and new strategies for combating BPH are required. To understand how BPHs respond towards BPH‐resistant plants, we systematically analysed the metabolic differences between BPHs feeding on the resistant and susceptible plants using NMR and GC‐FID/MS. We also measured the expression of some related genes involving glycolysis and biosyntheses of trehalose, amino acids, chitin and fatty acids using real‐time PCR. BPH metabonome was dominated by more than 60 metabolites including fatty acids, amino acids, carbohydrates, nucleosides/nucleotides and TCA cycle intermediates. After initial 12 h, BPHs feeding on the resistant plants had lower levels of amino acids, glucose, fatty acids and TCA cycle intermediates than on the susceptible ones. The levels of these metabolites recovered after 24 h feeding. This accompanied with increased level in trehalose, choline metabolites and nucleosides/nucleotides compared with BPH feeding on the susceptible plants. Decreased levels of BPH metabolites at the early feeding probably resulted from less BPH uptakes of sap from resistant plants and recovery of BPH metabolites at the later stage probably resulted from their adaptation to the adverse environment with their increased hopping frequency to ingest more sap together with contributions from yeast‐like symbionts in BPHs. Throughout 96 h, BPH feeding on the resistant plants showed significant up‐regulation of chitin synthase catalysing biosynthesis of chitin for insect exoskeleton, peritrophic membrane lining gut and tracheae. These findings provided useful metabolic information for understanding the BPH–rice interactions and perhaps for developing new BPH‐combating strategies.     

Lipid profiles reveal different responses to brown planthopper infestation for pest susceptible and resistant rice plants

Introduction

Brown planthopper (BPH) is the most destructive insect pest for rice, causing major reductions in rice yield and large economic losses. More than 31 BPH-resistance genes have been located, and several of them have been isolated. Nevertheless, the metabolic mechanism related to BPH-resistance genes remain uncharacterized.

Objectives

To elucidate the resistance mechanism of the BPH-resistance gene Bph6 at the metabolic level, a Bph6-transgenic line R6 (BPH-resistant) and the wild-type Nipponbare (BPH-susceptible) were used to investigate their lipid profiles under control and BPH treatments.

Methods

In conjunction with multivariate statistical analysis and quantitative real-time PCR, BPH-induced lipid changes in leaf blade and leaf sheath were investigated by GC–MS-based lipidomics.

Results

Forty-five lipids were identified in leaf sheath extracts. Leaf sheath lipidomics analysis results show that BPH infestation induces significant differences in the lipid profiles of Nipponbare and R6. The levels of hexadecanoic acid, methyl ester, linoleic acid, methyl ester, linolenic acid, methyl ester, glycidyl palmitate, eicosanoic acid, methyl ester, docosanoic acid, methyl ester, beta-monolinolein, campesterol, beta-sitosterol, cycloartenol, phytol and phytyl acetate had undergone enormous changes after BPH feeding. These results illustrate that BPH feeding enhances sterol biosynthetic pathway in Nipponbare plants, and strengthens wax biosynthesis and phytol metabolism in R6 plants. The results of quantitative real-time PCR of 5 relevant genes were consistent with the changes in metabolic level. Forty-five lipids were identified in the leaf blade extracts. BPH infestation induces distinct changes in the lipid profiles of the leaf blade samples of Nipponbare and R6. Although the lipid changes in Nipponbare are more drastic, the changes within the two varieties are similar. Lipid profiles in leaf sheath brought out significant differences than in leaf blade within Nipponbare and R6. We propose that Bph6 mainly affects the levels of lipids in leaf sheath, and mediates resistance by deploying metabolic re-programming during BPH feeding.

Conclusion

The results indicate that wax biosynthesis, sterol biosynthetic pathway and phytol metabolism play vital roles in rice response to BPH infestation. This finding demonstrated that the combination of lipidomics and quantitative real-time PCR is an effective approach to elucidating the interactions between brown planthopper and rice mediated by resistance genes.

     

Evaluation of tricin,a stylet probing stimulant of brown planthopper,in infested and non‐infested rice plants

The flavone, tricin (5,7,4′‐trihydroxy‐3′,5′‐dimethoxyflavone), is a valuable secondary metabolite that is common in gramineous plants, including cultivated rice (Oryza sativa). It can defend the rice plant against infestation by the brown planthopper (BPH), Nilaparvata lugens Stål, one of the most important pests of rice. This study evaluated the tricin concentration in infested and non‐infested rice plants. The results of the liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) quantitative analysis showed that the tricin concentration in rice leaves was significantly higher than in the stems and roots. The mass concentration of tricin in the leaves at the leaf stage was significantly higher than at the tiller and booting stages. The relationship between rice variety, BPH resistance and tricin concentration was investigated. There was a significant negative correlation between tricin concentration and the injury severity scores for rice varieties. Moreover, BPH infestation caused variations in tricin concentration among rice plants. High BPH infestation levels can significantly reduce the tricin concentration in rice plants. However, there is no significant effect of the length of infestation times on tricin concentrations in rice leaves. These results suggest that there may be an elicitor in BPH saliva, which is injected into rice plants during BPH infestation and triggers the tricin metabolic system. Future studies need to identify the elicitor and clarify the mechanism underlying tricin reduction in infested rice plants.     

不同浓度氯化钙浸种处理对水稻防御酶活性和抗褐飞虱的影响

[目的]研究不同浓度氯化钙(calcium chloride,CaCl2)浸种处理对水稻防御酶活性和抗褐飞虱Nilaparvata lugens的影响.[方法]分别用10,20,30,40和50 mmol/L CaCl2溶液浸泡水稻种子48 h,以蒸馏水浸种为对照,待水稻长至分蘖期时,检测褐飞虱3龄若虫取食胁迫下各浓度...     

Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes of Fusarium graminearum induced by Tri5 gene deletion

Fusarium graminearum (FG) is a serious plant pathogen causing huge losses in global production of wheat and other cereals. Tri5-gene encoded trichodiene synthase is the first key enzyme for biosynthesis of trichothecene mycotoxins in FG. To further our understandings of FG metabolism which is essential for developing novel strategies for controlling FG, we conducted a comprehensive investigation on the metabolic changes caused by Tri5-deletion by comparing metabolic differences between the wild-type FG5035 and an FG strain, Tri5(-), with Tri5 deleted. NMR methods identified more than 50 assigned fungal metabolites. Combined metabonomic and quantitative RT-PCR (qRT-PCR) analyses revealed that Tri5 deletion caused significant and comprehensive metabolic changes for FG apart from mycotoxin biosynthesis. These changes involved both carbon and nitrogen metabolisms including alterations in GABA shunt, TCA cycle, shikimate pathway, and metabolisms of lipids, amino acids, inositol, choline, pyrimidine, and purine. The hexose transporter has also been affected. These findings have shown that Tri5 gene deletion induces widespread changes in FG primary metabolism and demonstrated the combination of NMR-based metabonomics and qRT-PCR analyses as a useful way to understand the systems metabolic changes resulting from a single specific gene knockout in an eukaryotic genome and thus Tri5 gene functions.     

Nitric oxide production is associated with response to brown planthopper infestation in rice

Nilaparvata lugens Stål, the brown planthopper (BPH), is one of the most destructive phloem-feeding insects of rice (Oryza sativa L.) throughout Asia. Here, we show that BPH feeding increases the level of endogenous nitric oxide (NO) in the leaf and sheath tissue of both resistant and susceptible rice cultivars. However, in the roots, the NO level increased in the resistant cultivar, but decreased in the susceptible one. A burst of NO production occurred in the sheath within 1 h of infestation with BPH. The production of NO in response to BPH feeding appears to be dependent primarily on the activity of nitric oxide synthase. The application of exogenous NO reduced plant water loss by its effect on both stomatal opening and root architecture. It also stimulated the expression of certain drought stress-related genes, reduced plant height and delayed leaf senescence. Over the short term, NO supplementation reduced the seedling mortality caused by BPH feeding. This suggests that NO signaling plays a role in the rice tolerance response to BPH feeding.     

Role of hydroperoxide lyase in white-backed planthopper (Sogatella furcifera Horváth)-induced resistance to bacterial blight in rice, Oryza sativa L.

A pre-infestation of the white-backed planthopper (WBPH), Sogatella furcifera Horváth, conferred resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae ( Xoo ) in rice ( Oryza sativa  L.) under both laboratory and field conditions. The infestation of another planthopper species, the brown planthopper (BPH) Nilaparvata lugens Stål, did not significantly reduce the incidence of bacterial blight symptoms. A large-scale screening using a rice DNA microarray and quantitative RT-PCR revealed that WBPH infestation caused the upregulation of more defence-related genes than did BPH infestation. Hydroperoxide lyase 2 ( OsHPL2 ), an enzyme for producing C₆ volatiles, was upregulated by WBPH infestation, but not by BPH infestation. One C₆ volatile, ( E )-2-hexenal, accumulated in rice after WBPH infestation, but not after BPH infestation. A direct application of ( E )-2-hexenal to a liquid culture of Xoo inhibited the growth of the bacterium. Furthermore, a vapour treatment of rice plants with ( E )-2-hexenal induced resistance to bacterial blight. OsHPL2 -overexpressing transgenic rice plants exhibited increased resistance to bacterial blight. Based on these data, we conclude that OsHPL2 and its derived ( E )-2-hexenal play some role in WBPH-induced resistance in rice.     

水稻对褐飞虱抗性相关蛋白的双向电泳分析

以药用野生稻(Oryza officinalis)的转育后代B5(高抗褐飞虱(Nilaparvata lugens Stl))与感虫品种明恢63 (Oryza sativa L.)为亲本,构建了一个重组自交系群体.通过抗褐飞虱鉴定,筛选出极端抗虫株系和极端感虫株系,运用分群分析法(bulked segregant analysis,BSA)分别建成了极端抗虫集团(resistant bulk)和极端感虫集团(susceptible bulk)的蛋白质池.利用双向电泳技术,分别分析了极端抗虫集团和极端感虫集团受虫害与未受虫害的秧苗蛋白质的变化.结果发现,虫害48 h后,感虫集团的一个分子量为40 kD的蛋白质P40 (pI=6.3)的表达明显减弱甚至消失,而在抗虫集团中,P40的表达未受影响.与褐飞虱为害后抗虫株系和感虫株系不同的生理反应相联系,推测P40与水稻受褐飞虱虫害后引起的应答反应相关.     

水稻对褐飞虱抗性相关蛋白的双向电泳分析

以药用野生稻 (Oryzaofficinalis)的转育后代B5 (高抗褐飞虱 (NilaparvatalugensSt l) )与感虫品种明恢 6 3(OryzasativaL .)为亲本 ,构建了一个重组自交系群体。通过抗褐飞虱鉴定 ,筛选出极端抗虫株系和极端感虫株系 ,运用分群分析法 (bulkedsegregantanalysis ,BSA)分别建成了极端抗虫集团 (resistantbulk)和极端感虫集团 (susceptiblebulk)的蛋白质池。利用双向电泳技术 ,分别分析了极端抗虫集团和极端感虫集团受虫害与未受虫害的秧苗蛋白质的变化。结果发现 ,虫害 48h后 ,感虫集团的一个分子量为 40kD的蛋白质P40 (pI=6 .3)的表达明显减弱甚至消失 ,而在抗虫集团中 ,P40的表达未受影响。与褐飞虱为害后抗虫株系和感虫株系不同的生理反应相联系 ,推测P40与水稻受褐飞虱虫害后引起的应答反应相关     

Feeding‐induced changes in defence enzymes and PR proteins and their implications in host resistance to Nilaparvata lugens

Six rice genotypes showing susceptible and resistant reactions to brown planthopper (BPH), Nilaparvata lugens were studied for feeding‐induced changes in defence enzymes and pathogenesis‐related (PR) proteins. The high resistant genotypes PTB 33, ADT 45 and ASD 7 and moderately resistant genotypes CO 43 and KAU 1661 recorded the greater expression of defence enzymes peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, total phenol and β‐1,3 glucanase in response to N. lugens feeding at 1 day after infestation (DAI) compared with susceptible genotype TN1. The greater activity of chitinase was observed in resistant cultivars at 3 DAI and the activity was sustained for more than 1 week compared with susceptible TN1. In conclusion, the current study revealed that these defence enzymes and PR proteins might attribute to the resistance mechanisms in rice plants against BPH infestation.     

褐飞虱和白背飞虱的取食为害对水稻营养生长的影响

对塑料钵栽培的水稻进行罩宠试验,研究了褐飞虱和白背飞虱在不同若虫密度下取食为害对水稻营养生长的影响．结果表明,两种飞虱的成虫干重、水稻叶面积和其地上部干重因若虫密度的增加而下降．叶片干重占地上部干重的比例和稻株分配给叶片干物质量随为害程度的加重而增大;褐飞虱和白背飞虱总干重（X）与稻株地上部损失量（Y）之间存在着极显著的线性关系．两种飞虱干重每增加1mg,水稻地上部干重则分别损失26.01mg和21．90mg．讨论了稻飞虱取食为害对水稻致害的可能机制．     

Penetration into rice tissues by brown planthopper and fine structure of the salivary sheaths

The fine structure of the salivary sheaths in plant tissues can provide important information on homopteran probing and ingestion behaviors. Salivary sheaths secreted by the brown planthopper (BPH), Nilaparvata lugens (Stål) (Homoptera: Delphacidae), and their tissue pathway were investigated using light, scanning electron, and transmission electron microscopy. About half of the salivary flanges on the surface of the food substrate were connected with internal salivary sheaths. Only 43% of the salivary sheaths showed side branches. Many sculpture‐like protuberances and small cavities had been formed on the outer surface of the salivary sheath, but the sheath lumen circumferences were sealed. Brown planthoppers showed a preference for probing and leaving salivary sheaths in the susceptible rice variety TN1 rather than in the resistant variety B5 during the first 2 days of the experiments. The salivary sheaths in rice tissues reached the inner tissue layer of the leaf sheaths and stems, but were mostly observed to end in the first and second layer of the leaf sheaths. Brown planthoppers also preferred to probe into the thick segment of the outer leaf sheath. After ingestion by the insect, the cytoplasm in both phloem and companion cells degraded and the main organelles were lost. Numerous small vesicles were found in most of the phloem cells, but cell walls remained intact. Large numbers of symbiont‐like structures were observed inside the salivary sheath lumen. These results indicated that BPH has complicated feeding behaviors, which warrants further investigation.     

Jasmonic acid carboxyl methyltransferase regulates development and herbivory‐induced defense response in rice

Jasmonic acid(JA) and related metabolites play a key role in plant defense and growth. JA carboxyl methyltransferase(JMT) may be involved in plant defense and development by methylating JA to methyl jasmonate(Me JA) and thus influencing the concentrations of JA and related metabolites. However, no JMT gene has been well characterized in monocotyledon defense and development at the molecular level. After we cloned a rice JMT gene,Os JMT1, whose encoding protein was localized in the cytosol, we found that the recombinant Os JMT1 protein catalyzed JA to Me JA. Os JMT1 is up-regulated in response to infestation with the brown planthopper(BPH; Nilaparvata lugens). Plants in which Os JMT1 had been overexpressed(oeJMT plants) showed reduced height and yield. These oe-JMT plants also exhibited increased Me JA levels but reduced levels of herbivore-induced JA and jasmonoyl-isoleucine(JAIle). The oe-JMT plants were more attractive to BPH female adults but showed increased resistance to BPH nymphs,probably owing to the different responses of BPH female adults and nymphs to the changes in levels of H_2O_2 and Me JA in oe-JMT plants. These results indicate that Os JMT1,by altering levels of JA and related metabolites, plays a role in regulating plant development and herbivore-induced defense responses in rice.     

小粒野生稻导入系对稻飞虱的抗性鉴定与分析

稻飞虱是水稻生产最严重的害虫之一。野生稻拥有丰富的抗虫基因资源,导入系是鉴定和利用野生稻有利基因的有效途径。本研究通过对371份小粒野生稻导入系进行抗褐飞虱和白背飞虱接虫鉴定,分别筛选出了11份抗、72份中抗褐飞虱的材料和7份抗、45份中抗白背飞虱的材料,其中有5份材料兼抗褐飞虱和白背飞虱,这是从小粒野生稻中鉴定出抗白背飞虱材料的首次报道。通过对2份抗性导入系材料与感虫亲本杂交构建的F1和F2群体的抗虫鉴定和分析表明:K41对褐飞虱和白背飞虱的抗性受2对显性抗虫基因通过互补作用所控制;P114对褐飞虱和白背飞虱的抗性都是由1对主效的隐性基因控制。这些结果必将有利于小粒野生稻抗稻飞虱的基因定位和育种利用。