基于广泛靶向代谢组学分析黑老虎不同部位成分差异

为提高黑老虎(Kadsura coccinea)资源的综合利用率，采用广泛靶向代谢组学技术鉴定并分析了根、茎、叶代谢组分差异及高度富集成分。结果表明，在根、茎和叶中分别鉴定出642、650和619个代谢物，以酚酸、脂质、类黄酮和有机酸为主；叶与根、茎与根的共有成分分别为566和650个，显著差异成分有442和393个，主要为酚酸、类黄酮和脂质，差异代谢物在苯丙烷生物合成、黄酮与黄酮醇生物合成通路中显著富集。代谢物总丰度和次生代谢物丰度均表现为叶>根>茎，叶中酚酸、类黄酮和脂质及茎中酚酸积累量显著高于根，而氨基酸及其衍生物、萜类、木脂素、香豆素、生物碱的丰度在根中显著上调。因此，黑老虎根、茎、叶有大量共有成分，叶和茎中酚酸、叶中类黄酮和脂质高度富集，含有新绿原酸、绿原酸、槲皮素等多个丰度较高且具有重要生物活性化合物，具有较高利用价值。     

Metabolic changes in transgenic maize mature seeds over-expressing the <Emphasis Type="Italic">Aspergillus niger phyA2</Emphasis>

Key message

Non-targeted metabolomics analysis revealed only intended metabolic changes in transgenic maize over-expressing the Aspergillus niger phyA2.

Abstract

Genetically modified (GM) crops account for a large proportion of modern agriculture worldwide, raising increasingly the public concerns of safety. Generally, according to substantial equivalence principle, if a GM crop is demonstrated to be equivalently safe to its conventional species, it is supposed to be safe. In this study, taking the advantage of an established non-target metabolomic profiling platform based on the combination of UPLC-MS/MS with GC–MS, we compared the mature seed metabolic changes in transgenic maize over-expressing the Aspergillus niger phyA2 with its non-transgenic counterpart and other 14 conventional maize lines. In total, levels of nine out of identified 210 metabolites were significantly changed in transgenic maize as compared with its non-transgenic counterpart, and the number of significantly altered metabolites was reduced to only four when the natural variations were taken into consideration. Notably, those four metabolites were all associated with targeted engineering pathway. Our results indicated that although both intended and non-intended metabolic changes occurred in the mature seeds of this GM maize event, only intended metabolic pathway was found to be out of the range of the natural metabolic variation in the metabolome of the transgenic maize. Therefore, only when natural metabolic variation was taken into account, could non-targeted metabolomics provide reliable objective compositional substantial equivalence analysis on GM crops.

     

Extreme rates and heterogeneity in insect DNA evolution

Summary DNA-DNA hybridization studies of insects, more specificallyDrosophila and cave crickets, have revealed interesting patterns of genome evolution that contrast markedly with what has been seen in other taxa, especially mammals and birds. Insect genomes are composed of sections of single-copy DNA with extreme variation in rates of evolutionary change. This variation is more extreme than between introns and exons; introns fall into the relatively conserved fraction of the genome. Attempts to calculate absolute rates of change inDrosophila DNA have all led to estimates some 5–10 times faster than those found in most vertebrates; this is true even for the more conservative part of the nuclear genome. Finally we point out that morphological similarity, chromosomal similarity, and/or ability to form interspecific hybrids is often associated with quite high levels of single-copy DNA divergence in insects as compared to mammals and birds.     

Does past evolutionary history under different mating regimes influence the demographic dynamics of interspecific competition?

Interspecific interactions are contingent upon organism phenotypes, and thus phenotypic evolution can modify interspecific interactions and affect ecological dynamics. Recent studies have suggested that male–male competition within a species selects for capability to reproductively interfere with a closely related species. Here, we examine the effect of past evolutionary history under different mating regimes on the demographic dynamics of interspecific competition in Callosobruchus seed beetles. We used previously established experimental evolution lines of Callosobruchus chinensis that evolved under either forced lifelong monogamy or polygamy for 17 generations, and examined the demographic dynamics of competition between these C. chinensis lines and a congener, Callosobruchus maculatus. Callosobruchus chinensis was competitively excluded by C. maculatus in all trials. Time series data analyses suggested that reproductive interference from C. chinensis was relatively more important in the trials involving polygamous C. chinensis than those involving monogamous C. chinensis, in accordance with the potentially higher reproductive interference capability of polygamous C. chinensis. However, the estimated signs and magnitudes of interspecific interactions were not fully consistent with this explanation, implying the evolution of not only reproductive interference but also other interaction mechanisms. Our study thus suggests multifaceted effects of sexually selected traits on interspecific competitive dynamics.     

Metabolomics approach to investigate phytotoxic effects of Wedelia trilobata leaves,litter and soil

Although release and accumulation of plant metabolites from plant into soil can influence allelopathy, little information is known about metabolite changes that occur in leaf, litter and soil. In this study, seed germination bioassay tests and metabolomics analysis were performed to investigate the phytotoxic effects and metabolic variations (measured as buckets) in the ethanolic extracts of leaf, leaf litter and soil of Wedelia trilobata. Increasing the ethanolic extracts concentration of all extracts significantly inhibited Lactuca sativa germination rate (GR), shoot height (SH) and root length (RL). Soil exerted the strongest inhibition but contained the lowest number of buckets relative to those of leaf and leaf litter extracts. An overlap overview on the metabolome revealed a poor bucket overlap and redundancy among the leaf, leaf litter and soil extracts. Canonical correspondence analysis concluded that the SH of L. sativa was more sensitive to leaf litter extract and the leaf extract exerted a strong influence on the GR and RL of L. sativa. Multivariate analysis suggested that the metabolome of the leaf, leaf litter and soil differ substantially. Finally, putative identification using MS/MS data demonstrated various plant metabolites with phytotoxic effects that can contribute to the allelopathy of W. trilobata.     

Comparative analysis of metabolome of rice seeds at three developmental stages using a recombinant inbred line population

Plants are considered an important food and nutrition source for humans. Despite advances in plant seed metabolomics, knowledge about the genetic and molecular bases of rice seed metabolomes at different developmental stages is still limited. Here, using Zhenshan 97 (ZS97) and Minghui 63 (MH63), we performed a widely targeted metabolic profiling in seeds during grain filling, mature seeds and germinating seeds. The diversity between MH63 and ZS97 was characterized in terms of the content of metabolites and the metabolic shifting across developmental stages. Taking advantage of the ultra‐high‐density genetic map of a population of 210 recombinant inbred lines (RILs) derived from a cross between ZS97 and MH63, we identified 4681 putative metabolic quantitative trait loci (mQTLs) in seeds across the three stages. Further analysis of the mQTLs for the codetected metabolites across the three stages revealed that the genetic regulation of metabolite accumulation was closely related to developmental stage. Using in silico analyses, we characterized 35 candidate genes responsible for 30 structurally identified or annotated compounds, among which LOC_Os07g04970 and LOC_Os06g03990 were identified to be responsible for feruloylserotonin and l ‐asparagine content variation across populations, respectively. Metabolite?agronomic trait association and colocation between mQTLs and phenotypic quantitative trait loci (pQTLs) revealed the complexity of the metabolite?agronomic trait relationship and the corresponding genetic basis.     

Evolutionary relationship of plant catalase genes inferred from exon-intron structures: isozyme divergence after the separation of monocots and dicots

In order to understand the molecular evolution of catalase genes in higher plants, we compared the exon-intron structures of 12 genomic sequences from six plant species. It was assumed that the putative single primordial catalase gene had seven introns, because only those catalase genes having this structure are found in the monocotyledonae and dicotyledonae classes. After the evolutionary divergence of monocots from dicots, consecutive duplication of the primordial gene followed by the differential loss of introns occurred in each class to form three (or possibly four in dicots) diverse isozyme genes. In monocots, three ancestral isozyme genes were formed before the divergence of ancestral rice and maize. One of the rice genes, CatA, has an entirely new short intron which was not found in any other plant catalase gene examined. We have investigated the existence of the intron in the CatA homolog in other rice species by polymerase chain reaction (PCR) analysis. One major PCR product was found with the genomic DNAs from O. sativa (indica and japonica types), O. rufipogon and O. glaberrima. DNAs from several accessions of O. longistaminata showed variation in both the number and size of the DNA fragments amplified. PCR analyses and sequencing of the PCR products revealed that there are several CatA homologs having different sequences in some accessions of O. longistaminata. We have extended our study to other species in the Poaceae. The results suggest that the gain of the intron, most likely by insertion of a retroposon, took place in the ancestral genome of rice after its evolutionary divergence from other ancestral cereals such as barley, wheat and oat. Received: 20 November 1997 / Accepted: 5 January 1998     

Revealing different systems responses to brown planthopper infestation for pest susceptible and resistant rice plants with the combined metabonomic and gene-expression analysis

Brown planthopper (BPH) is a notorious pest of rice plants attacking leaf sheaths and seriously affecting global rice production. However, how rice plants respond against BPH remains to be fully understood. To understand systems metabolic responses of rice plants to BPH infestation, we analyzed BPH-induced metabolic changes in leaf sheaths of both BPH-susceptible and resistant rice varieties using NMR-based metabonomics and measured expression changes of 10 relevant genes using quantitative real-time PCR. Our results showed that rice metabonome was dominated by more than 30 metabolites including sugars, organic acids, amino acids, and choline metabolites. BPH infestation caused profound metabolic changes for both BPH-susceptible and resistant rice plants involving transamination, GABA shunt, TCA cycle, gluconeogenesis/glycolysis, pentose phosphate pathway, and secondary metabolisms. BPH infestation caused more drastic overall metabolic changes for BPH-susceptible variety and more marked up-regulations for key genes regulating GABA shunt and biosynthesis of secondary metabolites for BPH-resistant variety. Such observations indicated that activation of GABA shunt and shikimate-mediated secondary metabolisms was vital for rice plants to resist BPH infestation. These findings filled the gap of our understandings in the mechanistic aspects of BPH resistance for rice plants and demonstrated the combined metabonomic and qRT-PCR analysis as an effective approach for understanding plant-herbivore interactions.     

Climatic adaptation and ecological divergence between two closely related pine species in Southeast China

Climate is one of the most important drivers for adaptive evolution in forest trees. Climatic selection contributes greatly to local adaptation and intraspecific differentiation, but this kind of selection could also have promoted interspecific divergence through ecological speciation. To test this hypothesis, we examined intra‐ and interspecific genetic variation at 25 climate‐related candidate genes and 12 reference loci in two closely related pine species, Pinus massoniana Lamb. and Pinus hwangshanensis Hisa, using population genetic and landscape genetic approaches. These two species occur in Southeast China but have contrasting ecological preferences in terms of several environmental variables, notably altitude, although hybrids form where their distributions overlap. One or more robust tests detected signals of recent and/or ancient selection at two‐thirds (17) of the 25 candidate genes, at varying evolutionary timescales, but only three of the 12 reference loci. The signals of recent selection were species specific, but signals of ancient selection were mostly shared by the two species likely because of the shared evolutionary history. F_ST outlier analysis identified six SNPs in five climate‐related candidate genes under divergent selection between the two species. In addition, a total of 24 candidate SNPs representing nine candidate genes showed significant correlation with altitudinal divergence in the two species based on the covariance matrix of population history derived from reference SNPs. Genetic differentiation between these two species was higher at the candidate genes than at the reference loci. Moreover, analysis using the isolation‐with‐migration model indicated that gene flow between the species has been more restricted for climate‐related candidate genes than the reference loci, in both directions. Taken together, our results suggest that species‐specific and divergent climatic selection at the candidate genes might have counteracted interspecific gene flow and played a key role in the ecological divergence of these two closely related pine species.     

Dissection of genotype-phenotype associations in rice grains using metabolome quantitative trait loci analysis

A comprehensive and large‐scale metabolome quantitative trait loci (mQTL) analysis was performed to investigate the genetic backgrounds associated with metabolic phenotypes in rice grains. The metabolome dataset consisted of 759 metabolite signals obtained from the grains of 85 lines of rice (Oryza sativa, Sasanishiki × Habataki back‐crossed inbred lines). Metabolome analysis was performed using four mass spectrometry pipelines to enhance detection of different classes of metabolites. This mQTL analysis of a wide range of metabolites highlighted an uneven distribution of 802 mQTLs on the rice genome, as well as different modes of metabolic trait (m‐trait) control among various types of metabolites. The levels of most metabolites within rice grains were highly sensitive to environmental factors, but only weakly associated with mQTLs. Coordinated control was observed for several groups of metabolites, such as amino acids linked to the mQTL hotspot on chromosome 3. For flavonoids, m‐trait variation among the experimental lines was tightly governed by genetic factors that alter the glycosylation of flavones. Many loci affecting levels of metabolites were detected by QTL analysis, and plausible gene candidates were evaluated by in silico analysis. Several mQTLs profoundly influenced metabolite levels, providing insight into the control of rice metabolism. The genomic region and genes potentially responsible for the biosynthesis of apigenin‐6,8‐di‐C‐α‐l‐ arabinoside are presented as an example of a critical mQTL identified by the analysis.     

Interspecific plant functional variation prevails over intraspecific variation in driving spider beta diversity

1. Non-trophic interactions between plants and animals can affect community structure and species trait composition. However, it is unclear how changes in intra- and interspecific morphological traits of plant species affect non-trophic interactions at a metacommunity scale. Additionally, whether plant evolutionary history determines taxonomic and functional diversity of plant-dwelling predators is an open question. 2. To address these gaps, this study used a published dataset with spiders dwelling exclusively on bromeliads to investigate if: (i) intra- and interspecific variability in host plant morphological traits affects spider taxonomic and functional diversity; and (ii) bromeliad trait evolution determines present-day patterns of spider trait diversity. 3. Spider and bromeliad traits were measured, and a new statistical framework was used to quantify the response of spider beta diversity to intra- and interspecific variation in bromeliad traits. In addition, bromeliad traits were decomposed across its phylogenetic tree to check whether the current variation in morphological traits of bromeliads is a result of either ancestral or recent diversification. 4. Bromeliad intraspecific variation did not affect spiders, but leaf length variation between bromeliad species had a positive effect on spider functional beta diversity. Interestingly, the most ancestral split between two subfamilies explained most of the variation in bromeliad species, which suggests that spider functional diversity could represent an outcome of bromeliad evolutionary history. 5. Overall, the results of this study suggest that interactions between plants and organisms that do not feed directly on their tissues could be shaped by plant evolutionary history, which in turn suggests that non-trophic interactions can be maintained over time.     

GENETIC ARCHITECTURE OF METABOLIC RATE: ENVIRONMENT SPECIFIC EPISTASIS BETWEEN MITOCHONDRIAL AND NUCLEAR GENES IN AN INSECT

The extent to which mitochondrial DNA (mtDNA) variation is involved in adaptive evolutionary change is currently being reevaluated. In particular, emerging evidence suggests that mtDNA genes coevolve with the nuclear genes with which they interact to form the energy producing enzyme complexes in the mitochondria. This suggests that intergenomic epistasis between mitochondrial and nuclear genes may affect whole‐organism metabolic phenotypes. Here, we use crossed combinations of mitochondrial and nuclear lineages of the seed beetle Callosobruchus maculatus and assay metabolic rate under two different temperature regimes. Metabolic rate was affected by an interaction between the mitochondrial and nuclear lineages and the temperature regime. Sequence data suggests that mitochondrial genetic variation has a role in determining the outcome of this interaction. Our genetic dissection of metabolic rate reveals a high level of complexity, encompassing genetic interactions over two genomes, and genotype × genotype × environment interactions. The evolutionary implications of these results are twofold. First, because metabolic rate is at the root of life histories, our results provide insights into the complexity of life‐history evolution in general, and thermal adaptation in particular. Second, our results suggest a mechanism that could contribute to the maintenance of nonneutral mtDNA polymorphism.     

Compartmentalization of specialized metabolites across vegetative and reproductive tissues in two sympatric Psychotria species

Premise

The specialized metabolites of plants are recognized as key chemical traits in mediating the ecology and evolution of sundry plant–biotic interactions, from pollination to seed predation. Intra- and interspecific patterns of specialized metabolite diversity have been studied extensively in leaves, but the diverse biotic interactions that contribute to specialized metabolite diversity encompass all plant organs. Focusing on two species of Psychotria shrubs, we investigated and compared patterns of specialized metabolite diversity in leaves and fruit with respect to each organ's diversity of biotic interactions.

Methods

To evaluate associations between biotic interaction diversity and specialized metabolite diversity, we combined UPLC-MS metabolomic analysis of foliar and fruit specialized metabolites with existing surveys of leaf- and fruit-centered biotic interactions. We compared patterns of specialized metabolite richness and variance among vegetative and reproductive tissues, among plants, and between species.

Results

In our study system, leaves interact with a far larger number of consumer species than do fruit, while fruit-centric interactions are more ecologically diverse in that they involve antagonistic and mutualistic consumers. This aspect of fruit-centric interactions was reflected in specialized metabolite richness—leaves contained more than fruit, while each organ contained over 200 organ-specific specialized metabolites. Within each species, leaf- and fruit-specialized metabolite composition varied independently of one another across individual plants. Contrasts in specialized metabolite composition were stronger between organs than between species.

Conclusions

As ecologically disparate plant organs with organ-specific specialized metabolite traits, leaves and fruit can each contribute to the tremendous overall diversity of plant specialized metabolites.     

Metabolic Characterization of the Common Marmoset (Callithrix jacchus)

High-resolution metabolomics has created opportunity to integrate nutrition and metabolism into genetic studies to improve understanding of the diverse radiation of primate species. At present, however, there is very little information to help guide experimental design for study of wild populations. In a previous non-targeted metabolomics study of common marmosets (Callithrix jacchus), Rhesus macaques, humans, and four non-primate mammalian species, we found that essential amino acids (AA) and other central metabolites had interspecies variation similar to intraspecies variation while non-essential AA, environmental chemicals and catabolic waste products had greater interspecies variation. The present study was designed to test whether 55 plasma metabolites, including both nutritionally essential and non-essential metabolites and catabolic products, differ in concentration in common marmosets and humans. Significant differences were present for more than half of the metabolites analyzed and included AA, vitamins and central lipid metabolites, as well as for catabolic products of AA, nucleotides, energy metabolism and heme. Three environmental chemicals were present at low nanomolar concentrations but did not differ between species. Sex and age differences in marmosets were present for AA and nucleotide metabolism and warrant additional study. Overall, the results suggest that quantitative, targeted metabolomics can provide a useful complement to non-targeted metabolomics for studies of diet and environment interactions in primate evolution.     

Correlated evolution of fruit and leaf size in bird‐dispersed plants: species‐level variance in fruit traits explained a bit further?

The astounding morphological diversity exhibited by the fruits of vertebrate-dispersed plants has been traditionally interpreted as the adaptive outcome of divergent selective pressures exerted on plants by the broad array of frugivorous animals involved in seed dispersal. Although the selective capacity of frugivores provides support to this interpretation, recent studies have challenged it by documenting a strong phylogenetic component associated to interspecific variation in most fruit characteristics. Size-related fruit traits provide a conspicuous exception to this pattern, because they exhibit considerable variation at the between-species level which is largely independent of phylogeny and is correlated with consumption by differently-sized dispersal agents. Substantial species-level variance in size-related traits may reflect genuine disperser-driven diversification, but may also be partly influenced by correlated evolution of fruit size with the size of other plant structures. This latter possibility is tested here for bird-dispersed plants of the Iberian Peninsula using phylogenetically independent contrasts. Results demonstrate the existence of correlated evolution of fruit and leaf size at the species level. As all the plant taxa considered have their fruits eaten, and seeds dispersed, by the same relatively reduced set of frugivorous bird species, results suggest that a significant fraction of the variation in fruit size represented in the species sample may be explained as an indirect consequence of variation in leaf size, rather than being associated with adaptive divergence related to seed dispersal agents.     

Comparative genomic analysis of the Sm gene family in rice and maize

Sm proteins are a group of ubiquitous ring-shaped oligomers that function in multiple aspects of RNA metabolism. However, until this study, no comprehensive study incorporating phylogeny, chromosomal location, gene organization, adaptive evolution, expression profiling and functional networks has been reported for rice and maize. In this study, twenty-five and thirty-three Sm genes have been identified in rice and maize, respectively. Phylogenetic analyses identified eighteen gene groups. Results by gene locations indicated that segmental duplication contributes to the expansion of this gene family in rice and maize. Gene organization and motif compositions of the Sm members are highly conserved in each group, indicative of their functional conservation. Expression profiles have provided insights into the possible functional divergence among members of the Sm gene family. Adaptive evolution analyses suggested that purifying selection was the main force driving Sm evolution, but some critical sites might be responsible for functional divergence. In addition, four hundred and seventy-nine interactions were identified by functional network analyses, and most of which were associated with binding, cellular macromolecule biosynthesis, pre-mRNA processing and transferase activity. Overall, the data contribute to a better understanding of the complexity of Sm gene family in rice and maize and will provide a solid foundation for future functional studies.     

The role of interspecific interference competition in character displacement and the evolution of competitor recognition

The extent to which interspecific interference competition has contributed to character evolution is one of the most neglected problems in evolutionary biology. When formerly allopatric species come into secondary contact, aggressive interactions between the species can cause selection on traits that affect interspecific encounter rates (e.g. habitat preferences, activity schedules), competitor recognition (e.g. colouration, song), and fighting ability (e.g. weaponry, body size). We define agonistic character displacement (ACD) as the process of phenotypic evolution in a population caused by interference competition with one or more sympatric species and which results in shifts in traits that affect the rate, intensity or outcome of interspecific aggression. After clarifying the relationships between ACD and other evolutionary processes that may occur when species come into secondary contact, we develop an individual‐based, quantitative genetic model to examine how traits involved in competitor recognition would be expected to evolve under different secondary contact scenarios. Our simulation results show that both divergence and convergence are possible outcomes, depending on the intensity of interspecific exploitative competition, the costs associated with mutual versus unilateral recognition, and the extent of phenotypic differences prior to secondary contact. We then devise a set of eight criteria for evaluating putative examples of ACD and review the empirical literature to assess the strength of existing evidence and to identify promising avenues for future research. Our literature search revealed 33 putative examples of ACD across insects, fishes, bats, birds, lizards, and amphibians (15 divergence examples; 18 convergence examples). Only one example satisfies all eight criteria for demonstrating ACD, but most case studies satisfy four or more criteria. The current state of the evidence for ACD is similar to the state of the evidence for ecological character displacement just 10 years ago. We conclude by offering suggestions for further theoretical and empirical research on ACD.     

Measuring the contribution of evolution to community trait structure in freshwater zooplankton

There are currently few predictions about when evolutionary processes are likely to play an important role in structuring community features. Determining predictors that indicate when evolution is expected to impact ecological processes in natural landscapes can help researchers identify eco-evolutionary ‘hotspots', where eco-evolutionary interactions are more likely to occur. Using data collected from a survey in freshwater cladoceran communities, landscape population genetic data and phenotypic trait data measured in a common garden, we applied a Bayesian linear model to assess whether the impact of local trait evolution in the keystone species Daphnia magna on cladoceran community trait values could be predicted by population genetic properties (within-population genetic diversity, genetic distance among populations), ecological properties (Simpson's diversity, phenotypic divergence) or environmental divergence. We found that the impact of local trait evolution varied among communities. Moreover, community diversity and phenotypic divergence were found to be better predictors of the contribution of evolution to community trait values than environmental features or genetic properties of the evolving species. Our results thus indicate the importance of ecological context for the impact of evolution on community features. Our study also demonstrates one way to detect signatures of eco-evolutionary interactions in communities inhabiting heterogeneous landscapes using survey data of contemporary ecological and evolutionary structure.     

A simultaneous extraction method for metabolome and lipidome and its application in cry1Ac and sck-transgenic rice leaf treated with insecticide based on LC–MS analysis

Abiotic stress caused by insecticide treatment is an interesting and challenging topic in plant research. Here a simultaneous extraction method with methyl tert-butyl ether for metabolomics and lipidomics analysis by using rapid resolution liquid chromatography quadrupole time-of-flight mass spectrometry was developed and validated. The extraction efficiency of lipidome and metabolome based on the current developed method showed an obvious improvement compared with literatures. About 300 metabolites were identified in rice leaf. Method validation was performed and analytical properties including the linearity (R², 0.991–1.000), repeatability (over 87 % peaks with CV < 20 % accounting for over 92 % of total response) and recovery (74.4–119.6 %) were satisfactory. The method was then applied to investigate time-course changes caused by insecticide treatment in transgenic rice with cry1Ac and sck genes and its wild counterpart. Antioxidants including ferulic acid and sinapic acid were down-regulated at 24 h after insecticide treatment. Signaling metabolites including salicylic acid and nicotinamide adenine dinucleotide were significantly up-regulated at 12–24 h after treatment in transgenic rice. More flavonoids were significantly changed in transgenic plants. Results indicated that gene transformation affected the metabolic response of rice to insecticide stress.