Combined application of Trichoderma harzianum SH2303 and difenoconazole-propiconazolein controlling Southern corn leaf blight disease caused by Cochliobolus heterostrophus in maize

Southern corn leaf blight(SCLB)disease caused by Cochliobolus heterostrophus is one of the major threats to corn production worldwide.The synergistic application of low toxic chemical fungicide and biocontrol agents could improve biocontrol stability and efficiency against plant diseases,which ultimately reduce use of chemical fungicide.Trichoderma spp.,well-known biocontrol fungi have been used to control some foliar diseases.However,few works have been reported on synergistic application of chemical fungicide and Trichoderma against foliar diseases.This study was aimed to investigate the control effect on the synergistic application of Trichoderma harzianum SH2303 and difenoconazole-propiconazole(DP)against SCLB.Results showed that the synergistic application of DP and SH2303 reduced the leaf spot area compared to the control.The efficacy of synergistic application of DP+SH2303 against SCLB could last for 15–20 d in pot trial under the greenhouse condition.Under the natural field condition,maize treated with DP+DP and DP+SH2303 showed 60%control,which was higher than that of SH2303+DP(45%)and SH2303+SH2303(35%).All these treatments induced the synthesis of defense-related enzymes(phenylalanine ammonia lyase(PAL),catalase(CAT),and superoxide dismutase(SOD))and the defence-related gene expression of SA pathway(PR1).Taken together the in-vitro leaf test and field trial,the control of SCLB by synergistic application of DP+SH2303 was similar to that of DP+DP.Among synergistic application,the sequential application of DP+SH2303 showed better control than the sequential application of SH2303+DP.It was concluded that the synergistic application of chemical fungicide(DP)and biocontrol agent(T.harzianum SH2303)could be used to reduce the chemical fungicide and to reduce the SCLB diseases in maize,which provided alternative approach to realize an eco-friendly controlling of the foliar disease.

Soil application of Trichoderma asperellum GDFS1009 granules promotes growth and resistance to Fusarium graminearum in maize

Of diseases affecting maize(Zea mays), Fusarium graminearum is one of the most common pathogenic fungi that cause stalk rot. In the present study, the Trichoderma asperellum GDFS1009 strain was shown to be an effective biocontrol agent against stalk rot. In a confrontation culture test, Trichoderma strain displayed an approximately 60% inhibition rate on the mycelial growth of F. graminearum. In pot trials, the application of 2 g/pot of T. asperellum GDFS1009 granules had the best control effect on stalk rot at the seedling stage(up to 53.7%), while the average plant height and fresh weight were also significantly improved. Additionally when fertilizer was added at 8 g/pot, the application of 3 g/pot of Trichoderma granules had the best control effect on maize stalk rot(40.95%). In field trials, when inoculating F. graminearum alone, the disease index for inoculating was 62.45, but only 31.43 after treatment with T. asperellum GDFS1009 granules, suggesting a control efficiency of 49.67%. Furthermore, in a naturally F. graminearum-infected field, Trichoderma granules, when applied for 3 consecutive years, showed significant control of stalk rot and increased yields.

Sod gene of Curvularia lunata is associated with the virulence in maize leaf

Curvularia leaf spot, caused mainly by Curvularia lunata, is a widespread plant disease in China. In the recent years, di- rectional host selection by the pathogen, which likely results in the virulence differentiation in pathogens, is widely reported. Among the hallmarks potentially associated to pathogen variation in virulence, superoxide dismutase gene Sod has been found to be closely related to the enhancement of virulence. In the present study, the full-length of Sod was obtained via Blastn alignment against GenBank and the whole genome of C. lunata. In order to understand the role of Sod in the vir- ulence variation in C. lunata, targeted gene disruption was performed to construct Sod mutants. The cell wall degrading enzyme （CWDE） activities and toxin production of ASod were not distinctly different from wild-type strain CX-3 and its complon. However, at an early stage of infection, 3Sod virulence appeared to be lower than CX-3 and the complon, while at a later stage, its virulence gradually returned to the level of CX-3 and the complon. Furthermore, the melanin production of ASod was significantly reduced compared to CX-3 and the complon, suggesting that Sod gene influences the virulence by regulating melanin production at an early stage of infection but is not essential for pathogenicity. However, the disruption of Sod did not significantly affect the transcriptional expression of the melanin biosynthesis-associated genes, bml and scd. Therefore, we infer that Sod in C. lunata are involved, to some extent, with the virulence in maize leaf, but still needs further studies to have a clear understanding of its mechanism.

Comparative Analysis of Gene Expression in Two Muskmelon Cultivars(Cucumis melo L.) Under Salt Stress

Salinity is one of the most important abiotic stresses that adversely affects crop growth and productivity. A subtractive suppression hybridization （SSH） library were constructed from the roots of salt-sensitive Yulu cultivar melon seedlings under salt stress; 557 high-quality expressed sequence tags （ESTs） were randomly sequenced, with an average size of 428 bp, which assembled into 68 contigs and 315 singletons. Compared with our previous SSH library generated from the salt-tolerant Bingxuecui cultivar, the proportion of transcripts involved in metabolism, protein fate, cellular communication/signal transduction mechanisms, and cell rescue/defense were 4, 1.46, 0.94, and 0.4% higher, respectively, in the salt-tolerant cultivar than the in salt-sensitive cultivar. Quantitative real-time PCR analysis of eleven transcripts revealed temporal variations in their expression in the two cultivars under salt stress. One NAC gene （JZ477011） was heterologously expressed in yeast for functional characterization, and enhanced the sensitivity of yeast cells to high-salinity to salt stress and inhibited their growth. Information regards to their functions would aid in the understanding of response mechanisms to saline stress and in the development of molecular markers for selecting salt-tolerant melon cultivars.

Increased artemisinin production by promoting glandular secretory trichome formation and reconstructing the artemisinin biosynthetic pathway in Artemisia annua

Dear Editor,Artemisinin,which has potent antimalarial properties,is a sesquiterpene endoperoxide originally isolated from the traditional Chinese medicinal plant Artemisia annua.However,the artemisinin content in wild-type(WT)A.annua is low(1-10 mg/g dry weight),leading to its erratic supply and price fluctuations[1].

Characteristics of carbon storage in Shanghai's urban forest

Urban forest has undergone rapid development in China over the last three decades because of the acceleration of urbanization.Urban forest thus plays an increasingly important role in carbon sequestration at a regional and national scale.As one of the most urbanized cities in China,Shanghai showed an increase of forest coverage from 3% in the 1990s to 13% in 2009.Based on CITY-green model and the second soil survey of Shanghai,the forest biomass carbon(FBC) was estimated to be 0.48 Tg in the urban area and,forest soil organic carbon(SOC)(0-100 cm soil depth) is 2.48 Tg in the urban and suburban areas,respectively.These values are relatively within the median and lower level compared with other Chinese megacities,with the FBC of 0.02 Tg in Harbin to 47.29 Tg in Chongqing and the forest SOC of 1.74 Tg in Nanjing to 418.67 Tg in Chongqing.For the different land-use types in Shanghai,the SOC density ranges from 13.8(tidal field) to 38.6 t ha-1(agricultural land).On average,the forest SOC density(31.5 t ha-1) in Shanghai is lower than that in agricultural lands(38.6 t ha-1) and higher than that in lawns(26.5 t ha-1) and gardens(21.3 t ha-1).In Shanghai,the SOC density in newly established urban parks is generally lower than that in older parks.In the northern and southeastern suburban areas(e.g.,Baoshan,Yangpu,and Nanhui districts),greenspace SOC density is higher than that in the central commercial areas(Hongkou,Putuo,Changning,and Zhabei districts) and in newly developed district(Pudong District).Uncertainties still exist in the estimation of urban forest carbon in Shanghai,as well as in other Chinese cities.Thus,future research directions are also discussed in this paper.

MAPKs and acetyl-CoA are associated with Curvularia lunata pathogenicity and toxin production in maize

Mitogen-activated protein kinase （MAPK） cascades play an important role in extracellular signal transduction and are involved in the pathogenicity of fungal pathogens to host plants. In Curvularia lunata, the roles of two MAPK genes, Clkl and CIm 1, have already been studied. Clkl is involved in conidia formation and pathogenicity, and Clmf is closely related to pathogen cell wall formation and pathogenicity to maize leaves. In this study, a third C. lunata MAPK gene, Clhl, which is homologous to hog1, was successfully cloned. We found that a Clhl deletion mutant had lower intracellular glycerol accumulation than the wild-type stain and was unable to grow normally under osmotic stress conditions. Furthermore, the deletion mutants of three C. lunata MAPK genes （Clkl, Clml and Clhl） had lower levels of acetyI-CoA, which is an important intermediate product in the synthesis of melanin and furan toxin, and down-regulated expression of pathogenicity-associated genes. Furthermore, pathogenicity and the ability to produce toxin were restored after adding acetyI-CoA to the culture medium, suggesting that acetyI-CoA is closely involved in the pathogen MAPK signaling pathway.

The Genome of Artemisia annua Provides Insight into the Evolution of Asteraceae Family and Artemisinin Biosynthesis

Artemisia annua, commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A. annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A. annua, which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A. annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemi- sinin biosynthetic pathway. We further revealed by transcriptome profiling that A. annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on compre- hensive genomic and transcriptomic analyses we generated transgenic A. annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the chal- lenge of increasing global demand of artemisinin.

Red-Light-Dependent Interaction of phyB with SPA1 Promotes COP1-SPA1 Dissociation and Photomorphogenic Development in Arabidopsis

Arabidopsis phytochromes （phyA-phyE） are photoreceptors dedicated to sensing red/far-red light. Phyto- chromes promote photomorphogenic developments upon light irradiation via a signaling pathway that involves rapid degradation of PIFs （PHYTOCHROME INTERACTING FACTORS） and suppression of COP1 （CONSTITUTIVE PHOTOMORPHOGENIC 1） nuclear accumulation, through physical interactions with PIFs and COP1, respectively. Both phyA and phyB, the two best characterized phytochromes, regulate plant photomorphogenesis predominantly under far-red light and red light, respectively. It has been demonstrated that SPA1 （SUPPRESSOR OF PHYTOCHROME A 1） associates with COP1 to promote COP1 activity and suppress photomorphogenesis. Here, we report that the mechanism underlying phyB- promoted photomorphogenesis in red light involves direct physical and functional interactions between red-light-activated phyB and SPA1. We found that SPA1 acts genetically downstream of PHYB to repress photomorphogenesis in red light. Protein interaction studies in both yeast and Arabidopsis demonstrated that the photoactivated phyB represses the association of SPA1 with COP1, which is mediated, at least in part, through red-light-dependent interaction of phyB with SPA1. Moreover, we show that phyA physically interacts with SPA1 in a Pfr-form-dependent manner, and that SPA1 acts downstream of PHYA to regulate photomorphogenesis in far-red light. This study provides a genetic and biochemical model of how photo- activated phyB represses the activity of COP1-SPA1 complex through direct interaction with SPA1 to promote photomorphogenesis in red light.

Effects of plant species coexistence on soil enzyme activities and soil microbial community structure under Cd and Pb combined pollution

The relationship between plant species coexistence and soil microbial communities under heavy metal pollution has attracted much attention in ecology. However, whether plant species coexistence could offset the impacts of heavy metal combined pollution on soil microbial community structure and soil enzymes activities is not well studied. The modified ecological dose model and PCR-RAPD method were used to assess the effects of two plant species coexistence on soil microbial community and enzymes activities subjected to Cd and Ph combined stress. The results indicated that monoculture and mixed culture would increased microbe populations under Cd and Pb combined stress, and the order of sensitivity of microbial community responding to heavy metal stress was： actinomycetes 〉 bacteria 〉 fungi. The respirations were significantly higher in planted soil than that in unplanted soil. The plant species coexistence could enhance soil enzyme activities under Cd and Pb combined. Furthermore, planted soil would be helpful to enhance soil genetic polymorphisms, but Cd and Pb pollution would cause a decrease on soil genetic polymorphisms. Mixed culture would increase the ecological dose 50% （ED50） values, and the ED50 values for soil enzyme activities decreased with increasing culture time. The dehydrogenase was most sensitive to metal addition and easily loses activity under low dose of heavy metal. However, it was difficult to fully inhibit the phoshpatase activity, and urease responded similarly with phosphatase.

Transcriptional regulation of artemisinin biosynthesis in Artemisia annua L.

Artemisinin, also known as qinghaosu, a sesquiterpene endoperoxide lactone isolated from the Chinese medicinal plant Artemisia annua L., is the most effective antimalarial drug which has saved millions of lives.Due to its great antimalarial activity and low content in wild A. annua plants, researches focused on enhancing the artemisin yield in plants became a hotspot. Several families of transcription factors have been reported to participate in regulating the biosynthesis and accumulation of artemisinin.In this review, we summarize recent investigations in these fields, with emphasis on newly identified transcription factors and their functions in artemisinin biosynthesis regulation, and provide new insight for further research.

Advances in fungal-assisted phytoremediation of heavy metals:A review

Trace metals such as manganese(Mn),copper(Cu),zinc(Zn),and iron(Fe)are essential for many biological processes in plant life cycles.However,in excess,they can be toxic and disrupt plant growth processes,which is economically undesirable for crop production.For this reason,processes such as homeostasis and transport control of these trace metals are of constant interest to scientists studying heavily contaminated habitats.Phytoremediation is a promising cleanup technology for soils polluted with heavy metals.However,this technique has some disadvantages,such as the slow growth rate of metal-accumulating plant species,low bioavailability of heavy metals,and long duration of remediation.Microbial-assisted phytoremediation is a promising strategy for hyperaccumulating,detoxifying,or remediating soil contaminants.Arbuscular mycorrhizal fungi(AMF)are found in association with almost all plants,contributing to their healthy performance and providing resistance against environmental stresses.They colonize plant roots and extend their hyphae to the rhizosphere region,assisting in mineral nutrient uptake and regulation of heavy metal acquisition.Endophytic fungi exist in every healthy plant tissue and provide enormous services to their host plants,including growth enhancement by nutrient acquisition,detoxification of heavy metals,secondary metabolite regulation,and enhancement of abiotic/biotic stress tolerance.The aim of the present work is to review the recent literature regarding the role of AMF and endophytic fungi in plant heavy metal tolerance in terms of its regulation in highly contaminated conditions.

The CNT1 Domain of Arabidopsis CRY1 Alone Is Sufficient to Mediate Blue Light Inhibition of Hypocotyl Elongation

Dear Editor Cryptochromes （CRY） are photolyase-like blue light receptors that mediate various light responses in plants and animals. InAra- bidopsis, there are two homologous cryptochromes, CRY1 and CRY2, which mainly regulate photomorphogenesis and photope- riodic flowering, respectively （Guo et al., 1998; Linet al., 1998）. Cryptochromes are structurally divided into an N-terminal domain related to the photolyase and a C-terminal extension domain （Yang et al., 2000; Cashmore, 2003）. The C-terminal domains of CRY1 and CRY2 （CCT1 [residues 490-681] and CCT2 [residues 486-612], also known as CCE1 and CCE2） are shown to mediate blue light signaling, whereas the N-terminal domains of CRY1 and CRY2 （CNT1 [residues 1-489] and CNT2 [residues 1-485]） are known to bind chromophores to sense blue light and mediate CRY dimerization （Yang et al., 2000; Brautigam et al., 2004; Sang et al., 2005）. Specifically, CRY1- mediated blue light signaling involves the interactions of CCT1 with COP1 and SPA1 （Wang et al., 2001; Yang et al., 2001; Lian et al., 2011; Liu et al., 2011）. CCT2 also mediates CRY2-COP1 interaction （Wang et al., 2001）. The functional region of CCT2 is further confined to the NC80 motif （residues 486-565）, which is proposed to function through similar mechanisms as for CCT2 （Yu et al., 2007）. Interestingly, CCT2 does not mediate CRY2-SPA1 interaction, which is mediated by CNT2 （Zuo et al., 2011）. CRY2 is also found to interact with a family of bHLH transcription factors, cryptochrome-interacting bHLH （CIBs） （Liu et al., 2008）, through a photolyase homology region （PHR,

Diversity and versatile functions of metallothioneins produced by plants:A review

Metal ions are essential for plant growth and development,but in excess,these compounds can become highly toxic.Plants have adopted numerous ways to maintain metal homeostasis while mitigating adverse effects of excess metal ions,including phytochelatin and the metal-chelating proteins metallothioneins(MTs).A family of cysteine(Cys)-rich,intracellular,and low-molecular-weight(4-8 kDa)MTs are proteins found in nearly all phyla including plants,animals,and fungi,and they have the potential to scavenge reactive oxygen species and detoxify toxic metals including copper,cadmium,and zinc.Based on their Cys numbers and residues,MTs have been categorized into three major classes.Class I MTs,which have highly conserved Cys residues,are found in animals,while class II MTs,with less conserved Cys residues,are present in plants and are classified further into four groups.Class III MTs include phytochelatins,a group of enzymatically synthesized Cys-rich proteins.The MTs have been an area of interest for five decades with extensive studies,which have been facilitated by advancements in instrumental techniques,protein science,and molecular biology tools.Here,we reviewed current advances in our understanding of the regulation of MT biosynthesis,their expression,and their potential roles in the alleviation of abiotic stresses(i.e.,drought,salinity,and oxidative stresses)and heavy metal detoxification and homeostasis.

Classification and comparison of physical and chemical properties of corn stalk from three regions in China

Corn stalk samples from Anhui,Jiangxi and Shanghai were used as test materials.Their physical,chemical and thermo-chemical engineering characteristics were analyzed.The similarities and differences in properties of corn stalk from the three regions were determined using SIMCA-P and SPSS software in order to obtain a proper energy utilization method of corn stalk.The results show that the corn stalk from Shanghai has significant differences from the samples of Jiangxi and Anhui.In particular,the following properties of corn stalk from Shanghai such as the contents of cellulose,calcium(Ca),iron(Fe),crude ash,volatile matter,carbon(C),nitrogen(N),and oxygen(O)are significantly different from those of Jiangxi and Anhui samples(P<0.05).While other properties such as the contents of magnesium(Mg),copper(Cu),zinc(Zn),moisture,hydrogen(H),and sulfur(S)have no significant difference among samples of three regions.Compared with the corn stalk in Anhui and Jiangxi,the Shanghai samples are more suitable for the production of ethanol because of their higher ratio of cellulose to hemi-cellulose content.Because of its high content of ash and low calorific value,the Shanghai corn stalk is suitable for the gasification process instead of for direct combustion or bio-oil production.The research can provide a reference for raw material selection for biomass energy production and utilization.

Triggering the biocontrol of Botrytis cinerea by Trichoderma harzianum through inhibition of pathogenicity and virulence related proteins

This study reports a strain of Trichoderma harzianum CCTCC-SBW0162 with potential to enhance biocontrol activity against gray mold pathogen, Botrytis cinerea, and with a pivotal role in tomato(Solanum esculentum) plant growth enhancement. A total of 254 Trichoderma isolates were screened by in vitro antagonistic assay. Of these, 10 were selected for greenhouse experiments based on their greater inhibition of B. cinerea.The in vitro antagonistic assay and greenhouse experiments indicated that T. harzianum CCTCC-SBW0162 gave the highest inhibition rate(90.6%) and disease reduction(80.7%). Also, to study the possible mechanism associated with antifungal activity of CCTCC-SBW0162 against B. cinerea, molecular docking was used to assess the interactions between CCTCC-SBW0162-derived metabolites, and pathogencity and virulence related proteins of B. cinerea. The molecular docking results indicated that the combination of harzianopyridone,harzianolide and anthraquinone C derived from CCTCCSBW0162 could synergistically improve antifungal activity against B. cinerea through the inhibition/modification of pathogenicity and virulence related proteins.However, this computerized modeling work emphasized the need for further study in the laboratory to confirm the effect T. harzianum-derived metabolites against the proteins of B. cinerea and their interactions.

Effects of packaging materials and types on postharvest nutritional quality of mini Pakchoi Brassica chinensis

The baby leaves of mini Pakchoi Brassica chinensis are gaining popularity due to its pleasant appearance,flavour,tender texture and nutrition.Effects of retail packaging on the nutritional quality of Brassica chinensis at 4℃were studied.Samples were packaged using polyethylene bags(PE-B),polystyrene foam tray covered with polyethylene film(PE-C/PS-T),perforated oriented polypropylene bags(POPP-B)and biaxially oriented polystyrene box(BOPS-B).Weight losses,respiration rate,chlorophyll and carotenoid,vitamin C and nitrate were determined during storage period.After 10 d storage,the weight loss for samples packaged in PE-B was 0.89%,followed by PE-C/PS-T with weight losses of 6%.A significant reduction(>10%)in moisture was observed for the samples packaged in POPP-B and BOPS-B.The variation in respiration rate during storage seemed to form a trend with a sharp decline up to the fourth day.The difference in the reduction of chlorophyll and carotenoid was not significant among the four packages.BOPS-B had a better protective effect on vitamin C loss.The nitrate concentration during storage was within the official limit.

Mutualistic fungus Piriformospora indica modulates cadmium phytoremediation properties of host plant via concerted action of enzymatic and non-enzymatic biochemicals

Soils and ecosystems contaminated with cadmium (Cd) threaten human health and adversely affect morphological,physiological,and biochemical parameters of plants.The symbiotic association of endophytic fungi with their host plants is the best strategy to improve various plant characteristics and remediate soils polluted with heavy metal(loid)s (HMs).Being a well-known plant growth-promoting fungus,Piriformospora indica confers resistance against a number of abiotic stresses,including HM stress.This pot experiment explored the potential and ameliorative effects of P.indica on Artemisia annua L.plants treated with different concentrations (0,40,80,and 120 mg kg-1) of Cd.Inoculation with P.indica significantly increased plant performance,especially by enhancing chlorophyll concentration and water potential and by decreasing electrolytic leakage,when compared with un-inoculated plants,despite the high Cd levels.Similarly,P.indica enhanced antioxidant enzyme activities,thereby reducing the drastic effects of Cd in inoculated plants.In addition,P.indica accumulated Cd in the roots of colonized plants,as revealed by atomic absorption spectroscopy,and restricted Cd translocation to aerial parts.Furthermore,P.indica showed in vitro resistance (up to a certain level) to Cd stress;however,fungus growth was inhibited at very high Cd concentrations,proving it an excellent candidate for use as a potential phytoremediator in fields affected by Cd contamination.The transcriptional analysis showed that the signaling genes and artemisinin and flavonoid biosynthetic pathway genes were significantly upregulated in P.indica-co-cultivated plants when compared with un-inoculated plants,suggesting a fine collaboration between primary and secondary metabolisms to modulate resistance capacity and to enhance the phytoremediation capability of A.annua against Cd toxicity.

Physicochemical properties,flavor intensity and oxidative stability of different camellia oils

Comparison and analysis of physicochemical properties and oxidative stability of 10 brands of camellia oils were conducted in this study.Results showed that more attention should be paid to the iodine(IV),saponification(SV)and peroxide(PV)values of camellia oils during sampling inspection as they were more likely to be out the range of quality standards.Regression analysis between physicochemical indices and oxidative stability showed individual index could not affect the stability of camellia oils significantly(p>0.05).However,very high correlations were found between physical indices such as optical rotation(OR)and turbidity(R=-0.929),turbidity and color(R=-0.930).High correlations were found between chemical indices such as IV and moisture and volatile matter(MVM)(R=-0.853),IV and PV(R=0.831),MVM and PV(R=-0.809).Package with nitrogen could retard the oxidation of camellia oil.These results may be useful for rapid evaluation,differentiation and quality improvement of camellia oils.

Progress in NMR-based metabolomics of Catharanthus roseus

Metabolomics has been rapidly developed as an important field in plant sciences and natural products chemistry.As the only natural source for a diversity of monoterpenoid indole alkaloids(MIAs),especially the low-abundance antitumor agents vinblastine and vincristine,Catharanthus roseus is highly valued and has been studied extensively as a model for medicinal plants improvement.Due to multistep enzymatic biosynthesis and complex regulation,genetic modification in the MIA pathway has resulted in complicated changes of both secondary and primary metabolism in C.roseus,affecting not only the MIA pathway but also other pathways.Research at the metabolic level is necessary to increase knowledge on the genetic regulation of the whole metabolic network connected to MIA biosynthesis.Nuclear magnetic resonance(NMR)is a very suitable and powerful complementary technique for the identification and quantification of metabolites in the plant matrix.NMR-based metabolomics has been used in studies of C.roseus for pathway elucidation,understanding stress responses,classification among different cultivars,safety and quality controls of transgenic plants,cross talk between pathways,and diversion of carbon fluxes,with the aim of fully unravelling MIA biosynthesis,its regulation and the function of the alkaloids in the plant from a systems biology point of view.