Identification of a damage-associated molecular pattern (DAMP) receptor and its cognate peptide ligand in sweet potato (Ipomoea batatas).

Sweet potato (Ipomoea batatas) is an important tuber crop, but also target of numerous insect pests. Intriguingly, the abundant storage protein in tubers, sporamin, has intrinsic trypsin protease inhibitory activity. In leaves, sporamin is induced by wounding or a volatile homoterpene and enhances insect resistance. While the signalling pathway leading to sporamin synthesis is partially established, the initial event, perception of a stress-related signal is still unknown. Here, we identified an IbLRR-RK1 that is induced upon wounding and herbivory, and related to peptide-elicitor receptors (PEPRs) from tomato and Arabidopsis. We also identified a gene encoding a precursor protein comprising a peptide ligand (IbPep1) for IbLRR-RK1. IbPep1 represents a distinct signal in sweet potato, which might work in a complementary and/or parallel pathway to the previously described hydroxyproline-rich systemin (HypSys) peptides to strengthen insect resistance. Notably, an interfamily compatibility in the Pep/PEPR system from Convolvulaceae and Solanaceae was identified.

The diurnal emission of floral scent in Oncidium hybrid orchid is controlled by CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) through the direct regulation on terpene synthase.

BACKGROUND: To adapt the periodic fluctuation of environmental factors, plants are subtle to monitor the natural variation for the growth and development. The daily activities and physiological functions in coordination with the natural variation are regulated by circadian clock genes. The circadian emission of floral scents is one of the rhythmic physiological activities controlled by circadian clock genes. Here, we study the molecular mechanism of circadian emission pattern of ocimene and linalool compounds in Oncidium Sharry Baby (Onc. SB) orchid. RESULTS: GC-Mass analysis revealed that Onc. SB periodically emitted ocimene and linalool during 6 to 14 o'clock daily. Terpene synthase, one of the key gene in the terpenoid biosynthetic pathway is expressed in coordination with scent emission. The promoter structure of terpene synthase revealed a circadian binding sequence (CBS), 5'-AGATTTTT-3' for CIRCADIAN CLOCK ASSOCIATED1 (CCA1) transcription factor. EMSA data confirms the binding affinity of CCA1. Transactivation assay further verified that TPS expression is regulated by CCA1. It suggests that the emission of floral scents is controlled by CCA1. CONCLUSIONS: The work validates that the mechanism of circadian emission of floral scents in Onc. Sharry Baby is controlled by the oscillator gene, CCA1(CIRCADIAN CLOCK ASSOCIATED 1) under light condition. CCA1 transcription factor up-regulates terpene synthase (TPS) by binding on CBS motif, 5'-AGATTTTT-3' of promoter region to affect the circadian emission of floral scents in Onc. SB.

Piriformospora indica colonization increases the growth, development, and herbivory resistance of sweet potato (Ipomoea batatas L.).

KEY MESSAGE: Piriformospora indica symbiosis promoted the growth and photosynthesis, and simultaneously enhanced the resistance against insect herbivory by regulating sporamin-dependent defense in sweet potato. Piriformospora indica (P. indica), a versatile endophytic fungus, promotes the growth and confers resistance against multiple stresses by root colonization in plant hosts. In this study, the effects of P. indica colonization on the growth, physiological change, and herbivore resistance of leaf-vegetable sweet potato cultivar were investigated. P. indica symbiosis significantly improved the biomass in both above- and under-ground parts of sweet potato plants. In comparison with the non-colonized plants, the content of photosynthetic pigments and the efficiency of photosynthesis were increased in P. indica-colonized sweet potato plants. Further investigation showed that the activity of catalase was enhanced in both leaves and roots of sweet potato plants after colonization, but ascorbate peroxidase, peroxidase, and superoxide dismutase were not enhanced. Furthermore, the interaction between P. indica and sweet potato plants also showed the biological function in jasmonic acid (JA)-mediated defense. The plants colonized by P. indica had greatly increased JA accumulation and defense gene expressions, including IbNAC1, IbbHLH3, IbpreproHypSys, and sporamin, leading to elevated trypsin inhibitory activity, which was consistent with a reduced Spodoptera litura performance when larvae fed on the leaves of P. indica-colonized sweet potato plants. The root symbiosis of P. indica is helpful for the plant promoting growth and development and has a strong function as resistance inducers against herbivore attack in sweet potato cultivation by regulating sporamin-dependent defense.

The Endophytic Fungus Piriformospora indica Reprograms Banana to Cold Resistance.

Banana (Musa spp.), one of the most important fruits worldwide, is generally cold sensitive. In this study, by using the cold-sensitive banana variety Tianbaojiao (Musa acuminate) as the study material, we investigated the effects of Piriformospora indica on banana cold resistance. Seedlings with and without fungus colonization were subjected to 4 °C cold treatment. The changes in plant phenotypes, some physiological and biochemical parameters, chlorophyll fluorescence parameters, and the expression of eight cold-responsive genes in banana leaves before and after cold treatment were measured. Results demonstrated that P. indica colonization reduced the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) but increased the activities of superoxide dismutase (SOD) and catalase (CAT) and the contents of soluble sugar (SS) and proline. Noteworthily, the CAT activity and SS content in the leaves of P. indica-colonized banana were significant (p < 0.05). After 24 h cold treatment, the decline in maximum photochemistry efficiency of photosystem II (Fv/Fm), photochemical quenching coefficient (qP), efficient quantum yield [Y(II)], and photosynthetic electron transport rate (ETR) in the leaves of P. indica-colonized banana was found to be lower than in the non-inoculated controls (p < 0.05). Moreover, although the difference was not significant, P. indica colonization increased the photochemical conversion efficiency and electron transport rate and alleviated the damage to the photosynthetic reaction center of banana leaves under cold treatment to some extent. Additionally, the expression of the most cold-responsive genes in banana leaves was significantly induced by P. indica during cold stress (p < 0.05). It was concluded that P. indica confers banana with enhanced cold resistance by stimulating antioxidant capacity, SS accumulation, and the expression of cold-responsive genes in leaves. The results obtained from this study are helpful for understanding the P. indica-induced cold resistance in banana.

Colonisation of Oncidium orchid roots by the endophyte Piriformospora indica restricts Erwinia chrysanthemi infection, stimulates accumulation of NBS-LRR resistance gene transcripts and represses their targeting micro-RNAs in leaves.

BACKGROUND: Erwinia chrysanthemi (Ec) is a destructive pathogen which causes soft-rot diseases in diverse plant species including orchids. We investigated whether colonization of Oncidium roots by the endophytic fungus Piriformospora indica (Pi) restricts Ec-induced disease development in leaves, and whether this might be related to the regulation of nucleotide binding site-leucine rich repeat (NBS-LRR) Resistance (R) genes. RESULTS: Root colonization of Oncidium stackings by Pi restricts progression of Ec-induced disease development in the leaves. Since Pi does not inhibit Ec growth on agar plates, we tested whether NBS-LRR R gene transcripts and the levels of their potential target miRNAs in Oncidium leaves might be regulated by Pi. Using bioinformatic tools, we first identified NBS-LRR R gene sequences from Oncidium, which are predicted to be targets of miRNAs. Among them, the expression of two R genes was repressed and the accumulation of several regulatory miRNA stimulated by Ec in the leaves of Oncidium plants. This correlated with the progression of disease development, jasmonic and salicylic acid accumulation, ethylene synthesis and H2O2 production after Ec infection of Oncidium leaves. Interestingly, root colonization by Pi restricted disease development in the leaves, and this was accompanied by higher expression levels of several defense-related R genes and lower expression level of their target miRNA. CONCLUSION: Based on these data we propose that Pi controls the levels of NBS-LRR R mRNAs and their target miRNAs in leaves. This regulatory circuit correlates with the protection of Oncidium plants against Ec infection, and molecular and biochemical investigations will demonstrate in the future whether, and if so, to what extent these two observations are related to each other.

Growth promotion and disease resistance induced in Anthurium colonized by the beneficial root endophyte Piriformospora indica.

BACKGROUND: Anthurium andraeanum, an important ornamental flower, has to go through a growth-delaying period after transfer from tissue culture to soil, which requires time and extra costs. Furthermore, during this period, the plantlets are highly susceptible to bacterial infections, which results in impaired development and severe losses. Here, we aimed to address whether application of the endophytic fungus, Piriformospora indica protects the A. andraeanum root system during the critical propagation period, and whether P. indica reduce the mortality rate by stimulating the host's resistance against diseases. RESULTS: We demonstrate that P. indica shortens the recovery period of Anthurium, promotes growth and confers disease resistance. The beneficial effect of P. indica results in faster elongation of Anthurium roots early in the interaction. P. indica-colonized plants absorb more phosphorus and exhibit higher photosynthesis rates than uncolonized control plants. Moreover, higher activities of stress-related enzymes, of jasmonic acid levels and mRNA levels of jasmonic acid-responsive genes suggest that the fungus prepares the plant to respond more efficiently to potentially upcoming threats, including bacterial wilt. CONCLUSION: These results suggest that P. indica is a helpful symbiont for promoting Anthurium rooting and development. All our evidences are sufficient to support the disease resistance conferred by P. indica through the plant-fungal symbiosis. Furthermore, it implicates that P. indica has strong potential as bio-fertilizer for utilization in ornamental plant cultivation.

The Sweet Potato NAC-Domain Transcription Factor IbNAC1 Is Dynamically Coordinated by the Activator IbbHLH3 and the Repressor IbbHLH4 to Reprogram the Defense Mechanism against Wounding.

IbNAC1 is known to activate the defense system by reprogramming a genetic network against herbivory in sweet potato. This regulatory activity elevates plant defense potential but relatively weakens plants by IbNAC1-mediated JA response. The mechanism controlling IbNAC1 expression to balance plant vitality and survival remains unclear. In this study, a wound-responsive G-box cis-element in the IbNAC1 promoter from -1484 to -1479 bp was identified. From a screen of wound-activated transcriptomic data, one transcriptional activator, IbbHLH3, and one repressor, IbbHLH4, were selected that bind to and activate or repress, respectively, the G-box motif in the IbNAC1 promoter to modulate the IbNAC1-mediated response. In the early wound response, the IbbHLH3-IbbHLH3 protein complex binds to the G-box motif to activate IbNAC1 expression. Thus, an elegant defense network is activated against wounding stress. Until the late stages of wounding, IbbHLH4 interacts with IbbHLH3, and the IbbHLH3-IbbHLH4 heterodimer competes with the IbbHLH3-IbbHLH3 complex to bind the G-box and suppress IbNAC1 expression and timely terminates the defense network. Moreover, the JAZs and IbEIL1 proteins interact with IbbHLH3 to repress the transactivation function of IbbHLH3 in non-wounded condition, but their transcription is immediately inhibited upon early wounding. Our work provides a genetic model that accurately switches the regulatory mechanism of IbNAC1 expression to adjust wounding physiology and represents a delicate defense regulatory network in plants.

Sweet potato NAC transcription factor, IbNAC1, upregulates sporamin gene expression by binding the SWRE motif against mechanical wounding and herbivore attack.

Sporamin is a tuberous storage protein with trypsin inhibitory activity in sweet potato (Ipomoea batatas Lam.), which accounts for 85% of the soluble protein in tubers. It is constitutively expressed in tuberous roots but is expressed in leaves only after wounding. Thus far, its wound-inducible signal transduction mechanisms remain unclear. In the present work, a 53-bp DNA region, sporamin wound-response cis-element (SWRE), was identified in the sporamin promoter and was determined to be responsible for the wounding response. Using yeast one-hybrid screening, a NAC domain protein, IbNAC1, that specifically bound to the 5'-TACAATATC-3' sequence in SWRE was isolated from a cDNA library from wounded leaves. IbNAC1 was constitutively expressed in root tissues and was induced earlier than sporamin following the wounding of leaves. Transgenic sweet potato plants overexpressing IbNAC1 had greatly increased sporamin expression, increased trypsin inhibitory activity, and elevated resistance against Spodoptera litura. We further demonstrated that IbNAC1 has multiple biological functions in the jasmonic acid (JA) response, including the inhibition of root formation, accumulation of anthocyanin, regulation of aging processes, reduction of abiotic tolerance, and overproduction of reactive oxygen species (ROS). Thus, IbNAC1 is a core transcription factor that reprograms the transcriptional response to wounding via the JA-mediated pathway in sweet potato.

Advances of selectable marker genes in plastid genetic engineering.

Plastid genetic engineering is a safer, more precise, and more efficient transgene expression system than the nuclear genetic transformation system. It has been widely used in basic research and biotechnology applications as the next-generation transgenic technology in plants. Similar to nuclear genetic transformation, selection markers are needed in plastid genetic engineering to identify 'true' transformants and acquire homoplasmy. Because of the high copy number of plastids, maternal inheritance of the plastid genome, and the long process of homogenization of transplastomic plants, the selection markers for plastid genetic engineering are different from those used in the nuclear transformation system. At present, antibiotic resistance genes are the most commonly used selectable markers in the transplastomic selections. However for biosafety reasons, they needed to be replaced with either alternative markers or marker-free systems for the plastid genetic engineering. In this review, we have evaluated and summarized the positive and negative features of the selectable markers and marker elimination strategies commonly used in the plastid engineering research in the literature on plastid genetic engineering research. In addition, we have reviewed the features of the reporter genes used in plastid genetic engineering. We hope this review can help improving the current and developing new selectable markers and marker removal systems, and further promote the development of plastid genetic engineering, especially on the monocotyledonous plants.

A Chinese cabbage (Brassica campetris subsp. Chinensis) τ-type glutathione-S-transferase stimulates Arabidopsis development and primes against abiotic and biotic stress.

The beneficial root-colonizing fungus Piriformospora indica stimulates root development of Chinese cabbage (Brassica campestris subsp. Chinensis) and this is accompanied by the up-regulation of a τ-class glutathione (GSH)-S-transferase gene (BcGSTU) (Lee et al. 2011) in the roots. BcGSTU expression is further promoted by osmotic (salt and PEG) and heat stress. Ectopic expression of BcGSTU in Arabidopsis under the control of the 35S promoter results in the promotion of root and shoot growth as well as better performance of the plants under abiotic (150 mM NaCl, PEG, 42 °C) and biotic (Alternaria brassicae infection) stresses. Higher levels of glutathione, auxin and stress-related (salicylic and jasmonic acid) phytohormones as well as changes in the gene expression profile result in better performance of the BcGSTU expressors upon exposure to stress. Simultaneously the plants are primed against upcoming stresses. We propose that BcGSTU is a target of P. indica in Chinese cabbage roots because the enzyme participates in balancing growth and stress responses, depending on the equilibrium of the symbiotic interaction. A comparable function of BcGST in transgenic Arabidopsis makes the enzyme a valuable tool for agricultural applications.

A Low Glutathione Redox State Couples with a Decreased Ascorbate Redox Ratio to Accelerate Flowering in Oncidium Orchid.

Glutathione (GSH) plays multiple roles in plants, including stress defense and regulation of growth/development. Previous studies have demonstrated that the ascorbate (AsA) redox state is involved in flowering initiation in Oncidium orchid. In this study, we discovered that a significantly decreased GSH content and GSH redox ratio are correlated with a decline in the AsA redox state during flowering initiation and high ambient temperature-induced flowering. At the same time, the expression level and enzymatic activity of GSH redox-regulated genes, glutathione reductase (GR1), and the GSH biosynthesis genes γ-glutamylcysteine synthetase (GSH1) and glutathione synthase (GSH2), are down-regulated. Elevating dehydroascorbate (DHA) content in Oncidium by artificial addition of DHA resulted in a decreased AsA and GSH redox ratio, and enhanced dehydroascorbate reductase (DHAR) activity. This demonstrated that the lower GSH redox state could be influenced by the lower AsA redox ratio. Moreover, exogenous application of buthionine sulfoximine (BSO), to inhibit GSH biosynthesis, and glutathione disulfide (GSSG), to decrease the GSH redox ratio, also caused early flowering. However, spraying plants with GSH increased the GSH redox ratio and delayed flowering. Furthermore, transgenic Arabidopsis overexpressing Oncidium GSH1, GSH2 and GR1 displayed a high GSH redox ratio as well as delayed flowering under high ambient temperature treatment, while pad2, cad2 and gr1 mutants exhibited early flowering and a low GSH redox ratio. In conclusion, our results provide evidence that the decreased GSH redox state is linked to the decline in the AsA redox ratio and mediated by down-regulated expression of GSH metabolism-related genes to affect flowering time in Oncidium orchid.

WRKY6 restricts Piriformospora indica-stimulated and phosphate-induced root development in Arabidopsis.

BACKGROUND: Arabidopsis root growth is stimulated by Piriformospora indica, phosphate limitation and inactivation of the WRKY6 transcription factor. Combinations of these factors induce unexpected alterations in root and shoot growth, root architecture and root gene expression profiles. RESULTS: The results demonstrate that P. indica promotes phosphate uptake and root development under Pi limitation in wrky6 mutant. This is associated with the stimulation of PHOSPHATE1 expression and ethylene production. Expression profiles from the roots of wrky6 seedlings identified genes involved in hormone metabolism, transport, meristem, cell and plastid proliferation, and growth regulation. 25 miRNAs were also up-regulated in these roots. We generated and discuss here a list of common genes which are regulated in growing roots and which are common to all three growth stimuli investigated in this study. CONCLUSION: Since root development of wrky6 plants exposed to P. indica under phosphate limitation is strongly promoted, we propose that common genes which respond to all three growth stimuli are central for the control of root growth and architecture. They can be tested for optimizing root growth in model and agricultural plants.

Prolonged exposure to elevated temperature induces floral transition via up-regulation of cytosolic ascorbate peroxidase 1 and subsequent reduction of the ascorbate redox ratio in Oncidium hybrid orchid.

The bolting time of the Oncidium hybrid orchid is not season dependent and so it is a useful year-round model system to study thermal-induced flowering mechanisms in planta. Previously, we reported that a low ascorbate (AsA) content is essential for floral transition in Oncidium; however, the environmental factors governing initiation of the flowering process remained to be elucidated. The current study revealed that a prolonged elevated temperature treatment (30°C over a 14 d period) induces floral transition. This floral induction in response to thermal stress was associated with a significantly increased reactive oxygen species (ROS) level and a lowered AsA redox ratio, as well as prominently up-regulated expression of cytosolic ascorbate peroxidase (cytAPX1). Transcriptome analysis confirmed that increased temperature affected the differential expression of genes involved in antioxidant metabolism. Likewise, transgenic Arabidopsis ectopically overexpressing Oncidium cytAPX1 displayed an early-flowering phenotype and low AsA redox ratio under thermal stress, while cytAPX1 mutants, apx1-1 and apx1-2, exhibited a delayed-flowering phenotype and a high AsA redox ratio. Our present data illustrate that the floral transition response to thermal stress is mediated by the AsA redox ratio, and that CytAPX plays a pivotal role in modulating the AsA redox ratio in Oncidium hybrid orchid. Taken together, the results from this investigation of the thermal-induced flowering mechanism indicated that the AsA redox ratio is a master switch to mediate phase transition from the vegetative to reproductive stage.

Differential activation of sporamin expression in response to abiotic mechanical wounding and biotic herbivore attack in the sweet potato.

BACKGROUND: Plants respond differently to mechanical wounding and herbivore attack, using distinct pathways for defense. The versatile sweet potato sporamin possesses multiple biological functions in response to stress. However, the regulation of sporamin gene expression that is activated upon mechanical damage or herbivore attack has not been well studied. RESULTS: Biochemical analysis revealed that different patterns of Reactive oxygen species (ROS) and antioxidant mechanism exist between mechanical wounding (MW) and herbivore attack (HA) in the sweet potato leaf. Using LC-ESI-MS (Liquid chromatography electrospray ionization mass spectrometry analysis), only the endogenous JA (jasmonic acid) level was found to increase dramatically after MW in a time-dependent manner, whereas both endogenous JA and SA (salicylic acid) increase in parallel after HA. Through yeast one-hybrid screening, two transcription factors IbNAC1 (no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), and cup-shaped cotyledon (CUC)) and IbWRKY1 were isolated, which interact with the sporamin promoter fragment of SWRE (sporamin wounding-responsive element) regulatory sequences. Exogenous application of MeJA (methyl jasmonate), SA and DIECA (diethyldithiocarbamic acid, JAs biosynthesis inhibitor) on sweet potato leaves was employed, and the results revealed that IbNAC1 mediated the expression of sporamin through a JA-dependent signaling pathway upon MW, whereas both IbNAC1 and IbWRKY1 coordinately regulated sporamin expression through JA- and SA-dependent pathways upon HA. Transcriptome analysis identified MYC2/4 and JAZ2/TIFY10A (jasmonate ZIM/tify-domain), the repressor and activator of JA and SA signaling among others, as the genes that play an intermediate role in the JA and SA pathways, and these results were further validated by qRT-PCR (quantitative real-time polymerase chain reaction). CONCLUSION: This work has improved our understanding of the differential regulatory mechanism of sporamin expression. Our study illustrates that sweet potato sporamin expression is differentially induced upon abiotic MW and biotic HA that involves IbNAC1 and IbWRKY1 and is dependent on the JA and SA signaling pathways. Thus, we established a model to address the plant-wounding response upon physical and biotic damage.

The beneficial fungus Piriformospora indica protects Arabidopsis from Verticillium dahliae infection by downregulation plant defense responses.

BACKGROUND: Verticillium dahliae (Vd) is a soil-borne vascular pathogen which causes severe wilt symptoms in a wide range of plants. The microsclerotia produced by the pathogen survive in soil for more than 15 years. RESULTS: Here we demonstrate that an exudate preparation induces cytoplasmic calcium elevation in Arabidopsis roots, and the disease development requires the ethylene-activated transcription factor EIN3. Furthermore, the beneficial endophytic fungus Piriformospora indica (Pi) significantly reduced Vd-mediated disease development in Arabidopsis. Pi inhibited the growth of Vd in a dual culture on PDA agar plates and pretreatment of Arabidopsis roots with Pi protected plants from Vd infection. The Pi-pretreated plants grew better after Vd infection and the production of Vd microsclerotia was dramatically reduced, all without activating stress hormones and defense genes in the host. CONCLUSIONS: We conclude that Pi is an efficient biocontrol agent that protects Arabidopsis from Vd infection. Our data demonstrate that Vd growth is restricted in the presence of Pi and the additional signals from Pi must participate in the regulation of the immune response against Vd.

Transplastomic Nicotiana benthamiana plants expressing multiple defence genes encoding protease inhibitors and chitinase display broad-spectrum resistance against insects, pathogens and abiotic stresses.

Plastid engineering provides several advantages for the next generation of transgenic technology, including the convenient use of transgene stacking and the generation of high expression levels of foreign proteins. With the goal of generating transplastomic plants with multiresistance against both phytopathogens and insects, a construct containing a monocistronic patterned gene stack was transformed into Nicotiana benthamiana plastids harbouring sweet potato sporamin, taro cystatin and chitinase from Paecilomyces javanicus. Transplastomic lines were screened and characterized by Southern/Northern/Western blot analysis for the confirmation of transgene integration and respective expression level. Immunogold localization analyses confirmed the high level of accumulation proteins that were specifically expressed in leaf and root plastids. Subsequent functional bioassays confirmed that the gene stacks conferred a high level of resistance against both insects and phytopathogens. Specifically, larva of Spodoptera litura and Spodoptera exigua either died or exhibited growth retardation after ingesting transplastomic plant leaves. In addition, the inhibitory effects on both leaf spot diseases caused by Alternaria alternata and soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum were markedly observed. Moreover, tolerance to abiotic stresses such as salt/osmotic stress was highly enhanced. The results confirmed that the simultaneous expression of sporamin, cystatin and chitinase conferred a broad spectrum of resistance. Conversely, the expression of single transgenes was not capable of conferring such resistance. To the best of our knowledge, this is the first study to demonstrate an efficacious stacked combination of plastid-expressed defence genes which resulted in an engineered tolerance to various abiotic and biotic stresses.

The maturation zone is an important target of Piriformospora indica in Chinese cabbage roots.

The mutualistic symbiont Piriformospora indica exhibits a great potential in agriculture. The interaction between P. indica and Chinese cabbage (Brassica campestris cv. Chinensis) results in growth and biomass promotion of the host plant and in particular in root hair development. The resulting highly bushy root phenotype of colonized Chinese cabbage seedlings differs substantially from reports of other plant species, which prompted the more detailed study of this symbiosis. A large-scale expressed sequence tag (EST) data set was obtained from a double-subtractive EST library, by subtracting the cDNAs of Chinese cabbage root tissue and of P. indica mycelium from those of P. indica-colonized root tissue. The analysis revealed ~700 unique genes rooted in 141 clusters and 559 singles. A total of 66% of the sequences could be annotated in the NCBI GenBank. Genes which are stimulated by P. indica are involved in various types of transport, carbohydrate metabolism, auxin signalling, cell wall metabolism, and root development, including the root hair-forming phosphoinositide phosphatase 4. For 20 key genes, induction by fungal colonization was confirmed kinetically during the interaction by real-time reverse transcription-PCR. Moreover, the auxin concentration increases transiently after exposure of the roots to P. indica. Microscopic analyses demonstrated that the development of the root maturation zone is the major target of P. indica in Chinese cabbage. Taken together, the symbiotic interaction between Chinese cabbage and P. indica is a novel model to study root growth promotion which, in turn, is important for agriculture and plant biotechnology.

Methylation effect on chalcone synthase gene expression determines anthocyanin pigmentation in floral tissues of two Oncidium orchid cultivars.

The anthocyanin-biosynthetic pathway was studied in flowers of Oncidium Gower Ramsey with yellow floral color and mosaic red anthocyanin in lip crests, sepals and petals, and compared with the anthocyanin biosynthesis in flowers of Oncidium Honey Dollp, a natural somatoclone derived from tissue culture of Gower Ramsey, with a yellow perianth without red anthocyanins in floral tissues. HPLC analysis revealed that the red anthocyanin in lip crests of the Gower Ramsey cultivar comprised peonidin-3-O-glucoside, delphinidin-3-O-glucoside and cyanidin-3-O-glucoside, whereas Honey Dollp was devoid of anthocyanin compounds. Among the five anthocyanin-biosynthetic genes, OgCHS was actively expressed in lip crests of Gower Ramsey flowers, but no transcripts of OgCHS were detected in Honey Dollp floral tissues. Transient expression of OgCHS by bombardment confirmed that recovery of the OgCHS gene expression completed the anthocyanin pathway and produced anthocyanin compounds in lip crests of Honey Dollp flowers. Transcription factor genes regulating anthocyanin biosynthesis showed no distinctive differences in the expression level of OgMYB1, OgbHLH and OgWD40 between the two cultivars. A methylation assay revealed that the promoter of OgCHS was not methylated in Gower Ramsey, while a positive methylation effect was present in the upstream promoter region of OgCHS in Honey Dollp. Overall, our results suggest that the failure of anthocyanin accumulation in Honey Dollp floral tissues may be attributed to inactivation of the OgCHS gene resulting from the epigenetic methylation of 5'-upstream promoter region.

Simultaneous determination of sulphur metabolites in Arabidopsis thaliana via LC-ESI-MS/MS and ³4S-metabolic labelling.

INTRODUCTION: Sulphur-containing metabolites play an important role in metabolism and homeostasis. Determination of these metabolites is challenging owing to their low concentrations and the interference in mass spectrometry analysis. OBJECTIVE: To develop a sensitive and accurate method based on liquid chromatography, electrospray ionisation, tandem mass spectrometry (LC-ESI-MS/MS) and ³4S-metabolic labelling for quantification of methionine, reduced glutathione, oxidised glutathione in Arabidopsis thaliana. METHODOLOGY: A hydroponic set-up was used for the in vivo ³4S-metabolic labelling of A. thaliana. The ³4S-labelled metabolites biosynthesised in plant were extracted and used as internal standards. Tissue was extracted with perchloric acid (PCA) or PCA containing a known amount of the analytes for recovery analysis. Tissue extract mixed with extract of ³4S-labelled A. thaliana in an appropriate ratio was subjected to a LC system and electrospray ionisation-mass spectrometric (ESI-MS) analysis. Quantification of metabolites was measured by comparing the ³4S/³4S ratios obtained for samples with the calibration curves. RESULTS: Calibration curves showed linearity with regression coefficients in the range of 0.9994-0.9999. Analyte recoveries were approximately 100%. The coefficients of variation of intra-assay and inter-assay were less than 4.2% and 5%, respectively. The ranges for the limits of detection determined for Met, GSSG and GSH were 10 fmol, < 10 fmol and 1.12 fmol and the limits of quantification determined for Met, GSSG and GSH were 0.44 pmol, 0.16 pmol and 34 fmol, respectively. CONCLUSION: The validated method for determination of methionine, reduced glutathione and oxidised glutathione was effectively applied to measure metabolite dynamics of sulphur-containing metabolites at the whole-plant level.