Boosting species evenness, productivity and weed control in a mixed meadow by promoting arbuscular mycorrhizas.

Lowland meadows represent aboveground and belowground biodiversity reservoirs in intensive agricultural areas, improving water retention and filtration, ensuring forage production, contrasting erosion and contributing to soil fertility and carbon sequestration. Besides such major ecosystem services, the presence of functionally different plant species improves forage quality, nutritional value and productivity, also limiting the establishment of weeds and alien species. Here, we tested the effectiveness of a commercial seed mixture in restoring a lowland mixed meadow in the presence or absence of inoculation with arbuscular mycorrhizal (AM) fungi and biostimulation of symbiosis development with the addition of short chain chito-oligosaccharides (CO). Plant community composition, phenology and productivity were regularly monitored alongside AM colonization in control, inoculated and CO-treated inoculated plots. Our analyses revealed that the CO treatment accelerated symbiosis development significantly increasing root colonization by AM fungi. Moreover, the combination of AM fungal inoculation and CO treatment improved plant species evenness and productivity with more balanced composition in forage species. Altogether, our study presented a successful and scalable strategy for the reintroduction of mixed meadows as valuable sources of forage biomass; demonstrated the positive impact of CO treatment on AM development in an agronomic context, extending previous observations developed under controlled laboratory conditions and leading the way to the application in sustainable agricultural practices.

Long-lasting impact of chitooligosaccharide application on strigolactone biosynthesis and fungal accommodation promotes arbuscular mycorrhiza in Medicago truncatula.

The establishment of arbuscular mycorrhiza (AM) between plants and Glomeromycotina fungi is preceded by the exchange of chemical signals: fungal released Myc-factors, including chitooligosaccharides (CO) and lipo-chitooligosaccharides (LCO), activate plant symbiotic responses, while root-exuded strigolactones stimulate hyphal branching and boost CO release. Furthermore, fungal signaling reinforcement through CO application was shown to promote AM development in Medicago truncatula, but the cellular and molecular bases of this effect remained unclear. Here, we focused on long-term M. truncatula responses to CO treatment, demonstrating its impact on the transcriptome of both mycorrhizal and nonmycorrhizal roots over several weeks and providing an insight into the mechanistic bases of the CO-dependent promotion of AM colonization. CO treatment caused the long-lasting regulation of strigolactone biosynthesis and fungal accommodation-related genes. This was mirrored by an increase in root didehydro-orobanchol content, and the promotion of accommodation responses to AM fungi in root epidermal cells. Lastly, an advanced downregulation of AM symbiosis marker genes was observed at the latest time point in CO-treated plants, in line with an increased number of senescent arbuscules. Overall, CO treatment triggered molecular, metabolic, and cellular responses underpinning a protracted acceleration of AM development.

Can Blebbistatin block the hypertrophy status in the zebrafish ex vivo cardiac model?

Ex-vivo simple models are powered tools to study cardiac hypertrophy. It is possible to control the activation of critical genes and thus test the effects of drug therapies before the in vivo tests. A zebrafish cardiac hypertrophy developed by 500 µM phenylephrine (PE) treatment in ex vivo culture has been demonstrated to activate the essential expression of the embryonal genes. These genes are the same as those described in several previous pieces of research on hypertrophic pathology in humans. The efficacy of the chemical drug Blebbistatin (BL) on hypertrophy induced ex vivo cultured hearts is studied in this research. BL can inhibit the myosins and the calcium wave in counteracting the hypertrophy status caused by PE. Samples treated with PE, BL and PE simultaneously, or pre/post-treatment with BL, have been analysed for the embryonal gene activation concerning the hypertrophy status. The qRTPCR has shown an inhibitory effect of BL treatments on the microRNAs downregulation with the consequent low expression of essential embryonal genes. In particular, BL seems to be effective in blocking the hyperplasia of the epicardium but less effective in myocardium hypertrophy. The model can make it possible to obtain knowledge on the transduction pathways activated by BL and investigate the potential use of this drug in treating cardiac hypertrophy in humans.

Extraction of short chain chitooligosaccharides from fungal biomass and their use as promoters of arbuscular mycorrhizal symbiosis.

Short chain chitooligosaccharides (COs) are chitin derivative molecules involved in plant-fungus signaling during arbuscular mycorrhizal (AM) interactions. In host plants, COs activate a symbiotic signalling pathway that regulates AM-related gene expression. Furthermore, exogenous CO application was shown to promote AM establishment, with a major interest for agricultural applications of AM fungi as biofertilizers. Currently, the main source of commercial COs is from the shrimp processing industry, but purification costs and environmental concerns limit the convenience of this approach. In an attempt to find a low cost and low impact alternative, this work aimed to isolate, characterize and test the bioactivity of COs from selected strains of phylogenetically distant filamentous fungi: Pleurotus ostreatus, Cunninghamella bertholletiae and Trichoderma viride. Our optimized protocol successfully isolated short chain COs from lyophilized fungal biomass. Fungal COs were more acetylated and displayed a higher biological activity compared to shrimp-derived COs, a feature that-alongside low production costs-opens promising perspectives for the large scale use of COs in agriculture.

On line kinetic analysis of permeation profiles for UV filter loaded microemulsions using an automatic system with spectroscopic detection and a chemometric approach.

The analysis of the permeation kinetics of new UV filter formulations is of great importance since the kinetic parameters are related to the effectiveness of the product over time. The dynamics of this process can be evaluated by means of the calculation of the permeation kinetic constants, which can be obtained from the respective permeation profiles. This paper is aimed at improving the analytical performance of permeation assays using an on-line automatic system with spectrometric detection avoiding the chromatographic procedure and the usually manual sampling steps required using the traditional Franz diffusion cell. Then, the kinetics of permeation of octyl p-methoxycinnamate loaded in different microemulsions through a synthetic membrane (polyamide) was analysed at real time by UV-Vis and fluorescence spectroscopies. The spectral data were obtained at regular intervals of time (5 min) during 60 min, and the concentration of the permeated UV-filter was at each time calculated using univariate linear calibration. The interference caused by the presence of basil essential oil (oily phase) in some microemulsion samples was overcome using synchronous fluorescence spectroscopy (Δλ = 60 nm) and partial least squares. In all cases, the permeation profiles were obtained (first-order kinetics) and the respective permeation kinetic constants were calculated. The validation of the proposed method was assessed by gas chromatographic-mass spectroscopy and non-significant differences for the obtained permeation kinetic constants were found between methods (p = 0.05). Additionally, a commercial sample was analysed with the proposed methods and the results were validated by high performance liquid chromatography technique.

Short chain chito-oligosaccharides promote arbuscular mycorrhizal colonization in Medicago truncatula.

During the establishment of arbuscular mycorrhizal (AM) symbiosis, the fungus and the host plant exchange chemical signals that are crucial to reciprocal recognition. Short-chain chitin oligomers (CO) released by AM fungi are known to trigger symbiotic signaling in all host plant species tested. Here we applied exogenous CO, derived from crustacean exoskeleton, to pot-grown Medicago truncatula inoculated with the AM fungus Funneliformis mosseae and investigated root colonization, plant gene regulation and biomass production. CO treatment strongly promoted AM colonization with significant increases in arbuscule development, biomass production and photosynthetic surface compared to untreated mycorrhizal plants. Gene expression analyses indicated that CO treatment anticipated the expression of MtBCP and MtPT4 plant symbiotic markers, during the first two weeks post inoculation. Altogether, our results provide evidence that plant treatment with symbiotic fungal elicitors, anticipated and enhanced AM development, encouraging the use of CO to promote AM establishment in sustainable agricultural practices.

Size matters: three methods for estimating nuclear size in mycorrhizal roots of Medicago truncatula by image analysis.

BACKGROUND: The intracellular accommodation of arbuscular mycorrhizal (AM) fungi involves a profound molecular reprogramming of the host cell architecture and metabolism, based on the activation of a symbiotic signaling pathway. In analogy with other plant biotrophs, AM fungi are reported to trigger cell cycle reactivation in their host tissues, possibly in support of the enhanced metabolic demand required for the symbiosis. RESULTS: We here compare the efficiency of three Fiji/ImageJ image analysis plugins in localizing and quantifying the increase in nuclear size - a hallmark of recursive events of endoreduplication - in M. truncatula roots colonized by the AM fungus Gigaspora margarita. All three approaches proved to be versatile and upgradeable, allowing the investigation of nuclear changes in a complex tissue; 3D Object Counter provided more detailed information than both TrackMate and Round Surface Detector plugins. On this base we challenged 3D Object Counter with two case studies: verifying the lack of endoreduplication-triggering responses in Medicago truncatula mutants with a known non-symbiotic phenotype; and analysing the correlation in space and time between the induction of cortical cell division and endoreduplication upon AM colonization. Both case studies revealed important biological aspects. Mutant phenotype analyses have demonstrated that the knock-out mutation of different key genes in the symbiotic signaling pathway block AM-associated endoreduplication. Furthermore, our data show that cell divisions occur during initial stages of root colonization and are followed by recursive activation of the endocycle in preparation for arbuscule accommodation. CONCLUSIONS: In conclusion, our results indicate 3D Object Counter as the best performing Fiji/ImageJ image analysis script in plant root thick sections and its application highlighted endoreduplication as a major feature of the AM pre-penetration response in root cortical cells.

Local endoreduplication as a feature of intracellular fungal accommodation in arbuscular mycorrhizas.

The intracellular accommodation of arbuscular mycorrhizal (AM) fungi is a paradigmatic feature of this plant symbiosis that depends on the activation of a dedicated signaling pathway and the extensive reprogramming of host cells, including striking changes in nuclear size and transcriptional activity. By combining targeted sampling of early root colonization sites, detailed confocal imaging, flow cytometry and gene expression analyses, we demonstrate that local, recursive events of endoreduplication are triggered in the Medicago truncatula root cortex during AM colonization. AM colonization induces an increase in ploidy levels and the activation of endocycle specific markers. This response anticipates the progression of fungal colonization and is limited to arbusculated and neighboring cells in the cortical tissue. Furthermore, endoreduplication is not induced in M. truncatula mutants for symbiotic signaling pathway genes. On this basis, we propose endoreduplication as part of the host cell prepenetration responses that anticipate AM fungal accommodation in the root cortex.

The apocarotenoid metabolite zaxinone regulates growth and strigolactone biosynthesis in rice.

Carotenoid cleavage dioxygenases (CCDs) form hormones and signaling molecules. Here we show that a member of an overlooked plant CCD subfamily from rice, that we name Zaxinone Synthase (ZAS), can produce zaxinone, a novel apocarotenoid metabolite in vitro. Loss-of-function mutants (zas) contain less zaxinone, exhibit retarded growth and showed elevated levels of strigolactones (SLs), a hormone that determines plant architecture, mediates mycorrhization and facilitates infestation by root parasitic weeds, such as Striga spp. Application of zaxinone can rescue zas phenotypes, decrease SL content and release and promote root growth in wild-type seedlings. In conclusion, we show that zaxinone is a key regulator of rice development and biotic interactions and has potential for increasing crop growth and combating Striga, a severe threat to global food security.

TPLATE Recruitment Reveals Endocytic Dynamics at Sites of Symbiotic Interface Assembly in Arbuscular Mycorrhizal Interactions.

Introduction: Arbuscular mycorrhizal (AM) symbiosis between soil fungi and the majority of plants is based on a mutualistic exchange of organic and inorganic nutrients. This takes place inside root cortical cells that harbor an arbuscule: a highly branched intracellular fungal hypha enveloped by an extension of the host cell membrane-the perifungal membrane-which outlines a specialized symbiotic interface compartment. The perifungal membrane develops around each intracellular hypha as the symbiotic fungus proceeds across the root tissues; its biogenesis is the result of an extensive exocytic process and shows a few similarities with cell plate insertion which occurs at the end of somatic cytokinesis. Materials and Methods: We here analyzed the subcellular localization of a GFP fusion with TPLATE, a subunit of the endocytic TPLATE complex (TPC), a central actor in plant clathrin-mediated endocytosis with a role in cell plate anchoring with the parental plasma membrane. Results: Our observations demonstrate that Daucus carota and Medicago truncatula root organ cultures expressing a 35S::AtTPLATE-GFP construct accumulate strong fluorescent green signal at sites of symbiotic interface construction, along recently formed perifungal membranes and at sites of cell-to-cell hyphal passage between adjacent cortical cells, where the perifungal membrane fuses with the plasmalemma. Discussion: Our results strongly suggest that TPC-mediated endocytic processes are active during perifungal membrane interface biogenesis-alongside exocytic transport. This novel conclusion, which might be correlated to the accumulation of late endosomes in the vicinity of the developing interface, hints at the involvement of TPC-dependent membrane remodeling during the intracellular accommodation of AM fungi.

Ectopic activation of cortical cell division during the accommodation of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizas (AMs) between plants and soil fungi are widespread symbioses with a major role in soil nutrient uptake. In this study we investigated the induction of root cortical cell division during AM colonization by combining morphometric and gene expression analyses with promoter activation and protein localization studies of the cell-plate-associated exocytic marker TPLATE. Our results show that TPLATE promoter is activated in colonized cells of the root cortex where we also observed the appearance of cells that are half the size of the surrounding cells. Furthermore, TPLATE-green fluorescent protein recruitment to developing cell plates highlighted ectopic cell division events in the inner root cortex during early AM colonization. Lastly, transcripts of TPLATE, KNOLLE and Cyclinlike 1 (CYC1) are all upregulated in the same context, alongside endocytic markers Adaptor-Related Protein complex 2 alpha 1 subunit (AP2A1) and Clathrin Heavy Chain 2 (CHC2), known to be active during cell plate formation. This pattern of gene expression was recorded in wild-type Medicago truncatula roots, but not in a common symbiotic signalling pathway mutant where fungal colonization is blocked at the epidermal level. Altogether, these results suggest the activation of cell-division-related mechanisms by AM hosts during the accommodation of the symbiotic fungus.

The Association With Two Different Arbuscular Mycorrhizal Fungi Differently Affects Water Stress Tolerance in Tomato.

Arbuscular mycorrhizal (AM) fungi are very widespread, forming symbiotic associations with ∼80% of land plant species, including almost all crop plants. These fungi are considered of great interest for their use as biofertilizer in low-input and organic agriculture. In addition to an improvement in plant nutrition, AM fungi have been reported to enhance plant tolerance to important abiotic and biotic environmental conditions, especially to a reduced availability of resources. These features, to be exploited and applied in the field, require a thorough identification of mechanisms involved in nutrient transfer, metabolic pathways induced by single and multiple stresses, physiological and eco-physiological mechanisms resulting in improved tolerance. However, cooperation between host plants and AM fungi is often related to the specificity of symbiotic partners, the environmental conditions and the availability of resources. In this study, the impact of two AM fungal species (Funneliformis mosseae and Rhizophagus intraradices) on the water stress tolerance of a commercial tomato cultivar (San Marzano nano) has been evaluated in pots. Biometric and eco-physiological parameters have been recorded and gene expression analyses in tomato roots have been focused on plant and fungal genes involved in inorganic phosphate (Pi) uptake and transport. R. intraradices, which resulted to be more efficient than F. mosseae to improve physiological performances, was selected to assess the role of AM symbiosis on tomato plants subjected to combined stresses (moderate water stress and aphid infestation) in controlled conditions. A positive effect on the tomato indirect defense toward aphids in terms of enhanced attraction of their natural enemies was observed, in agreement with the characterization of volatile organic compound (VOC) released. In conclusion, our results offer new insights for understanding the molecular and physiological mechanisms involved in the tolerance toward water deficit as mediated by a specific AM fungus. Moreover, they open new perspectives for the exploitation of AM symbiosis to enhance crop tolerance to abiotic and biotic stresses in a scenario of global change.

The phosphate transporters LjPT4 and MtPT4 mediate early root responses to phosphate status in non mycorrhizal roots.

Arbuscular mycorrhizal (AM) symbiosis improves host plant phosphorous (P) status and elicits the expression of AM-inducible phosphate transporters (PTs) in arbuscule-containing cells, where they control arbuscule morphogenesis and P release. We confirmed such functions for LjPT4 in mycorrhizal Lotus japonicus. Promoter-GUS experiments showed LjPT4 transcription not only in arbusculated cells but also in root tips, in the absence of the fungus: here LjPT4 transcription profile depended on the phosphate level. In addition, quantitative RT-PCR confirmed the expression of Lotus and Medicago truncatula PT4 in the tips of non-mycorrhizal roots. Starting from these observations, we hypothesized that AM-inducible PTs may have a regulatory role in plant development, irrespective of the fungal presence. Firstly, we focused on root development responses to different phosphate treatments in both plants demonstrating that phosphate starvation induced a higher number of lateral roots. By contrast, Lotus PT4i plants and Medicago mtpt4 mutants did not show any differential response to phosphate levels, suggesting that PT4 genes affect early root branching. Phosphate starvation-induced genes and a key auxin receptor, MtTIR1, showed an impaired expression in mtpt4 plants. We suggest PT4 genes as novel components of the P-sensing machinery at the root tip level, independently of AM fungi.

Identification and functional characterization of a sulfate transporter induced by both sulfur starvation and mycorrhiza formation in Lotus japonicus.

Arbuscular mycorrhizas (AMs) are one of the most widespread symbioses in the world. They allow plants to receive mineral nutrients from the symbiotic fungus which in turn gets back up to 20% of plant carbon and completes its life cycle. Especially in low-nutrient conditions, AM fungi are capable of significantly improving plant phosphate and nitrogen acquisition, but fewer data are available about sulfur (S) nutrition. We focused on S metabolism in Lotus japonicus upon mycorrhizal colonization under sulfur starvation or repletion. We investigated both tissue sulfate concentrations and S-related gene expression, at cell-type or whole-organ level. Gene expression and sulfate tissue concentration showed that Rhizophagus irregularis colonization can improve plant S nutritional status under S starvation. A group 1 sulfate transporter, LjSultr1;2, induced by both S starvation and mycorrhiza formation, was identified. Its transcript was localized in arbuscule-containing cells, which was confirmed with a promoter-GUS assay, and its function was verified through phenotyping of TILLING mutants in nonmycorrhizal seedlings. LjSultr1;2 thus appears to encode a key protein involved in plant sulfate uptake. In contrast to phosphate transporters, a single gene, LjSultr1;2, seems to mediate both direct and symbiotic pathways of S uptake in L. japonicus.

The Lotus japonicus MAMI gene links root development, arbuscular mycorrhizal symbiosis and phosphate availability.

The arbuscular mycorrhizal-induced LjMAMI gene is phylogenetically related to GARP transcription factors involved in Pi-starvation responses such as AtPHR1. The gene is strongly upregulated in arbusculated cells from mycorrhizal plants and in root meristems, irrespectively of the fungal presence. A further expression analysis revealed a similar expression pattern for LjPT4, considered a marker gene for mycorrhizal functionality. Here we show that the LjPT4 promoter contains two conserved cis-acting elements typically found in Pi-starvation induced Pi transporters. One of these is strongly related to the binding site of AtPHR1, suggesting a direct regulation of LjPT4 by LjMAMI. The expression of both genes in non-mycorrhizal tissues leads to the hypothesis that these symbiosis-inducible genes are also involved in Pi starvation responses in root meristems in an AM-independent manner.

An AM-induced, MYB-family gene of Lotus japonicus (LjMAMI) affects root growth in an AM-independent manner.

The interaction between legumes and arbuscular mycorrhizal (AM) fungi is vital to the development of sustainable plant production systems. Here, we focus on a putative MYB-like (LjMAMI) transcription factor (TF) previously reported to be highly upregulated in Lotus japonicus mycorrhizal roots. Phylogenetic analyses revealed that the protein is related to a group of TFs involved in phosphate (Pi) starvation responses, the expression of which is independent of the Pi level, such as PHR1. GUS transformed plants and quantitative reverse transcription PCR revealed strong gene induction in arbusculated cells, as well as the presence of LjMAMI transcripts in lateral root primordia and root meristems, even in the absence of the fungus, and independently of Pi concentration. In agreement with its putative identification as a TF, an eGFP-LjMAMI chimera was localized to the nuclei of plant protoplasts, whereas in transgenic Lotus roots expressing the eGFP-LjMAMI fusion protein under the control of the native promoter, the protein was located in the nuclei of the arbusculated cells. Further expression analyses revealed a correlation between LjMAMI and LjPT4, a marker gene for mycorrhizal function. To elucidate the role of the LjMAMI gene in the mycorrhizal process, RNAi and overexpressing root lines were generated. All the lines retained their symbiotic capacity; however, RNAi root lines and composite plants showed an important reduction in root elongation and branching in the absence of the symbiont. The results support the involvement of the AM-responsive LjMAMI in non-symbiotic functions: i.e. root growth.

Two putative-aquaporin genes are differentially expressed during arbuscular mycorrhizal symbiosis in Lotus japonicus.

BACKGROUND: Arbuscular mycorrhizas (AM) are widespread symbioses that provide great advantages to the plant, improving its nutritional status and allowing the fungus to complete its life cycle. Nevertheless, molecular mechanisms that lead to the development of AM symbiosis are not yet fully deciphered. Here, we have focused on two putative aquaporin genes, LjNIP1 and LjXIP1, which resulted to be upregulated in a transcriptomic analysis performed on mycorrhizal roots of Lotus japonicus. RESULTS: A phylogenetic analysis has shown that the two putative aquaporins belong to different functional families: NIPs and XIPs. Transcriptomic experiments have shown the independence of their expression from their nutritional status but also a close correlation with mycorrhizal and rhizobial interaction. Further transcript quantification has revealed a good correlation between the expression of one of them, LjNIP1, and LjPT4, the phosphate transporter which is considered a marker gene for mycorrhizal functionality. By using laser microdissection, we have demonstrated that one of the two genes, LjNIP1, is expressed exclusively in arbuscule-containing cells. LjNIP1, in agreement with its putative role as an aquaporin, is capable of transferring water when expressed in yeast protoplasts. Confocal analysis have demonstrated that eGFP-LjNIP1, under its endogenous promoter, accumulates in the inner membrane system of arbusculated cells. CONCLUSIONS: Overall, the results have shown different functionality and expression specificity of two mycorrhiza-inducible aquaporins in L. japonicus. One of them, LjNIP1 can be considered a novel molecular marker of mycorrhizal status at different developmental stages of the arbuscule. At the same time, LjXIP1 results to be the first XIP family aquaporin to be transcriptionally regulated during symbiosis.

An outbreak of Klebsiella pneumoniae and Enterobacter aerogenes bacteremia after interventional pain management procedures, New York City, 2008.

BACKGROUND AND OBJECTIVES: In October 2008, an investigation was conducted into a cluster of gram-negative bloodstream infections after invasive pain management procedures at an outpatient facility to identify additional cases and determine the source of illness. METHODS: We conducted a retrospective cohort study to determine exposures associated with illness. Eligible patients had an invasive procedure in the 4 days before or after the procedure date of the initial case-patients. Infection control assessments were made, and environmental specimens collected. RESULTS: Four laboratory-confirmed case-patients (3 with Klebsiella pneumoniae and 1 with Enterobacter aerogenes) and 5 suspect case-patients were identified. In addition to the 9 confirmed and suspect case-patients, 45 patients were interviewed. All confirmed and suspect case-patients had a sacroiliac joint steroid injection procedure; injection into the sacroiliac joint was associated with illness (9/22 versus 0/31; P < 0.0001). Multiple breaches in infection control were noted including the reuse of single-use vials for multiple patients. The 3 K. pneumoniae with positive blood cultures were indistinguishable by pulse-field gel electrophoresis, and the E. aerogenes-positive blood culture was indistinguishable by pulse-field gel electrophoresis to the culture from an open vial of 100-mL iodixanol contrast solution. CONCLUSION: Infection was associated with pain management procedures, specifically those involving injection to the sacroiliac joint. Lapses in infection control likely led to the contamination of single-use vials that were then used for multiple patients. Reuse of medication vials should be restricted, and affordable single-dose vials should be made available.

Effects of feeding Spodoptera littoralis on lima bean leaves: IV. Diurnal and nocturnal damage differentially initiate plant volatile emission.

Continuous mechanical damage initiates the rhythmic emission of volatiles in lima bean (Phaseolus lunatus) leaves; the emission resembles that induced by herbivore damage. The effect of diurnal versus nocturnal damage on the initiation of plant defense responses was investigated using MecWorm, a robotic device designed to reproduce tissue damage caused by herbivore attack. Lima bean leaves that were damaged by MecWorm during the photophase emitted maximal levels of beta-ocimene and (Z)-3-hexenyl acetate in the late photophase. Leaves damaged during the dark phase responded with the nocturnal emission of (Z)-3-hexenyl acetate, but with only low amounts of beta-ocimene; this emission was followed by an emission burst directly after the onset of light. In the presence of (13)CO(2), this light-dependent synthesis of beta-ocimene resulted in incorporation of 75% to 85% of (13)C, demonstrating that biosynthesis of beta-ocimene is almost exclusively fueled by the photosynthetic fixation of CO(2) along the plastidial 2-C-methyl-D-erythritol 4-P pathway. Jasmonic acid (JA) accumulated locally in direct response to the damage and led to immediate up-regulation of the P. lunatus beta-ocimene synthase gene (PlOS) independent of the phase, that is, light or dark. Nocturnal damage caused significantly higher concentrations of JA (approximately 2-3 times) along with enhanced expression levels of PlOS. Transgenic Arabidopsis thaliana transformed with PlOS promoter :: beta-glucuronidase fusion constructs confirmed expression of the enzyme at the wounded sites. In summary, damage-dependent JA levels directly control the expression level of PlOS, regardless of light or dark conditions, and photosynthesis is the major source for the early precursors of the 2-C-methyl-D-erythritol 4-P pathway.