Terpene emission in tissue culture

Tissue cultured plants\u27 vessel headspace is subject to changes during subculture, and the analysis of its variation offers a non-destructive approach for monitoring plant physiology. Among the volatile organic compounds (VOCs) that can be released by plants and be potentially recovered in the airspace of plant tissue cultures, terpenes are very important since they can offer a snapshot of the physiological status of the plant under in vitro cultivation. Terpenes are synthesized from carbon directly shunted from the photosynthetic carbon fixation cycle and their emission is under genetic and environmental control. The experiments described in this paper propose the evaluation of the plant terpene profile in the culture\u27s headspace as an early indicator of plant stress through the characterization of plant terpene production. Monitoring of terpene emission as a plant response to mechanical stress such as plant wounding showed an increased isoprene and monoterpene emission rates in the first hour after cutting. The comparison of headspace composition of cultures of two fruit rootstocks, Colt and GF677, showed the former having higher emissions of isoprene, alpha-pinene and limonene than the latter. A decreasing emission trend was observed during subculture, apparently as a result of culture aging. The in vitro headspace analysis of different myrtle (Myrtus communis L.) clones showed a specific and distinctive terpene emission profile. VOC monitoring of in vitro culture headspace is discussed as a non-destructive approach useful for its relation with major physiological activities of culture and for the determination of the potential production of terpenes

Isoprenoid emission in trees of Quercus pubescens and Quercus ilex with lifetime exposure to naturally high CO2 environment

The long-term effect of elevated atmospheric CO2 on isoprenoid emissions from adult trees of two Mediterranean oak species (the monoterpene-emitting Quercus ilex L. and the isoprene-emitting Quercus pubescens Willd.) native to a high-CO2 environment was investigated. During two consecutive years, isoprenoid emission was monitored both at branch level, measuring the actual emissions under natural conditions, and at leaf level, measuring the basal emissions under the standard conditions of 30 degreesC and at light intensity of 1000 mumol m(-2) s(-1). Long-term exposure to high atmospheric levels of CO2 did not significantly affect the actual isoprenoid emissions. However, when leaves of plants grown in the control site were exposed for a short period to an elevated CO2 level by rapidly switching the CO2 concentration in the gas-exchange cuvette, both isoprene and monoterpene basal emissions were clearly inhibited. These results generally confirm the inhibitory effect of elevated CO2 on isoprenoid emission. The absence of a CO2 effect on actual emissions might indicate higher leaf temperature at elevated CO2, or an interaction with multiple stresses some of which (e.g. recurrent droughts) may compensate for the CO2 effect in Mediterranean ecosystems. Under elevated CO2, isoprene emission by Q. pubescens was also uncoupled from the previous day\u27s air temperature. In addition, pronounced daily and seasonal variations of basal emission were observed under elevated CO2 underlining that correction factors may be necessary to improve the realistic estimation of isoprene emissions with empirical algorithms in the future. A positive linear correlation of isoprenoid emission with the photosynthetic electron transport and in particular with its calculated fraction used for isoprenoid synthesis was found. The slope of this relationship was different for isoprene and monoterpenes, but did not change when plants were grown in either ambient or elevated CO2. This suggests that physiological algorithms may usefully predict isoprenoid emission also under rising CO2 levels

Role of xanthophyll cycle-mediated photoprotection in Arbutus unedo plants exposed to water stress during the Mediterranean summer

We analyzed the response of potted strawberry tree (Arbutus unedo L.) seedlings exposed to water stress by withholding water for 10 d (WS). Leaf water potential, net CO2 assimilation, and stomatal conductance decreased with increasing water deficit. A 30 % reduction of chlorophyll (Chl) content in the antenna complexes was observed in WS-plants. Simultaneously, a decline of photochemical efficiency (F-v/F-m) occurred as a result of an excess of solar radiation energy when carbon assimilation was limited by stomata closure due to soil water deficit. The non-photochemical quenching of Chl fluorescence (Phi(NPQ)) significantly increased, as well as the leaf contents of zeaxanthin (Z) and antheraxanthin (A) at the expense of violaxanthin during the WS-period. Elevated predawn contents of de-epoxidized xanthophyll cycle components were associated with a sustained lowering of predawn photosystem 2 efficiency; this suggested an engagement of Z+A in a state primed for energy dissipation. Thus, the ability of strawberry trees to maintain the functionality of the xanthophyll cycle during the Mediterranean summer is an efficient mechanism to prevent irreversible damages to the photosynthetic machinery through thermal energy dissipation in the antenna and the reduction in photochemical efficiency

Quantitative predictions on auxin-induced polar distribution of PIN proteins during vein formation in leaves

The dynamic patterning of the plant hormone auxin and its efflux facilitator the PIN protein are the key regulator for the spatial and temporal organization of plant development. In particular auxin induces the polar localization of its own efflux facilitator. Due to this positive feedback auxin flow is directed and patterns of auxin and PIN arise. During the earliest stage of vein initiation in leaves auxin accumulates in a single cell in a rim of epidermal cells from which it flows into the ground meristem tissue of the leaf blade. There the localized auxin supply yields the successive polarization of PIN distribution along a strand of cells. We model the auxin and PIN dynamics within cells with a minimal canalization model. Solving the model analytically we uncover an excitable polarization front that triggers a polar distribution of PIN proteins in cells. As polarization fronts may extend to opposing directions from their initiation site we suggest a possible resolution to the puzzling occurrence of bipolar cells, such we offer an explanation for the development of closed, looped veins. Employing non-linear analysis we identify the role of the contributing microscopic processes during polarization. Furthermore, we deduce quantitative predictions on polarization fronts establishing a route to determine the up to now largely unknown kinetic rates of auxin and PIN dynamics.Comment: 9 pages, 4 figures, supplemental information included, accepted for publication in Eur. Phys. J.

Effect of arbuscular mycorrhizal (AM) colonization on terpene emission and content of Artemisia annua L.

Plant roots interact with a wide variety of rhizospheric microorganisms, including bacteria and the symbiontic arbuscular mycorrhizal (AM) fungi. The mycorrhizal symbiosis represents a series of complex feedbacks between plant and fungus regulated by their physiology and nutrition. Despite the widespread distribution and ecological significance of AM symbiosis, little is known about the potential of AM fungi to affect plant VOC metabolism. The purpose of this study was to investigate whether colonization of plant roots by AM fungi and associated soil microorganisms affects VOC emission and content of Artemisia annua L. plants (Asteraceae). Two inoculum types were evaluated: one consisted of only an arbuscular mycorrhizal (AM) fungus species (Glomus spp.), and the other was a mixture of different Glomus species and associated soil bacteria. Inoculated plants were compared with non-inoculated plants and with plants supplemented with extra phosphorus (P) to obtain plants of the same size as mycorrhizal plants, thus excluding potentially-confounding mycorrhizal effects on shoot growth. VOC emissions of Artemisia annua plants were analyzed by leaf cuvette sampling followed by off-line measurements with pre-concentration and gas chromatography mass spectrometry (GC-MS). Measurements of CO2 and H2O exchanges were conducted simultaneously. Several volatile monoterpenes were identified and characterized from leaf emissions of Artemisia annua L. by GC-MS analysis. The main components identified belong to different monoterpene structures: alpha-pinene, beta-pinene, camphor, 1,8-cineole, limonene, and artemisia ketone. A good correlation between monoterpene leaf concentration and leaf emission was found. Leaf extracts included also several sesquiterpenes. Total terpene content and emission was not affected by AM inoculation with or without bacteria, while emission of limonene and artemisia ketone was stimulated by this treatment. No differences were found among treatments for single monoterpene content, while accumulation of specific sesquiterpenes in leaves was altered in mycorrhizal plants compared to control plants. Growth conditions seemed to have mainly contributed to the outcome of the symbiosis and influenced the magnitude of the plant response. These results highlight the importance of considering the below-ground interaction between plant and soil for estimating VOC emission rates and their ecological role at multitrophic level

A tethered-balloon PTRMS sampling approach for surveying of landscape-scale biogenic VOC fluxes

Landscape-scale fluxes of biogenic gases were surveyed by deploying a 100 m Teflon tube attached to a tethered balloon as a sampling inlet for a fast-response proton-transfer-reaction mass spectrometer (PTRMS). Along with meteorological instruments deployed on the tethered balloon and a 3 m tripod and outputs from a regional weather model, these observations were used to estimate landscape-scale biogenic volatile organic compound fluxes with two micrometeorological techniques: mixed layer variance and surface layer gradients. This highly mobile sampling system was deployed at four field sites near Barcelona to estimate landscape-scale biogenic volatile organic compound (BVOC) emission factors in a relatively short period (3 weeks). The two micrometeorological techniques were compared with emissions predicted with a biogenic emission model using site-specific emission factors and land-cover characteristics for all four sites. The methods agreed within the uncertainty of the techniques in most cases, even though the locations had considerable heterogeneity in species distribution and complex terrain. Considering the wide range in reported BVOC emission factors for individual vegetation species (more than an order of magnitude), this temporally short and inexpensive flux estimation technique may be useful for constraining BVOC emission factors used as model inputs

The Effects of Arbuscular Mycorrhizal Fungi on Direct and Indirect Defense Metabolites of Plantago lanceolata L.

Arbuscular mycorrhizal fungi can strongly influence the metabolism of their host plant, but their effect on plant defense mechanisms has not yet been thoroughly investigated. We studied how the principal direct defenses (iridoid glycosides) and indirect defenses (volatile organic compounds) of Plantago lanceolata L. are affected by insect herbivory and mechanical wounding. Volatile compounds were collected and quantified from mycorrhizal and non-mycorrhizal P. lanceolata plants that underwent three different treatments: 1) insect herbivory, 2) mechanical wounding, or 3) no damage. The iridoids aucubin and catalpol were extracted and quantified from the same plants. Emission of terpenoid volatiles was significantly higher after insect herbivory than after the other treatments. However, herbivore-damaged mycorrhizal plants emitted lower amounts of sesquiterpenes, but not monoterpenes, than herbivore-damaged non-mycorrhizal plants. In contrast, mycorrhizal infection increased the emission of the green leaf volatile (Z)-3-hexenyl acetate in untreated control plants, making it comparable to emission from mechanically wounded or herbivore-damaged plants whether or not they had mycorrhizal associates. Neither mycorrhization nor treatment had any influence on the levels of iridoid glycosides. Thus, mycorrhizal infection did not have any effect on the levels of direct defense compounds measured in P. lanceolata. However, the large decline in herbivore-induced sesquiterpene emission may have important implications for the indirect defense potential of this species

Does mycorrhization influence herbivore-induced volatile emission in Medicago truncatula?

Symbiosis with mycorrhizal fungi substantially impacts secondary metabolism and defensive traits of colonised plants. In the present study, we investigated the influence of mycorrhization (Glomus intraradices) on inducible indirect defences against herbivores using the model legume Medicago truncatula. Volatile emission by mycorrhizal and non-mycorrhizal plants was measured in reaction to damage inflicted by Spodoptera spp. and compared to the basal levels of volatile emission by plants of two different cultivars. Emitted volatiles were recorded using closed-loop stripping and gas chromatography/mass spectrometry. The documented volatile patterns were evaluated using multidimensional scaling to visualise patterns and stepwise linear discriminant analysis to distinguish volatile blends of plants with distinct physiological status and genetic background. Volatile blends emitted by different cultivars of M. truncatula prove to be clearly distinct, whereas mycorrhization only slightly influenced herbivore-induced volatile emissions. Still, the observed differences were sufficient to create classification rules to distinguish mycorrhizal and non-mycorrhizal plants by the volatiles emitted. Moreover, the effect of mycorrhization turned out to be opposed in the two cultivars examined. Root symbionts thus seem to alter indirect inducible defences of M. truncatula against insect herbivores. The impact of this effect strongly depends on the genetic background of the plant and, hence, in part explains the highly contradictory results on tripartite interactions gathered to date