Regulation of isoprene synthase promoter by environmental and internal factors

Isoprene synthase (ISPS) catalyzes the formation of isoprene, an important volatile terpenoid with strong effects on global atmospheric chemistry and protective physiological functions in plant leaves. Many terpene synthase genes including isoprene synthase, a member of the TPS-b cluster of this numerous gene family, were already functionally analysed but much less is known about regulation of their promoters. To study regulation of the PcISPS gene in detail we developed transgenic Grey poplar (Populus × canescens) and Arabidopsis thaliana plants in which the PcISPS promoter is fused to enhanced green fluorescent protein (E-GFP) and β-glucuronidase (GUS) reporter genes. We analysed these reporters during plant development, for organ specificity and in plants subjected to different light and temperature regimes. We observed low promoter activity in non-isoprene emitting tissue like roots where ISPS gene is transcribed but no active enzyme is detectable. In leaves we demonstrate that light and temperature directly modulate ISPS promoter activity. Moreover, with confocal laser scanning microscopy we show a cell specific gradient of ISPS promoter activity within the leaf parenchyma depending on light direction. Our results indicate that ISPS promoter activity, which correlates with basal isoprene emission capacity, is not uniformly distributed within leaf tissue and that it can adapt rapidly towards internal as well as external environmental stimuli.     

Isoprene synthesis protects transgenic tobacco plants from oxidative stress

Isoprene emission represents a significant loss of carbon to those plant species that synthesize this highly volatile and reactive compound. As a tool for studying the role of isoprene in plant physiology and biochemistry, we developed transgenic tobacco plants capable of emitting isoprene in a similar manner to and at rates comparable to a naturally emitting species. Thermotolerance of photosynthesis against transient high-temperature episodes could only be observed in lines emitting high levels of isoprene; the effect was very mild and could only be identified over repetitive stress events. However, isoprene-emitting plants were highly resistant to ozone-induced oxidative damage compared with their non-emitting azygous controls. In ozone-treated plants, accumulation of toxic reactive oxygen species (ROS) was inhibited, and antioxidant levels were higher. Isoprene-emitting plants showed remarkably decreased foliar damage and higher rates of photosynthesis compared to non-emitting plants immediately following oxidative stress events. An inhibition of hydrogen peroxide accumulation in isoprene-emitting plants may stall the programmed cell death response which would otherwise lead to foliar necrosis. These results demonstrate that endogenously produced isoprene provides protection from oxidative damage.     

An ectomycorrhizal fungus alters sensitivity to jasmonate,salicylate, gibberellin,and ethylene in host roots

The phytohormones jasmonate, gibberellin, salicylate, and ethylene regulate an interconnected reprogramming network integrating root development with plant responses against microbes. The establishment of mutualistic ectomycorrhizal symbiosis requires the suppression of plant defense responses against fungi as well as the modification of root architecture and cortical cell wall properties. Here, we investigated the contribution of phytohormones and their crosstalk to the ontogenesis of ectomycorrhizae (ECM) between grey poplar (Populus tremula x alba) roots and the fungus Laccaria bicolor. To obtain the hormonal blueprint of developing ECM, we quantified the concentrations of jasmonates, gibberellins, and salicylate via liquid chromatography–tandem mass spectrometry. Subsequently, we assessed root architecture, mycorrhizal morphology, and gene expression levels (RNA sequencing) in phytohormone-treated poplar lateral roots in the presence or absence of L. bicolor. Salicylic acid accumulated in mid-stage ECM. Exogenous phytohormone treatment affected the fungal colonization rate and/or frequency of Hartig net formation. Colonized lateral roots displayed diminished responsiveness to jasmonate but regulated some genes, implicated in defense and cell wall remodelling, that were specifically differentially expressed after jasmonate treatment. Responses to salicylate, gibberellin, and ethylene were enhanced in ECM. The dynamics of phytohormone accumulation and response suggest that jasmonate, gibberellin, salicylate, and ethylene signalling play multifaceted roles in poplar L. bicolor ectomycorrhizal development.     

Non‐specific phospholipase C5 and diacylglycerol promote lateral root development under mild salt stress in Arabidopsis

Developing a robust root system is crucial to plant survival and competition for soil resources. Here we report that the non‐specific phospholipase C5 (NPC5) and its derived lipid mediator diacylglycerol (DAG) mediate lateral root (LR) development during salt stress in Arabidopsis thaliana. T‐DNA knockout mutant npc5‐1 produced few to no LR under mild NaCl stress, whereas overexpression of NPC5 increased LR number. Roots of npc5‐1 contained a lower level of DAG than wild type, whereas NPC5 overexpressor exhibited an increase in DAG level. Application of DAG, but not phosphatidic acid, fully restored LR growth of npc5‐1 to that of wild type under NaCl stress. NPC5 expression was significantly induced in Arabidopsis seedlings treated with NaCl. Npc5‐1 was less responsive to auxin‐mediated root growth than the wild type. These results indicate that NPC5 mediates LR development in response to salt stress and suggest that DAG functions as a lipid mediator in the stress signalling.     

Within‐plant isoprene oxidation confirmed by direct emissions of oxidation products methyl vinyl ketone and methacrolein

Isoprene is emitted from many terrestrial plants at high rates, accounting for an estimated 1/3 of annual global volatile organic compound emissions from all anthropogenic and biogenic sources combined. Through rapid photooxidation reactions in the atmosphere, isoprene is converted to a variety of oxidized hydrocarbons, providing higher order reactants for the production of organic nitrates and tropospheric ozone, reducing the availability of oxidants for the breakdown of radiatively active trace gases such as methane, and potentially producing hygroscopic particles that act as effective cloud condensation nuclei. However, the functional basis for plant production of isoprene remains elusive. It has been hypothesized that in the cell isoprene mitigates oxidative damage during the stress‐induced accumulation of reactive oxygen species (ROS), but the products of isoprene‐ROS reactions in plants have not been detected. Using pyruvate‐2‐¹³C leaf and branch feeding and individual branch and whole mesocosm flux studies, we present evidence that isoprene (i) is oxidized to methyl vinyl ketone and methacrolein (i_ox) in leaves and that i_ox/i emission ratios increase with temperature, possibly due to an increase in ROS production under high temperature and light stress. In a primary rainforest in Amazonia, we inferred significant in plant isoprene oxidation (despite the strong masking effect of simultaneous atmospheric oxidation), from its influence on the vertical distribution of i_ox uptake fluxes, which were shifted to low isoprene emitting regions of the canopy. These observations suggest that carbon investment in isoprene production is larger than that inferred from emissions alone and that models of tropospheric chemistry and biota–chemistry–climate interactions should incorporate isoprene oxidation within both the biosphere and the atmosphere with potential implications for better understanding both the oxidizing power of the troposphere and forest response to climate change.     

A new role for glutathione in the regulation of root architecture linked to strigolactones

Reduced glutathione (GSH) is required for root development, but its functions are not characterized. The effects of GSH depletion on root development were therefore studied in relation to auxin and strigolactone (SL) signalling using a combination of molecular genetic approaches and pharmacological techniques. Lateral root (LR) density was significantly decreased in GSH synthesis mutants (cad2‐1, pad2‐, rax1‐), but not by the GSH synthesis inhibitor, buthionine sulfoximine (BSO). BSO‐induced GSH depletion therefore did not influence root architecture in the same way as genetic impairment. Root glutathione contents were similar in the wild‐type seedlings and max3‐9 and max4‐1 mutants that are deficient in SL synthesis and in the SL‐signalling mutant, max2‐1. BSO‐dependent inhibition of GSH synthesis depleted the tissue GSH pool to a similar extent in the wild‐type and SL synthesis mutants, with no effect on LR density. The application of the SL analogue GR24 increased root glutathione in the wild‐type, max3‐9 and max4‐1 seedlings, but this increase was absent from max2‐1. Taken together, these data establish a link between SLs and the GSH pool that occurs in a MAX2‐dependent manner.     

Use of carbon-11 in Populus shows that exogenous jasmonic acid increases biosynthesis of isoprene from recently fixed carbon

A new approach for pulse labelling of plants using the short-lived positron emitting radioisotope carbon-11 (half-life: 20.4 min) as ¹¹CO₂ is reported together with its application to measuring [¹¹C]isoprene emissions from intact leaves capturing information associated with: (1) rate of emission; (2) the relative contribution of recently fixed carbon to isoprene biosynthesis; and (3) the transit time for tracer movement through the leaf and biochemical pathways associated with isoprene biosynthesis. This approach was applied to study the response of certain Populus species to exogenous treatments of jasmonic acid (JA), a plant hormone implicated in signal transduction linked to defence response against herbivory. Twelve hours after treatment of single intact leaves of aspen (Populus tremuloides) with a 1 m m JA spray, isoprene emissions from those leaves increased 1.5 times the controls from 35.4 ± 2.2 to 53.1 ± 4.8 nmol m⁻² s⁻¹. [¹¹C]Isoprene emissions from the same leaves, reflecting the isoprene that was derived from recently fixed carbon, increased much more, to 2.2 times the controls. This increase coincided with a change in emitted [¹¹C]isoprene from 0.31 to 0.68% of ¹¹C fixed in the leaf tissue, while the tracer transit time remained constant at 12.5 min. These results suggest that JA had no effect on enzyme kinetics involved in isoprene biosynthesis, but did impact the source of recent carbon feeding that pathway. Studies with poplar (Populus nigra clone NC 5271) showed similar trends in systemic emissions (from an untreated leaf on the same plant).     

Poplar PtabZIP1‐like enhances lateral root formation and biomass growth under drought stress

Developing drought‐resistance varieties is a major goal for bioenergy crops, such as poplar (Populus), which will be grown on marginal lands with little or no water input. Root architecture can affect drought resistance, but few genes that affect root architecture in relation to water availability have been identified. Here, using activation tagging in the prime bioenergy crop poplar, we have identified a mutant that overcomes the block of lateral root (LR) formation under osmotic stress. Positioning of the tag, validation of the activation and recapitulation showed that the phenotype is caused by the poplar PtabZIP1‐like (PtabZIP1L) gene with highest homology to bZIP1 from Arabidopsis. PtabZIP1L is predominantly expressed in roots, particularly in zones where lateral root primordia (LRP) initiate and LR differentiate and emerge. Transgenics overexpressing PtabZIP1L showed precocious LRP and LR development, while PtabZIP1L suppression significantly delayed both LRP and LR formation. Transgenic overexpression and suppression of PtabZIP1L also resulted in modulation of key metabolites like proline, asparagine, valine and several flavonoids. Consistently, expression of both of the poplar Proline Dehydrogenase orthologs and two of the Flavonol Synthases genes was also increased and decreased in overexpressed and suppressed transgenics, respectively. These findings suggest that PtabZIP1L mediates LR development and drought resistance through modulation of multiple metabolic pathways.     

Nitric oxide mediates strigolactone signaling in auxin and ethylene-sensitive lateral root formation in sunflower seedlings

Strigolactones (SLs) play significant role in shaping root architecture whereby auxin-SL crosstalk has been observed in SL-mediated responses of primary root elongation, lateral root formation and adventitious root (AR) initiation. Whereas GR24 (a synthetic strigolactone) inhibits LR and AR formation, the effect of SL biosynthesis inhibitor (fluridone) is just the opposite (root proliferation). Naphthylphthalamic acid (NPA) leads to LR proliferation but completely inhibits AR development. The diffusive distribution of PIN1 in the provascular cells in the differentiating zone of the roots in response to GR24, fluridone or NPA treatments further indicates the involvement of localized auxin accumulation in LR development responses. Inhibition of LR formation by GR24 treatment coincides with inhibition of ACC synthase activity. Profuse LR development by fluridone and NPA treatments correlates with enhanced [Ca²⁺]_cyt in the apical region and differentiating zones of LR, indicating a critical role of [Ca²⁺] in LR development in response to the coordinated action of auxins, ethylene and SLs. Significant enhancement of carotenoid cleavage dioxygenase (CCD) activity (enzyme responsible for SL biosynthesis) in tissue homogenates in presence of cPTIO (NO scavenger) indicates the role of endogenous NO as a negative modulator of CCD activity. Differences in the spatial distribution of NO in the primary and lateral roots further highlight the involvement of NO in SL-modulated root morphogenesis in sunflower seedlings. Present work provides new report on the negative modulation of SL biosynthesis through modulation of CCD activity by endogenous nitric oxide during SL-modulated LR development.     

Early signalling pathways in rice roots under vanadate stress

Vanadate is beneficial to plant growth at low concentration. However, plant exposure to high concentrations of vanadate has been shown to arrest cell growth and lead to cell death. We are interested in understanding the signalling pathways of rice roots in response to vanadate stress. In this study, we demonstrated that vanadate induced rice root cell death and suppressed root growth. In addition, we found that vanadate induced ROS accumulation, increased lipid peroxidation and elicited a remarkable increase of MAPKs and CDPKs activities in rice roots. In contrast, pre-treatment of rice roots with ROS scavenger (sodium benzoate), serine/threonine protein phosphatase inhibitor (endothall), and CDPK antagonist (W7), reduced the vanadate-induced MAPKs activation. Furthermore, the expression of a MAPK gene (OsMPK3) and four tyrosine phosphatase genes (OsDSP3, OsDSP5, OsDSP6, and OsDSP10) were regulated by vanadate in rice roots. Collectively, these results strongly suggest that ROS, protein phosphatase, and CDPK may function in the vanadate-triggered MAPK signalling pathway cause cell death and retarded growth in rice roots.     

Effects of growth under elevated UV-B on photosynthesis and isoprene emission in Quercus gambelii and Mucuna pruriens

Increasing surface levels of UV-B resulting from stratospheric ozone reduction directly affect tropospheric photochemistry. There may also be indirect tropospheric effects due to changes in emission of organic compounds from vegetation. We treated woody and herbaceous isoprene-emitting species in the field with supplemental UV-B simulating 30% ozone depletion. For Quercus gambelii, photosynthesis and isoprene emission were significantly greater in elevated UV-B treatments when expressed on a leaf area basis, but not on a leaf mass basis. Leaves of Mucuna pruriens, however, showed no significant differences in photosynthesis or isoprene emission between treatments, nor when exposed for 45 min to acute high levels of UV-B. Elevated UV-B during growth did not elicit significant isoprene emission from Acer platanoides, a non-emitting species. Other potential UV-B effects, such as changes in leaf area or species composition, which may influence regional isoprene emissions, should be examined.     

Do cluster roots of Hakea actities (Proteaceae) acquire complex organic nitrogen?

Although there has been speculation that cluster roots play a role in plant nitrogen (N) acquisition there have been few experimental studies, demonstrating this. We grew the subtropical wet heathland plant Hakea actities in sand and in axenic vermiculite-sand cultures to investigate its use of different N sources. Seedlings produced greater quantities of cluster roots when grown with NO₃ ^–, glutathione or protein as N sources than with NH₄ ⁺, glutamine or without N addition, suggesting that N source influences cluster root initiation and development. Axenically grown seedlings acquired more N when grown with 4.5 mM than with 0.5 mM glutathione or protein as N sources suggesting that seedlings are able to assimilate complex organic N. However, control seedlings that did not receive N other than seed storage N (approximately 0.2 mg N) acquired 2 mg N from an unknown source, so that data are not unequivocal. Peptidase activity in extracts derived from non-axenic cluster root tissue changed with age of cluster roots. Developing and mature cluster roots had higher peptidase activity than young and senescing cluster roots. To determine whether peptidases are associated with the outer surface of cluster roots, entire cluster roots were gently centrifuged and the resulting solution analysed for peptidase activity using gelatine-containing PAGE. Different peptidases were active at different times of cluster root development, suggesting that plant or microbially derived peptidases could play a role in organic N acquisition by cluster roots. Roots and cluster roots expressed a putative amino acid transporter (HaAAT4-1) and peptide transporter (HaPepT1) as determined by northern blot analysis. Expression of HaPepT1 in cluster roots increased throughout cluster root development although further studies need to confirm this.     

Phaseolus vulgaris RbohB functions in lateral root development

Respiratory burst oxidase homologs (RBOHs) catalyze the reduction of oxygen to generate superoxide anion, a kind of reactive oxygen species (ROS). The ROS produced by RBOHs play essential roles in diverse processes, such as root hair development, stomata closure and signaling mechanisms in response to abiotic stimuli and during plant-pathogen interactions. Recently, we found that PvRbohB silencing in transgenic Phaseolus vulgaris roots had a negative impact on lateral root density. In this work, we show that the downregulation of PvRbohB affects both the growth and ROS levels in recently emerged lateral roots. In addition, we found that the PvRbohB promoter was activated during lateral root primordium initiation in the pericycle, and remained active throughout lateral root development. This study identifies RBOHs as potentially important players in lateral root development in P. vulgaris.     

Accumulation of reactive oxygen species in arbuscular mycorrhizal roots

We investigated the accumulation of reactive oxygen species (ROS) in arbuscular mycorrhizal (AM) roots from Medicago truncatula, Zea mays and Nicotiana tabacum using three independent staining techniques. Colonized root cortical cells and the symbiotic fungal partner were observed to be involved in the production of ROS. Extraradical hyphae and spores from Glomus intraradices accumulated small levels of ROS within their cell wall and produced ROS within the cytoplasm in response to stress. Within AM roots, we observed a certain correlation of arbuscular senescence and H₂O₂ accumulation after staining by diaminobenzidine (DAB) and a more general accumulation of ROS close to fungal structures when using dihydrorhodamine 123 (DHR 123) for staining. According to electron microscopical analysis of AM roots from Z. mays after staining by CeCl₃, intracellular accumulation of H₂O₂ was observed in the plant cytoplasm close to intact and collapsing fungal structures, whereas intercellular H₂O₂ was located on the surface of fungal hyphae. These characteristics of ROS accumulation in AM roots suggest similarities to ROS accumulation during the senescence of legume root nodules.     

Isolation and Purification of Protein Responsible for the Conversion of Dimethylallylpyrophosphate from Poplar Leaves into Isoprene

The protein converting dimethylallylpyrophosphate (DMAPP) into isoprene in vitrowas isolated and purified 3000-fold from leaves of berry-bearing poplar (Populus deltoidesMarsh.). As the enzyme was purified, its specific activity increased and at the final stage reached 266 nmol/(min mg protein). The enzyme was eluted by anion-exchange chromatography in a 120–170 mM NaCl gradient and by chromatography on the hydroxyapatite column in 170 mM sodium phosphate. The active molecular weight of the protein determined by gel filtration was 100–110 kD. As the enzyme was purified, the K _Mvalue increased from 2 to 9 mM. A parallelism isoprene emission from DMAPP and an increase in the specific activity of the enzyme as it was purified proved that the enzyme catalyzed isoprene emission.     

Lateral root stimulation in the early interaction between Arabidopsis thaliana and the ectomycorrhizal fungus Laccaria bicolor: Is fungal auxin the trigger?

Lateral root (LR) stimulation during early signal exchange between plant roots and ectomycorrhizal (ECM) fungi has recently been shown to be achieved by modulation of auxin gradients. We suggested that this modulation could occur through altered polar auxin transport (PAT) and through activation of auxin signalling pathways in the root. However, it remains unclear, which fungal molecules alter auxin pathways inside the plant partner. It has been suggested in previous studies that auxin released by the fungus could trigger observed plant responses during early signal exchange and later on during root colonization. Here we focus on the early interaction and we provide evidence for an alternative mechanism. Indeed, LR stimulation by the fungus in Arabidopsis thaliana followed a totally different timing than with exogenously applied auxin. Furthermore, experimental conditions that excluded the exchange of soluble molecules while allowing exchange of volatile(s) between the plant and the fungus were sufficient for LR induction, therefore questioning the role of secreted fungal auxin. These data suggest that volatiles released by the fungus and sensed by the plant may act upstream of altered auxin signaling in the plant.Key words: mycorrhiza, ectomycorrhiza, lateral root, auxin, volatiles, ethylene, jasmonic acidInteractions of plant roots with symbiotic, ectomycorrhizal soil fungi lead to lateral root (LR) stimulation during the very early interaction phase.¹ This LR stimulation has recently been shown to be independent of root colonization and to occur as well in non-mycorrhizal plants, such as Arabidopsis suggesting that fungal signals have a broad perception spectrum.^1,2 However, little is known about the type of signals exchanged between fungi and their plant partners during this early interaction phase. Several studies have proposed a role for the phytohormone auxin produced and secreted by ECM fungi as the signalling molecule during ECM fungus/plant signaling.^2–7 Recently we studied changes in auxin response and auxin transport in poplar and Arabidopsis thaliana roots during contact with the ECM fungus Laccaria bicolor.¹ We demonstrated that the presence of the fungus enhances the auxin response and distribution at the root apex and that this, as well as LR stimulation, is reliant on polar auxin transport through AtPIN2 and probably through PtPIN9 in poplar. Here, using Arabidopsis thaliana, whose LR stimulation by Laccaria bicolor has been demonstrated, we propose that not yet identified fungal volatiles may regulate auxin homeostasis in the plant, questioning the contribution of the auxin released by the fungus on the induction of LR.