Time-series transcriptomics reveals a BBX32-directed control of acclimation to high light in mature Arabidopsis leaves.

The photosynthetic capacity of mature leaves increases after several days' exposure to constant or intermittent episodes of high light (HL) and is manifested primarily as changes in chloroplast physiology. How this chloroplast-level acclimation to HL is initiated and controlled is unknown. From expanded Arabidopsis leaves, we determined HL-dependent changes in transcript abundance of 3844 genes in a 0-6 h time-series transcriptomics experiment. It was hypothesized that among such genes were those that contribute to the initiation of HL acclimation. By focusing on differentially expressed transcription (co-)factor genes and applying dynamic statistical modelling to the temporal transcriptomics data, a regulatory network of 47 predominantly photoreceptor-regulated transcription (co-)factor genes was inferred. The most connected gene in this network was B-BOX DOMAIN CONTAINING PROTEIN32 (BBX32). Plants overexpressing BBX32 were strongly impaired in acclimation to HL and displayed perturbed expression of photosynthesis-associated genes under LL and after exposure to HL. These observations led to demonstrating that as well as regulation of chloroplast-level acclimation by BBX32, CRYPTOCHROME1, LONG HYPOCOTYL5, CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA-105 are important. In addition, the BBX32-centric gene regulatory network provides a view of the transcriptional control of acclimation in mature leaves distinct from other photoreceptor-regulated processes, such as seedling photomorphogenesis.

Plant environmental sensing relies on specialized plastids.

In plants, plastids are thought to interconvert to various forms that are specialized for photosynthesis, starch and oil storage, and diverse pigment accumulation. Post-endosymbiotic evolution has led to adaptations and specializations within plastid populations that align organellar functions with different cellular properties in primary and secondary metabolism, plant growth, organ development, and environmental sensing. Here, we review the plastid biology literature in light of recent reports supporting a class of 'sensory plastids' that are specialized for stress sensing and signaling. Abundant literature indicates that epidermal and vascular parenchyma plastids display shared features of dynamic morphology, proteome composition, and plastid-nuclear interaction that facilitate environmental sensing and signaling. These findings have the potential to reshape our understanding of plastid functional diversification.

Cadmium induces reactive oxygen species-dependent pexophagy in Arabidopsis leaves.

Cadmium treatment induces transient peroxisome proliferation in Arabidopsis leaves. To determine whether this process is regulated by pexophagy and to identify the mechanisms involved, we analysed time course-dependent changes in ATG8, an autophagy marker, and the accumulation of peroxisomal marker PEX14a. After 3 hr of Cd exposure, the transcript levels of ATG8h, ATG8c, a, and i were slightly up-regulated and then returned to normal. ATG8 protein levels also increased after 3 hr of Cd treatment, although an opposite pattern was observed in PEX14. Arabidopsis lines expressing GFP-ATG8a and CFP-SKL enabled us to demonstrate the presence of pexophagic processes in leaves. The Cd-dependent induction of pexophagy was demonstrated by the accumulation of peroxisomes in autophagy gene (ATG)-related Arabidopsis knockout mutants atg5 and atg7. We show that ATG8a colocalizes with catalase and NBR1 in the electron-dense peroxisomal core, thus suggesting that NBR1 may be an autophagic receptor for peroxisomes, with catalase being possibly involved in targeting pexophagy. Protein carbonylation and peroxisomal redox state suggest that protein oxidation may trigger pexophagy. Cathepsine B, legumain, and caspase 6 may also be involved in the regulation of pexophagy. Our results suggest that pexophagy could be an important step in rapid cell responses to cadmium.

Time-Series Transcriptomics Reveals That AGAMOUS-LIKE22 Affects Primary Metabolism and Developmental Processes in Drought-Stressed Arabidopsis.

In Arabidopsis thaliana, changes in metabolism and gene expression drive increased drought tolerance and initiate diverse drought avoidance and escape responses. To address regulatory processes that link these responses, we set out to identify genes that govern early responses to drought. To do this, a high-resolution time series transcriptomics data set was produced, coupled with detailed physiological and metabolic analyses of plants subjected to a slow transition from well-watered to drought conditions. A total of 1815 drought-responsive differentially expressed genes were identified. The early changes in gene expression coincided with a drop in carbon assimilation, and only in the late stages with an increase in foliar abscisic acid content. To identify gene regulatory networks (GRNs) mediating the transition between the early and late stages of drought, we used Bayesian network modeling of differentially expressed transcription factor (TF) genes. This approach identified AGAMOUS-LIKE22 (AGL22), as key hub gene in a TF GRN. It has previously been shown that AGL22 is involved in the transition from vegetative state to flowering but here we show that AGL22 expression influences steady state photosynthetic rates and lifetime water use. This suggests that AGL22 uniquely regulates a transcriptional network during drought stress, linking changes in primary metabolism and the initiation of stress responses.

To defend or to grow: lessons from Arabidopsis C24.

The emergence of Arabidopsis as a model species and the availability of genetic and genomic resources have resulted in the identification and detailed characterization of abiotic stress signalling pathways. However, this has led only to limited success in engineering abiotic stress tolerance in crops. This is because there needs to be a deeper understanding of how to combine resistances to a range of stresses with growth and productivity. The natural variation and genomic resources of Arabidopsis thaliana (Arabidopsis) are a great asset to understand the mechanisms of multiple stress tolerances. One natural variant in Arabidopsis is the accession C24, and here we provide an overview of the increasing research interest in this accession. C24 is highlighted as a source of tolerance for multiple abiotic and biotic stresses, and a key accession to understand the basis of basal immunity to infection, high water use efficiency, and water productivity. Multiple biochemical, physiological, and phenological mechanisms have been attributed to these traits in C24, and none of them constrains productivity. Based on the uniqueness of C24, we postulate that the use of variation derived from natural selection in undomesticated species provides opportunities to better understand how complex environmental stress tolerances and resource use efficiency are co-ordinated.

Arabidopsis HEAT SHOCK TRANSCRIPTION FACTORA1b regulates multiple developmental genes under benign and stress conditions.

In Arabidopsis thaliana, HEAT SHOCK TRANSCRIPTION FACTORA1b (HSFA1b) controls resistance to environmental stress and is a determinant of reproductive fitness by influencing seed yield. To understand how HSFA1b achieves this, we surveyed its genome-wide targets (ChIP-seq) and its impact on the transcriptome (RNA-seq) under non-stress (NS), heat stress (HS) in the wild type, and in HSFA1b-overexpressing plants under NS. A total of 952 differentially expressed HSFA1b-targeted genes were identified, of which at least 85 are development associated and were bound predominantly under NS. A further 1780 genes were differentially expressed but not bound by HSFA1b, of which 281 were classified as having development-associated functions. These genes are indirectly regulated through a hierarchical network of 27 transcription factors (TFs). Furthermore, we identified 480 natural antisense non-coding RNA (cisNAT) genes bound by HSFA1b, defining a further mode of indirect regulation. Finally, HSFA1b-targeted genomic features not only harboured heat shock elements, but also MADS box, LEAFY, and G-Box promoter motifs. This revealed that HSFA1b is one of eight TFs that target a common group of stress defence and developmental genes. We propose that HSFA1b transduces environmental cues to many stress tolerance and developmental genes to allow plants to adjust their growth and development continually in a varying environment.

Integrating transcriptomic techniques and k-means clustering in metabolomics to identify markers of abiotic and biotic stress in Medicago truncatula.

INTRODUCTION: Nitrogen-fixing legumes are invaluable crops, but are sensitive to physical and biological stresses. Whilst drought and infection from the soil-borne pathogen Fusarium oxysporum have been studied individually, their combined effects have not been widely investigated. OBJECTIVES: We aimed to determine the effect of combined stress using methods usually associated with transcriptomics to detect metabolic differences between treatment groups that could not be identified by more traditional means, such as principal component analysis and partial least squares discriminant analysis. METHODS: Liquid chromatography-high resolution mass spectrometry data from the root and leaves of model legume Medicago truncatula were analysed using Gaussian Process 2-Sample Test, k-means cluster analysis and temporal clustering by affinity propagation. RESULTS: Metabolic differences were detected: we identified known stress markers, including changes in concentration for sucrose and citric acid, and showed that combined stress can exacerbate the effect of drought. Changes in roots were found to be smaller than those in leaves, but differences due to Fusarium infection were identified. The transfer of sucrose from leaves to roots can be seen in the time series using transcriptomic techniques with the metabolomics time series. Other metabolite concentrations that change as a result of treatment include phosphoric acid, malic acid and tetrahydroxychalcone. CONCLUSIONS: Probing metabolomic data with transcriptomic tools provides new insights and could help to identify resilient plant varieties, thereby increasing future crop yield and improving food security.

The temporal foliar transcriptome of the perennial C3 desert plant Rhazya stricta in its natural environment.

BACKGROUND: The perennial species Rhazya stricta (R. stricta) grows in arid zones and carries out typical C3 photosynthesis under daily extremes of heat, light intensity and low humidity. In order to identify processes attributable to its adaptation to this harsh environment, we profiled the foliar transcriptome of apical and mature leaves harvested from the field at three time periods of the same day. RESULTS: Next generation sequencing was used to reconstruct the transcriptome and quantify gene expression. 28018 full length transcript sequences were recovered and 45.4% were differentially expressed (DE) throughout the day. We compared our dataset with microarray experiments in Arabidopsis thaliana (Arabidopsis) and other desert species to identify trends in circadian and stress response profiles between species. 34% of the DE genes were homologous to Arabidopsis circadian-regulated genes. Independent of circadian control, significant overlaps with Arabidopsis genes were observed only with heat and salinity/high light stress-responsive genes. Also, groups of DE genes common to other desert plants species were identified. We identified protein families specific to R. stricta which were found to have diverged from their homologs in other species and which were over -expressed at midday. CONCLUSIONS: This study shows that temporal profiling is essential to assess the significance of genes apparently responsive to abiotic stress. This revealed that in R. stricta, the circadian clock is a major regulator of DE genes, even of those annotated as stress-responsive in other species. This may be an important feature of the adaptation of R. stricta to its extreme but predictable environment. However, the majority of DE genes were not circadian-regulated. Of these, some were common to other desert species and others were distinct to R. stricta, suggesting that they are important for the adaptation of such plants to arid environments.

C3 photosynthesis in the desert plant Rhazya stricta is fully functional at high temperatures and light intensities.

The C3 plant Rhazya stricta is native to arid desert environment zones, where it experiences daily extremes of heat, light intensity (PAR) and high vapour pressure deficit (VPD). We measured the photosynthetic parameters in R. stricta in its native environment to assess the mechanisms that permit it to survive in these extreme conditions. Infrared gas exchange analysis examined diel changes in assimilation (A), stomatal conductance (gs ) and transpiration (E) on mature leaves of R. stricta. A/ci analysis was used to determine the effect of temperature on carboxylation capacity (Vc,max ) and the light- and CO2 -saturated rate of photosynthesis (Amax ). Combined chlorophyll fluorescence and gas exchange light response curve analysis at ambient and low oxygen showed that both carboxylation and oxygenation of Rubisco acted as the major sinks for the end products of electron transport. Physiological analysis in conjunction with gene expression analysis suggested that there are two isoforms of Rubisco activase which may provide an explanation for the ability of R. stricta to maintain Rubisco function at high temperatures. The potential to exploit this ability to cope with extreme temperatures is discussed in the context of future crop improvement.

Isoprene emission protects photosynthesis but reduces plant productivity during drought in transgenic tobacco (Nicotiana tabacum) plants.

Isoprene protects the photosynthetic apparatus of isoprene-emitting plants from oxidative stress. The role of isoprene in the response of plants to drought is less clear. Water was withheld from transgenic isoprene-emitting and non-emitting tobacco (Nicotiana tabacum) plants, to examine: the response of isoprene emission to plant water deficit; a possible relationship between concentrations of the drought-induced phytohormone abscisic acid (ABA) and isoprene; and whether isoprene affected foliar reactive oxygen species (ROS) and lipid peroxidation levels. Isoprene emission did not affect whole-plant water use, foliar ABA concentration or leaf water potential under water deficit. Compared with well-watered controls, droughted non-emitting plants significantly increased ROS content (31-46%) and lipid peroxidation (30-47%), concomitant with decreased operating and maximum efficiencies of photosystem II photochemistry and lower leaf and whole-plant water use efficiency (WUE). Droughted isoprene-emitting plants showed no increase in ROS content or lipid peroxidation relative to well-watered controls, despite isoprene emission decreasing before leaf wilting. Although isoprene emission protected the photosynthetic apparatus and enhanced leaf and whole-plant WUE, non-emitting plants had 8-24% more biomass under drought, implying that isoprene emission incurred a yield penalty.

Cadmium exposure induced light/dark- and time-dependent redox changes at subcellular level in Arabidopsis plants.

Cadmium (Cd) is one of the most toxic heavy metals for plants and humans. Reactive oxygen species (ROS) are some of the primary signaling molecules produced after Cd treatment in plants but the contribution of different organelles and specific cell types, together with the impact of light is unknown. We used Arabidopsis lines expressing GRX1-roGFP2 (glutaredoxin1-roGFP) targeted to different cell compartments and analysed changes in redox state over 24 h light/dark cycle in Cd-treated leaf discs. We imaged redox state changes in peroxisomes and chloroplasts in leaf tissue. Chloroplasts and peroxisomes were the most affected organelles in the dark and blocking the photosynthetic electron transport chain (pETC) by DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) promotes higher Cd-dependent oxidation in all organelles. Peroxisomes underwent the most rapid changes in redox state in response to Cd and DCMU and silencing chloroplastic NTRC (NADPH thioredoxin reductase C) considerably increases peroxisome oxidation. Total NAD(P)H and cytosolic NADH decreased during exposure to Cd, while Ca+2 content in chloroplasts and cytosol increased in the dark period. Our results demonstrate a Cd-, time- and light-dependent increase of oxidation of all organelles analysed, that could be in part triggered by disturbances in pETC and photorespiration, the decrease of NAD(P)H availability, and differential antioxidants expression at subcellular level.

Photosynthetic adaptation to length of day is dependent on S-sulfocysteine synthase activity in the thylakoid lumen.

Arabidopsis (Arabidopsis thaliana) chloroplasts contain two O-acetyl-serine(thiol)lyase (OASTL) homologs, OAS-B, which is an authentic OASTL, and CS26, which has S-sulfocysteine synthase activity. In contrast with OAS-B, the loss of CS26 function resulted in dramatic phenotypic changes, which were dependent on the light treatment. We have performed a detailed characterization of the photosynthetic and chlorophyll fluorescence parameters in cs26 plants compared with those of wild-type plants under short-day growth conditions (SD) and long-day growth conditions (LD). Under LD, the photosynthetic characterization, which was based on substomatal CO(2) concentrations and CO(2) concentration in the chloroplast curves, revealed significant reductions in most of the photosynthetic parameters for cs26, which were unchanged under SD. These parameters included net CO(2) assimilation rate, mesophyll conductance, and mitochondrial respiration at darkness. The analysis also showed that cs26 under LD required more absorbed quanta per driven electron flux and fixed CO(2). The nonphotochemical quenching values suggested that in cs26 plants, the excess electrons that are not used in photochemical reactions may form reactive oxygen species. A photoinhibitory effect was confirmed by the background fluorescence signal values under LD and SD, which were higher in young leaves compared with mature ones under SD. To hypothesize the role of CS26 in relation to the photosynthetic machinery, we addressed its location inside of the chloroplast. The activity determination and localization analyses that were performed using immunoblotting indicated the presence of an active CS26 enzyme exclusively in the thylakoid lumen. This finding was reinforced by the observation of marked alterations in many lumenal proteins in the cs26 mutant compared with the wild type.

Arabidopsis HEAT SHOCK TRANSCRIPTION FACTORA1b overexpression enhances water productivity, resistance to drought, and infection.

Heat-stressed crops suffer dehydration, depressed growth, and a consequent decline in water productivity, which is the yield of harvestable product as a function of lifetime water consumption and is a trait associated with plant growth and development. Heat shock transcription factor (HSF) genes have been implicated not only in thermotolerance but also in plant growth and development, and therefore could influence water productivity. Here it is demonstrated that Arabidopsis thaliana plants with increased HSFA1b expression showed increased water productivity and harvest index under water-replete and water-limiting conditions. In non-stressed HSFA1b-overexpressing (HSFA1bOx) plants, 509 genes showed altered expression, and these genes were not over-represented for development-associated genes but were for response to biotic stress. This confirmed an additional role for HSFA1b in maintaining basal disease resistance, which was stress hormone independent but involved H2O2 signalling. Fifty-five of the 509 genes harbour a variant of the heat shock element (HSE) in their promoters, here named HSE1b. Chromatin immunoprecipitation-PCR confirmed binding of HSFA1b to HSE1b in vivo, including in seven transcription factor genes. One of these is MULTIPROTEIN BRIDGING FACTOR1c (MBF1c). Plants overexpressing MBF1c showed enhanced basal resistance but not water productivity, thus partially phenocopying HSFA1bOx plants. A comparison of genes responsive to HSFA1b and MBF1c overexpression revealed a common group, none of which harbours a HSE1b motif. From this example, it is suggested that HSFA1b directly regulates 55 HSE1b-containing genes, which control the remaining 454 genes, collectively accounting for the stress defence and developmental phenotypes of HSFA1bOx.

The Passage of H2O2 from Chloroplasts to Their Associated Nucleus during Retrograde Signalling: Reflections on the Role of the Nuclear Envelope.

The response of chloroplasts to adverse environmental cues, principally increases in light intensity, stimulates chloroplast-to-nucleus retrograde signalling, which leads to the induction of immediate protective responses and longer-term acclimation. Hydrogen peroxide (H2O2), generated during photosynthesis, is proposed to both initiate and transduce a retrograde signal in response to photoinhibitory light intensities. Signalling specificity achieved by chloroplast-sourced H2O2 for signal transduction may be dependent upon the oft-observed close association of a proportion of these organelles with the nucleus. In this review, we consider more precisely the nature of the close association between a chloroplast appressed to the nucleus and the requirement for H2O2 to cross both the double membranes of the chloroplast and nuclear envelopes. Of particular relevance is that the endoplasmic reticulum (ER) has close physical contact with chloroplasts and is contiguous with the nuclear envelope. Therefore, the perinuclear space, which transducing H2O2 molecules would have to cross, may have an oxidising environment the same as the ER lumen. Based on studies in animal cells, the ER lumen may be a significant source of H2O2 in plant cells arising from the oxidative folding of proteins. If this is the case, then there is potential for the ER lumen/perinuclear space to be an important location to modify chloroplast-to-nucleus H2O2 signal transduction and thereby introduce modulation of it by additional different environmental cues. These would include for example, heat stress and pathogen infection, which induce the unfolded protein response characterised by an increased H2O2 level in the ER lumen.

Arbuscular mycorrhizal symbiosis limits foliar transcriptional responses to viral infection and favors long-term virus accumulation.

Tomato (Solanum lycopersicum) can establish symbiotic interactions with arbuscular mycorrhizal (AM) fungi, and can be infected by several pathogenic viruses. Here, we investigated the impact of mycorrhization by the fungus Glomus mosseae on the Tomato spotted wilt virus (TSWV) infection of tomato plants by transcriptomic and hormones level analyses. In TSWV-infected mycorrhizal plants, the AM fungus root colonization limited virus-induced changes in gene expression in the aerial parts. The virus-responsive upregulated genes, no longer induced in infected mycorrhizal plants, were mainly involved in defense responses and hormone signaling, while the virus-responsive downregulated genes, no longer repressed in mycorrhizal plants, were involved in primary metabolism. The presence of the AM fungus limits, in a salicylic acid-independent manner, the accumulation of abscissic acid observed in response to viral infection. At the time of the molecular analysis, no differences in virus concentration or symptom severity were detected between mycorrhizal and nonmycorrhizal plants. However, in a longer period, increase in virus titer and delay in the appearance of recovery were observed in mycorrhizal plants, thus indicating that the plant's reaction to TSWV infection is attenuated by mycorrhization.

Isoprene synthesis in plants: lessons from a transgenic tobacco model.

Isoprene is a highly reactive gas, and is emitted in such large quantities from the biosphere that it substantially affects the oxidizing potential of the atmosphere. Relatively little is known about the control of isoprene emission at the molecular level. Using transgenic tobacco lines harbouring a poplar isoprene synthase gene, we examined control of isoprene emission. Isoprene synthase required chloroplastic localization for catalytic activity, and isoprene was produced via the methyl erythritol (MEP) pathway from recently assimilated carbon. Emission patterns in transgenic tobacco plants were remarkably similar to naturally emitting plants under a wide variety of conditions. Emissions correlated with photosynthetic rates in developing and mature leaves, and with the amount of isoprene synthase protein in mature leaves. Isoprene synthase protein levels did not change under short-term increase in heat/light, despite an increase in emissions under these conditions. A robust circadian pattern could be observed in emissions from long-day plants. The data support the idea that substrate supply and changes in enzyme kinetics (rather than changes in isoprene synthase levels or post-translational regulation of activity) are the primary controls on isoprene emission in mature transgenic tobacco leaves.

Assessing Phototoxicity in a Mammalian Cell Line: How Low Levels of Blue Light Affect Motility in PC3 Cells.

Phototoxicity is a significant constraint for live cell fluorescence microscopy. Excessive excitation light intensities change the homeostasis of the observed cells. Erroneous and misleading conclusions may be the problematic consequence of observing such light-induced pathophysiology. In this study, we assess the effect of blue light, as commonly used for GFP and YFP excitation, on a motile mammalian cell line. Tracking PC3 cells at different light doses and intensities, we show how motility can be used to reliably assess subtle positive and negative effects of illumination. We further show that the effects are a factor of intensity rather than light dose. Mitotic delay was not a sensitive indicator of phototoxicity. For early detection of the effect of blue light, we analysed the expression of genes involved in oxidative stress. This study addresses the need for relatively simple and sensitive methods to establish a dose-response curve for phototoxicity in mammalian cell line models. We conclude with a working model for phototoxicity and recommendations for its assessment.

Quantitative measurement of specific biomarkers for protein oxidation, nitration and glycation in Arabidopsis leaves.

Higher plants are continually exposed to reactive oxygen and nitrogen species during their lives. Together with glucose and reactive dicarbonyls, these can modify proteins spontaneously, leading to protein oxidation, nitration and glycation. These reactions have the potential to damage proteins and have an impact on physiological processes. The levels of protein oxidation, nitration and glycation adducts were assayed, using liquid chromatography coupled with tandem mass spectrometry, in total leaf extracts over a diurnal cycle and when exposed to conditions that promote oxidative stress. Changes in the levels of oxidation, glycation and nitration adducts were found between the light and dark phases under non-stress conditions. A comparison between wild-type plants and a mutant lacking peptide methionine sulfoxide reductase (pmsr2-1) showed increased protein oxidation, nitration and glycation of specific amino acid residues during darkness in pmsr2-1. Short-term excess light exposure, which promoted oxidative stress, led to increased protein glycation, specifically by glyoxal. This suggested that any increased oxidative damage to proteins was within the repair capacity of the plant. The methods developed here provide the means to simultaneously detect a range of protein oxidation, nitration and glycation adducts within a single sample. Thus, these methods identify a range of biomarkers to monitor a number of distinct biochemical processes that have an impact on the proteome and therefore the physiological state of the plant.

Genetic structure and regulation of isoprene synthase in Poplar (Populus spp.).

Isoprene is a volatile 5-carbon hydrocarbon derived from the chloroplastic methylerythritol 2-C-methyl-D: -erythritol 4-phosphate isoprenoid pathway. In plants, isoprene emission is controlled by the enzyme isoprene synthase; however, there is still relatively little known about the genetics and regulation of this enzyme. Isoprene synthase gene structure was analysed in three poplar species. It was found that genes encoding stromal isoprene synthase exist as a small gene family, the members of which encode virtually identical proteins and are differentially regulated. Accumulation of isoprene synthase protein is developmentally regulated, but does not differ between sun and shade leaves and does not increase when heat stress is applied. Our data suggest that, in mature leaves, isoprene emission rates are primarily determined by substrate (dimethylallyl diphosphate, DMADP) availability. In immature leaves, where isoprene synthase levels are variable, emission levels are also influenced by the amount of isoprene synthase protein. No thylakoid isoforms could be identified in Populus alba or in Salix babylonica. Together, these data show that control of isoprene emission at the genetic level is far more complicated than previously assumed.

Effects of fosmidomycin on plant photosynthesis as measured by gas exchange and chlorophyll fluorescence.

In higher plants, many isoprenoids are synthesised via the chloroplastic 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Attempts to elucidate the function of individual isoprenoids have used the antibiotic/herbicidal compound fosmidomycin (3-[N-formyl-N-hydroxy amino] propyl phosphonic acid) to inhibit this pathway. Examination of the effect of fosmidomycin on the major components of photosynthesis in leaves of white poplar (Populus alba) and tobacco (Nicotiana tabacum) was made. Fosmidomycin reduced net photosynthesis in both species within 1 h of application, but only when photosynthesis was light-saturated. In P. alba, these reductions were confounded by high light and fosmidomycin inducing stomatal patchiness. In tobacco, this was caused by significant reductions in PSII chlorophyll fluorescence and reductions in V(cmax) and J(max). Our data indicate that the diminution of photosynthesis is likely a complex effect resulting from the inhibition of multiple MEP pathway products, resulting in photoinhibition and photo-damage. These effects should be accounted for in experimental design and analysis when using fosmidomycin to avoid misinterpretation of results as measured by gas exchange and chlorophyll fluorescence.