Engineered poplar for bioproduction of the triterpene squalene.

Building sustainable platforms to produce biofuels and specialty chemicals has become an increasingly important strategy to supplement and replace fossil fuels and petrochemical-derived products. Terpenoids are the most diverse class of natural products that have many commercial roles as specialty chemicals. Poplar is a fast growing, biomassdense bioenergy crop with many species known to produce large amounts of the hemiterpene isoprene, suggesting an inherent capacity to produce significant quantities of other terpenes. Here we aimed to engineer poplar with optimized pathways to produce squalene, a triterpene commonly used in cosmetic oils, a potential biofuel candidate, and the precursor to the further diversified classes of triterpenoids and sterols. The squalene production pathways were either re-targeted from the cytosol to plastids or co-produced with lipid droplets in the cytosol. Squalene and lipid droplet co-production appeared to be toxic, which we hypothesize to be due to disruption of adventitious root formation, suggesting a need for tissue specific production. Plastidial squalene production enabled up to 0.63 mg/g fresh weight in leaf tissue, which also resulted in reductions in isoprene emission and photosynthesis. These results were also studied through a technoeconomic analysis, providing further insight into developing poplar as a production host.

The isoprene-responsive phosphoproteome provides new insights into the putative signalling pathways and novel roles of isoprene.

Many plants, especially trees, emit isoprene in a highly light- and temperature-dependent manner. The advantages for plants that emit, if any, have been difficult to determine. Direct effects on membranes have been disproven. New insights have been obtained by RNA sequencing, proteomic and metabolomic studies. We determined the responses of the phosphoproteome to exposure of Arabidopsis leaves to isoprene in the gas phase for either 1 or 5 h. Isoprene effects that were not apparent from RNA sequencing and other methods but were apparent in the phosphoproteome include effects on chloroplast movement proteins and membrane remodelling proteins. Several receptor kinases were found to have altered phosphorylation levels. To test whether potential isoprene receptors could be identified, we used molecular dynamics simulations to test for proteins that might have strong binding to isoprene and, therefore might act as receptors. Although many Arabidopsis proteins were found to have slightly higher binding affinities than a reference set of Homo sapiens proteins, no specific receptor kinase was found to have a very high binding affinity. The changes in chloroplast movement, photosynthesis capacity and so forth, found in this work, are consistent with isoprene responses being especially useful in the upper canopy of trees.

Hydroxymethylbutenyl diphosphate accumulation reveals MEP pathway regulation for high CO2-induced suppression of isoprene emission.

Isoprene is emitted by some plants and is the most abundant biogenic hydrocarbon entering the atmosphere. Multiple studies have elucidated protective roles of isoprene against several environmental stresses, including high temperature, excessive ozone, and herbivory attack. However, isoprene emission adversely affects atmospheric chemistry by contributing to ozone production and aerosol formation. Thus, understanding the regulation of isoprene emission in response to varying environmental conditions, for example, elevated CO2, is critical to comprehend how plants will respond to climate change. Isoprene emission decreases with increasing CO2 concentration; however, the underlying mechanism of this response is currently unknown. We demonstrated that high-CO2-mediated suppression of isoprene emission is independent of photosynthesis and light intensity, but it is reduced with increasing temperature. Furthermore, we measured methylerythritol 4-phosphate (MEP) pathway metabolites in poplar leaves harvested at ambient and high CO2 to identify why isoprene emission is reduced under high CO2. We found that hydroxymethylbutenyl diphosphate (HMBDP) was increased and dimethylallyl diphosphate (DMADP) decreased at high CO2. This implies that high CO2 impeded the conversion of HMBDP to DMADP, possibly through the inhibition of HMBDP reductase activity, resulting in reduced isoprene emission. We further demonstrated that although this phenomenon appears similar to abscisic acid (ABA)-dependent stomatal regulation, it is unrelated as ABA treatment did not alter the effect of elevated CO2 on the suppression of isoprene emission. Thus, this study provides a comprehensive understanding of the regulation of the MEP pathway and isoprene emission in the face of increasing CO2.

Characterization of promoter elements of isoprene-responsive genes and the ability of isoprene to bind START domain transcription factors.

Isoprene has recently been proposed to be a signaling molecule that can enhance tolerance of both biotic and abiotic stress. Not all plants make isoprene, but all plants tested to date respond to isoprene. We hypothesized that isoprene interacts with existing signaling pathways rather than requiring novel mechanisms for its effect on plants. We analyzed the cis-regulatory elements (CREs) in promoters of isoprene-responsive genes and the corresponding transcription factors binding these promoter elements to obtain clues about the transcription factors and other proteins involved in isoprene signaling. Promoter regions of isoprene-responsive genes were characterized using the Arabidopsis cis-regulatory element database. CREs bind ARR1, Dof, DPBF, bHLH112, GATA factors, GT-1, MYB, and WRKY transcription factors, and light-responsive elements were overrepresented in promoters of isoprene-responsive genes; CBF-, HSF-, WUS-binding motifs were underrepresented. Transcription factors corresponding to CREs overrepresented in promoters of isoprene-responsive genes were mainly those important for stress responses: drought-, salt/osmotic-, oxidative-, herbivory/wounding and pathogen-stress. More than half of the isoprene-responsive genes contained at least one binding site for TFs of the class IV (homeodomain leucine zipper) HD-ZIP family, such as GL2, ATML1, PDF2, HDG11, ATHB17. While the HD-zipper-loop-zipper (ZLZ) domain binds to the L1 box of the promoter region, a special domain called the steroidogenic acute regulatory protein-related lipid transfer, or START domain, can bind ligands such as fatty acids (e.g., linolenic and linoleic acid). We tested whether isoprene might bind in such a START domain. Molecular simulations and modeling to test interactions between isoprene and a class IV HD-ZIP family START-domain-containing protein were carried out. Without membrane penetration by the HDG11 START domain, isoprene within the lipid bilayer was inaccessible to this domain, preventing protein interactions with membrane bound isoprene. The cross-talk between isoprene-mediated signaling and other growth regulator and stress signaling pathways, in terms of common CREs and transcription factors could enhance the stability of the isoprene emission trait when it evolves in a plant but so far it has not been possible to say what how isoprene is sensed to initiate signaling responses.

Isoprene measurements to assess plant hydrocarbon emissions and the methylerythritol pathway.

Isoprene is the most abundant non-methane hydrocarbon emitted to the atmosphere and a target of biotechnology as a source of biofuels or chemical feedstock. Measurements of the amount of isoprene or the rate of production of isoprene are important for atmospheric chemistry, evaluating biotechnology processes, and can provide information on the capacity and regulation of the methyl erythritol 4-phosphate pathway found in plants and bacteria. In this chapter we discuss techniques, and their strengths and weaknesses, of methods in common use for measuring isoprene. There are many sources of isoprene for measurements including emissions from leaves and head space analysis of reactions involving recombinant enzymes or bacterial/fungal cultures. Similarly, there are a variety of detection methods including several mass spectrometer methods that are useful for examining rates of labeling of isoprene when carbon isotopes are used.

Spatial Profiles of Phosphate in Roots Indicate Developmental Control of Uptake, Recycling, and Sequestration.

The availability of inorganic phosphate (Pi) limits plant growth and crop productivity on much of the world's arable land. To better understand how plants cope with deficient and variable supplies of this essential nutrient, we used Pi imaging to spatially resolve and quantify cytosolic Pi concentrations and the respective contributions of Pi uptake, metabolic recycling, and vacuolar sequestration to cytosolic Pi homeostasis in Arabidopsis (Arabidopsis thaliana) roots. Microinjection coupled with confocal microscopy was used to calibrate a FRET-based Pi sensor to determine absolute, rather than relative, Pi concentrations in live plants. High-resolution mapping of cytosolic Pi concentrations in different cells, tissues, and developmental zones of the root revealed that cytosolic concentrations varied between developmental zones, with highest levels in the transition zone, whereas concentrations were equivalent in epidermis, cortex, and endodermis within each zone. Pi concentrations in all zones were reduced, at different rates, by Pi starvation, but the developmental pattern of Pi concentration persisted. Pi uptake, metabolic recycling, and vacuolar sequestration were distinguished in each zone by using cyanide to block Pi assimilation in wild-type plants and a vacuolar Pi transport mutant, and then measuring the subsequent change in cytosolic Pi concentration over time. Each of these processes exhibited distinct spatial profiles in the root, but only vacuolar Pi sequestration corresponded with steady-state cytosolic Pi concentrations. These results highlight the complexity of Pi dynamics in live plants and revealed developmental control of root Pi homeostasis, which has potential implications for plant sensing and signaling of Pi.

ATP compartmentation in plastids and cytosol of Arabidopsis thaliana revealed by fluorescent protein sensing.

Matching ATP:NADPH provision and consumption in the chloroplast is a prerequisite for efficient photosynthesis. In terms of ATP:NADPH ratio, the amount of ATP generated from the linear electron flow does not meet the demand of the Calvin-Benson-Bassham (CBB) cycle. Several different mechanisms to increase ATP availability have evolved, including cyclic electron flow in higher plants and the direct import of mitochondrial-derived ATP in diatoms. By imaging a fluorescent ATP sensor protein expressed in living Arabidopsis thaliana seedlings, we found that MgATP2- concentrations were lower in the stroma of mature chloroplasts than in the cytosol, and exogenous ATP was able to enter chloroplasts isolated from 4- and 5-day-old seedlings, but not chloroplasts isolated from 10- or 20-day-old photosynthetic tissues. This observation is in line with the previous finding that the expression of chloroplast nucleotide transporters (NTTs) in Arabidopsis mesophyll is limited to very young seedlings. Employing a combination of photosynthetic and respiratory inhibitors with compartment-specific imaging of ATP, we corroborate the dependency of stromal ATP production on mitochondrial dissipation of photosynthetic reductant. Our data suggest that, during illumination, the provision and consumption of ATP:NADPH in chloroplasts can be balanced by exporting excess reductants rather than importing ATP from the cytosol.