Ziru Li

Metabolic profiling of the methylerythritol phosphate pathway reveals the source of post-illumination isoprene burst from leaves.

The methylerythritol phosphate (MEP) pathway in plants produces the prenyl precursors for all plastidic isoprenoids, including carotenoids and quinones. The MEP pathway is also responsible for synthesis of approximately 600 Tg of isoprene per year, the largest non-methane hydrocarbon flux into the atmosphere. There have been few studies of the regulation of the MEP pathway in plants under physiological conditions. In this study, we combined gas exchange techniques and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) and measured the profile of MEP pathway metabolites under different conditions. We report that in the MEP pathway, metabolites immediately preceding steps requiring reducing power were in high concentration. Inhibition of the MEP pathway by fosmidomycin caused deoxyxylulose phosphate accumulation in leaves as expected. Evidence is presented that accumulation of MEP pathway intermediates, primarily methylerythritol cyclodiphosphate, is responsible for the post-illumination isoprene burst phenomenon. Pools of intermediate metabolites stayed at approximately the same level 10 min after light was turned off, but declined eventually under prolonged darkness. In contrast, a strong inhibition of the second-to-last step of the MEP pathway caused suppression of isoprene emission in pure N(2). Our study suggests that reducing equivalents may be a key regulator of the MEP pathway and therefore isoprene emission from leaves.

Molecular and Pathway Controls on Biogenic Volatile Organic Compound Emissions

Plants make a number of volatile organic compounds (BVOCs), many of which are emitted in a light- and temperature-dependent manner. The vast majority of these BVOCs are isoprenoids including isoprene, monoterpenes, and sesquiterpenes. The total BVOC flux into the atmosphere is on the order of a petagram (1015 g) and has multiple effects on atmospheric chemistry. Understanding the biochemical and molecular regulation of BVOC emissions allows us to build prediction models that better reflect the underlying physiological and biochemical processes. In this chapter we review the enzymes and pathways involved in the biosynthesis of various BVOCs that originate from plants, using isoprene as a model. The biochemical and molecular control of BVOC emission in response to short-term environment drivers such as temperature, light, CO2, and O2, and long-term factors such as circadian, seasonal, and developmental effects are discussed. An emerging theme in the regulation of isoprene emission is that the enzyme isoprene synthase controls the basal emission rate in the long term, while the responses of isoprene emission to short-term factors are regulated by levels of the substrate (dimethylallyl diphosphate), which is in turn determined by upstream enzymes. In addition, we propose a new hypothesis to explain the high-CO2 suppression of isoprene emission. At high CO2 concentrations, a high cytosolic inorganic phosphate (Pi) gradient needed to transport triose phosphates out of the chloroplasts could work against the transport of phosphoenol pyruvate into the chloroplasts. This altered partitioning of phosphoenol pyruvate would then reduce the supply of pyruvate into the MEP pathway. Much work is still needed to understand the CO2 response of BVOC emissions but we expect to see significant progress in the near future.

Engineering starch accumulation by manipulation of phosphate metabolism of starch

A new understanding of leaf starch degradation has emerged in the last 10 years. It has been shown that starch phosphorylation and dephosphorylation are critical components of this process. Glucan, water dikinase (GWD) (and phosphoglucan, water dikinase) adds phosphate to starch, and phosphoglucan phosphatase (SEX4) removes these phosphates. To explore the use of this metabolism to manipulate starch accumulation, Arabidopsis (Arabidopsis thaliana) plants were engineered by introducing RNAi constructs designed to reduce expression of AtGWD and AtSEX4. The timing of starch build-up was altered with ethanol-inducible and senescence-induced gene promoters. Ethanol induction of RNAi lines reduced transcript for AtGWD and AtSEX4 by 50%. The transgenic lines had seven times more starch than wild type at the end of the dark period but similar growth rates and total biomass. Elevated leaf starch content in maize leaves was engineered by making an RNAi construct against a gene in maize that appeared to be homologous to AtGWD. The RNAi construct was expressed using the constitutive ubiquitin promoter. Leaf starch content at the end of a night period in engineered maize plants was 20-fold higher than in untransformed plants with no impact on total plant biomass. We conclude that plants can be engineered to accumulate starch in the leaves with little impact on vegetative biomass.

Effect of Temperature on Postillumination Isoprene Emission in Oak and Poplar

Isoprene emission from broadleaf trees is highly temperature dependent, accounts for much of the hydrocarbon emission from plants, and has a profound effect on atmospheric chemistry. We studied the temperature response of postillumination isoprene emission in oak (Quercus robur) and poplar (Populus deltoides) leaves in order to understand the regulation of isoprene emission. Upon darkening a leaf, isoprene emission fell nearly to zero but then increased for several minutes before falling back to nearly zero. Time of appearance of this burst of isoprene was highly temperature dependent, occurring sooner at higher temperatures. We hypothesize that this burst represents an intermediate pool of metabolites, probably early metabolites in the methylerythritol 4-phosphate pathway, accumulated upstream of dimethylallyl diphosphate (DMADP). The amount of this early metabolite(s) averaged 2.9 times the amount of plastidic DMADP. DMADP increased with temperature up to 35°C before starting to decrease; in contrast, the isoprene synthase rate constant increased up to 40°C, the highest temperature at which it could be assessed. During a rapid temperature switch from 30°C to 40°C, isoprene emission increased transiently. It was found that an increase in isoprene synthase activity is primarily responsible for this transient increase in emission levels, while DMADP level stayed constant during the switch. One hour after switching to 40°C, the amount of DMADP fell but the rate constant for isoprene synthase remained constant, indicating that the high temperature falloff in isoprene emission results from a reduction in the supply of DMADP rather than from changes in isoprene synthase activity.

Measuring dimethylallyl diphosphate available for isoprene synthesis.

Dimethylallyl diphosphate (DMADP) is a central metabolite in isoprenoid metabolism, but it is difficult to measure. Three different methods for measuring DMADP are compared, and a new method based on the conversion of DMADP to isoprene using recombinant isoprene synthase is introduced. Mass spectrometry is reliable but does not distinguish between DMADP and isopentenyl diphosphate. Acid hydrolysis is reliable for measuring DMADP in bacterial extracts but overestimates DMADP in plant samples. To measure the DMADP in chloroplasts, light minus dark measurements are normally used. Chloroplast DMADP amounts measured using acid hydrolysis and a mass spectrometric method were comparable in this assay. Post-illumination isoprene emission tended to slightly overestimate chloroplast DMADP concentration. The DMADP pool size in bacteria is highly regulated, consistent with previous observations made with plants. DMADP is a very labile metabolite, but four methods described here allow measurements of samples from plants and bacteria. The use of recombinant isoprene synthase can greatly simplify the analysis. The various techniques tested here have advantages and disadvantages, and it is useful to have more than one method available when studying biological isoprene production.

Isopentenyl diphosphate and dimethylallyl diphosphate/isopentenyl diphosphate ratio measured with recombinant isopentenyl diphosphate isomerase and isoprene synthase.

Isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP) are building units for all isoprenoids; thus, intracellular pool sizes of IDP and DMADP play important roles in living organisms. Several methods have been used to quantify the amount of DMADP or the combined amount of IDP plus DMADP, but measuring the DMADP/IDP ratio has been difficult. In this study, a method was developed to measure the ratio of DMADP/IDP. Catalyzed by a recombinant IDP isomerase (IDI) together with a recombinant isoprene synthase (IspS), IDP was converted to isoprene, which was then detected by chemiluminescence. With this method, the in vitro equilibrium ratio of DMADP/IDP was found to be 2.11:1. IDP and DMADP pools were significantly increased in Escherichia coli transformed with methylerythritol 4-phosphate pathway genes; the ratio of DMADP/IDP was 3.85. An E. coli strain transformed with IspS but no additional IDI had a lower DMADP level and a DMADP/IDP ratio of 1.05. Approximately 90% of the IDP and DMADP pools in light-adapted kudzu leaves were light dependent and so presumably were located in the chloroplasts; the DMADP/IDP ratios in chloroplasts and cytosol were the same as the in vitro ratio (2.04 in the light and 2.32 in the dark).

Triose phosphate use limitation of photosynthesis: short-term and long-term effects.

AbstractMain conclusionThe triose phosphate use limitation was studied using long-term and short term changes in capacity. The TPU limitation caused increased proton motive force; long-term TPU limitation additionally reduced other photosynthetic components. Photosynthetic responses to CO2 can be interpreted primarily as being limited by the amount or activity of Rubisco or the capacity for ribulose bisphosphate regeneration, but at high rates of photosynthesis a third response is often seen. Photosynthesis becomes insensitive to CO2 or even declines with increasing CO2, and this behavior has been associated with a limitation of export of carbon from the Calvin–Benson cycle. It is often called the triose phosphate use (TPU) limitation. We studied the long-term consequences of this limitation using plants engineered to have reduced capacity for starch or sucrose synthesis. We studied short-term consequences using temperature as a method for changing the balance of carbon fixation capacity and TPU. A long-term and short-term TPU limitation resulted in an increase in proton motive force (PMF) in the thylakoids. Once a TPU limitation was reached, any further increases in CO2 was met with a further increase in the PMF but no increase or little increase in net assimilation of CO2. A long-term TPU limitation resulted in reduced Rubisco and RuBP regeneration capacity. We hypothesize that TPU, Rubisco activity, and RuBP regeneration are regulated so that TPU is normally in slight excess of what is required, and that this results in more effective regulation than if TPU were in large excess.

Effects of heat and drought stress on post‐illumination bursts of volatile organic compounds in isoprene‐emitting and non‐emitting poplar

Abstract Over the last decades, post‐illumination bursts (PIBs) of isoprene, acetaldehyde and green leaf volatiles (GLVs) following rapid light‐to‐dark transitions have been reported for a variety of different plant species. However, the mechanisms triggering their release still remain unclear. Here we measured PIBs of isoprene‐emitting (IE) and isoprene non‐emitting (NE) grey poplar plants grown under different climate scenarios (ambient control and three scenarios with elevated CO2 concentrations: elevated control, periodic heat and temperature stress, chronic heat and temperature stress, followed by recovery periods). PIBs of isoprene were unaffected by elevated CO2 and heat and drought stress in IE, while they were absent in NE plants. On the other hand, PIBs of acetaldehyde and also GLVs were strongly reduced in stress‐affected plants of all genotypes. After recovery from stress, distinct differences in PIB emissions in both genotypes confirmed different precursor pools for acetaldehyde and GLV emissions. Changes in PIBs of GLVs, almost absent in stressed plants and enhanced after recovery, could be mainly attributed to changes in lipoxygenase activity. Our results indicate that acetaldehyde PIBs, which recovered only partly, derive from a new mechanism in which acetaldehyde is produced from methylerythritol phosphate pathway intermediates, driven by deoxyxylulose phosphate synthase activity.

Facing the Future: Effects of Short-Term Climate Extremes on Isoprene-Emitting and Nonemitting Poplar

The ability to emit isoprene does not protect poplar trees from realistic short-term and periodic drought and heat waves under proposed future conditions. Isoprene emissions from poplar (Populus spp.) plantations can influence atmospheric chemistry and regional climate. These emissions respond strongly to temperature, [CO2], and drought, but the superimposed effect of these three climate change factors are, for the most part, unknown. Performing predicted climate change scenario simulations (periodic and chronic heat and drought spells [HDSs] applied under elevated [CO2]), we analyzed volatile organic compound emissions, photosynthetic performance, leaf growth, and overall carbon (C) gain of poplar genotypes emitting (IE) and nonemitting (NE) isoprene. We aimed (1) to evaluate the proposed beneficial effect of isoprene emission on plant stress mitigation and recovery capacity and (2) to estimate the cumulative net C gain under the projected future climate. During HDSs, the chloroplastidic electron transport rate of NE plants became impaired, while IE plants maintained high values similar to unstressed controls. During recovery from HDS episodes, IE plants reached higher daily net CO2 assimilation rates compared with NE genotypes. Irrespective of the genotype, plants undergoing chronic HDSs showed the lowest cumulative C gain. Under control conditions simulating ambient [CO2], the C gain was lower in the IE plants than in the NE plants. In summary, the data on the overall C gain and plant growth suggest that the beneficial function of isoprene emission in poplar might be of minor importance to mitigate predicted short-term climate extremes under elevated [CO2]. Moreover, we demonstrate that an analysis of the canopy-scale dynamics of isoprene emission and photosynthetic performance under multiple stresses is essential to understand the overall performance under proposed future conditions.

Characterization of photosynthesis in Arabidopsis ER-to-plastid lipid trafficking mutants

Vascular plants use two pathways to synthesize galactolipids, the predominant lipid species in chloroplasts—a prokaryotic pathway that resides entirely in the chloroplast, and a eukaryotic pathway that involves assembly in the endoplasmic reticulum. Mutants deficient in the endoplasmic reticulum pathway, trigalactosyldiacylglycerol (tgd1-1 and tgd2-1) mutants, had been previously identified with reduced contents of monogalactosyldiacylglycerol and digalactosyldiacylglycerol, and altered lipid molecular species composition. Here, we report that the altered lipid composition affected photosynthesis in lipid trafficking mutants. It was found that proton motive force as measured by electrochromic shift was reduced by ~40 % in both tgd mutants. This effect was accompanied by an increase in thylakoid conductance attributable to ATPase activity and so the rate of ATP synthesis was nearly unchanged. Thylakoid conductance to ions also increased in tgd mutants. However, gross carbon assimilation in tgd mutants as measured by gas exchange was only marginally affected. Rubisco activity, electron transport rate, and photosystem I and II oxidation status were not altered. Despite the large differences in proton motive force, responses to heat and high light stress were similar between tgd mutants and the wild type.