Stephanie Long

Separation and quantification of monothiols and phytochelatins from a wide variety of cell cultures and tissues of trees and other plants using high performance liquid chromatography

The HPLC method presented here for the quantification of metal-binding thiols is considerably shorter than most previously published methods. It is a sensitive and highly reproducible method that separates monobromobimane tagged monothiols (cysteine, glutathione, gamma-glutamylcysteine) along with polythiols (PC(2), PC(3), PC(4) and PC(5)) within 23min from a wide variety of samples. Total run time of the method is 35min. Detection limits for thiols is 33fmol for 10microlL injection. This method will be applicable to study the metal detoxification mechanisms for a wide variety of cell cultures and tissues of plants and trees including algae, Arabidopsis, crambe, rice, and red spruce.

Foliar free polyamine and inorganic ion content in relation to soil and soil solution chemistry in two fertilized forest stands at the Harvard Forest, Massachusetts

Polyamines (putrescine, spermidine, and spermine) are low molecular weight, open-chained, organic polycations which are found in all organisms and have been linked with stress responses in plants. The objectives of our study were to investigate the effects of chronic N additions to pine and hardwood stands at Harvard Forest, Petersham, MA on foliar polyamine and inorganic ion contents as well as soil and soil solution chemistry. Four treatment plots were established within each stand in 1988: control, low N (50 kg N ha-1 yr-1 as NH4NO3), low N + sulfur (74 kg S ha-1 yr-1 as Na2SO4), and high N (150 kg N ha-1 yr-1 as NH4NO3). All samples were analyzed for inorganic elements; foliage samples were also analyzed for polyamines and total N. In the pine stand putrescine and total N levels in the foliage were significantly higher for all N treatments as compared to the control plot. Total N content was positively correlated with polyamines in the needles (P ≤ 0.05). Both putrescine and N contents were also negatively correlated with most exchangeable cations and total elements in organic soil horizons and positively correlated with Ca and Mg in the soil solution (P ≤ 0.05). In the hardwood stand, putrescine and total N levels in the foliage were significantly higher for the high N treatment only as compared to the control plot. Here also, total foliar N content was positively correlated with polyamines (P ≤ 0.05). Unlike the case with the pine stand, in the hardwood stand foliar polyamines and N were significantly and negatively correlated with foliar total Ca, Mg, and Mn (P ≤ 0.05). Additional significant (P ≤ 0.05) relationships in hardwoods included: negative correlations between foliar polyamines and N content to exchangeable K and P and total P in the organic soil horizon; and positive correlations between foliar polyamines and N content to Mg in soil solution. With few exceptions, low N + S treatment had effects similar to the ones observed with low N alone for both stands. The changes observed in the pine stand for polyamine metabolism, N uptake, and element leaching from the soil into the soil solution in all treatment plots provide additional evidence that the pine stand is more nitrogen saturated than the hardwood stand. These results also indicate that the long-term addition of N to these stands has species specific and/or site specific effects that may in part be explained by the different land use histories of the two stands.

Putrescine overproduction negatively impacts the oxidative state of poplar cells in culture

While polyamines (PAs) have been suggested to protect cells against Reactive Oxygen Species (ROS), their catabolism is known to generate ROS. We compared the activities of several enzymes and cellular metabolites involved in the ROS scavenging pathways in two isogenic cell lines of poplar (Populus nigraxmaximowiczii) differing in their PA contents. Whereas the control cell line was transformed with beta-glucuronidase (GUS), the other, called HP (High Putrescine), was transformed with a mouse ornithine decarboxylase (mODC) gene. The expression of mODC resulted in several-fold increased production of putrescine as well its enhanced catabolism. The two cell lines followed a similar trend of growth over the seven-day culture cycle, but the HP cells had elevated levels of soluble proteins. Accumulation of H(2)O(2) was higher in the HP cells than the control cells, and so were the activities of glutathione reductase and monodehydroascorbate reductase; the activity of ascorbate peroxidase was lower in the former. The contents of reduced glutathione and glutamate were significantly lower in the HP cells but proline was higher on some days of analysis. There was a small difference in mitochondrial activity between the two cell lines, and the HP cells showed increased membrane damage. In the HP cells, increased accumulation of Ca was concomitant with lower accumulation of K. We conclude that, while increased putrescine accumulation may have a protective role against ROS in plants, enhanced turnover of putrescine actually can make them vulnerable to increased oxidative damage.

Ornithine: The Overlooked Molecule in the Regulation of Polyamine Metabolism

We overexpressed a mouse ornithine decarboxylase gene under the control of a constitutive and an estradiol-inducible promoter in Arabidopsis thaliana to increase our understanding of the regulation of polyamine metabolism. Of particular interest was the role of the substrate ornithine not only in the regulation of polyamine biosynthesis, but also in the accumulation of related amino acids in response to short-term induction of this enzyme. We hypothesized that the inducible expression of the transgene would mimic the natural responses of plants to changing conditions, e.g. under stress conditions and during rapid growth. Our results reveal that ornithine, even though present in relatively small quantities (compared with other amino acids of the glutamate-arginine-proline pathway), may not only be the key regulator of polyamine biosynthesis in Arabidopsis, but it may also regulate the entire subset of pathways for glutamate to arginine and to proline. Indirectly, it could also regulate putrescine catabolism, therefore contributing to the γ-aminobutyric acid content of the cells. Furthermore, the induction of mouse ornithine decarboxylase resulted in up- and down-regulation of several amino acids in the transgenic plants. It was learned that the turnover of putrescine in both the wild type and the transgenic plants occurs rapidly, with a half-life of 6-8 h.

Development of a standardized methodology for quantifying total chlorophyll and carotenoids from foliage of hardwood and conifer tree species.

Despite the availability of several protocols for the extraction of chlorophylls and carotenoids from foliage of forest trees, information regarding their respective extraction efficiencies is scarce. We compared the efficiencies of acetone, ethanol, dimethyl sulfoxide (DMSO), and N, N-dimethylformamide (DMF) over a range of incubation times for the extraction of chlorophylls and carotenoids using small amounts of unmacerated tissue. Of the 11 species studied, comparable amounts of chlorophyll were extracted by all four solvents from three species and by ethanol and DMF from nine species. In four species, acetone, ethanol, and DMF extracted comparable chlorophyll amounts, while in another two species comparable amounts were extracted by ethanol, DMSO, and DMF. In one species, ethanol extracted significantly greater amounts of chlorophyll compared with all other solvents. The brown coloration of DMSO extracts for some species compromised the calculations of chlorophylls and carotenoids, making DMSO a poor ...

Reduced Silver Nanoparticle Phytotoxicity in Crambe abyssinica with Enhanced Glutathione Production by Overexpressing Bacterial γ-Glutamylcysteine Synthase

Silver nanoparticles (Ag NPs) are widely used in consumer products, and their release has raised serious concerns about the risk of their exposure to the environment and to human health. However, biochemical mechanisms by which plants counteract NP toxicity are largely unknown. We have previously engineered Crambe abyssinica plants expressing the bacterial γ-glutamylecysteine synthase (γ-ECS) for enhancing glutathione (GSH) levels. In this study, we investigated if enhanced levels of GSH and its derivatives can protect plants from Ag NPs and AgNO3 (Ag(+) ions). Our results showed that transgenic lines, when exposed to Ag NPs and Ag(+) ions, were significantly more tolerant, attaining a 28%-46% higher biomass and 34-49% more chlorophyll content, as well as maintaining 35-46% higher transpiration rates as compared to those of wild type (WT) plants. Transgenic γ-ECS lines showed 2-6-fold Ag accumulation in shoot tissue and slightly lower or no difference in root tissue relative to levels in WT plants. The levels of malondialdehyde (MDA) in γ-ECS lines were also 27.3-32.5% lower than those in WT Crambe. These results indicate that GSH and related peptides protect plants from Ag nanotoxicity. To our knowledge, this is the first direct report of Ag NP detoxification by GSH in transgenic plants, and these results will be highly useful in developing strategies to counteract the phytotoxicty of metal-based nanoparticles in crop plants.

Assays for the activities of polyamine biosynthetic enzymes using intact tissues

Traditionally, most enzyme assays utilize homogenized cell extracts with or without dialysis. Homogenization and centrifugation of large numbers of samples for screening of mutants and transgenic cell lines is quite cumbersome and generally requires sufficiently large amounts (hundreds of milligrams) of tissue. However, in situations where the tissue is available in small quantities, or one needs to study changes in enzyme activities during development (e.g. somatic embryogenesis), it is desirable to have rapid and reproducible assay methods that utilize only a few milligrams of tissue and can be conducted without homogenization. Here, we report a procedure for the measurement of enzyme activities of the three key decarboxylases involved in polyamine biosynthesis utilizing small quantities of plant tissue without the homogenization and centrifugation steps. Suspension cultures of red spruce (Picea rubens (Sarg.)), hybrid poplar (Populus nigra x maximowiczii), and wild carrot (Daucus carota) were used directly to measure decarboxylation of omithine, arginine and S-adenosylmethionine. Our results demonstrate that this procedure can be used to quantify the activities of arginine decarboxylase (EC 4.1.1.19), omithine decarboxylase (EC 4.1.1.17) and S-adenosylmethionine decarboxylase (EC 4.1 .lSO) in a manner quite comparable to the traditional assays for these enzymes that involve laborious steps of homogenization and centrifugation. 0 Elsevier, Paris ADC / carrot I enzyme assays / ODC / polyamines / SAMDC / trees ADC, arginine decarboxylase / DFMA, DL a-difluoromethylarginine / DFMO, DL a-difluoromethylornithine / EDTA, Na,-ethylenediamine-tetraacetate / MGBG, methylglyoxal bis(guanylhydrazone) / ODC, ornithine decarboxylase / SAMDC, S-adenosylmethionine decarboxylase

Fuel treatment effects on soil chemistry and foliar physiology of three coniferous species at the Teakettle Experimental Forest, California, USA

A full factorial design crossing overstory (O) and understory (U) thinning and prescribed burning (B) was started at Teakettle Experimental Forest, California, in 2001 with the aim of achieving shifts in species composition to favor fire-resistant pines over fir. The goal of the present study was to evaluate the use of metabolic changes as early indicators for predicting the effects of these treatments on species-specific growth and long-term productivity of Pinus jeffreyi, Pinus lambertiana, and Abies concolor. Foliar polyamines, amino acids, soluble proteins and chlorophyll were quantified and related with foliar and soil chemistry. Most changes in soil chemistry occurred only with combined treatments (BU or BO). Changes in foliar metabolism were not driven by soil chemistry. In general, the pines were more sensitive to water stress as compared to fir as indicated by proline content. Understory thinning had no effect on any species. Both O and BO had species-specific effects on foliar amino acids, chlorophyll, soluble proteins, and inorganic ions. In addition, both O and BO were the most extreme thinning treatments for pines as they greatly increase the time needed to replenish carbon stocks. With the exception of some amino acids, B by itself had no major effects on foliar inorganic ions, chlorophyll content, and polyamines for any of the three species while BU caused a decrease in chlorophyll in white fir, suggesting a decline in photosynthetic capacity. These data point to BU as the best management option to specifically decrease the growth of fire-sensitive white fir without a major impact on pines.

Extraction and estimation of the quantity of calcium oxalate crystals in the foliage of conifer and hardwood trees

The main goal of this study was to develop a method for the extraction and indirect estimation of the quantity of calcium oxalate (CaOx) in the foliage of trees. Foliar tissue was collected from a single tree of each species (five conifers and five hardwoods) for comparison of extractions in different solvents using 10 replicates per species from the same pool of tissue. For each species, calcium (Ca) and oxalate were extracted sequentially in double deionized water and 2N acetic acid, and finally, five replicate samples were extracted in 5% (0.83N) perchloric acid (PCA) and the other five in 2N hydrochloric acid (HCl); three cycles of freezing and thawing were used for each solvent. Total ions were extracted by microwave digestion. Calcium was quantified with an inductively coupled plasma emission spectrophotometer method and oxalate was eluted and quantified using a high performance liquid chromatography method. This experiment was repeated again with two conifer and two hardwood species using four trees per species, and two analytical replicates for each tree. We report here that, regardless of age of individual trees within a species, time of collection or species type, the third extraction in PCA or HCl resulted in near equimolar quantities of Ca and oxalate (r(2) ≥ 0.99). This method provides an easy estimate of the quantity of CaOx crystals using a small sample of foliar tissue. An additional benefit of PCA is that it precipitates the nucleic acids and proteins, allowing the quantification of several free/soluble metabolites such as amino acids, polyamines, organic acids and inorganic elements all from a single sample extract.