The Development of Polyamines throughout Brassica rapa over its Lifecycle

Polyamines are naturally produced chemicals in plants involved in growth, development and stress response. The primary objective of my study is to create a profile of changes in the entire life of the plant, in every organ at all stages of development from seed germination to seed formation. We have analyzed polyamines putrescine, spermidine and spermine in all parts of Brassica rapa, a small, rapid growing plant. Parallel to the polyamines, we will also study changes in the activities of the polyamine biosynthetic enzymes and the expression of their genes in different organs at different times. In the next stage of the study, the expression of selected genes will be inhibited by RNAi constructs, allowing further analysis of their role in growth and stress response. Because polyamines play are important in development and lifecycle of plants, altering their presence may be useful in altering plant growth patterns, such as in seasonal crops

Long-term trends of changes in pine and oak foliar nitrogen metabolism in response to chronic nitrogen amendments at Harvard Forest, MA

We evaluated the long-term (1995–2008) trends in foliar and sapwood metabolism, soil solution chemistry and tree mortality rates in response to chronic nitrogen (N) additions to pine and hardwood stands at the Harvard Forest Long Term Ecological Research (LTER) site. Common stress-related metabolites like polyamines (PAs), free amino acids (AAs) and inorganic elements were analyzed for control, low N (LN, 50 kg NH4NO3 ha−1 year−1) and high N (HN, 150 kg NH4NO3 ha−1 year−1) treatments. In the pine stands, partitioning of excess N into foliar PAs and AAs increased with both N treatments until 2002. By 2005, several of these effects on N metabolites disappeared for HN, and by 2008 they were mostly observed for LN plot. A significant decline in foliar Ca and P was observed mostly with HN for a few years until 2005. However, sapwood data actually showed an increase in Ca, Mg and Mn and no change in PAs in the HN plot for 2008, while AAs data revealed trends that were generally similar to foliage for 2008. Concomitant with these changes, mortality data revealed a large number of dead trees in HN pine plots by 2002; the mortality rate started to decline by 2005. Oak trees in the hardwood plot did not exhibit any major changes in PAs, AAs, nutrients and mortality rate with LN treatment, indicating that oak trees were able to tolerate the yearly doses of 50 kg NH4NO3 ha−1 year−1. However, HN trees suffered from physiological and nutritional stress along with increased mortality in 2008. In this case also, foliar data were supported by the sapwood data. Overall, both low and high N applications resulted in greater physiological stress to the pine trees than the oaks. In general, the time course of changes in metabolic data are in agreement with the published reports on changes in soil chemistry and microbial community structure, rates of soil carbon sequestration and production of woody biomass for this chronic N study. This correspondence of selected metabolites with other measures of forest functions suggests that the metabolite analyses are useful for long-term monitoring of the health of forest trees

Soil bacterial communities of a calcium-supplemented and a reference watershed at the Hubbard Brook Experimental Forest (HBEF), New Hampshire, USA

Soil Ca depletion because of acidic deposition-related soil chemistry changes has led to the decline of forest productivity and carbon sequestration in the northeastern USA. In 1999, acidic watershed (WS) 1 at the Hubbard Brook Experimental Forest (HBEF), NH, USA was amended with Ca silicate to restore soil Ca pools. In 2006, soil samples were collected from the Ca-amended (WS1) and reference watershed (WS3) for comparison of bacterial community composition between the two watersheds. The sites were about 125 m apart and were known to have similar stream chemistry and tree populations before Ca amendment. Ca-amended soil had higher Ca and P, and lower Al and acidity as compared with the reference soils. Analysis of bacterial populations by PhyloChip revealed that the bacterial community structure in the Ca-amended and the reference soils was significantly different and that the differences were more pronounced in the mineral soils. Overall, the relative abundance of 300 taxa was significantly affected. Numbers of detectable taxa in families such as Acidobacteriaceae, Comamonadaceae, and Pseudomonadaceae were lower in the Ca-amended soils, while Flavobacteriaceae and Geobacteraceae were higher. The other functionally important groups, e.g. ammonia-oxidizing Nitrosomonadaceae, had lower numbers of taxa in the Ca-amended organic soil but higher in the mineral soil

Oligotyping reveals stronger relationship of organic soil bacterial community structure with N-amendments and soil chemistry in comparison to that of mineral soil at Harvard Forest, MA, USA

The impact of chronic nitrogen amendments on bacterial communities was evaluated at Harvard Forest, Petersham, MA, USA. Thirty soil samples (3 treatments × 2 soil horizons × 5 subplots) were collected in 2009 from untreated (control), low nitrogen-amended (LN; 50 kg NH4NO3 ha-1 yr-1) and high nitrogen-amended (HN; 150 kg NH4NO3 ha-1 yr-1) plots. PCR-amplified partial 16S rRNA gene sequences made from soil DNA were subjected to pyrosequencing (Turlapati et al., 2013) and analyses using oligotyping. The parameters M (the minimum count of the most abundant unique sequence in an oligotype) and s (the minimum number of samples in which an oligotype is expected to be present) had to be optimized for forest soils because of high diversity and the presence of rare organisms. Comparative analyses of the pyrosequencing data by oligotyping and operational taxonomic unit clustering tools indicated that the former yields more refined units of taxonomy with sequence similarity of ≥99.5%. Sequences affiliated with four new phyla and 73 genera were identified in the present study as compared to 27 genera reported earlier from the same data (Turlapati et al., 2013). Significant rearrangements in the bacterial community structure were observed with N-amendments revealing the presence of additional genera in N-amended plots with the absence of some that were present in the control plots. Permutational MANOVA analyses indicated significant variation associated with soil horizon and N treatment for a majority of the phyla. In most cases soil horizon partitioned more variation relative to treatment and treatment effects were more evident for the organic (Org) horizon. Mantel test results for Org soil showed significant positive correlations between bacterial communities and most soil parameters including NH4 and NO3. In mineral soil, correlations were seen only with pH, NH4, and NO3. Regardless of the pipeline used, a major hindrance for such a study remains to be the lack of reference databases for forest soils

Glutamate, Ornithine, Arginine, Proline, and Polyamine Metabolic Interactions: The Pathway Is Regulated at the Post-Transcriptional Level

The metabolism of glutamate into ornithine, arginine, proline, and polyamines is a major network of nitrogen-metabolizing pathways in plants, which also produces intermediates like nitric oxide, and γ-aminobutyric acid (GABA) that play critical roles in plant development and stress. While the accumulations of intermediates and the products of this network depend primarily on nitrogen assimilation, the overall regulation of the interacting sub-pathways is not well understood. We tested the hypothesis that diversion of ornithine into polyamine biosynthesis (by transgenic approach) not only plays a role in regulating its own biosynthesis from glutamate but also affects arginine and proline biosynthesis. Using two high putrescine producing lines of Arabidopsis thaliana (containing a transgenic mouse ornithine decarboxylase gene), we studied the: (1) effects of exogenous supply of carbon and nitrogen on polyamines and pools of soluble amino acids; and, (2) expression of genes encoding key enzymes in the interactive pathways of arginine, proline and GABA biosynthesis as well as the catabolism of polyamines. Our findings suggest that: (1) the overall conversion of glutamate to arginine and polyamines is enhanced by increased utilization of ornithine for polyamine biosynthesis by the transgene product; (2) proline and arginine biosynthesis are regulated independently of polyamines and GABA biosynthesis; (3) the expression of most genes (28 that were studied) that encode enzymes of the interacting sub-pathways of arginine and GABA biosynthesis does not change even though overall biosynthesis of Orn from glutamate is increased several fold; and (4) increased polyamine biosynthesis results in increased assimilation of both nitrogen and carbon by the cells

Polyamines and abiotic stress in plants: a complex relationship1

The physiological relationship between abiotic stress in plants and polyamines was reported more than 40 years ago. Ever since there has been a debate as to whether increased polyamines protect plants against abiotic stress (e.g., due to their ability to deal with oxidative radicals) or cause damage to them (perhaps due to hydrogen peroxide produced by their catabolism). The observation that cellular polyamines are typically elevated in plants under both short-term as well as long-term abiotic stress conditions is consistent with the possibility of their dual effects, i.e., being protectors from as well as perpetrators of stress damage to the cells. The observed increase in tolerance of plants to abiotic stress when their cellular contents are elevated by either exogenous treatment with polyamines or through genetic engineering with genes encoding polyamine biosynthetic enzymes is indicative of a protective role for them. However, through their catabolic production of hydrogen peroxide and acrolein, both strong oxidizers, they can potentially be the cause of cellular harm during stress. In fact, somewhat enigmatic but strong positive relationship between abiotic stress and foliar polyamines has been proposed as a potential biochemical marker of persistent environmental stress in forest trees in which phenotypic symptoms of stress are not yet visible. Such markers may help forewarn forest managers to undertake amelioration strategies before the appearance of visual symptoms of stress and damage at which stage it is often too late for implementing strategies for stress remediation and reversal of damage. This review provides a comprehensive and critical evaluation of the published literature on interactions between abiotic stress and polyamines in plants, and examines the experimental strategies used to understand the functional significance of this relationship with the aim of improving plant productivity, especially under conditions of abiotic stress

Genetic manipulation of polyamine metabolism in poplar II: effects on ethylene biosynthesis

Possible competition between polyamine and ethylene metabolisms was studied in two types of transgenic poplar (Populus nigra × maximowiczii) cells: (a) constitutively expressing a mouse ornithine decarboxylase (ODC, EC 4.1.1.17) cDNA under the control of double 35S cauliflower mosaic virus (CaMV) promoter (cell line 2E), and (b) constitutively expressing a Datura S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) cDNA under the control of a single 35S CaMV promoter (line PS-18). The 2E cells contained significantly higher putrescine (Put) as well as spermidine (Spd) contents than the non-transgenic (NT) cells. The PS-18 cells contained three- to five-fold lower amounts of Put than the NT cells; their Spd content was either comparable to NT cells (at 3 d of culture) or it was higher than the NT cells (at 6 d of culture). The production of ethylene in the 2E cells was generally higher than in the NT cells throughout the 7-d culture period. Ethylene production in the PS-18 cells was comparable to NT cells. The cellular content of 1-aminocyclo-propane-1-carboxylic acid in the NT and 2E cells was quite similar, while it was slightly lower in the PS-18 cells. It is concluded that in poplar cells the cellular pool of S-adenosylmethionine is probably large enough to satisfy the demand for both polyamine and ethylene production and no competition between the two pathways is apparent

Changes in polyamines, inorganic ions and glutamine synthetase activity in response to nitrogen availability and form in red spruce (Picea rubens)

We analyzed effects of nitrogen availability and form on growth rates, concentrations of polyamines and inorganic ions and glutamine synthetase activity in in-vitro-cultured red spruce (Picea rubens Sarg.) cells. Growth rates, concentrations of polyamines and glutamine synthetase activity declined when either the amount of nitrate or the total amount of N in the culture medium was reduced. When total N in the medium was increased, cell mass increased without significant changes in glutamine synthetase activity or polyamine concentration. Reductions in the amount of nitrate or total N in the culture medium resulted in increased accumulations of Ca, Mn and Zn in the cells, and K accumulation decreased in response to decreasing nitrate:ammonium ratios. The data indicate that changes in total N availability as well as the forms of N play important roles in the physiological responses of in-vitro-grown red spruce cells that mimic the observed responses of forest trees to soil N deficiency and N fertilization

High-performance liquid chromatographic method for the determination of dansyl-polyammines

This paper describes a fast reliable, and a sensitive technique for the separation and quantification of dansylated polyamines by high-performance liquid chromatography. Using a small 33 × 4.6 mm I.D., 3 μm particle size, C18 reversed-phase cartridge column and a linear gradient of acetonitrile—heptanesulfonate (10 mM, Ph 3.4), at a flow-rate of 2.5 ml/min, the retention time for different polyamines was: N8-acetyl-spermidine, 1.79 min; N1-acetylspermidine, 1.82 min;putrescine, 2.26 min; cadaverine, 2.43 min; heptanediamine, 2.83 min; spermidine, 3.42 min; and spermine, 4.41 min. With an additional column regeneration time of 3—4 min, the complete cycle per sample took less than 8 min at room temperture. Using a fluorescence detector, the lower limit of detection was less than 1 pmol per 6 μl injection volume. The fluorescence response was linear up to 200 pmol per 6 μl for each polyamine. The method is suitable for separation of polyamines from animal, plant and fungal sources