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ABSTRACT: We over-expressed 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 in the regulation of not only polyamine biosynthesis, but also 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 (vs. 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 γ-aminobutyric acid content of the cells. Furthermore, the induction of the 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 to 8 hours.
Plant and Cell Physiology 04/2013; · 4.70 Impact Factor
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ABSTRACT: At the Harvard Forest, Petersham, MA, the impact of 20 years of annual ammonium nitrate application to the mixed hardwood stand on soil bacterial communities was studied using 16S rRNA genes pyrosequencing. Amplification of 16S rRNA genes was done using DNA extracted from 30 soil samples (three treatments × two horizons × five subplots) collected from untreated (control), low N-amended (50 kg ha(-1) year(-1) ) and high N-amended (150 kg ha(-1) year(-1) ) plots. A total of 1.3 million sequences were processed using qiime. Although Acidobacteria represented the most abundant phylum based on the number of sequences, Proteobacteria were the most diverse in terms of operational taxonomic units (OTUs). UniFrac analyses revealed that the bacterial communities differed significantly among soil horizons and treatments. Microsite variability among the five subplots was also evident. Nonmetric multidimensional scaling ordination of normalized OTU data followed by permutational manova further confirmed these observations. Richness indicators and indicator species analyses revealed higher bacterial diversity associated with N amendment. Differences in bacterial diversity and community composition associated with the N treatments were also observed at lower phylogenetic levels. Only 28-35% of the 6 936 total OTUs identified were common to three treatments, while the rest were specific to one treatment or common to two.
FEMS Microbiology Ecology 09/2012; · 3.41 Impact Factor
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ABSTRACT: Cell suspension cultures of red spruce (Picea rubens Sarg.) were selected to study the effects of cadmium (Cd) and zinc (Zn) on phytochelatins (PCs) and related metabolites after
24h exposure. The PC2 and its precursor, γ-glutamylcysteine (γ-EC) increased two to fourfold with Cd concentrations ranging from 12.5 to 200μM
as compared to the control. However, Zn-treated cells showed a less than twofold increase in γ-EC and PC2 levels as compared to the control even at the highest concentration of 800μM. In addition, unidentified higher chain PCs
were also found in both the Cd and Zn treated cells and they increased significantly with increasing concentrations of Cd
and Zn. The cellular ratio of PC2:Cd or Zn content clearly indicated that Cd (with ratios ranging from 0.131 to 0.546) is a more effective inducer of PC2 synthesis/accumulation than Zn (with ratios ranging from 0.032 to 0.102) in red spruce cells. A marginal decrease in glutathione
(GSH) was observed in both Cd and Zn treated cells. However, the GSH precursor, cysteine, declined twofold with all Cd concentrations
while the decrease with Zn was 1.5–2-fold only at the higher treatment concentrations of Zn as compared to control. In addition,
changes in other free amino acids, polyamines, and inorganic ions were also studied. These results suggest that PCs and their
biosynthetic intermediates play a significant role in red spruce cells protecting against Cd and Zn toxicity.
Plant Cell Tissue and Organ Culture 04/2012; 88(2):201-216. · 3.09 Impact Factor
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ABSTRACT: 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.
FEMS Microbiology Ecology 11/2011; 79(3):728-40. · 3.41 Impact Factor
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ABSTRACT: Plants in nutrient-poor environments typically have low foliar nitrogen (N) concentrations, long-lived tissues with leaf traits designed to use nutrients efficiently, and low rates of photosynthesis. We postulated that increasing N availability due to atmospheric deposition would increase photosynthetic capacity, foliar N, and specific leaf area (SLA) of bog shrubs. We measured photosynthesis, foliar chemistry and leaf morphology in three ericaceous shrubs (Vaccinium myrtilloides, Ledum groenlandicum and Chamaedaphne calyculata) in a long-term fertilization experiment at Mer Bleue bog, Ontario, Canada, with a background deposition of 0.8 g N m(-2) a(-1). While biomass and chlorophyll concentrations increased in the highest nutrient treatment for C. calyculata, we found no change in the rates of light-saturated photosynthesis (A(max)), carboxylation (V(cmax)), or SLA with nutrient (N with and without PK) addition, with the exception of a weak positive correlation between foliar N and A(max) for C. calyculata, and higher V(cmax) in L. groenlandicum with low nutrient addition. We found negative correlations between photosynthetic N use efficiency (PNUE) and foliar N, accompanied by a species-specific increase in one or more amino acids, which may be a sign of excess N availability and/or a mechanism to reduce ammonium (NH(4)) toxicity. We also observed a decrease in foliar soluble Ca and Mg concentrations, essential minerals for plant growth, but no change in polyamines, indicators of physiological stress under conditions of high N accumulation. These results suggest that plants adapted to low-nutrient environments do not shift their resource allocation to photosynthetic processes, even after reaching N sufficiency, but instead store the excess N in organic compounds for future use. In the long term, bog species may not be able to take advantage of elevated nutrients, resulting in them being replaced by species that are better adapted to a higher nutrient environment.
Oecologia 05/2011; 167(2):355-68. · 3.41 Impact Factor
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ABSTRACT: Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied changes in the expression of all genes of the Orn/Arg biosynthetic pathway in response to up-regulation [via transgenic expression of mouse Orn decarboxylase (mODC)] of PA biosynthesis in poplar (Populus nigra × maximowiczii) cells grown in culture. Cloning and sequencing of poplar genes involved in the Orn/Arg biosynthetic pathway showed that they have high homology with similar genes in other plants. The expression of the genes of Orn, Arg and PA biosynthetic pathway fell into two hierarchical clusters; expression of one did not change in response to high putrescine, while members of the other cluster showed a shift in expression pattern during the 7-day culture cycle. Gene expression of branch point enzymes (N-acetyl-Glu synthase, Orn aminotransferase, Arg decarboxylase, and spermidine synthase) in the sub-pathways, constituted a separate cluster from those involved in intermediary reactions of the pathway (N-acetyl-Glu kinase, N-acetyl-Glu-5-P reductase, N-acetyl-Orn aminotransferase, N (2)-acetylOrn:N-acetyl-Glu acetyltransferase, N (2)-acetyl-Orn deacetylase, Orn transcarbamylase, argininosuccinate synthase, carbamoylphosphate synthetase, argininosuccinate lyase, S-adenosylmethionine decarboxylase, spermine synthase). We postulate that expression of all genes of the Glu-Orn-Arg pathway is constitutively coordinated and is not influenced by the increase in flux rate through this pathway in response to increased utilization of Orn by mODC; thus the pathway involves mostly biochemical regulation rather than changes in gene expression. We further suggest that Orn itself plays a major role in the regulation of this pathway.
Amino Acids 11/2010; 42(1):295-308. · 3.25 Impact Factor
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ABSTRACT: Distribution of biogenic amines-the diamine putrescine (Put), triamine spermidine (Spd), and tetraamine spermine (Spm)-differs between species with Put and Spd being particularly abundant and Spm the least abundant in plant cells. These amines are important for cell viability and their intracellular levels are tightly regulated, which have made it difficult to characterize individual effects of Put, Spd and Spm on plant growth and developmental processes. The recent transgenic intervention and mutational genetics have made it possible to stably alter levels of naturally occurring polyamines and study their biological effects. We bring together an analysis of certain metabolic changes, particularly in amino acids, to infer the responsive regulation brought about by increased diamine or polyamine levels in actively growing poplar cell cultures (transformed with mouse ornithine decarboxylase gene to accumulate high Put levels) and ripening tomato pericarp (transformed with yeast S-adenosylmethionine decarboxylase gene to accumulate high Spd and Spm levels at the cost of Put). Our analysis indicates that increased Put has little effect on increasing the levels of Spd and Spm, while Spd and Spm levels are inter-dependent. Further, Put levels were positively associated with Ala (alpha and beta), Ile and GABA and negatively correlated with Gln and Glu in both actively growing poplar cell cultures and non-dividing tomato pericarp tissue. Most amino acids showed positive correlations with Spd and Spm levels in actively growing cells. Collectively these results suggest that Put is a negative regulator while Spd-Spm are positive regulators of cellular amino acid metabolism.
Amino Acids 12/2009; 38(2):405-13. · 3.25 Impact Factor
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ABSTRACT: The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and gamma-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra x maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and gamma-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.
Amino Acids 09/2009; 38(4):1117-29. · 3.25 Impact Factor
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ABSTRACT: 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.
Tree Physiology 01/2009; 28(12):1793-803. · 2.88 Impact Factor
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ABSTRACT: 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.
Journal of Chromatography 09/2008; 1207(1-2):72-83. · 4.53 Impact Factor
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ABSTRACT: We have studied the effects of overproduction of putrescine (Put) via transgenic expression of a mouse ornithine decarboxylase (ODC) gene on the expression of native genes for four enzymes involved in polyamine biosynthesis in hybrid poplar (Populus nigra×maximowiczii) cells. An examination of the transcript levels of arginine decarboxylase (ADC), ODC, S-adenosylmethionine decarboxylase (SAMDC) and spermidine synthase (SPDS), as well as their enzyme activities (except SPDS), showed that the expression of different members of the SAMDC and SPDS gene families was affected differently in response to alteration of the cellular Put content. It was further observed that there was a strong correlation between transcript levels and the activity of the respective enzyme in the cells. Moreover, there was no feedback inhibition of the expression of the native ODC or the ADC genes or their enzyme activities by increased Put in the cells.
Physiologia Plantarum 03/2007; 129(4):707 - 724. · 3.11 Impact Factor
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ABSTRACT: Interactions among external (soil) pH, cellular pH, and their effects on respiratory metabolism are complex. While the effects
of changes in the apoplastic pH on the cytosolic pH are not clearly understood, pH directly affects enzymatic reactions in
the cell, and pH-regulated ion uptake has profound indirect effects on cellular respiratory metabolism. A major consequence
of soil acidification is the release of aluminum in solubilized forms from its insoluble forms, which, in turn, adversely
affects the uptake of cations, causes organic acid secretion, and inhibits cell division and growth in the roots. Consequently,
the respiratory metabolism is redirected to meet the needs of organic acid efflux from the roots. The effects of changes in
external pH on cellular pH and consequent effects of this change on respiratory metabolism, particularly through effects on
soil aluminum are summarized.
03/2006: pages 159-176;
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ABSTRACT: The major objective of this study was to determine if the observed changes in polyamines and their biosynthetic enzymes during
somatic embryo development were specifically related to either the stage of the embryo development or to the duration of time
spent on the maturation medium. Somatic embryos of red spruce (Picea rubens) at different developmental stages, grown in the embryo development and maturation media for various lengths of time, were
separated from the associated subtending tissue (embryogenic and the suspensor cell masses) and analyzed for their polyamine
content as well as for polyamine biosynthetic enzyme activities. Polyamine content was also analyzed in embryos representing
different stages of developmentthat were collected from the sam culture plate at the same time and the subtending tissue surrouding
them. Putrescine was the predominant polyamine in the pro-embryogenic tissue, while spermidine was predominant during embryo
development. Significant changes in spermidine/putrescine and spermine/putrescine ratios were observed at all stages of embryo
development as compared to the pro-embryogenic cell mass. Changes in the ratios of various polyamines were clearly correlated
with the developmental stage of the embryo rather than the period of growth in the maturation medium. Whereas the activities
of both ornithine decarboxylase and arginine decarboxylase increased by week 3 or 4 and stayed high during the subsequent
6 wk of growth, the activity of S-adenosylmethionine decarboxylase steadily declined during embryo development.
In Vitro Cellular & Developmental Biology - Plant 10/2004; 40(6):572-580. · 1.50 Impact Factor
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ABSTRACT: We determined: (a) the physiological consequences of overproduction of putrescine in transgenic poplar (Populus nigra x maximoviczii) cells expressing an ornithine decarboxylase transgene; and (b) effects of variation in nitrogen (N) concentration of the medium on cellular polyamine concentration in transgenic and non-transgenic cells. Cells grown in the presence of supplemental (to the normal concentrations of N sources in the growth medium) and reduced amounts of NH4NO3 and KNO3 were used to study effects on membrane permeability, mitochondrial respiratory activity, protein accumulation, growth rates and changes in cellular polyamine concentration. The N concentration of the MS medium was not a limiting factor for continued overproduction of putrescine in transgenic cells. However, continued supplies of NH4+ and NO3- were required to maintain homeostatic amounts of putrescine in both cell lines. The presence of high amounts of putrescine in transgenic cells had significant effects on the physiological parameters measured. Compared with non-transgenic cells, transgenic cells had greater plasma membrane permeability, less tolerance to NH4NO3, more tolerance to KNO3, and accumulated higher amounts of soluble protein.
Tree Physiology 06/2004; 24(5):551-60. · 2.88 Impact Factor
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ABSTRACT: The objective of the present study was to develop a rapid HPLC method for simultaneous separation and quantitation of dansylated amino acids and common polyamines in the same matrix for analyzing forest tree tissues and cell cultures. The major modifications incorporated into this method as compared to previously published HPLC methods for separation of only dansyl amino acids include: use of a 10 cm column to reduce the total run time by approximately 15 min; modification of the dansyl derivatization process and gradient profile to elute amino acids and common polyamines within the same run; addition of steps for column cleaning within each run; shorter re-equilibration time; and finally, column cleaning and physically reversing the column at the end of a loop of samples. These changes improved peak resolution and increased column longevity by several-fold. Over 1000 foliar samples from mature forest trees could be analyzed with the same column as compared to only 200-250 samples before the incorporation of these changes. This method eluted 22 amino acids within 40 min plus all three common polyamines between 44 and 47 min. The total run time is 53.6 min for amino acids only and 55.6 min for both amino acids and polyamines.
Journal of Chromatography 05/2004; 1035(1):63-73. · 4.53 Impact Factor
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ABSTRACT: In the absence of added Al, the concentration of succinate in cultured red spruce (Picea rubens Sarg.) cells was 15-20 times higher (> 600 nmol g-1FW) than that of citrate or oxalate and 4-6 times higher than that of malate. Addition of AlCl3 (effective monomeric Al concentrations of 0.23 and 0.48 mM) to 3-day-old suspension cultures significantly increased cellular succinate concentrations with a concomitant decrease in cellular oxalate concentrations. However, in the medium of Al-treated cell cultures, both succinate and oxalate concentrations were significantly higher than in the medium of cell cultures without added Al, and oxalate concentrations were several times higher than succinate concentrations. Aluminum did not significantly affect the cellular concentrations of malate, ascorbate and citrate, and none of these organic acids was present in detectable quantities in the medium. Exogenous succinate alone or with Al had no effect on cellular free polyamine concentrations or cell mass. Aluminum caused a significant increase in cellular putrescine concentrations. Addition of malate had a positive effect on growth and completely reversed the effects of Al on cell physiology. In contrast, the addition of oxalate and citrate only partly reversed the effects of Al.
Tree Physiology 01/2004; 24(1):55-64. · 2.88 Impact Factor
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ABSTRACT: We investigated the catabolism of putrescine (Put) in a non-transgenic (NT) and a transgenic cell line of poplar (Populus nigra x maximowiczii) expressing a mouse (Mus musculus) ornithine (Orn) decarboxylase (odc) cDNA. The transgenic cells produce 3- to 4-fold higher amounts of Put than the NT cells. The rate of loss of Put from the cells and the initial half-life of cellular Put were determined by feeding the cells with [U-(14)C]Orn and [1,4-(14)C]Put as precursors and following the loss of [(14)C]Put in the cells at various times after transfer to label-free medium. The amount of Put converted into spermidine as well as the loss of Put per gram fresh weight were significantly higher in the transgenic cells than the NT cells. The initial half-life of exogenously supplied [(14)C]Put was not significantly different in the two cell lines. The activity of diamine oxidase, the major enzyme involved in Put catabolism, was comparable in the two cell lines even though the Put content of the transgenic cells was severalfold higher than the NT cells. It is concluded that in poplar cells: (a) exogenously supplied Orn enters the cells and is rapidly converted into Put, (b) the rate of Put catabolism is proportional to the rate of its biosynthesis, and (c) the increased Put degradation occurs without significant changes in the activity of diamine oxidase.
Plant physiology 05/2002; 128(4):1455-69. · 6.53 Impact Factor
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ABSTRACT: Changes in the cellular content of three polyamines (putrescine, spermidine and spermine) were compared at different stages of development in zygotic and somatic embryos of Pinus radiata D. Don. During embryo development, both the zygotic and the somatic embryos showed a steady increase in spermidine content, with either a small decrease or no significant change in putrescine. This led to a several-fold increase in spermidine/putrescine ratios during development of both types of embryos. Cell cultures of plant-forming and non-plant-forming lines derived from the same clone and growing on proliferation (maintenance) medium differed significantly in their polyamine levels. Mature, cotyledonary stage somatic embryos capable of germination and formation of plants could be distinguished by their higher spermidine/putrescine ratios from abnormal cotyledonary stage somatic embryos which were incapable of forming plants.
Physiologia Plantarum 12/1998; 105(1):155 - 164. · 3.11 Impact Factor
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ABSTRACT: Forest trees are constantly exposed to various types of natural and anthropogenic stressors. A major long-term goal of our research is to develop a set of early physiological and biochemical markers of stress in trees before the appearance of visual symptoms. Six red spruce (t Picea rubens Sarg.) stands from the northeastern United States were selected for collection of soil and foliage samples. All of the chosen sites had soil solution pH values below 4.0 in the Oa horizon but varied in their geochemistry. Some of these sites were apparently under some form of environmental stress as indicated by a large number of dead and dying red spruce trees. Samples of soil and needles (from apparently healthy red spruce trees) were collected from these sites four times during a two-year period. The needles were analyzed for perchloric acid-soluble polyamines and exchangeable inorganic ions. Soil and soil solution samples from the Oa and B horizons were analyzed for their exchange chemistry. The data showed a strong positive correlation between Ca and Mg concentrations in the needles and in the Oa horizon of the soil. However, needles from trees growing on relatively Ca-rich soils with a low exchangeable Al concentration and a low Al:Ca soil solution ratio had significantly lower concentrations of putrescine and spermidine than those growing on Ca-poor soils with a high exchangeable Al concentration and a high Al:Ca soil solution in the Oa horizon. The magnitude of this change was several fold higher for putrescine concentrations than for spermidine concentrations. Neither putrescine nor spermidine were correlated with soil solution Ca, Mg, and Al concentrations in the B horizon. The putrescine concentrations of the needles always correlated significantly with exchangeable Al (r2=0.73, t p0.05) and soil solution Al:Ca ratios (r2=0.91, t p0.01) of the Oa horizon. This suggests that in conjunction with soil chemistry, putrescine and/or spermidine may be used as a potential early indicator of Al stress before the appearance of visual symptoms in red spruce trees.
Plant and Soil 03/1997; 191(1):109-122. · 2.73 Impact Factor
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ABSTRACT: A fast and reliable method for the extraction of cellular polyamines and major inorganic ions (Ca, Mg, Mn, K, and P) from
several plant tissues is described. The method involves repeated freezing and thawing of samples instead of homogenization.
The efficiency of extraction of both the polyamines and inorganic ions by these two methods was compared for 10 different
tissues. In each case, the freeze-thaw procedure resulted in a precise and quantitatively equal, or greater, yield than homogenization.
Freeze-thawing not only eliminates the need for various tissue homogenizers (such as polytrons, tissumizers, and mortars and
pestles), but it is so simple that a large number of samples can be processed simultaneously. We routinely processed 50–80
samples for quantitation of polyamines and inorganic ions. Freeze-thawing was equally useful for the extraction of polyamines
from liver, spleen, and kidney tissues of mice.
Journal of Plant Growth Regulation 04/1994; 13(4):187-193. · 2.86 Impact Factor
