Rakesh Minocha

RESPONSE OF SUGAR MAPLE TO CALCIUM ADDITION TO NORTHERN HARDWOOD FOREST

Watershed budget studies at the Hubbard Brook Experimental Forest (HBEF), New Hampshire, USA, have demonstrated high calcium depletion of soil during the 20th century due, in part, to acid deposition. Over the past 25 years, tree growth (especially for sugar maple) has declined on the experimental watersheds at the HBEF. In October 1999, 0.85 Mg Ca/ha was added to Watershed 1 (W1) at the HBEF in the form of wollastonite (CaSiO3), a treatment that, by summer 2002, had raised the pH in the Oie horizon from 3.8 to 5.0 and, in the Oa horizon, from 3.9 to 4.2. We measured the response of sugar maple to the calcium fertilization treatment on W1. Foliar calcium concentration of canopy sugar maples in W1 increased markedly beginning the second year after treatment, and foliar manganese declined in years four and five. By 2005, the crown condition of sugar maple was much healthier in the treated watershed as compared with the untreated reference watershed (W6). Following high seed production in 2000 and 2002, the density of sugar maple seedlings increased significantly on W1 in comparison with W6 in 2001 and 2003. Survivorship of the 2003 cohort through July 2005 was much higher on W1 (36.6%) than W6 (10.2%). In 2003, sugar maple germinants on W1 were approximately 50% larger than those in reference plots, and foliar chlorophyll concentrations were significantly greater (0.27 g/m2 vs. 0.23 g/m2 leaf area). Foliage and fine-root calcium concentrations were roughly twice as high, and manganese concentrations twice as low in the treated than the reference seedlings in 2003 and 2004. Mycorrhizal colonization of seedlings was also much greater in the treated (22.4% of root length) than the reference sites (4.4%). A similar, though less dramatic, difference was observed for mycorrhizal colonization of mature sugar maples (56% vs. 35%). These results reinforce and extend other regional observations that sugar maple decline in the northeastern United States and southern Canada is caused in part by anthropogenic effects on soil calcium status, but the causal interactions among inorganic nutrition, physiological stress, mycorrhizal colonization, and seedling growth and health remain to be established.

Polyamines and abiotic stress in plants: a complex relationship.

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.

Polyamines and cellular metabolism in plants: transgenic approaches reveal different responses to diamine putrescine versus higher polyamines spermidine and spermine

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 (α and β), 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.

A Rapid and Reliable Procedure for Extraction of Cellular Polyamines and Inorganic Ions from Plant Tissues

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.

Genetic Manipulation of the Metabolism of Polyamines in Poplar Cells. The Regulation of Putrescine Catabolism

We investigated the catabolism of putrescine (Put) in a non-transgenic (NT) and a transgenic cell line of poplar (Populus nigra × 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-14C]Orn and [1,4-14C]Put as precursors and following the loss of [14C]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 [14C]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.

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.

Relationships among foliar chemistry, foliar polyamines, and soil chemistry in red spruce trees growing across the northeastern United States

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 p≤0.05) and soil solution Al:Ca ratios (r2=0.91, t p≤0.01) 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.

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.