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Graphs of putrescine relationship with Al:Ca ratios
  Figure 1. A significant positive relationship occurs between free putrescine (a biochemical stress indicator) in one-year-old red spruce foliage and exchangeable Al:Ca ratios in forest floor soils that were collected from six different sites within New England area (Minocha et al., 1997). Ratios were derived from mean concentrations (cmolc kg-1) of Al and Ca for each location.
  Graphs of changes in putrescine
  Figure 2. Putrescine as an indicator of remediation from nutritional stress (Ca deficiency) or plant competition. Data presented show changes in putrescine (A) and (B) soil and foliar exchangeable Ca after application of lime and/or herbicide indicating reduced stress in sugar maple trees at Allegheny State Forest, PA (Wargo et al., 2002).
Environmental stress physiology of trees


  Contact Info:
  Rakesh Minocha, Ph.D
Research Biochemist
USDA Forest Service, NRS
271 Mast Road
Durham, NH 03824
phone: (603) 868-7622
fax: (603) 868-7604

This research was funded jointly by Northeastern States Research Cooperative (NSRC) and USDA Forest Service, Research Work Unit, NRS-4505.

LONG-TERM STUDIES of nutrient cycling in forested watersheds across the Eastern United States have raised concerns about the depletion of available soil calcium (Ca) due to nutrient removals by forest harvesting and leaching induced by acid deposition and aluminum (Al) mobilization in acidified soils. This has led to a heightened interest in the role of base cations, such as Ca, in forest health and productivity. Calcium is an essential plant nutrient that serves to stabilize wood structure and cell membranes, and it is involved in numerous cellular processes including responses to abiotic and biotic stress. Depletion of soil Ca could disrupt nutrition and predispose forests to decline in tree health and growth.

Biochemical and physiological indicators of plant stress can be used to assess the changes in tree health before visible symptoms of damage occur. Putrescine (small organic cation) concentrations in foliage have been proposed as one of these early indicators. A variety of stimuli such as high levels of ozone, acidic deposition, pathogen infections, SO2 fumigation, high salt concentrations, high Al, and mineral nutrient (K, Ca, and Mg) deficiencies can lead to an accumulation of polyamines (PAs), particularly putrescine (Figures 1 and 2). PAs have been observed in foliage in response to perturbations in soil Ca attributed to acidic deposition, cation depletion, Al mobilization, nitrogen fertilization and ice storm injury. A link between putrescine and carbon and nitrogen metabolism has been shown previously (Figure 3). The fact that PAs enhance a tolerance to a wide variety of abiotic stresses suggests that they may interact with common stress remediation mechanisms such as excess generation of reactive oxygen species (ROS) causing oxidative damage. We are determining (i) whether there is a correlation between changes in foliar biochemistry/physiology and soil Ca concentrations and (ii) whether this relationship is species-specific.

The work in progress will provide needed information about the physiological effects of Ca fertilization. Our long-term objectives include: (1) to identify the basic physiological mechanisms that relate tree health to Ca availability and Al mobilization; and (2) to develop techniques and additional data that will contribute to the use of biochemical indicators in forest-health monitoring and assessment. Work toward meeting these objectives is being carried out as part of two long-term studies: a large-scale Ca replacement study and a plot-scale fertilization study (Nupert study, described below).

  Polyamine biosynthetic pathway diagram
  Figure 3. Pathways of biosynthesis for polyamines (putrescine, spermidine and spermine) are linked with with C and N cycling in plants.

  Graphs of calcium addition effects on putrescine
  Figure 4. Effect of wollastonite (calcium silicate) addition in WS1 on (A) free foliar putrescine (an indicator of stress remediation) and (B) foliar exchangeable Ca. Note a significant reduction in putrescine with increase in foliar Ca indicating a reduction in stress caused by Ca deficiency only at mid and high elevations. Asterisk(s) indicate a significant difference between treatment and reference sites within each elevation. Manuscript in preparation.

Calcium replacement study

A large-scale experiment was begun in 1999 to evaluate the role of Ca supply in regulating the structure and function of a base-poor forest ecosystem. The base status of the soil was improved by adding wollastonite (calcium silicate) to Watershed 1 of the Hubbard Brook Experimental Forest. The specific objective of the proposed study is to investigate the effect of Ca addition on foliar biochemistry and physiology, and ultimately tree growth and health. Foliar samples from mature trees located near established lysimeters (for sampling ground water from different soil horizons) were collected in July or August from 4 - 6 species (up to 20 trees/species if available) at Watershed 1 during 2000-2006. Samples were also collected from east of Watershed 3 boundary to be used as a reference during 2004-2006. These samples were analyzed for exchangeable inorganic elements (including Ca, Mg, K, Mn, Fe, P, and Zn) as well as free polyamines, free amino acids, soluble proteins, and chlorophyll by using published methods that have been developed by our group and are routinely used in our laboratory (Minocha et. al., 2000). These foliar data will be tested for correlations with each other and with soil solution chemistry and soil nitrification rates that are being gathered by other scientists who are also a part of the ongoing study at this site. Foliar free putrescine (a physiological marker of stress) significantly decreased in sugar maple (Figure 4) only at mid and high elevations and this change was inversely proportional to foliar Ca in these trees.

In the summers of 2005-2006, tiny wood plugs were also collected using a Swedish hammer (Figure 5) and analyzed for exchangeable inorganic ions, free polyamines and amino acids. The goal was to determine if wood, like foliage, can be used to study stress physiology of trees within an ecosystem. Preliminary data indicates that changes in putrescine and Ca could be reliably detected in wood tissue from several sites though the magnitude of these changes was several fold lower than in foliage (data not shown).

Collection and processing of samples
Figure 5. Collection and processing of foliage and wood plugs for biochemical analyses.


Nupert study
This study was initiated west of Watershed 6 in 1995 where Ca and Al are being applied to the soil. Three plots per treatment were set up making a total of 12 plots (including control plots). Every year 0.9 g m-2 Al (AlCl3) has been added with two exceptions (1997 the dose was doubled and 1998 no treatments were applied). Calcium (2-3 g m-2) was added as CaCl2 until 1997 and then it was replaced with a one time wollastonite treatment of 112 g m-2 in 1999. Foliar samples were collected from sugar maple trees during 1997-2002 for stress physiology research. Aluminum addition caused an increase in putrescine accompanied by a decrease in Ca in sugar maple (data not shown).

Putrescine levels respond to several types of abiotic stress including nutritional deficiencies and ozone as well as biotic stress such as exposure of hemlock trees to the pathogen, hemlock woolly adelgid (preliminary data, not shown). Putrescine levels were often found to be significantly related to foliar or soil Ca deficiency, increased soil Al and/or excess soil N caused by fertilization or acidic deposition. Foliar putrescine is therefore a reliable indicator of the current health of red spruce and sugar maple in forests across the Northern United States.

Minocha R, Shortle WC, Lawrence GB, David MB and Minocha SC. 1997. Relationships among foliar chemistry, foliar polyamines, and soil chemistry in red spruce trees growing across the northeastern United States . Plant Soil. 191:109-122.

Minocha R, Long S, Magill AH, Aber J and McDowell WH. 2000. 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. Plant Soil. 222:119-137.

Wargo PM, Minocha R, Wong BL, Long RP, Horsley SB and Hall TJ. 2002. Measuring changes in stress and vitality indicators in limed sugar maple on the Allegheny Plateau in north-central Pennsylvania . Can. J. For. Res. 32:629-641.

Date Prepared: October 2006