Salinity Trials for the Induction of PeroxisomesJennifer Griffith

IntroductionSoil salinity is one of the major limiting factors of plants to be able to spread across their

natural habitats, especially in arid and semi arid regions. Tolerance of salinity in plants depends on numerous physiological interactions (Qados). It is estimated that approximately 20% of agricultural land and 50% of cropland is salt-stressed in the world. This stress limits the caloric and nutritional potential of crops (Yokoi et al). Changes in plant metabolism can be seen in salt stressed environment. Reduction in water potential and CO2 assimilation of the plant are affected, and also ion imbalance and toxicity are seen (Hernandez et al). Piscum sativum is the common garden pea. Hernandez et al. showed in previous studies that long-term exposure to salt at 70 mM caused changes in expression of foliar ascorbate-glutathione cycle enzymes, superoxide dismutase isozymes, and mRNAs. This was especially seen in the NaCl-tolerant variety used in the study, suggesting that induction of antioxidant defenses is important to tolerance in salt-stressed environments. Qados (2011) looked at the effects of sodium chloride (NaCl) on growth, osmotic potential, chlorophyll content, and protein content in Vicia faba L. using for concentrations. The concentrations were 0.0, 60, 120, and 240 mM. Plant height increased in the low and medium concentrations and decreased in the heightest. Chlorophyll a/b, total chlorophyll, and carotenoid content was reduced, which also had an effect on protein content. Also a decrease of foliage was seen in the two highest concentrations. Plants use numerous processes to respond to increased salinity to alleviate cellular hyperosmolarity and ion disequilibrium (Yokoi et al). Peroxidases and carotenoids are one part of plants defense against salt stress and its way to try to repair damage (Hernandez et al).

Peroxidases are part of a large family of enzymes that are oxidoreductases that use organic and inorganic substrates alike as hydrogen donors when hydrogen peroxide is present. They are 25% carbohydrate, consisting of ~300 amino acid residues of two glucosamines, eight true sugars, two bound calcium bonds, and two to six glycosylation sites. They are thermostable proteins with and isoelectric point that ranges from 3.5 to 10 (Vicuna). They are ubiquitous in all life forms. They function in signaling and maintain oxygen tolerance. Heme peroxidases are divided into two superfamilies and three families. The two superfamilies are peroxidase-cyclooxygenase and peroxidase-catalase. The peroxidase-catalase superfamily consists of three classes that are found in bacteria, fungi, and plants. Class I is responsible for the evolution of bifunctional catalase-peroxidases. Class II are found in fungi and are involved in carbon recycling by degradation of lignin. Class III are secretory peroxidases, found in algae and plants, that are part of a wide range of secondary metabolism. (Zamocky et al). They are found in all parts of the plant. They play a critical role in oxygen levels in the plant due to their reduction of hydrogen peroxide (Dalton et al). The other major role they play in plants is being involved in the polymerization of the precursors of lignin (Vicuna).

In this study, Piscum sativum and Wisconsin Fast Plants were used to look at the possibility of increased peroxidases due to salt stresses. Piscum sativum was chosen due to previous studies in responses to salinity and the ability to follow previously set working concentrations. Wisconsin Fast Plants, Brassica rapa, is commonly used in the laboratory for experiments due to their rapid cycling, self-incompatibility for pollination, and genetic diversity.

They can complete a life cycle in 35-45 d. Growth conditions are favorable for indoor temperatures, and lighting when placed by a window or by using intense fluorescent lighting (Wendel et al). Three salinity concentrations were chosen from previous literature; 0, 60, and 240 mM. Increasing levels of salinity is theorized to cause peroxisome levels to increase. Peas will not show as much negative effects to increased soil salinity as Fast Plants, but both will increase levels of peroxidases with increasing soil salinity.

Materials and MethodsThirty-six pots, approximately 350 ml in volume, were obtained for use. Before adding

soil to pots, paper towels were measured and cut to fit the bottom of the pots. A single layer of paper towel was placed into each pot. 100 ml sand and 300 ml potting soil was mixed and 200 ml of mixture was placed into each pot. Six pots were placed in a tray. Two to three peas or two to three Fast Plant seeds were randomly placed into each pot. Six Fast Plants and six peas were labeled as control plants. These were watered with dH2O through the trial. Two salinity levels were chosen for the experiment, 60 mM and 240 mM, twelve pots, or six pots of peas and six pots of Fast Plants, were labeled for each of these salinities.

Salt solutions were prepared in 1 L Erlenmeyer flasks. To prepare the 60 mM salt solution, 3.510 g NaCl was weighed and placed in flask with 1000 ml dH2O. Flask was then shook to mix into solution. The 240 mM salt solution was prepared by adding 14.04 g NaCl to 1000 ml dH2O. Mixture was heated at 1200ºC for ~10 min with a stir bar at 700 rpm to create solution.

Trays were set on rack beside eastward facing window. Each pot was watered initially with 30 mL of the appropriate treatment. Plants were watered three times a week.

ResultsOver a three week period, a salt layer was observed to collect, first on 240 mM plants

and then on the 60 mM plants. A thick layer of salt crystals was also observed in the tray holding the six pots receiving the 240 mM treatment. A slightly thinner layer was also seen in the tray for the 60 mM treatment. No growth was seen in any of these pots. The control plants showed normal growth. Fast Plants obtained a height of ~1’ and peas reached a height of ~4-5”.

DiscussionDue to no growth of either type of plant in the 60 mM or 240 mM treatment,

peroxisome identification was impossible. It was theorized that a combination of pot size, salinity concentration, and frequency of watering with the salt solutions caused non-growth in the two treatments. Control plants showed no indications of growth impairment from the soil concentration, lighting, or amount and frequency of watering. This supports the idea that the salt concentration was too high per volume. In the future, salt concentration of the solutions should be at least cut in half. It is also suggestible to water plants with regular dH2O twice a week and with the salt solutions one time a week.

References1. Dalton, D. A., F. J. Hanus, S. A. Russell, and H. J. Evans. 1987. Purification, properties, and

distribution of ascorbate peroxidase in legume root nodules. Plant Physiol. 83, 789-794.2. Hernandez, J. A., A. Jimenez, P. Mullineaux, and F. Sevilla. 2000. Tolerance of pea (Pisum

sativum L.) to long-term salt stress is associated with induction of antioxidant defenses. Plant, Cell and Environment. 23, 853-862.

3. Qados, A. M. S. A. 2011. Effect of salt stress on plant growth and metabolism of bean plant Vicia faba (L.). Journal of the Saudi Society of Agricultural Sciences. 10, 7-15.

4. Torres, E., and M. Ayala. 2010. Biocatalysis Based on Heme Peroxidases. 11th ed., pages 8-32. Springer.

5. Vicuna, D. (Thesis) 2005. The role of peroxidases in the development of plants and their responses to abiotic stresses. Dublin Institute of Technology. Doctoral Paper 15.

6. Wendell, D. L., and D. Pickard. 2007. Teaching human genetics with Mustard: Rapid cycling Brassica rapa (Fast Plants type) as a model for human genetics in the classroom laboratory. CBE-Life Sciences Education. 6, 179-185.

7. Yokoi, S., R. A. Bressan, and P. M. Hasegawa. 2002. Salt stress tolerance of plants. JIRCAS Working Report. 25-33.