Kader MAL, S. (2010 March) Cytosolic calcium and ph signaling in plants under salinity stress. Plant Signal Behav. 5(3):

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1 Effects of NaCl on pepper seed germination, and whole plant recovery after extensive fertilization Nicole Newell, with Katie Kuefler and Mike Neufeld Fall 213 ABSTRACT In these experiments, we conducted seed germination trials in solutions of NaCl or mannitol with varying osmolarities in order to observe and identify the effects of salinity on pepper plant seeds. We found that there is a significant delay in germination caused by the increasing osmolarity of the solutions and the decrease of water absorption. In addition to this osmotic effect, we found that at higher than 4mOsm of NaCl the germinated seeds experienced ionic stress from the toxic Na+ ion. We also conducted whole pepper plant experiments to determine their ability to recover from extensive fertilization. After four weeks, 11 out of 12 plants had recovered from visible wilt and showed new growth. INTRODUCTION Salt stress is one of the major abiotic threats to plant life and plant agriculture worldwide and significantly reduces crop yield in affected areas. Excessive salt above what plants need limits plant growth and productivity and can lead to plant death. About 2% of all irrigated land is affected by soil salinity, resulting in a decrease of crop yields (Kader, 21 March). Plants are affected by salt stress in two main ways: osmotic stress and ionic toxicity. These stresses affect all major plant processes, including photosynthesis, cellular metabolism, and plant nutrition. Salinity affects seed germination as well as normal plant processes in a growing plant (Malcolm et al., 23). Salt has two major effects on plants: osmotic stress and ionic toxicity, both of which affect all major plant processes (Yadav et al., 211). Plants are able to take up water and essential minerals because under normal conditions they have a higher osmotic pressure than the soil. When salt stress occurs, the osmotic pressure of the soil solution is greater than that in the plant cells. Thus, the plant cannot get enough water (Kader, 21 March). In addition, its cells will have decreased turgor and stomata will close to conserve water. Stomatal closing can lead to less carbon fixation and cause production of Reactive Oxygen Species (ROS) such as superoxide and singlet oxygen. ROS disrupts cell processes through damage to lipids, proteins, and nucleic acids (Parida and Das, 25). Ionic toxicity occurs when concentrations of salts are imbalanced inside cells and inhibit cellular processes, including metabolism. Sodium ions at the root surface disrupt plant nutrition by inhibiting the uptake of the similar cation, potassium, as well as enzymatic activities within the cell. Potassium is an important nutrient in a plant, regulating over 5 enzymes (Kader, 21 March). Essential for maintaining cell turgor pressure, creating membrane potential, and regulating enzymatic activities, potassium must be maintained at 1-2mM in the cytosol. Sodium, on the other hand, causes stress at concentrations higher than 1mM in the cytosol (Kader, 21 March). Na+ is a cation similar to K+ and easily crosses the cell membrane. It also acts as an inhibitor to many enzymes, affecting metabolic processes. Calcium cations, however, protect some plants through signaling pathways that regulate potassium sodium transporters (Parida and Das, 25). When a plant senses salt stress through transmembrane proteins or enzymes in the cytosol, the amount of calcium in the cytosol increases (Kader, 21 March). Calcium is a second messenger important to many biochemical pathways and can aid plants in responding to salt stress. The osmotic and ionic stress induced by salinity can halt plant growth as the plant focuses its energy on conserving water and improving ionic balance. In order for plants to return to normal functioning and photosynthesis, the plant must facilitate its own detoxification Newell - 1

2 damage must be prevented or lessened, homeostasis must be re-established, and growth must resume (Zhu, 21). Our research examines the ways in which salt inhibits plant function in regards to sodium chloride stress on pepper plant seed germination. In our studies, we look at seed germination using solutions of NaCl or mannitol, an osmoticum, to distinguish between osmotic and ionic effects. In addition, we researched the effect of extensive fertilization on whole pepper plants to observe their recovery potential. MATERIALS AND METHODS Using lab and greenhouse space, we conducted several experiments over the course of 16 weeks. We began with seed germination trials using Pepper Flamingo seeds (Harris seeds brand: lot #16142 and #11281). For the first experiment, we soaked 16 seeds in 5% Clorox/DI water solution for 1 minutes to sanitize the seeds. We then rinsed the seeds with DI water until they no longer smelled of Clorox. We prepared 16 petri dishes, rinsed them with DI water, set them out to dry, and used tweezers to place Fisher filter paper in each dish. Four dishes received 7mL of DI water each as controls. We prepared solutions with varying osmolarities of sodium chloride (measured with a Model 333 Advanced Micro-Osmometer): four dishes were treated with 7mL of 65mOsm NaCl, four with 7mL of 125mOsm NaCl, and four with 7mL of 22mOsm NaCl. We used tweezers to place 1 seeds in each dish and wrapped the closed petri dishes in brand M Parafilm to prevent water loss. We stored the dishes in a dark drawer at room temperature and collected germination data daily over the course of a week. We considered the seed to have germinated as soon as we could clearly see the white radicle exiting the seed. We repeated this procedure but instead used mannitol solutions of similar osmolarities. Using a very similar procedure but with only 15 seeds, we prepared just two solutions, one 437mOsm NaCl and another 416mOsm mannitol, to test the effects of higher osmolarities on seed germination. Again, we used a control of DI water with no salt or mannitol added. We did five repetitions of three trials for this experiment and took germination data daily for two weeks. For our whole plant studies, we seeded 3+ pepper seeds in a Schulz Moisture Plus potting mix and then transplanted 12 into separate pots after 3 weeks. The plants were watered daily with tap water and kept in a greenhouse with growth lights on a timer. At 7 weeks, we fertilized the peppers extensively with a 1½ qt. fertilizer solution (½ tsp. of Miracle Grow Tomato, N-P-K ratio of ) divided between 12 plants. We then watered them extensively over the next several weeks. RESULTS In our NaCl seed germination trials, we found that increased salt osmolarity caused a delay in pepper seed germination from the control (figure 1). The onset of germination began at day 4 for all the trials except in the 61mOsm solution in which 2.5% of seeds (1 seed) seemed to have germinated before day 4. At day 4, 92.5% of seeds germinated in the control, while only 57.5% germinated in the 61mOsm solution, 5% in the 124mOsm, and 27.5% in the 229mOsm. However by day 6, almost 1% of seeds germinated in each solution. A similar but more pronounced delay occurred in mannitol solutions with similar osmolarities (figure 2). At the onset of germination on day 4, 65% of seeds germinated in the control, while only 27.5% germinated in the 71mOsm solution, 7.5% in the 133mOsm, and % in the 24mOsm. By day 6 almost 1% of seeds germinated in each solution, though only 87.5% in the highest osmolarity mannitol solution. In another set of trials, we found that seed germination was also delayed in solutions of higher osmolarity (figure 3). In the mosm control solution, the onset of germination occurred on day 4, with 48% germination; on day 5, 98% had germinated, and on day 7, 1%. In the 437mOsm NaCl solution, the onset of germination began 3 days later than the control group on day 7, with 4% seeds germinated. On day 8, 9% had germinated, and on day nine, 98% germinated. In the 416mOsm mannitol solution, the onset of seed germination came a day sooner Newell - 2

3 than that in the NaCl solution, with 2% of seeds germinating on day 6, and a 1% by day 8. In the end, almost 1% of seeds germinated in all trials. For this experiment, we photographed seeds from each solution on day 14 (figure 4). In the control, the seed radicles grew a few centimeters and were fuzzy with root hairs. In addition, the seeds released the cotyledons. In the 437mOsm NaCl solution, the seed radicles were only a few millimeters long and had virtually no root hairs. Discoloration appeared on some radicle tips and the seeds themselves. A similar phenomenon occurred for the seeds in the 416mOsm mannitol solutions, but these seeds had longer radicles and slightly more prominent root hairs. For the whole plant studies, we found that 11 plants out of 12 remained alive four weeks after extensive fertilization (figure 5). Plants began to wilt after fertilization and leaves yellowed and fell off. Though most of the plants no longer looked wilted after four weeks, the leaves were still fragile. The leaves show signs of salt excretion in small ring spots. A few plants also had shriveled leaf tips. Only one plant was permanently wilted and died, drying out. Three plants had recovered enough to produce buds in addition to leaf and stem growth. We took height measurements and leaf counts on these plants at the same time as we took photos (figure 6). One plant recovered so much that it reached a height of 19cm and had 23 leaves while one plant dried out completely. The plant that recovered least was 1.6cm and had 7 leaves. The median height was 14.3cm, and the median number of leaves was 13. DISCUSSION We found that sodium chloride has both osmotic and ionic effects on plant growth in germinating seeds. Because water imbibition is essential for germination to occur, decreasing differences in osmotic potential between the seed and the solution cause a delay in germination (Kader, 21 March). Successive delays in seed germination occurred in NaCl solutions of increasing osmolarities. Mannitol studies confirmed that these delays are osmotic effects, because the seeds in mannitol solutions were delayed similarly in germination onset. This occurs because the difference in water potential between the seed and the solution decreased with increasing osmolarities in the solutions, inhibiting water uptake. Ionic effects of sodium chloride were most visible with the 437mOsm NaCl solution, in which seeds had very short radicles, discoloration, and very few root hairs. In contrast, the seeds in the mannitol solution had longer radicles and some fuzzy root hairs, although discoloration still occurred. This shows that sodium ions may have had an effect on radicle elongation and root hair formation two processes that require cellular machinery, communication, and metabolism processes that may be inhibited by sodium chloride. Sodium causes stress at concentrations higher than 1mM in the cytosol (Kader, 21 March). Glycophytes cope to a point by creating a high K+/Na+ ratio through active ion transport, shifting ionic and electrochemical gradients to be more favorable to cytosolic processes (Yadav et al., 211). The whole plants under salt stress from extensive fertilization exhibited clear osmotic and ionic effects. Wilted from water loss, they became yellowed, lost some lower leaves, and halted the growth process until the soil became less saline. In order to combat the negative effects of salt stress on glycophytes, soil salinity must decrease or glycophytes must slowly adapt through natural processes or anthropogenic breeding or genetic modification (Zhu, 21). We watered the plants intensively after fertilization to wash away the salts as much as possible. This caused a decrease in soil salinity over time that helped the plants recover. Under salt stress, salt accumulates in the reproductive organs and the leaves, and the plant focuses on mere survival rather than growth or reproduction (Zakharin and Panichkin, 29). As the plants recovered, salt spots on the leaves accumulated as the plant excreted the salt. This shows that the pepper plants, though severely affected by salt stress from over fertilization, were able to recover after salts were washed out of the soil and excreted through the leaves. Newell - 3

4 REFERENCES Aslam R, Bostan N, Nabgha-e-Amen, Maria M, Safdar W (211) A critical review on halophytes: Salt tolerant plants. J Med Plants Res 5: Kader MAL, S. (21 March) Cytosolic calcium and ph signaling in plants under salinity stress. Plant Signal Behav. 5(3): Kant S, Kant P, Raveh E, Barak S (26) Evidence that differential gene expression between the halophyte, Thellungiella halophila, and Arabidopsis thaliana is responsible for higher levels of the compatible osmolyte proline and tight control of Na+ uptake in T-halophila. Plant Cell Environ 29: Malcolm CV, Lindley VA, O'Leary JW, Runciman HV, Barrett-Lennard EG (23) Halophyte and glycophyte salt tolerance at germination and the establishment of halophyte shrubs in saline environments. Plant Soil 253: Parida AK, Das AB (25) Salt tolerance and salinity effects on plants: a review. Ecotox. Environ. Safe. 6: Radyukina NL, Kartashov AV, Ivanov YV, Shevyakova NI, Kuznetsov VV (27) Functioning of defense systems in halophytes and glycophytes under progressing salinity. Russ J Plant Physl+ 54: Yadav S, Irfan M, Ahmad A, Hayat S (211) Causes of salinity and plant manifestations to salt stress: A review. J Environ Biol 32: Zakharin AA, Panichkin LA (29) Glycophyte salt resistance. Russ J Plant Physl+ 56: Zhu JK (21) Plant salt tolerance. Trends Plant Sci. 6: Zhu JK (27) Plant salt stress. In Encylcopedia of Life Sciences. John Wiley & Sons, Ltd. FIGURES Percent Germinated (%) Day Osmolarity (mosm) Figure 1. Effects of NaCl on seed germination over time. Newell - 4

5 Percent Germination (%) Day Osmolarity (mosm) Figure 2. Effects of mannitol on seed germination over time. Percent Germination (%) Day Osmolarity (mosm) Figure 3. Effects of higher osmolarity NaCl or Mannitol solutions on seed germination. Newell - 5

6 Figure 4. Photos of seeds germinated in control, 437mOsm NaCl solution, and 416mOsm mannitol solution (from right to left) after 2 weeks. Figure 5. Photos of whole plants four weeks after extensive fertilization. Newell - 6

7 Table 1. Height and number of leaves of whole plants four weeks after extensive fertilization Height (cm) Number of Leaves Newell - 7