Water Strss on Plants

Table 1 – Experiment 1 Results

Table 2 – Experiment 2 Results

Interpretation:

Due to the continuous variation of results within each population, the mean was taken from each in both experiments so as to provide a summary value for the length of plant growth and number of new roots developed in each concentration of mannitol solution, respectively. The mean was the most appropriate method of averaging as there were a roughly equal number of values on either side of the mean itself, i.e. the data were not particularly skewed in either direction, and the few extreme values that were present in the results had corresponding extreme values on the other side of the mean – symmetrical distribution. This is likely linked to genetic variation within populations, despite efforts to ensure the populations were as homozygotic as possible.

In experiment 1, where the effect of water stress on plant stem height was investigated, and experiment 2, in which the effects water stress on the number of new secondary roots developed, plant development was arrested by water stress simulated by mannitol concentration within their water supply – a negative correlation, higher mannitol concentrations led to reduced plant growth. It is interesting to note that the control populations – those with no mannitol present in the water growth medium – gave averages that followed the general trend of negative correlation but developed significantly further than the populations treated even with the lowest concentration of mannitol (0.05mol dmˉ³). It seems that even slight water stress causes significant differences in Pisum sativum development, but increasing the water stress beyond low levels has a directly proportional relationship to the species development. This is especially obvious in stem height, but not so clear in the number of new roots developed.

This can be explained in the functioning of plant metabolism. The organisms develop best when water is not limiting the metabolic rate of the various life processes, i.e. the conditions are optimized for cell function. With increased mannitol concentration means a higher osmotic gradient over the root cell membranes, less water is taken up and essential processes, especially photosynthesis, become less efficient and growth is stunted. In photosynthesis, water is broken down by photolysis to provide oxygen molecules for direct respiration, and hydrogen ions which reduce nicotinamide adenine dinucleotide phosphate. Reduced NADP loses these hydrogen ions to produce glyceraldehyde 3-phosphate, some of which is used to form carbohydrates within which the hydrogen is fixed as an energy store but also to form other organic compounds, e.g. amino acids. Without this process continuing efficiently it is obvious that growth will not continue at an optimal rate.

There seems to be an anomalous result in experiment 2, in which the mean number of roots grown by the population treated with 0.2mol dmˉ³ aqueous mannitol does not quite fit in with the negative trend of the rest of the data pairs. This result was likely the result of mere chance possibly resulting from biological variance; all other variables were kept the same for this population as for the others and the anomaly does not occur far from the line of best fit, even if the large offset from the large divergence of the control population is taken into account.

 The means have been used in the Spearman Rank test to reject the null hypothesis and confirm that the results acquired were unlikely to have been the product of chance alone.

Table 3 – ranking table for experiment 1 data

rs = Spearman’s rank correlation coefficient

∑ = sum of

D = Difference

n = number of pairs of data

 

         

                6(0)

rs = 1- ––––––––––––

              9(81– 1)

rs =  1.000             

At a 5% significance level (p<0.05, or a 95% chance the null hypothesis is false), for a data set consisting of nine pairs of data the critical value is 0.683. Given that the spearman rank coefficient for this experiment exceeds this value, the null hypothesis is rejected and that there is a one in twenty chance that the two variables are not correlated.

Table 3 – ranking table for experiment 2 data

         

              6(∑D²)

rs = 1- ––––––––––––

            n(n ²– 1)

                6(2)

rs = 1- ––––––––––––

              9(81– 1)

rs =  0.8333             

As with experiment one, the spearman rank coefficient exceeds the critical value for this level of significance and the number of data sets used. The null hypothesis is rejected, and mannitol concentration/water stress is shown to be correlated with the number of roots developed to a certainty of 95%.

Conclusions:

Experiment 1 has shown stem heights to be limited by mannitol being dissolved in the water source. Across the population the average stem height was reduced, with overlap being common between populations but nonetheless with statistically significant results – an unusually strong correlation emerged between variables. Experiment 2  showed that that this particular plant species does not exhibit a positive feedback response to compensate for water shortage; it rather becomes less efficient at producing new roots as less water diffuses into the roots and less photosynthesis can occur within the plant’s chloroplasts. With increased concentrations fewer roots on average develop, and as with the experiment investigating stem heights, it appears even slight water stress has a larger effect on the dependent variable than when the concentration is increased in later increments. While less statistically significant than experiment 1 it was nonetheless a strong correlation. Water stresses clearly affect plant development significantly, even if these stresses are only slight.

The aim of this investigation has been accomplished, though the results are limited in that they only investigate a single plant species, and the investigation has not shown this pattern of plant development to be present within other species undergoing similar stresses. Also, the investigation does not take into account any direct negative effects of mannitol itself on P. sativum, though it is highly unlikely that any exist but this investigation does not provide any evidence to comment on this – prior research was used in suggesting no toxicity. The investigation also does not make use of direct calculations of osmotic pressure and water potential to see how their specific variance affects plant development; the results only detailed mannitol concentrations in aqueous solution.

In the stem height investigation no instrument more accurate than a ruler would have been needed as the differences in height were obvious without reading beyond centimetre units. Being subject to human error, perhaps a better dependent variable to measure would be dried mass, which could be measured with electronic scales. In the roots investigation the dependent variable required counting of individual roots and in this investigation human error is likely. A possible alternative to root counting would be to measure the dried mass of the roots after a set period of time, primary and secondary, much like the first experiment.  It is also unclear in experiment 2 whether the relationship between variables is linear or whether a different type of relationship exists. To provide more information an investigation could be carried out using even smaller concentrations of mannitol, and ‘fill the gap’ if possible, between the control population and those treated with mannitol sugar.

Appendix I - Plan:

Safety:

• Mannitol must not be ingested/breathed/allowed into eyes.
• Hands must be washed after handling mannitol and compost
• Breakages and spillages must be cleaned up immediately
• Water must not be allowed near the electric scales

Apparatus:

• Mannitol
• Distilled Water
• Pea seeds × 298
• 100ml glass beakers ×18
• Weighing Scales (sensitive to two decimal places minimum)
• Soil compost
• Plastic plant trays × 9
• Measuring cylinder
• 250ml glass beakers ×8
• Ruler

Ethics

Plants lack sentience and cannot suffer as such, nor are the species used endangered. There are no ethical issues relating to this experiment.

Variables

Only one variable will be purposefully modified in the two experiments, the concentration of mannitol per decimetre of water. Other possible variables such as the amount of sunlight, volume of solution in each beaker, species of pea - Pisum sativum,  and the source of the seeds, will all be kept the same so as to ensure fair testing and eliminating as many possible variables from affecting the test unfairly. For the first experiment, I shall measure the height of each plantlet’s stem from the highest tip (not including leaves) down to the seed itself, from where the plumule emerges from the seed coat. In the second experiment I shall measure the number of first-order lateral roots grown from the seminal root of the pea seeds, measuring other variables would be possible but increasingly complicated and difficult to measure with accuracy and fairness.

Statistics

Large amounts of data will be generated from this experiment, as is appropriate given that in both of the experiments a wide array of values is likely to result given the experiment is closely based on living organisms and minor genetic variation may have an effect. Also, in both experiments the seeds shall be germinated and grown in soil making uniform distribution of the water/mannitol solutions difficult. These results shall be averaged within each population so that all results will be taken account of, including those that are exceptionally high or low for the data-sets. However, it is unlikely that these results will stretch across too wide a range to make the arithmetic mean misleading or biased, and a large number of replicates (20 per solution concentration for experiment 1, 12 for each concentration in experiment 2) will be made for each concentration of mannitol. These averages will then be used in the Spearman Rank correlation test to correlate the variables for the two separate experiments, each with their own table and subsequent graph (which will display the relationship between mannitol concentration and the number/height of the plant parts that will be measured).

Method(s) – Experiment 1

Nine plastic plant trays must be filled with compost and 20 seeds placed in rows (at least 1.5-2cm apart from each other to allow growing space). Soil is used as it gives the plantlets a solid growth medium in which to develop and increase in height. These must then be watered with specific concentrations of aqueous mannitol solutions, in the order of: pure distilled water, 0.05M, 0.10M, 0.15M, 0.20M, 0.25M, 0.30M, 0.35M and 0.40M. These concentrations were mixed in 250ml beakers, with the following masses of solute and volumes of solution:

 

Mr of mannitol = 182.172

  0.05mol/dm³ = 182.172 × 5 = 9.11g        

                               100

                         = 54.65 = 2.28grams per 250ml water

                               4

   0.10mol/dm³ = 182.172 × 10 = 18.22g        

                               100

                         = 54.65 = 4.55grams per 250ml water

                               4

   0.15mol/dm³ = 182.172 × 15 = 27.33g        

                               100

                         = 109.30 = 6.83grams per 250ml water

                                4

   0.20mol/dm³ = 182.172 × 20 = 36.43g        

                               100

                         = 54.65 = 9.10grams per 250ml water

                               4

   0.25mol/dm³ = 182.172 × 25 = 45.54g        

                               100

                         = 54.65 = 11.39grams per 250ml water

                               4

   0.30mol/dm³ = 182.172 × 30 = 54.65g        

                               100

                         = 54.65 = 13.66grams per 250ml water

                               4

   0.35mol/dm³ = 182.172 × 35 = 63.76g        

                               100

                         = 54.65 = 15.94grams per 250ml water

                               4

   0.40mol/dm³ = 182.172 × 40 = 72.87g        

                               100

                         = 54.65 = 18.22grams per 250ml water

                               4

150ml aqueous mannitol solution must be measured out for each solution and the soil must be properly moistened throughout. Recycle any of the water that emerges through the drainage holes back into the soil until it is all wet, but do not allow it to become waterlogged – allow drainage if too wet. Label each tray. After the seeds are placed in rows, place them in a bright place but not in direct sunlight (unless all plantlets will have equal access), preferably on the same shelf/windowsill or other such place. Water at regular intervals, every 2 days approximately, as long as they all receive the same amount of solution and receive it on the same day – 100ml of solution should be sufficient. After 2-2½weeks measure the stems height from highest tip to the seed using a ruler. A more detailed measuring instrument will not be necessary nor practical should the differences in height be visible in millimetres.

Method(s) – Experiment 2

108 seeds will be grown (preferably more just to ensure enough actually germinate and develop – spares can be discarded) in plant trays just as in experiment 1, except all will be watered with distilled water for approximately 2 weeks, after which time they will be uprooted and have their primary root cut to 4 centimetres and all lateral roots removed. This shall involve cutting off the apical meristem, and all secondary roots. The pea plants will then be stored in beakers of distilled water for another two days to give the roots time to repair the damaged roots but not grow new roots, allowing for mannitol-filtering but giving all of the roots an equal standing in water absorption.

Without doing this, the primary roots would he of varying lengths, and the number of the secondary roots and the total surface areas for each plant’s root system would be vastly different. By doing this they are brought as close as possible to the same for each, even though the apical meristem has been removed and the seminal root that emerged as a radicle from the seed can no longer grow. This aspect of seed growth and development could not be investigated in this experiment anyway, as many plants lost the apical meristem when being removed from the soil, along with secondary roots as well. After the 2 days, the seeds must be placed in 100ml beakers which will be filled with 100ml of differing mannitol solution, two beakers per solution, and 6 plantlets in each beaker. If water evaporates, it must be replaced with distilled water, mannitol replacement will be unnecessary. The same concentrations with masses/volumes of mannitol and water shall be used in this experiment as was used in Experiment 1. After a further 2 weeks, the plantlets should be removed, their secondary roots counted. This is best done in a clean beaker filled with water, as the new roots will spread out in the water being more easily counted. Only roots in excess of 2 millimetres will be counted, one plant at a time in the beaker. There is a potentially significant margin of error at this stage as no actual instruments are being used to count

Appendix II - Trial Plan:

Before beginning the actual experimentation a trial will be conducted to ensure the experiment produces useful results or at least provides ways for the proper experiment to be modified for greater efficiency or more accurate results.

Safety:

• Mannitol must not be ingested/breathed/allowed into eyes.
• Hands must be washed after handling mannitol and coleus cuttings
• Breakages and spillages must be cleaned up immediately
• Water must not be allowed near the electric scales

Apparatus:

• Mannitol
• Distilled Water
• Coleus cuttings × 9
• 100ml glass beakers ×3
• Weighing Scales (sensitive to two decimal places minimum)

Variables

Only one variable will be purposefully modified, the concentration of mannitol per decimetre of water. Other possible variables such as the amount of sunlight, volume of solution in each beaker, species of coleus cutting (all cuttings will come from the same plant organism) will all be kept as close to identical as possible. There is a variable for which compensation is only possible slightly – the size of each cutting, while each cutting is composed of two leaves of approximately 4-5 centimetres each with lower leaves removed so as to allow root development due to the presence of auxins, there is some variation in the sizes of each cutting in terms of leaf surface area and stem length and thickness. However this is unlikely to significantly affect the results, the basic effects triggered by water stress should be apparent in all of the tested plants assuming such effects occur.

Method

Firstly the cuttings shall be prepared, severed from the stems of a larger coleus blumei with at least two medium sized leaves at the tip and roughly 5 centimetres stem below – lower leaves must be present so that they may be picked and roots may form in the aqueous solution. Once prepared, these must be placed in three separate solutions held in 100ml glass beaker. Three cuttings for each beaker, one will have pure distilled water, one will have a concentration of 0.3mol mannitol per dm³ water, and the last will have 0.6mol per dm³ water.  The mannitol should be weighed with electric scales and mixed with 100ml water in the following amounts to give these concentrations, mixing a full decimetre of each solution would be wasteful and impractical.

Mr of mannitol = 182.172

     0.3mol/dm³ = 182.172 × 30 = 54.65g        

                               100

                         = 54.65 = 5.47grams per 100ml water

                              10

     0.6mol/dm³ = 182.172 × 30 = 109.30g        

                               100

                         = 109.30 = 10.93grams per 100ml water

                               10

100cm³ of each solution should be mixed and placed in a single beaker each, and a third beaker should have 100cm³ distilled water. The cutting should be placed inside, the leaves should not be submerged but the point where the lower leaves were cut should be submerged. These should now be left for 7 days, if any of the water evaporates below the level of the stem additional distilled water should added so that the beaker reads 100ml. All three beakers should be stored in a bright place but not in direct sunlight – preferably in the same place so as to eliminate bias.

Coleus cuttings root relatively quickly, part of the reason they were chosen for this experiment. Being a trial experiment, statistics will not be needed hence the small population and small number of different concentrations tested. But this trial will inform as to whether or not mannitol does have an actual effect in simulating water stress, whether cuttings are suitable for the actual experiment as opposed to plantlets with roots already established or seeds being grown with the solutions present. The trial will also give a good idea as to the mass of mannitol needed to create significant water stress, e.g. using concentrations as high as 0.6mol/dm³ may be unnecessary.

Trial Review:

No measurable results were collected from the trial owing to the fact that Coleus cuttings placed in mannitol solutions showed symptoms of water stress but these results could not be quantified. After 9 days the cuttings placed in water were healthy and growing new fibrous roots. On account of the manner in which Coleus root systems develop it would have been of far more value scientifically to weigh their dried root mass as opposed to counting the roots individually, but given that the other cuttings grew no roots at all and withered, these results would be meaningless anyway. From the trial it is clear that lower concentrations of mannitol create less water stress (the cuttings in the 0.3mol solution were far less withered than the cuttings in the 0.6mol solution). The range of concentrations tested will vary from 0.05M to 0.40M mannitol per litre of water, 0.3M showed significant enough water stress and going too far beyond this would simply be unnecessary as enough statistical data will be available with a larger population. From this, I have decided to use Pisum sativum, or pea plants, in the actual experiment due to the fact that Coleus seeds are unavailable and it has a far more easily observed root structure (taproot as opposed to fibrous, the equipment available is not sensitive enough to measure the dried root mass of Coleus roots so actual counting will have to suffice instead).

Also, two experiments will be conducted, one to test growth and development from germination onwards when placed under water stress (this test shall be used to measure stem growth as a general indicator of plant success), and one to test for  developmental changes in plants with a root system already established, as part of the failure of the trial seems to have come from the fact that the cuttings did not already have roots to serve as filters for the mannitol, mannitol was taken up the coleus stems into the xylem and other tissues where the whole plant became unable to function normally as mannitol prevented water from diffusing across  the cell membranes – osmosis was not possible.

Bibliography

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