Regulation By Environmental And Biotic Stresses

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3.2.1 Environmental Stresses

A host of environmental factors are involved in the regulation of metabolite biosynthesis in plants. The need for this control of synthesis stems from the fact that plants must be able to adjust the production of metabolites according to changing factors if they are to survive. Light is obviously a key factor in the ultimate production of many compounds because it supplies the energy needed to fix carbon. It is also more directly necessary for the biosynthesis of compounds such as chlorophylls, as mentioned in Chapter 2. Here, photons trigger the enzymatic conversion of protochlorophyllide and phytol to chlorophylls a and b, and thence, to chlorophyll-protein complexes in chloroplasts.1 Light also catalyzes the synthesis of anthocyanin pigment, via the plant pigment, phyto-chrome, in many tissues of many plants such as cotyledon (seed leaf) epidermal cells and hypocotyl (stem portion below the cotyledons) subepidermal cells in mustard seedlings.1 Light intensity plays an important role in the biosynthesis of medicinally important metabolites. An excellent case in point is the tree of joy (Camptotheca accuminata) (Figure 3.1), where levels of the anti-prostate cancer drug, camptothecin (an alkaloid metabolite), significantly increase as the amount of light reaching the tops of the plants decreases. University of Michigan Biology students, Atul Rustgi and Ashish Goyal, provide the following essay on their Bachelor's research project on the effects of different light intensities on camptothecin levels in tree of joy plantlets.

Research Project

Objective

The objective of our experiment was to test the effects of light intensity on the production of camptothecin (CPT) in the tree of joy plants. It has been shown in previous experiments that a decrease in light intensity will increase the production of CPT. Our objective was to determine the effects of different light intensities on the biosynthesis of CPT.

Materials and Methods

Three trays containing seedlings of Camptotheca accuminata were grown in a greenhouse. Each tray contained plants of the same age and height. Each tray of plants was exposed to a particular light intensity different from that of the other two trays. In each tray, the seedlings were arranged in two rows. The first tray received no shading and had a light intensity at the top of the plants of 3000

FIGURE 3.1 Author Peter Kaufman is shown standing in a plantation of tree of joy (Camptotheca accuminata) trees planted in southern Louisiana at the Citrus Experiment Station, located near Port Sulphur, LA, as part of a research project sponsored by the Agricultural Experiment Station of the Louisiana State University at Baton Rouge, LA and XyloMed Research, Inc. (Photo provided by Tracy Moore, President of XyloMed Research, Inc.).

FIGURE 3.1 Author Peter Kaufman is shown standing in a plantation of tree of joy (Camptotheca accuminata) trees planted in southern Louisiana at the Citrus Experiment Station, located near Port Sulphur, LA, as part of a research project sponsored by the Agricultural Experiment Station of the Louisiana State University at Baton Rouge, LA and XyloMed Research, Inc. (Photo provided by Tracy Moore, President of XyloMed Research, Inc.).

pEm-2s-1. The second received 1x shading by means of a thin wire screen that was held above the plants by four posts at each corner of the tray. The light intensity measured at the top of this set of plants was 750 pEm-2s-1. The third tray received 2x shading by means of two wire screens. The light intensity measured at the top of this set of plants was 300 to 400 pEm-2s-1 (see Figures 3.2 and 3.3).

At the time of setup, a random sampling of the largest top leaves of the plants was taken. This was done in order to get a measurement of the initial concentration (T0) of CPT in these seedlings before any experimental variables were introduced. The following procedure was used in order to determine the concentration of CPT in this sample (T0) and in successive samples.

1. Freeze leaves in liquid nitrogen

2. Crush to a powder using a mortar and pestle

3. Add 1 g of crushed leaves to a beaker containing 50 ml of methanol (MeOH)

4. Cover beaker for 24 h

5. Vacuum filter

6. Transfer liquid portion to a clean beaker

7. Air dry

8. Add 0.01 g of dried filtrate to 400 pl of refrigerated MeOH in order to avoid evaporation

9. Cover using Parafilm™

10. Use a sonicator to fully dissolve

11. Analyze 10 pl with a high-pressure liquid chromatograph (HPLC)3

For the HPLC, 10 pl injections were used with a C18 column. The wavelength used was 347 nm, the temperature was 40°C and the flow rate was 1 mLmin-1. The mobile phase used was run in acetonitrile (ACN). The 10 pl injection was run

FIGURE 3.2 Students Ashish Goyal, Kathryn Timberlake, and Atul Rustgi measuring light intensity with a photo flux density meter (Ly-Cor, Inc.) in their shading experiment with seedlings of tree of joy, Camptotheca accuminata. (Photo courtesy of David Bay.)
FIGURE 3.3 Illustration of shading experiment with tree of joy (Camptotheca accumi-nata) seedlings grown at three different light intensities in the greenhouse at the University of Michigan. (Photo courtesy of David Bay.)

from a gradient of 20 to 80% ACN over the course of 60 min. The printer was set to an attenuation of 9 in order to get the best chromatograph.

In order to find out which peak on the graph from the HPLC represented CPT, a sample of T0 was spiked with extra CPT. For this run, 0.015 g of the air dried

Back Pressure Liquid Chromatography

FIGURE 3.4 High pressure liquid chromatography trace illustrating camptothecin peak from nonspiked sample extract from tree of joy (Camptotheca accuminata) seedlings.

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FIGURE 3.4 High pressure liquid chromatography trace illustrating camptothecin peak from nonspiked sample extract from tree of joy (Camptotheca accuminata) seedlings.

FIGURE 3.5 High pressure liquid chromatography trace illustrating camptothecin peak from a camptothecin-spiked sample extract from tree of joy (Camptotheca accuminata) seedlings.

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FIGURE 3.5 High pressure liquid chromatography trace illustrating camptothecin peak from a camptothecin-spiked sample extract from tree of joy (Camptotheca accuminata) seedlings.

filtrate and 0.0004 g of CPT were added to 500 pl of MeOH. When the graph obtained for this run was compared to a run without the extra CPT (see Figure 3.4), one peak was noticeably larger (see Figure 3.5), thus indicating this peak to be the one representing CPT. For that peak, the given area under it represented the amount of CPT in a given injection.

At T1 (week one) six leaves were taken from each of the three trays. The six leaves were a collection of the largest top three leaves of two different plants in the same row of a particular tray. For the next 4 weeks, leaves were taken from the top of the plants of a new row so as to avoid getting young buds from a plant whose leaves had been removed the preceding week. The six leaves then used in the procedure described above in order to obtain data. The CPT peaks for the chromatographs of these successive trials could be identified by comparing these chromatographs to that of T0 and searching for similarities in the shape of and the time of elution of that CPT peak.

Results

With a standard curve, the amount of CPT in unknown samples can be determined. A sample chromatogram for 2.87E-03 M sample is shown in Figure 3.4. The standard curve results and a graphical representation are shown in Table 3.1 and Figure 3.6, respectively.

TABLE 3.1

Data on Areas under Curves for Respective Camptothecin Concentrations

Area under Concentration of camptothecin the curve (moles)

11,348 2.87E-06

183,077 2.87E-05

952,883 2.87E-04

6,196,572 2.87E-03

Note: Also used for the calculation of the standard curve for camptothecin in Figure 3.6.

The results for the three different amounts of shading are shown in Tables 3.2, 3.3, and 3.4.

A sample chromatograph of Run 1 (T1) is in Figure 3.5. A graphical representation of a comparison of all three runs is shown in Figure 3.7.

Conclusion

Concentration of Camfjtothccin (mo<os|

FIGURE 3.6 Standard curve for concentration of camptothecin plotted against areas under the high pressure liquid chromatography peaks.

FIGURE 3.6 Standard curve for concentration of camptothecin plotted against areas under the high pressure liquid chromatography peaks.

TABLE 3.2

Time-Course Changes in Camptothecin Levels in Tree of Joy Seedlings Grown Without Artificial Shading

No Shading (Run 1)

TABLE 3.2

Time-Course Changes in Camptothecin Levels in Tree of Joy Seedlings Grown Without Artificial Shading

No Shading (Run 1)

[ ] of

Time

Area under

Camptothecin

(days)

the curve

(moles)

0 (T0)

2,469,311

1.10E-03

7 (T1)

2,384,273

1.06E-03

14 (T2)

2,101,377

9.22E-04

21(T3)

2,311,930

1.02E-03

28 (T4)

3,031,237

1.36E-03

35 (T5)

4,062,633

1.85E-03

Note: Simulated full sunlight conditions

Note: Simulated full sunlight conditions

The data shows that Run 1, which had no shading, had a slow decrease in the amount of CPT production up to week 1. Thereafter there was a continuous slow rise in the production. The rise in production must be due to the effects of leaf growth. Run 2, which had 1x shading, showed an initial decrease in production of CPT, but then after week 1 showed a dramatic increase in production. The dramatic increase in production of CPT must be due to the shading effect. Run 3, which had 2x shading, showed continued increase in the production of CPT after the onset of the run, but started to decrease production after week 1 until week 3. This decrease must be due to poor leaf growth. After week 3, Run 3 showed an increase of CPT production, which was due to new leaf growth. Going into

TABLE 3.3

Time-Course Changes in Camptothecin Levels in Tree of Joy Seedlings Grown Under 1x (Partial) Shading Conditions

1X Shading (Run 2)

TABLE 3.3

Time-Course Changes in Camptothecin Levels in Tree of Joy Seedlings Grown Under 1x (Partial) Shading Conditions

1X Shading (Run 2)

[ ] of

Time

Area under

Camptothecin

(days)

the curve

(moles)

0 (T0)

2,469,311

1.10E-03

7 (T1)

1,887,101

8.21E-04

14 (T2)

2,530,378

1.12E-03

21 (T3)

5,339,954

2.45E-03

28 (T4)

5,370,632

2.47E-03

35 (T5)

7,592,100

Time-Course Changes in Camptothecin Levels of Tree of Joy Seedlings Grown Under 2x (Deep) Shading Conditions

2X Shading (Run 3)

TABLE 3.4

Time-Course Changes in Camptothecin Levels of Tree of Joy Seedlings Grown Under 2x (Deep) Shading Conditions

2X Shading (Run 3)

[ ] of

Time

Area under

Camptothecin

(days)

the curve

(moles)

0 (T0)

2,469,311

1.10E-03

7 (T1)

5,153,219

2.36E-03

14 (T2)

4,425,086

2.02E-03

21 (T3)

2,546,829

1.13E-03

28 (T4)

3,770,064

1.71E-03

35 (T5)

7,230,344

3.34E-03

week 5, both Runs 2 and 3 were producing the same amount of CPT, but Run 2 showed a greater potential because its new growth was due to the shading effect.

A result that seemed to be surprising, was the fact that Run 3 had an initial rise in the production of CPT, while the other two runs showed an initial decrease in production. The results show that no shading produces the least amount of CPT. In the short run, it seems as through 2x shading produces the most amount of CPT. In the long run, it also seems as though both 1x and 2x shading produce the same amount of CPT, but 2x shading has more potential to produce greater amounts of CPT in the future due to its high growth time.

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