1. Farnaz Pournia; Jarnail Mehroke; Santokh Singh Results and Discussion
Effects of Cadmium on Morphology, Photosynthesis and Protein Profile of Bean Plants
Farnaz Pournia; Jarnail Mehroke; Santokh Singh
Department of Botany, Biology Program, University of British Columbia, Vancouver, B.C, Canada
Abstract
Figures
Results and Discussion
Morphology
High concentrations of Cd, 1000 and 100 uM inhibit growth and development of bean plants and cause wilted morphology in 24 hours and 48 hours respectively
The effects of low Cd concentration (10 uM) are observed after 96 hr
Cd effects are both time- and concentration-dependent.
Photosynthesis and Transpiration Rates
Photosynthesis is partially (approximately 50%) reduced in higher Cd concentrations, 100 and 1000 uM, after 24 hr
Cd completely inhibited photosynthesis rate at and after 96 hr
Transpiration is reduced (approximately 75%) in 100 and 1000 uM Cd by 24 hr and in 10 uM Cd by 96 hr
Transpiration is completely inhibited by 1000 uM Cd after 96 hr
A more rapid decline in photosynthesis rate compared to transpiration, suggests that factors other than stomatal closure are affecting photosynthesis. These could be due to changes in structure and/or function of photosynthetic proteins
Protein Profile
A new protein (18 KDa) appears in bean leaves treated with 1000 uM Cd at and after 48 hr, in 100 uM at and after 96 hr and in 10 uM at 168 hr
Appearance of the 18 KDa protein, a likely Cd stress response protein, is both time- and concentration-dependent
Dehydrin protein which is a stress protein and protects plants against dehydration is strongly present in Cd treatments of1000 uM at 48 hr and of 10 uM at and after 96 hr
Rubisco and LHC-IIB protein levels are not affected by Cd; however, their activity might be
Cd affects thylakoid membrane permeability which may lead to a decline in proton gradient across the membrane and a reduction in photosynthesis rate
Cadmium (Cd), a cancer-causing heavy metal ion, concentration in the environment has recently shown a rapid increase mainly as a result of human activity. Since many plants are used as medicine and food, it is crucial to investigate effects of Cd on plant growth and development. This study examines the time course and concentration-dependent effects of Cd on morphology, photosynthesis and protein profiles in hydroponically-grown bean plants (Phaseolus vulgaris). High concentrations of Cd (1000 and 100 uM) induced leaf wilting and reduced both leaf growth and photosynthesis rate within 24 and 48 hours respectively. Lower concentration of 10 uM Cd showed the wilted morphology after 96 hours. In addition, the Cd-induced changes in protein profiles especially the photosynthesis and stress proteins will be discussed in relation to morphology, leaf growth and photosynthesis in bean plants.
6 hr
24 hr
48 hr
96 hr
168 hr
Figure 1 -Time Course Changes in Morphology of Control and Cd Treated bean plants
Introduction
Environmental pollution has increased dramatically over the past few decades mainly because of human activities (1). Presence of Cd causes different types of problems to living beings including a well-known cancer causation in human (2). Since plants as primary producers are consumed by animals and human and also because of the usage of plants in drug production, it is necessary to study the effects of Cd on plants (1). Previous studies have shown negative effects on morphology and reduced photosynthesis and transpiration rate in presence of Cd (2). This has been attributed to the interference of the element with molecular pathways such as photosystem II and electron transport chain in thylakoid membrane (2). Also, stomatal closure in Cd stress conditions results in lower transpiration rate and lower CO2 intake which in turn causes lower photosynthesis rate (2). Cd is also known to disturb the structure and activities of enzymes and alter the lipid composition and permeability of membrane (3). Finally accumulation of Cd in plant organs such as shoot and root may introduce inhibitors to regulate processes of plants such as nitrate assimilation pathway (3).
The objective of this study is to examine the effect of cadmium on morphology, photosynthesis, transpiration, and the protein profiles. .
Figure 2 - Time Course Changes in photosynthesis Rate (umol.m-2 s-1 ) of Control and Cd Treated bean plants
Figure 3 - Time Course Changes in Transpiration Rate ( umol.m-2 s-1 ) of Control and Cd treated bean plants
Standard Proteins
Standard Proteins
66 KDa
Large subunit of Rubisco and ATPase (54 KDa)
45 KDa
66 KDa
Large subunit of Rubisco & ATPase ( 54KDa)
36 KDa
Conclusions
45 KDa
29 KDa
36 KDa
LHC- IIB
(25 KDa)
LHC- IIB
(25 KDa)
29 KDa
24 KDa
24 KDa
1. Morphological and physiological effects of Cd are both time- and concentration-dependent.
2. Photosynthesis and transpiration rates are inhibited by Cd
3. A new protein (18 KDa) is detected in response to Cd stress.
20 KDa
Newly synthesised protein (18 KDa)
20 KDa
Newly synthesised protein (18 KDa)
14.2 KDa
14.2 KDa
24 hr
Control
24 hr
10 uM
24 hr
100 uM
24 hr
1000 uM
48 hr
Control
48 hr
10 uM
48 hr
100 uM
48 hr
1000 uM
96 hr
Control
96 hr
10 uM
96 hr
100 uM
96 hr
1000 uM
Materials and Methods
168 hr
Control
168 hr
10 uM
Figure 4 - Polyacrylamide gel showing the major protein bands in 24 and 48 hr control and Cd treated bean plants
Figure 5 - Polyacrylamide gel showing the major protein bands in 24 and 48 hr control and Cd treated bean plants
Green Beans (Phaseolus vulgaris) were grown in the greenhouse for 7 days. The seedlings were then transferred onto a hydroponic system which is a growing medium with known concentrations of nutrients. After 7 more days (16 hr. light / day), the plants were treated with Cd at concentrations of 10, 100 and 1000 uM.
Growth and development of plants were monitored for seven days. Rates of photosynthesis and transpiration for each treatment were measured at the end of 4, 6, 24, 48, 96 and 168 hours after treatment using LI-6200 Portable Photosynthetic System. By the end of each specified period, leaves from control and the three treatments were collected for protein [Ribulose-1,5-biphosphate carboxylase/ oxygenase (Rubisco), Light Harvesting Complex-IIB (LHC-IIB) and Dehydrin)] analysis by SDS-PAGE and Western blot techniques.
Future Studies
Identify structure and function of the 18 KDa protein
Study Cd effects at the membrane permeability level
Investigate Cd effects on gene expression
Control
10 uM
100 uM
1000 uM
Control
10 uM
0 hr
Control
10 uM
100 uM
1000 uM
Control
10uM
100 uM
1000 uM
Acknowledgements
4 hr
6 hr
96 hr
168 hr
Figure 8 - Western Blot Dehydrin (60 KDa) in 96 and 168 hr control and Cd treated bean plants
Figure 6 - Western Blot, Dehydrin (60 KDa) in 4 and 6 hr control and Cd treated bean plants
I would like to thank my Biology 352 lab partners Breanne Johnson, Samantha Iversen and Rosanne Pipunic for help with a preliminary experiment. I would also like to thank Shing Zhan for helping with some data collection. Finally, I would like to thank Skylight Development Fund, Faculty of Science and the Department of Botany for financial support.
100 uM
1000 uM
Control
10 uM
100 uM
1000 uM
Control
10 uM
100 uM
1000 uM
Control
10 uM
References
24 hr
48 hr
Figure 7 -Western Blot,Dehydrin (60 KDa) in 24 and 48 hr control and Cd treated bean plants
Figure 9 -Western Blot LHC-IIB (25 KDa) in 48 hr control and Cd treated bean plants
Rai V.,Khatoon S., Bisht S. S. and Mehrotra S Effects of Cadmium on growth, ultramorphology of leaf and secondary metabolism of Phyllanthus amarus and Thorn. Chemosphere. 61 :
Clijsters H., Van Assche F Inhibition of photosynthesis by heavy metals. Photosynthesis Research. 7: 31-40, 1985.
Sandalio, L.M., Dalurzo, H.C., Gomez, M., Romero-Puertas, M.C. and del Rio, L.A Journal of Experimental Botany 52: