Lab Report

Revisiting Blackman’s limiting conditions for photosynthesis: influence of temperature on photosynthetic rate of leaf disksRevisiting Blackman’s limiting conditions for photosynthesis: influence of temperature on photosynthetic rate of leaf disks

 

Abstract
Among other factors, ambient temperature has been reported to significantly influence the apparent photosynthetic rate of plants.  In this paper, we demonstrate the influence of ambient temperature on apparent photosynthetic rate using a leaf disk model system. Our results indicate that apparent photosynthetic rate increases with temperature. We discuss the possible reasons for the observed pattern and discuss the implications that the relation may have for plant growth and productivity.

 

Introduction
Among  several  environmental  variables,  temperature  has  been  known  to  strongly influence photosynthetic rate of plants (Blackman, 1905). Classically the effects of temperature on photosynthetic rates follow a parabolic relationship, with rates being low at extremely low and high temperatures and high at moderate temperatures (Rabinowitch and Govindjee, 1969). Consequent to this relationship, plant growth and productivity is often  limited  by  extremes  of  temperature,  with  either  too  low  or  high  temperatures affecting the growth and productivity of plants (Rabinowitch and Govindjee, 1969; Govindjee, 1975). Obviously  it appears that extremes of temperature could adversely affect the apparent photosynthetic rate of leaves. Further, while tissue respiration rates may remain unaltered under extreme temperatures, decrease in the photosynthetic rates

 

can lead to a negative carbon balance in the plant (Hipkins, 1987). Under such conditions, the growth and productivity and consequently the adaptability of plants would be jeopardized. These arguments pre-suppose that plant species in nature have an optimum temperature at which their photosynthetic  rates are maximized. Extreme temperatures either too low or too high depress the photosynthetic rates. In recent years, there has been renewed interest in evaluating plant responses to elevated temperature owing to the green house effect.

 

In this paper we explicitly examine the hypothesis that within a limited range of temperatures, the photosynthetic rates of leaves would scale positively with temperature. We discuss the results in the light of existing knowledge on the role of temperature in influencing apparent photosynthetic rates in species and how such relation could have implications for plant growth and productivity.

 

Materials and Methods
Rationale of the experiment
The experiments were performed on spinach leaf disks.   Leaf disks contain spongy mesophyll layer of cells, which largely comprise of large air spaces. By depleting the air spaces under vacuum, the leaf disks sink in water. However, under sufficiently lighted conditions when the leaf disks photosynthesize, the air spaces are refilled with oxygen (a product of photosynthetic oxygen evolution) and tend to float again. The rate at which the leaf disks begin to float can be used as a surrogate measure of the photosynthetic rate of the leaf disks. Thus leaf disks that fail to float are those in which photosynthetic oxygen

 

evolution has not occurred (and hence in which photosynthesis is absent). On the other hand and keeping everything else constant, it can be inferred that leaf disks that float slowly are those were the photosynthetic rates are relatively slow compared to disks that float rapidly.

 

Preparation of leaf disks for incubation

 

Leaf disks were made using a hole puncher. About 60 leaf disks were prepared from 2 or
3 fresh leaves of spinach at each effort.   With the help of a surgical syringe, air was removed from the spongy tissue of the leaf disks by creating vacum and replaced with sodium bicarbonate solution (0.2 percent w/v). After this process, most of the disks sink to the bottom of the syringe, indicating that in these disks the air spaces were successfully evacuated and replaced with bicarbonate solution. Disks that remained afloat were discarded. The sunken disks were transferred to fresh sodium bicarbonate solution and maintained in dark.

 

Treatment details
The  study  comprised  of  three  temperature  treatments,  namely,  one  below  room temperature (7OC), one above room temperature (36O C) and yet another at room temperature  (28O   C).  Temperature  regimes  (28O   C  and  36O   C)  were  provided  by maintaining the Petri dishes in incubators with the respective temperatures pre-set; temperature  of  7OC  was  maintained  by  incubating  the  Petri  dishes  in  temperature controlled  refrigerated  incubators.    All  treatments  were  conducted  under  light  from

 

incandescent bulbs. For each treatment, four trials were conducted.  Each trial consisted of one Petri dish containing 20 leaf disks. Using a forceps, skillfully and rapidly, twenty disks were transferred from the sunken disks maintained in sodium bicarbonate solution in dark.   Care was taken  to  randomise  the allocation  of leaf  disks  into each  of the treatment blocks in order to avoid all systematic biases in the experiment. After equilibration for 10 sec, the lamps were turned on and the experiment begun.

 

Observation and data analysis
For each trial in each of the various treatments, the number of leaf disks floating at every two minute interval, for 10 minutes was recorded.  From this basic data set, we computed the following additional parameters: a) the average time to float, taken as the median value between two successive time intervals of observation, b) the total number of leaf disks floating at each interval of observation, c) the number of new leaf disks that floated in every successive interval of observation.

 

From the above observation and computations, we further computed the average time taken for leaf disks to float in each trial. This was calculated as the sum of the product of the new disks floating at each interval of time and the average time it took to float, normalized with the total number of disks found floating. We then computed the average time taken for the leaf disks to float, considering the total number of disks that finally was found floating.

 

The average time taken for leaf disk to float in each trial was used as an estimate of time it took to photosynthesize 2µl of oxygen (the average amount of oxygen gas in each floating disk). Accordingly, we calculated the amount of photosynthetic oxygen produced for every minute and expressed it as µl oxygen/min. This was expressed as the average photosynthetic rate of the system.  The mean of the average photosynthetic rate obtained for the four trials for each of the treatment provided an estimate of the overall photosynthetic  rate  of  the  system.  Finally  we  calculated  the  standard  deviation  and standard error of mean for each of the estimate.

 

Results
Based on the experiments performed, we present key results pertaining to the effect of temperature  on the photosynthetic  rate of leaf disks. Table 1 provides results on the average photosynthetic rate, percent floaters and the overall average photosynthetic rate of leaf disks exposed  to different  temperature  regimes.   At low temperature  (7O   C), everything else remaining constant, none of the leaf disks floated indicating that none of the leaf disks photosynthesized.Table 1: Average photosynthetic rate, percent floaters and overall average photosynthetic rate of leaf disks at different temperatures

 

Treatment       Average           Percent   Overall avgTemperature©                     photsynrate      floaters   photsynrate
degree C ul oxygen/min ul oxygen/min 7                      0             0                     0
28               0.164         100             0.1640.168         100             0.168

 

0.165 100 0.165 0.187 100 0.187Mean 0.171 0.171SD 0.010801234 0.0108 36 0.234 100 0.234 0.253 100 0.253 0.177 100 0.177 0.212 100 0.212Mean 0.219 0.219SD 0.03262923 0.0326

On the other hand, with increase in temperature, both at 28O  C and   36O  C, all disks floated (Table 1). The average time to float decreased from 11.87±0.85 min at 28O  C to9.28±1.458 min at 36O C (Fig 1).

Fig 1: Average time to float
141210864207                        28                       36
Temperature ( C)

 

 

At elevated temperature of 36O C, there was a greater rate of oxygen evolution compared to that at 28O C (Fig 2). The average overall photosynthetic rate (ul oxygen/min) at 36O C was 0.219 ±0.0326 compared to 0.171±0.0108 at  28O C .

 

 

 

 

0.3
0.25
0.2
0.15
0.1
0.05
0 Fig 2: Overall average photosynthetic rate in spinach leaf disks

 

 

 

 

 

 

7                   28                  36
T emperature ( C)

 

 

Discussion
About a hundred years ago, Blackman from the University of Cambridge, UK, demonstrated  that the rate of photosynthesis  in plants is strongly influenced by three factors, namely, carbon dioxide concentration, light intensity and temperature (Blackman,1905).    A  hundred  years  since,  and  after  innumerable  number  of  studies,  the  basic findings of Blackman  has been so well recognized  that the three variables  are often referred  to as the “limiting  factors”  for photosynthesis  in plants.  Crop physiologists, agronomists, ecologists, crop modelers and a score of others interested in plants have endlessly toyed with these variables in their pursuits to understand plant responses to extreme climatic conditions to, in their efforts to design and breed plants with higher productivity.  Obviously  the implications  of understanding  the role of these  “limiting factors” have important implications for crop improvement and in crop modeling.

 

 

Results obtained in this study using a simple leaf disk model system, clearly validate the hypothesis that photosynthetic rates are strongly influenced by ambient temperature conditions. The observed results are best explained by the sensitivity of photochemical and biochemical events involved in photosynthesis to temperature (Rabinowitch and Govindjee,   1969;   Govindjee,   1975).   Especially,   the   effect   of   temperature   on photosynthesis  will  depend  upon  how  stable  and  active  the  key  enzyme,  RUBP carboxylase is with changes in ambient temperature (Ellis, 1979).

 

At low temperatures (such as at 7O C), the rate of carboxylating reaction is slow or even absent, because of extremely low rates of molecular collisions of the enzyme with the substrate. Besides the lowered chemical kinetics, low temperatures are also known to change the chloroplast membrane fluidity leading to a poor photochemical function. On the other hand, at higher temperatures (such as at 28O  C and  36O  C), both because of a higher rate of molecular collision frequency (of enzyme with substrate) as well as due to greater chloroplast membrane and enzyme stability, the photosynthetic yields are higher (Rabinowitch and Govindjee, 1969; Govindjee, 1975).

 

In summary, our studies using the flotation technique, confirm the effect of temperature on photosynthetic rate, an observation first made over a hundred years ago by Blackman. The implications of these results are manifold, especially considering the consequences on plant growth and productivity. For example, the simple demonstration here indicates why on an average, the tropical forests are more productive than are temperate forests. In

 

other words, temperature as a limiting factor might therefore play an important role in species distribution and abundance. The results also imply that keeping everything else constant, increase in temperature such as due to global warming, can actually lead to a higher biomass productivity of a species.

 

References
Blackman, F.F. 1905. Optima and limiting factors. Ann. Bot. 19: 281-295.”
Ellis, R.J. 1979. The most abundant protein in the world. Trends Biochem. Sci.,
4:241-244.”
Govindjee, 1975. Bioenergetics of Photosynthesis. New York. Academic Press” Hipkins, M.F. Photosynthesis. 1987. In : Advanced Plant Physiology (Ed. M.B.Wilkins). England, ELBS/Longman”
Rabinowitch, E. and Govindjee. 1969. Photosynthesis. New York: John Wiley & Sons, Inc.”

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