446 



u viN and r. Massisi: TIip Tnth of C.nrl.nn in Photosynthesis 



IEXPERIENTI»V0I..VI 11/12] 



suitable oxidizing agents leading to the evolution of 

 oxygen. Furthermore, the experiments of Ruben* 

 showed that the molecule of oxygen evolved in photo- 

 synthesis had its proximate origin in the oxygen of 

 the water molecule and that the oxygen atom associ- 

 ated with the carbon dioxide must first pass through 

 water before arrivingatgaseousoxygcn. From the chart 

 it may be seen that the ultimate result, then, of- the 

 photochemical reaction initiated by the absorption of 

 light by the chlorophyll molecule is the division of the 

 water molecule into an oxidized part which ultimately 

 leads to molecular oxygen and some reduced parts 

 represented in the chart by [H], 



This reduced part [H] we have called "reducing 

 power" because as yet it is not possible to state specifi- 

 cally what form or forms it may be in. This reducing 

 power is capable of reducing carbon dioxide in the 

 absence of light; that is to say, that the reduction of 

 carbon dioxide itself is a dark reaction. This was indi- 

 cated first in the earlier experiment of McAlister" in 

 which he was able to show that following a period of 

 photosynthesis a number of plants continued to absorb 

 carbon dioxide for a short period (seconds to minutes) 

 after cessation of illumination. We were able to demon- 

 strate this in an even more direct and uneciuivocal 

 fashion and generalize it for all plants so far tried when 

 we were able to show that not only did all of these 

 plants absorb (|uantities of carbon dioxide in the dark 

 after illumination but that the products formed in the 

 dark were (jualitatively and under certain conditions 

 quantitatively similar to those formed in a fairly com- 

 parable light period'. The method used for this demon- 

 stration was the same as those to be described later in 

 the review. The lifetime in the dark of this reducing 

 power which is generated by light is also of the order 

 of seconds to minutes and almost certainly corresponds 

 to a concentration of one or more definite chemical 

 species. It is quite conceivable, as mentioned earlier, 

 that some of it might be in the form of reduced coen- 

 zymes. 



Very recently it has been reported* that both the 

 higher plants and isolated chloroplasts emit a chemi- 

 luminiscence following cessation of illumination. This 

 chemiluminiscence has a decay time which corresponds 

 very closely to that which we have observed for the 

 reducing power. In fact, it would seem almost surely 

 to represent the reversal of the conversion of electro- 

 magnetic into chemical energy, namely, the transfor- 

 mation of at least some of the chemical energy stored 

 in the reducing power into the electromagnetic energy 

 of luminiscence. Furthermore, the luminiscence is re- 



' S. UruKN, M. Randaij., M. 11. K\men, .ind J. Hvor. J. A\\\. 

 Chcin. Soc. C3, 877 (1941). 



' K. U. McAr.isTER and J. MvF.RS, J. Smithsonian Insl. I'uM 

 (Misc. Coll.) c, aa (1940). 



' M. Calvin, J. Chcui. Uducation J6, 030 (lOl'J). 



* B. L. STREHi.tR and W. .\rnoi.u, J. Gen. Physiol. 34, sua 

 (lull). - H. I.. STRtHUF.R, .^rch. BiochoiTi. Hiophys. 34, M9 (19:.l) 



duced by the presence of carbon dioxide in those cases 

 in which the carbon dioxide fixing system is still pre- 

 sent. However, when the carbon dioxide system has 

 been removed, as is true in the case of chloroplasts, the 

 luminiscence becomes independent of carbon dioxide. 

 While it thus appears that the unique problem of 

 photosynthesis lies in the right hand half of thechart 

 of Figure 1, the present discussion will be limited to 

 the other side of the chart, that is, the path through 

 which carbon passes on its way from carbon dioxide to 

 all the raw materials of the plant. It is essentially a 

 study of what we now believe to be entirely dark 

 reactions and might best be characterized as phyto- 

 synthesis. This area not only has a great interest for its 

 own sake but would almost certainly cast some light 

 upon the nature of the reducing agents which arrive 

 from the photochemical part of the reaction and drive 

 the carbon cycle toward reduction. The reason for this 

 particular interest lies in the fact that we have, in recent 

 years, come into possession of a tool which is especially 

 suited for this study, namely, labeled carbon atoms in 

 the form of a radioactive isotope of carbon, O*. All of 

 the results that will be described later were made 

 possible through the use of this labeled carbon dioxide. 

 With such a labeled molecule available, the design of 

 an experiment for determining the sequence of com- 

 pounds into which the carbon atoms of carbon dioxide 

 may pass during the course of their incorporation in the 

 plant is, in its first phase, a straightforward one. 



./ 



CO2 



We may visualize the problem in terms of the chart 

 in Figure 2 in which the green leaf is represented 

 schematically as a closed opaque container into which 

 stream the raw materials of photosynthesis, namely, 

 carbon dioxide, light and water containing the neces- 

 sary mineral elements. From this container are evolved 

 the products of photosynthesis- oxygen gas and the 

 reduced carbon compounds constituting the plant and 

 its stored reserves. Heretofore, it has been possible to 

 study in a quantitative way the nature of the process 

 going on inside the opaque container only by varying 

 external conditions and noting variations in the final 

 products. Although there has been no serious doubt 

 that the formation of sugar did not take place by the 

 aggregation of six molecules of carbon dioxide, six 



80 



