CHAPTER VII.

STARCH AND SUGAR FORMATION.

1. The Gases Concerned.

=121. Gas given off by green plants in the sunlight.=—Let us take some green alga, like spirogyra, which is in a fresh condition, and place one lot in a beaker or tall glass vessel of water and set this in the direct sunlight or in a well lighted place. At the same time cover a similar vessel of spirogyra with black cloth so that it will be in the dark, or at least in very weak light.

=122.= In a short time we note that in the first vessel small bubbles of gas are accumulating on the surface of the threads of the spirogyra, and now and then some free themselves and rise to the surface of the water. Where there is quite a tangle of the threads the gas is apt to become caught and held back in larger bubbles, which on agitation of the vessel are freed.

If we now examine the second vessel we see that there are no bubbles, or only a very few of them. We are led to believe then that sunlight has had something to do with the setting free of this gas from the plant.

=123.= We may now take another alga-like vaucheria and perform the experiment in the same way, or to save time the two may be set up at once. In fact if we take any of the green algæ and treat them as described above gas will be given off in a similar manner.

=124.= We may now take one of the higher green plants, an aquatic plant like elodea, callitriche, etc. Place the plant in the water with the cut end of the stem uppermost, but still immersed, the plant being weighted down by a glass rod or other suitable object. If we place the vessel of water containing these leafy stems in the bright sunlight, in a short time bubbles of gas will pass off quite rapidly from the cut end of the stem. If in the same vessel we place another stem, from which the leaves have been cut, the number of bubbles of gas given off will be very few. This indicates that a large part of the gas is furnished by the leaves.

=125.= Another vessel fitted up in the same way should be placed in the dark or shaded by covering with a box or black cloth. It will be seen here, as in the case of spirogyra, that very few or no bubbles of gas will be set free. Sunlight here also is necessary for the rapid escape of the gas.

=126.= We may easily compare the rapidity with which light of varying intensity effects the setting free of this gas. After cutting the end of the stem let us plunge the cut surface several times in melted paraffine, or spread over the cut surface a coat of varnish. Then prick with a needle a small hole through the paraffine or varnish. Immerse the plant in water and place in sunlight as before. The gas now comes from the puncture through the coating of the cut end, and the number of bubbles given off during a given period can be ascertained by counting. If we duplicate this experiment by placing one plant in weak light or diffused sunlight, and another in the shade, we can easily compare the rapidity of the escape of the gas under the different conditions, which represent varying intensities of light. We see then that not only is sunlight necessary for the setting free of this gas, but that in diffused light or in the shade the activity of the plant in this respect is less than in direct sunlight.

=127. What this gas is.=—If we take quite a quantity of the plants of elodea and place them under an inverted funnel which is immersed in water, the gas will be given off in quite large quantities and will rise into the narrow exit of the funnel. The funnel should be one with a short tube, or the vessel one which is quite deep so that a small test tube which is filled with water may in this condition be inverted over the opening of the funnel tube. With this arrangement of the experiment the gas will rise in the inverted test tube, slowly displace a portion of the water, and become collected in a sufficient quantity to afford us a test. When a considerable quantity has accumulated in the test tube, we may close the end of the tube in the water with the thumb, lift it from the water and invert. The gas will rise against the thumb. A dry soft-pine splinter should be then lighted, and after it has burned a short time, extinguish the flame by blowing upon it, when the still burning end of the splinter should be brought to the mouth of the tube as the thumb is quickly moved to one side. The glowing of the splinter shows that the gas is _oxygen_.

=128.= It is better to allow the apparatus to stand several days in the sunlight in order to catch a full tube of the gas. Or on a sunny day carbon dioxide gas can be led into the water in the jar from a generator, such an one as is used for the evolution of CO₂. The CO₂ can be produced by the action of hydrochloric acid on bits of marble. The CO₂ should not be run below the funnel. The test tube should be fastened so that the light oxygen gas will not raise it off the funnel. With the tube full of gas the test for oxygen can be made by lifting the tube with one hand and quickly thrusting the glowing end of the splinter in with the other hand. If properly handled, the splinter will flame again. If it is necessary to keep the apparatus standing for more than one day it is well to add fresh water in the place of most of the water in the jar. Do not use leaves of land plants in this experiment, since the bubbles which rise when these leaves are placed in water are not evidence that this process is taking place.

=129. Oxygen given off by green land plants also.=—If we should extend our experiments to land plants we should find that oxygen is given off by them under these conditions of light. Land plants, however, will not do this when they are immersed in water, but it is necessary to set up rather complicated apparatus and to make analyses of the gases at the beginning and at the close of the experiments. This has been done, however, in a sufficiently large number of cases so that we know that all green plants in the sunlight, if temperature and other conditions are favorable, give off oxygen.

=130. Absorption of carbon dioxide.=—We have next to inquire where the oxygen comes from which is given off by green plants when exposed to the sunlight, and also to learn something more of the conditions necessary for the process. We know that water which has been for some time exposed to the air and soil, and has been agitated, like running water of streams, or the water of springs, has mixed with it a considerable quantity of oxygen and carbon dioxide.

If we boil spring water or hydrant water which comes from a stream containing oxygen and carbon dioxide, for about 20 minutes, these gases are driven off. We should set this aside where it will not be agitated, until it has cooled sufficiently to receive plants without injury. Let us now place some spirogyra or vaucheria, and elodea, or other green water plant, in this boiled water and set the vessel in the bright sunlight under the same conditions which were employed in the experiments for the evolution of oxygen. No oxygen is given off.

Can it be that this is because the oxygen was driven from the water in boiling? We shall see. Let us take the vessel containing the water, or some other boiled water, and agitate it so that the air will be thoroughly mixed with it. In this way oxygen is again mixed with the water. Now place the plant again in the water, set in the sunlight, and in several minutes observe the result. No oxygen or but little is given off. There must be then some other requisite for the evolution of the oxygen.

=132. The gases are interchanged in the plants.=—We will now introduce carbon dioxide again in the water. This can be done by leading CO₂ from a gas generator into the water. Broken bits of marble are placed in the generator, acted upon by hydrochloric acid, and the gas is led over by glass tubing. Now if we place the plant in the water and set the vessel in the sunlight, in a few minutes the oxygen is given off rapidly.

=133. A chemical change of the gas takes place within the plant cell.=—This leads us to believe then that CO₂ is in some way necessary for the plant in this process. Since oxygen is given off while carbon dioxide, a different gas, is necessary, it would seem that a chemical change takes place in the gases within the plant. Since the process takes place in such simple plants as spirogyra as well as in the more bulky and higher plants, it appears that the changes go on within the cell, in fact within the protoplasm.

=134. Gases as well as water can diffuse through the protoplasmic membrane.=—Carbon dioxide then is absorbed by the plant while oxygen is given off. We see therefore that gases as well as water can diffuse through the protoplasmic membrane of plants under certain conditions.

2. Where Starch is Formed.

We have found by these simple experiments that some chemical change takes place within the protoplasm of the green cells of plants during the absorption of carbon dioxide and the giving off of oxygen. We should examine some of the green parts of those plants used in the experiments, or if they are not at hand we should set up others in order to make this examination.

=135. Starch formed as a result of this process.=—We may take spirogyra which has been standing in water in the bright sunlight for several hours. A few of the threads should be placed in alcohol for a short time to kill the protoplasm. From the alcohol we transfer the threads to a solution of iodine in potassium iodide. We find that at certain points in the chlorophyll band a bluish tinge, or color, is imparted to the ring or sphere which surrounds the pyrenoid. In our first study of the spirogyra cell we noted this sphere as being composed of numerous small grains of starch which surround the pyrenoid.

=136. Iodine used as a test for starch.=—This color reaction which we have obtained in treating the threads with iodine is the well-known reaction, or test, for starch. We have demonstrated then that starch is present in spirogyra threads which have stood in the sunlight with free access to carbon dioxide.

If we examine in the same way some threads which have stood in the dark for a few days we obtain no reaction for starch, or at best only a slight reaction. This gives us some evidence that a chemical change does take place during this process (absorption of CO₂ and giving off of oxygen), and that starch is a product of that chemical change.

=137. Schimper’s method of testing for the presence of starch.=—Another convenient and quick method of testing for the presence of starch is what is known as Schimper’s method. A strong solution of chloral hydrate is made by taking 8 grams of chloral hydrate for every 5_cc_ of water. To this solution is added a little of an alcoholic tincture of iodine. The threads of spirogyra may be placed directly in this solution, and in a few moments mounted in water on the glass slip and examined with the microscope. The reaction is strong and easily seen.

We should also examine the leaves of elodea, or one of the higher green plants which has been for some time in the sunlight. We may use here Schimper’s method by placing the leaves directly in the solution of chloral hydrate and iodine. The leaves are made transparent by the chloral hydrate so that the starch reaction from the iodine is easily detected.

The following is a convenient and safe method of extracting chlorophyll from leaves. Fill a large pan, preferably a dishpan, half full of hot water. This may be kept hot by a small flame. On the water float an evaporating dish partly filled with alcohol. The leaves should be first immersed in the hot water for several minutes, then placed in the alcohol, which will quickly remove the chlorophyll. Now immerse the leaves in the iodine solution.

=138. Green parts of plants form starch when exposed to light.=—Thus we find that in the case of all the green plants we have examined, starch is present in the green cells of those which have been standing for some time in the sunlight where the process of the absorption of CO₂ and the giving off of oxygen can go on, and that in the case of plants grown in the dark, or in leaves of plants which have stood for some time in the dark, starch is absent. We reason from this that starch is the product of the chemical change which takes place in the green cells under these conditions. The CO₂ which is absorbed by the plant mixes with the water (H₂O) in the cell and immediately forms carbonic acid. The chlorophyll in the leaf absorbs radiant energy from the sun which splits up the carbonic acid, and its elements then are put together into a more complex compound, starch. This process of putting together the elements of an organic compound is a _synthesis_, or a _synthetic assimilation_, since it is done by the living plant. It is therefore a synthetic assimilation of carbon dioxide. Since the sunlight supplies the energy it is also called _photosynthesis_, or _photosynthetic assimilation_. We can also say carbon dioxide assimilation, or CO₂ assimilation (see paragraph on assimilation at close of Chapter 10).

=139. Starch is formed only in the green parts of variegated leaves.=—If we test for starch in variegated leaves like the leaf of a coleus plant, we shall have an interesting demonstration of the fact that the green parts of plants only form starch. We may take a leaf which is partly green and partly white, from a plant which has been standing for some time in bright light. Fig. 68 is from a photograph of such a leaf. We should first boil it in alcohol to remove the green color. Now immerse it in the potassium iodide of iodine solution for a short time. The parts which were formerly green are now dark blue or nearly black, showing the presence of starch in those portions of the leaf, while the white part of the leaf is still uncolored. This is well shown in fig. 69, which is from a photograph of another coleus leaf treated with the iodine solution.

3. Chlorophyll and the Formation of Starch.

=140.= In our experiments thus far in treating of the absorption of carbon dioxide and the evolution of oxygen, with the accompanying formation of starch, we have used green plants.

=141. Fungi cannot form starch.=—If we should extend our experiments to the fungi, which lack the green color so characteristic of the majority of plants, we should find that photosynthesis does not take place even though the plants are exposed to direct sunlight. These plants cannot then form starch, but obtain carbohydrates for food from other sources.

=142. Photosynthesis cannot take place in etiolated plants.=—Moreover photosynthesis is usually confined to the green plants, and if by any means one of the ordinary green plants loses its green color this process cannot take place in that plant, even when brought into the sunlight, until the green color has appeared under the influence of light.

This may be very easily demonstrated by growing seedlings of the bean, squash, corn, pea, etc. (pine seedlings are green even when grown in the dark), in a dark room, or in a dark receiver of some kind which will shut out the rays of light. The room or receiver must be quite dark. As the seedlings are “coming up,” and as long as they remain in the dark chamber, they will present some other color than green; usually they are somewhat yellowed. Such plants are said to be _etiolated_. If they are brought into the sunlight now for a few hours and then tested for the presence of starch the result will be negative. But if the plant is left in the light, in a few days the leaves begin to take on a green color, and then we find that carbon dioxide assimilation begins.

=143. Chlorophyll and chloroplasts.=—The green substance in plants is then one of the important factors in this complicated process of forming starch. This green substance is _chlorophyll_, and it usually occurs in definite bodies, the chlorophyll bodies, or _chloroplasts_.

The material for new growth of plants grown in the dark is derived from the seed. Plants grown in the dark consist largely of water and protoplasm, the walls being very thin.

=144. Form of the chlorophyll bodies.=—Chlorophyll bodies vary in form in some different plants, especially in some of the lower plants. This we have already seen in the case of spirogyra, where the chlorophyll body is in the form of a very irregular band, which courses around the inner side of the cell wall in a spiral manner. In zygnema, which is related to spirogyra, the chlorophyll bodies are star-shaped. In the desmids the form varies greatly. In œdogonium, another of the thread-like algæ, illustrated in fig. 144, the chlorophyll bodies are more or less flattened oval disks. In vaucheria, too, a branched thread-like alga shown in fig. 138, the chlorophyll bodies are oval in outline. These two plants, œdogonium and vaucheria, should be examined here if possible, in order to become familiar with their form, since they will be studied later under morphology (see chapters on œdogonium and vaucheria, for the occurrence and form of these plants). The form of the chlorophyll body found in œdogonium and vaucheria is that which is common to many of the green algæ, and also occurs in the mosses, liverworts, ferns, and the higher plants. It is a more or less rounded, oval, flattened body.

=145. Chlorophyll is a pigment which resides in the chloroplast.=—That the chlorophyll is a coloring substance which resides in the chloroplastid, and does not form the body itself, can be demonstrated by dissolving out the chlorophyll when the framework of the chloroplastid is apparent. The green parts of plants which have been placed for some time in alcohol lose their green color. The alcohol at the same time becomes tinged with green. In sectioning such plant tissue we find that the chlorophyll bodies, or chloroplastids as they are more properly called, are still intact, though the green color is absent. From this we know that chlorophyll is a substance distinct from that of the chloroplastid.

=146. Chlorophyll absorbs energy from sunlight for photosynthesis.=—It has been found by analysis with the spectroscope that chlorophyll absorbs certain of the rays of the sunlight. The energy which is thus obtained from the sun, called _kinetic_ energy, acts on the molecules of CH₂O₃, separating them into molecules of C, H, and O. (When the CO₂ from the air enters the plant cell it immediately unites with some of the water, forming carbonic acid = CH₂O₃.) After a series of complicated chemical changes starch is formed by the union of carbon, oxygen, and hydrogen. In this process of the reduction of the CH₂O₃ and the formation of starch there is a surplus of oxygen, which accounts for the giving off of oxygen during the process.

=147. Rays of light concerned in photosynthesis.=—If a solution of chlorophyll be made, and light be passed through it, and this light be examined with the spectroscope, there appear what are called absorption bands. These are dark bands which lie across certain portions of the spectrum. These bands lie in the red, orange, yellow, green, blue, and violet, but the bands are stronger in the red, which shows that chlorophyll absorbs more of the red rays of light than of the other rays. These are the rays of low refrangibility. The kinetic energy derived by the absorption of these rays of light is transformed into potential energy. That is, the molecule of CH₂O₃ is broken up, and then by a different combination of certain elements starch is formed.[8]

[8] In the formation of starch during photosynthesis the separated molecules from the carbon dioxide and water unite in such a way that carbon, hydrogen, and oxygen are united into a molecule of starch. This result is usually represented by the following equation: CO₂ + H₂O = CH₂O + O₂. Then by polymerization 6(CH₂O) = C₆H₁₂O₆ = grape sugar. Then C₆H₁₂O₆-H₂O = C₆H₁₀O₅ = starch. It is believed, however, that the process is much more complicated than this, that several different compounds are formed before starch finally appears, and that the formula for starch is much higher numerically than is represented by C₆H₁₀O₅.

=148. Starch grains formed in the chloroplasts.=—During photosynthesis the starch formed is deposited generally in small grains within the green chloroplast in the leaf. We can see this easily by examining the leaves of some moss-like funaria which has been in the light, or in the chloroplasts of the prothallia of ferns, etc. Starch grains may also be formed in the chloroplasts from starch which was formed in some other part of the plant, but which has passed in solution. Thus the functions of the chloroplast are twofold, that of photosynthesis and the formation of starch grains.

=149.= In the translocation of starch when it becomes stored up in various parts of the plant, it passes from the state of solution into starch grains in connection with plastids similar to the chloroplasts, but which are not green. The green ones are sometimes called _chloroplasts_, while the colorless ones are termed _leucoplasts_, and those possessing other colors, as red and yellow, in floral leaves, the root of the carrot, etc., are called _chromoplasts_.

=150. Photosynthesis in other than green plants.=—While carbohydrates are usually only formed by green plants, there are some exceptions. Apparent exceptions are found in the blue-green algæ, like oscillatoria, nostoc, or in the brown and red sea weeds like fucus, rhabdonia, etc. These plants, however, possess chlorophyll, but it is disguised by another pigment or color. There are plants, however, which do not have chlorophyll and yet form carbohydrates with evolution of oxygen in the presence of light, as for example a purple bacterium, in which the purple coloring substance absorbs light, though the rays absorbed most energetically are not the red.

=151. Influence of light on the movement of chlorophyll bodies.=—_In fern prothallia_.—If we place fern prothallia in weak light for a few hours, and then examine them under the microscope, we find that the most of the chlorophyll bodies in the cells are arranged along the inner surface of the horizontal wall. If now the same prothallia are placed in a brightly lighted place for a short time most of the chlorophyll bodies move so that they are arranged along the surfaces of the perpendicular walls, and instead of having the flattened surfaces exposed to the light as in the former case, the edges of the chlorophyll bodies are now turned toward the light. (See figs. 70, 71.) The same phenomenon has been observed in many plants. Light then has an influence on chlorophyll bodies, to some extent determining their position. In weak light they are arranged so that the flattened surfaces are exposed to the incidence of the rays of light, so that the chlorophyll will absorb as great an amount as possible of kinetic energy; but intense light is stronger than necessary, and the chlorophyll bodies move so that their edges are exposed to the incidence of the rays. This movement of the chlorophyll bodies is different from that which takes place in some water plants like elodea. The chlorophyll bodies in elodea are free in the protoplasm. The protoplasm in the cells of elodea streams around the inside of the cell wall much as it does in nitella and the chlorophyll bodies are carried along in the currents, while in nitella they are stationary.