Transpiration – The Loss of Water from Plants

Transpiration – The Loss of Water from Plants!

Need of Water:

Of all the materials required by a plant for its existence and normal development that is taken up in the largest amount, is water.

Water is absolutely essential to animal and plant life. Plant and animal cells, deprived of water to any considerable extent soon die.

The bulk of this water absorbed, however, takes no permanent part in its develop­ment or in metabolic processes, but evaporates into the air from the leaves and other aerial parts. This loss of water in the form of vapour from living plants, particularly from the aerial parts, is known as transpiration. The process is in principle one of evaporation and diffusion.

Loss of water vapour may occur from any part of the plant which is exposed to the air. All aerial parts lose water by transpiration, although in some tissues due to the presence on some organs, of superficial layers which are impervious to water, e.g., cork cells, the rate of water loss is almost insignificant compared to the water lost from leaves through stomata.

Most of the water vapour lost from leaves occurs through the stomata, this is termed stomatal transpiration; smaller amounts of water vapour are also lost by direct evaporation from the epidermal cells, when the stomata are closed, through the cuticle; this is usually termed cuticular transpiration.

As a general rule transpiration rates are higher when the stomata are open, lower when they are closed. In other words, stomatal transpiration is usually many times higher than cuticular transpiration, commonly about 4 times as high during a warm sunny day.

But there are all gradations from one extreme to the other. In the case of succulents like cacti, cuticular transpira­tion is usually nil. On the other hand, in some plants, cuticular transpiration can be as high as stomatal transpiration.

The general need of water for plants can be discussed under the following heads:

It is best to point out here that water for its capacity to form hydrogen bonds is largely responsible for the maintenance of the three dimensional structures of such macromolecules as nucleic acids and proteins and of the various subcellular organelles —essential for their biological function.

Water scarcity or any other condition which destroys or disrupts their physical structures impairs the activity of these macromole­cules and organelles, which ultimately may have disastrous consequences for plant life. Among all natural compounds, water also gains and loses heat slower than any other known substance.

Because plants contain so much water, the rapid changes of tempera­ture that may take place in the surrounding atmosphere have less effect on the plant than they would, if water gained or lost heat quickly.

We know that chemical reactions are speeded up by heating and slowed down by cooling. The abundance of water in the plant maintains a fairly constant temperature so that chemical reactions inside the plant are not speeded up or slowed down so rapidly between the extremes of tempera­ture to which a plant cell may often be subjected.

In nearly all its physical properties, water is either unique or at the extreme end of the range of a property. From these physical and chemical characteristic of water, the biological importance of water is realised.

Among its thermal properties, water has the greatest specific heat known among liquids; the same is true of water’s latent heat of vaporisation. Finally, with the excep­tion of mercury, water has the greatest thermal conductivity of all liquids.

As Constituent of the Protoplasm:

It is by far the most important integral part of the protoplasm. Large amounts of water are necessary for the proper functioning of the protoplasm. The younger, the more vigorous, the more rapidly growing protoplasm is, the more water it contains.

In the actively growing meristematic regions of plants more than 90% of water is directly associated with protoplasm. Water deficiency during the conjugating phase of meiosis (reduction division) greatly decreases the number of the terminal junctions and chiasmata.

If the normal proportion of cell-water is removed from the protoplasm, it becomes less and less active until a point (below 30-50%) is reached, when death of the protoplasm by desiccation ensues.

In the Vacuoles of the Mature Cells:

As has been discussed previously a certain amount of water is absolutely necessary to maintain the normal condition of turgour essential for the growth of the cells also for giving rigidity and erectness to the young plant.

For Translocation of Minerals and Synthesized Food from One Part of the Plant to the Other:

All inorganic minerals which move upwards through xylem and the movement of foods synthesised in the leaves downwards through the phloem and all from cell to cell are translocated in the form of solution in water.

For Chemical Combination:

Many of the various complex organic substances synthesised in plant tissues are formed by chemical combination of water with certain inorganic materials that enter the plant from air and soil. Taking the fresh weight of a maize plant of average height (about 3-4 m) as for example, growing vigorously in well- watered and aerated soil, to be about 3,000 g, the total dry wt. was found to be about 850 g.

The water content is thus about 2,150 g, i.e., the amount of water the plant needed for the protoplasm, for maintenance of turgour of the cells and used in the trans­location of materials through the plants.

The amount of water necessary for all the chemical combinations in which water is known to take part would not certainly exceed 250 g. Thus, about 2,150 + 250 = 2,400 g (less than 2 1/2 litres) of water a plant would need during its entire growing season which will be sufficient for all its essential requirements.

In practice, however, under normal conditions much greater quantity of water is taken up by the plants from soil—-about 250 litres or approximately 100 times more than is needed. This large excess of water absorbed by the plants from soil is eliminated by the plant in the form of vapour into the atmosphere. How do plants achieve this?

Transpiration and Leaf Anatomy:

The occurrence of transpiration seems to be a natural consequence of the basic facts of leaf anatomy. The leaves consist of water-filled mesophyll cells, the water of which certainly also saturates the protoplasm lining the vacuoles and the cell walls. This water is supplied to the mesophyll cells of the leaves through the xylem bundles which form the network of veins of the leaves.

The wet cell walls of these cells of the leaves are in intimate contact with the intercellular spaces which cover from 5-75% of the total area of the leaf. Hence evaporation of water takes place by direct absorption of radiant energy by leaves from the wet cell walls into the internal atmosphere of the inter­cellular spaces just as it will occur from any wet surface into the surrounding air.

Recent evidences indicate that the mesophyll cell walls in the leaves are hydrophobic in nature, i.e., they are not rapidly wetted. Thus, it would seem that water would remain deep within the walls, leaving the wall surfaces relatively dry.

The intercellular spaces form a connected system extending throughout the leaf and in turn lead through the stomata to the outside atmosphere. Water evaporating from the wet mesophyll cells is con­ducted through the continuous intercellular spaces and if the stomata are open, to the external air.

If the stomata are closed, however, the only effect of evaporation from the cells walls will be gradual saturation of the entire internal atmosphere of the intercellular space with water vapour.

It must be clearly understood that the structure of leaf is adapted for absorption of gases that it needs and for excretion of those, which arise as end products in plant meta­bolism. It is certainly very poorly adapted for retention of water within the plant so that continuous threat of loss of water is a constant menace to the life of plant.

Under certain conditions, the intercellular spaces can become filled with liquid water (guttation); normally they are occupied by air.

Transpiration and its Relation with Radiant Energy:

Leaves exposed to direct sunlight absorb large quantities (as much as 80%) of radiant energy and unless the energy absorbed is dissipated, it will be converted to heat energy raising the temperature of the leaf to such an extent as to be lethal to the protoplasm of most plants in a very short period of time.

Only a very small proportion of the total absorbed energy (0.3-5%) is utilised under natural conditions (under best laboratory conditions, however, the efficiency of photosynthetic apparatus in converting absorbed light energy is at least 35%) in photosynthesis and stored in carbohydrate molecules.

But actually, the leaf temperature seldom exceeds atmospheric temperature by more than 2-5°C. Part of the absorbed energy after being changed into heat is used in transpiration, i.e., in changing water into water vapour.

Since transpiration like evaporation of water is an energy consuming process, it has been assumed that in the evaporation of water from the leaves most of the energy absorbed by them is dissipated.

A simple calculation, however, shows that even at maximal transpiration rates, transpiration can at the most account for the dissipation of only a part of the radiant energy absorbed by leaves in sunlight.

Even if transpiration could account for the dissipation of most of the absorbed solar radiation, in doing so it apparently plays no essential role since the absorbed energy could as well, if not better, be dissipated by purely physical means, just as an object heated above its environmental temperature, does so by conduction, convection and radiation.

Thus the function of transpiration seems to be redundant in protecting the leaves from a thermal death.

Transpiration During Day and Night: Amount of Water Lost:

Transpiration occurs both during day and night but in general by far the greater amount of water, about 95%, is lost during the daylight hours.

In general more water is transpired during the afternoon than during the fore­noon. The maximum rate of water loss takes place between 11 a.m.-3 p.m.

Amounts of water lost by plants per day undoubtedly depends upon the prevailing climatic conditions. A sunflower plant, under normal climatic conditions, may lose as much as 4 litres per day on the average.

The Significance of Transpiration:

Although transpiration has been studied extensively, very little is known concern­ing the significance of transpiration in the life of the plant. There is even now some controversy whether transpiration is a necessary and unavoidable evil or whether it is in some way essential for the welfare of the plant.

In the first category, opinions prevail that transpiration brings nothing but harm to the plant, and the harmful effects ascribed to transpiration far outweigh its supposed beneficial effects, while in the second, there are opinions which consider transpiration as important as photosynthesis and respira­tion.

From the former point of view the primary function of stomata is to permit exchange of CO₂ and O₂ between the plant and the atmosphere in the process of photo­synthesis and respiration and water is lost through the open stomata, because there is no stopping the passage of water vapour, while permitting CO₂ and O₂ exchange.

Thus the primary function of stomata is not to be thought of as one permitting water loss any more than the function of a main door in a busy office building is to let heat escape from the building in winter.

Just as loss of heat cannot be avoided if the door must be kept open to continuous streams of persons who enter and leave the building during business hours, similarly loss of water vapour from the leaf is inevitable when stomata are open to permit exchange of CO₂ and O₂ between the plant and the atmosphere.

From the other point of view transpiration fulfils perhaps, only partly, these main functions:

Transpiration Promotes Absorption and Translocation of Solutes:

It has often been assumed that the more rapid the rate of transpiration, the greater is the rate of absorption of solutes from the soil. This view certainly implies that the dissolved mineral salts are swept into the plant along with water.

This implication, however, completely ignores much evidence that the mechanism of water absorption and the absorption of mineral salts are very different and possibly independent of each other.

The results of certain experiments, however, do indicate that somewhat larger quantities of mineral salts accumulate in plants under conditions favouring high transpiration compared to the similar plants growing under conditions where transpiration rate is low.

But as there is certainly no direct relationship between the volume of water absorbed and the volume of water lost by transpiration, it is evident that at the most only a slight, if any, correlation exists between the rate of transpiration and the rate of absorption of solutes from the soil.

Since, however, plants grown even under conditions favouring low transpiration rates obtain their usual and normal requirements of various mineral salts, provided they are present in the soil, it is difficult to see how this effect of larger intake of salts is of any great particular advantage to the plant.

It is also generally considered that transpiration plays a significant role in the translocation of solute solution in the xylem vessels from the roots to the leaves. This factor has been the subject of the most controversy.

That mineral elements absorbed from the soil are translocated mainly through the xylem vessels and rise upward with the transpiration stream, is indisputable. The question is, however, sometimes raised whether or not the rate of translocation of solutes in the vessels depends on the rate of transpira­tion.

A critical examination, however, shows that if the solutes from the soil enter plants by active absorption, the amount of solute reaching the leaves per unit time will depend not on the rate or speed of the transpiration current through the xylem vessels but on the rate of absorption of mineral salts from the soil.

There is also considerable evidence that at least a part of mineral salts absorbed from soil may move through the phloem and not through the xylem and it is evident that transpiration can have little or no effect upon such movement of salts.

Transpiration Reduces Leaf Temperature:

It has frequently been stated that transpiration cools the leaves and prevents their death or injury by high temperatures. The change of water from the liquid stage to vapour does involve absorption of a consi­derable amount of heat energy and thus vaporisation would tend to lower the tempera­ture of the leaf.

It has been shown that the actual cooling effect of transpiration is not so great as some investigators have believed; it is generally not more than 3-5°C. com­pared with the temperature of the atmosphere prevailing at the time—a difference, which so far as our knowledge of protoplasm goes, would scarcely be of any marked benefit in preventing injurious effect of high temperature.

According to some investi­gators, however, a matter of even a very few degrees may mean at its worst, difference between life and death or between efficient and inefficient functioning of the cells.

In a few experiments it has been claimed that leaves exposed to sun becomes much hotter than air temperature, when transpiration is stopped artificially or slowed down by wilt­ing. The temperature of leaf undergoing rapid transpiration may remain, in exceptional cases, as much as 20° C. below the temperature of the surrounding air.

Stomata:

The stomata constitute the main route for the escape of water vapour from the leaves of land plants to the surrounding atmosphere.

Stomata are very minute openings or pores surrounded by two semilunar guard cells. They occur only in the superficial layer, in the epidermis of all the plant organs except in the roots, being much more numerous in the leaves.

They are formed by the ordinary cell division of a young elliptical epidermal cell forming two daughter cells. By the dissolution of the middle lamella between the two cells, a pore or intercellular space is formed. The two cells resulting from the division are known as the guard cells and the pore between them is the stomatal pore or aperture.

The guard cells differ from epidermal cells not only in shape but also in possession of rich protoplasmic material, chloroplastids and peculiar localised thickening of their cells walls. The chlorophyll of the guard cells can be formed either in light or in darkness in contrast to the chlorophyll of the mesophyll cells which requires light for its formation.

If a leaf is kept in dark the chlorophyll of the mesophyll is decomposed whereas chlorophyll of the guard cells is not. The parts of the guard cell wall which abut on the stomatal pore are much thicker than the remainder of the wall which is in contact with ordinary epidermal cell walls.

Because of this uneven thickening of the walls of the guard cells, increased turgour leads to opening, and decreased turgour leads to closure of stomata. The epidermal cells which border the guard cells are also somewhat different from the ordinary epidermal cells and are sometimes called subsidiary cells or accessory cells.

Distribution, Number and Size of Stomata:

The number of stomata per unit area of the leaf is characteristic for each species of plant. In most dorsiventral leaves, the distribution of stomata is usually restricted only on the lower epidermis of the leaves. In such leaves, approximately 95-97% of total gas exchange occurs through the lower epidermis and only about 3-5% occurs directly through the cuticle of the upper epidermis.

In isobilateral leaves of most monocotyledons, stomata are more or less evenly distributed on both the upper and lower epidermis. In wheat leaves, actually more stomata are found in the upper epidermis.

In floating leaves such as those of waterlily, stomata are restricted only on the upper epidermis, the lower epidermis being in contact with water of the medium. Stomata usually occur with a frequency of 5,000 to 30,000 per sq. cm. of the leaf area, although up to 130,000 per sq. cm have been frequently reported.

The area of the stomatal aperture or pore, when fully open, as in maize, can be as high as 100µ². There is no conclusive evidence as yet whether environmental conditions influence the number of stomata formed per unit area of the leaf.

Species, in which the stomata are relatively smaller in size, have more per unit aroa than species in which sto­mata are relatively large. The aggregate area of stomata, when the pore is fully open, is approximately 1-5% of the total leaf surface.

The number of stomata per unit area varies from leaf to leaf of the same plant and even in different parts of the same leaf. In any particular leaf, the greatest number of stomata per unit area is at the top, the lowest towards the base and the middle fre­quency is in the centre of the leaf. As a rule, the younger leaves in the tops of the plant have greater number of stomata per unit area than those situated below.

In general, if the stomata are open, no correlation direct or indirect is found between the rates of transpiration and the size, number and distribution of stomata per unit area, other factors, particularly environmental conditions, being much more important.

Capacity of Stomata for Diffusion of Gases:

Practically all the interchange of gases between the interior of the leaf and the outside atmosphere, takes place through stomata. Although stomata occupy only about 1-5% of the total area of the epidermal surface, nevertheless, they have a carrying capacity for gases greatly in excess of that needed by plants for all their metabolic activities.

Consequently the stomatal pore can close to a considerable degree and yet provide for all necessary gaseous exchange bet­ween the plant and the atmosphere.

The rapid rate of diffusion of gases and water vapour into and out of the leaf is understandable on the basis of fundamental laws of diffusion the diffusion constant of gases is high, about 10,000 times that of liquid water; the area across which diffusion occurs, is also large.

The difference in vapour pressure between intercellular spaces and outside atmosphere is large, and the distance between the internal air surface and outside atmosphere is small. But the area can only be considered large, if we consider the whole leaf area.

Although the area of stomata only seldom exceeds 1 % of the total leaf area, yet a leaf may lose almost as much water vapour as a free water surface of the same total area. This certainly means that the rate of water loss per unit stomatal area is of the order of about 100 times that from a unit area of free water surface.

It has been shown that the loss of water from a free water surface is practically unaltered when it is covered by a perforated sheet, even though the area across which diffusion occurs is reduced to only a small fraction of total area.

It has also been experimentally determined that if the openings or pores of the perforated sheet are as close together as 10-20 times their diameter, maximum diffusion occurs. Doubling the diameter of these openings only, has no effect on the rate of diffusion of water vapour. The same fact largely holds good for stomatal pore on the surface of the leaves.

Regulation or Control of Water Loss by Stomata:

The general view came to be held that the stomata by opening and closing control or regulate the transpirational water loss from the plant.

The earlier idea was that stomata could close in anticipation of wilting and thus conserve the water supply of the leaf and prevent it from wilting. When later it was found that the stomata do not close until the leaf has wilted to some extent, it was then rea­lised that the regulation, if any, was passive since under such conditions, the stomatal closure is not due to any special activity of the guard cells but to a decrease in their turgour caused by loss of water from the leaf cells.

The rate of transpiration is generally higher than the absorption of water by roots from soil during the day and the reverse is true at night. The water content of a leaf may vary over a considerable range during a 24-hour period without any visible signs of wilt­ing of the aerial organs.

We have discussed before that no correlation generally exists between the number of stomata per unit area of the leaf surface, and the rate of transpiration.

The general view now held by most investigations is that when the stomata are fully open or nearly so, the transpiration rate is primarily determined by the same physical factors which control evaporation from a free water surface.

Transpiration may actually increase when stomata begin to close and may decrease when they open wider. This is sometimes due to the fact, as we have seen before, that when the diameters of the stomatal pores are about 1 /10th to 1 /20th the distance between the two pores, no further opening of the pore can affect the rate of diffusion of water vapour through them.

As the stomata gradually close, influence of physical factors of evaporation is lessened until at 50% opening of stomatal pore, variable atmospheric conditions become the primary factors in controlling transpiration, physiological regulation by the stomata, of the water loss, taking only a subsidiary role.

Only when the pores are almost closed, the guard cells begin to exert a controlling influence on the water loss regardless of evaporation and climatic factors prevailing at the time.

Mechanism of Opening and Closing of Stomata:

Because of the uneven and peculiar thickening of the walls of the guard cells (guard cell walls are thick only on the side bordering the pore, the rest of the wall being thin), increased turgour of the cell sap of the guard cells leads to opening, loss of turgour leads to closure.

Since, we know that turgour is a hydrostatic pressure due to entry of water into the cells, stomatal movement is controlled evidently by water exchange and any factor that alters the water content of the guard cells will in turn affect their turgour and therefore the stomatal opening.

The turgour pressure, as we very well know, at its maximum can equalise the osmotic pressure of the cell sap. Any increase in osmotic pressure of the cell sap due to an increase in solutes will therefore favour opening of the stomata; any decrease will favour its closure. It must be remembered, however, that any increase in solute content of the cell sap can affect only the osmotic pressure directly and the actual change in turgour pressure can occur only as a result of movement of water into or out of the guard cells.

Any real change in the turgour pressure consequently is accompanied by a change in the degree of opening of the pore if the elastic cell wall is capable of a further stretch or shrinking.

The other epidermal cells, particularly the subsidiary cells in the neighbourhood of the guard cells if they are turgid, exert, so to say, a back pressure on the guard cells and if all the epidermal cells were to increase equally in osmotic pressure and turgour pressure, no stomatal opening would immedia­tely result.

Similarly, if all the epidermal cells decreased equally in osmotic and turgour pressures, no closure would occur. Under such conditions stomatal opening may actually result from decreased turgour in the adjacent epidermal cells (subsidiary cells), even in darkness.

In fact, a simultaneous water loss from all the epidermal cells may result in the opening of the stomata if the guard cells lose less water than the other epidermal cells, because the guard cells are the last of the epidermal cells to become flaccid and to reach zero turgour.

The importance of the osmotic pressure of the guard cells in stomatal movement is shown by the high values—20-100 atm. that have been recorded for open stomata and the corresponding low values, 5-10 atm. in the case of closed ones.

The classical idea about changes brought about in the guerd cells, was thought to be due to three possible causes of such change:

(a) accumulation of sugars due to photo­synthesis in the guard cells; (b) starch ↔ sugar transformation (starch → sugar lead­ing to opening and sugar → starch, to closure) and (c) active absorption of solutes by the guard cells from surrounding epidermal cells.

The following classical theory as to the mechanism of stomatal opening and closing in most dicotyledons is based on perhaps not much experimental evidence, but the theory, for a long time, certainly looked good enough on paper:

(1) When the plant is exposed to light, photosynthesis occurs in the green cells of the leaf, including the guard cells;

(2) As a result, the CO₂ content of the leaf is reduced due to its utilisation in photosynthesis;

(3) The pH of the guard cells rises, i.e., become alkaline;

(4) Starch is converted into sugar in the guard cells;

(5) The permeability of the plasma membrane of the walls of the guard cells increases in light;

(6) Osmotic pressure of the guard cells rises;

(7) Water enters the guard cells from the surrounding epidermal accessory cells;

(8) Turgour pressures increase in the guard cells;

(9) The guard cells are forced apart due to their uneven cell wall-thickening, opening the stomatal pore.

The starch ↔ sugar conversion is an enzymatically controlled chemical reaction and it is well known that enzymes are usually most active at some pH and inactive at others. The enzyme responsible for the reversible conversion of starch ↔ sugar— phosphorylase—is present in the guard cells.

It has been experimentally shown that in the presence of this enzyme at pH 5, the ratio of starch to sugar is about 4 times than it is at pH 7. More recent investigations indicate, however, that starch to sugar transforma­tion is absolutely insufficient to explain the speed with which stomata open or close in most plants.

As a result, the starch ↔ sugar balance theory of stomatal movement in light and darkness, given above, has always been suspect, to say the least.

The recently discovered fact that when the CO₂ content of the substomatal spaces is reduced artificially from 0.03% (normal percentage present in the air) to 0.01%, the stomata open. The closing in the dark is supposed to be caused by the increase in the CO₂-content in the intercellular spaces produced by dark respiration.

It has also been shown that sufficient photosynthesis can occur in the guard cells from internally derived respiratory CO₂– and not dependent on an external atmospheric CO₂-supply—>to permit usual rates of stomatal opening in light.

On the other hand, however, all the behaviour of the stomata is net fully explained by this theory. Thus the night opening of the stomata, when it takes place in many plants cannot be related either to photo­synthesis or to a reduction of CO₂-content as there is no photosynthesis during the hours of darkness.

Transpiration as a Metabolic Process:

In the light of recent much detailed and critical work in this long neglected field of research, it is now accepted by most investigators that transpiration is actually a metabolic process, like photosynthesis and respiration and like them needs considerable usage of ATP molecules, photosynthetically phosphorylated by the guard cell-chloroplasts.

It has never been shown that guard cells completely devoid of chloroplasts are cap­able of opening in light. It has been seen that the development of the stomatal light response, in etiolated wheat and onion leaves, closely followed the increase in chlorophyll-a content of the tissue.

It has been shown recently that the behaviour of epidermal strips of Senecio provides strong evidence that, photosynthesis in the guard cells themselves, is responsible for the maintenance of stomatal opening in light. When these epidermal strips are treated with DCMU (dichlorophenyl dimethyl urea) even at 10^-5 M dilution, there was complete closure of stomata in light and it is well known that DCMU is a particularly strong inhibitor of photosystem II in photosynthesis as also non-cyclic photophosphorylation.

Naturally all these observations raised the inevitable question of whether photo­synthesis of the guard cells was actually needed for the production of carbon com­pounds, as was supposed before or is primarily required for the production of ATP molecules only and that too non-cyclically.

A number of lines of evidence—and certainly there are contradictory evidences- indicated that glycolate (CH₂OH.COOH) metabolism plays an important part in the opening of stomata in light—either to re-oxidise NADPH, facilitating non-cyclic photophosphorylation or glycolate may possibly be an intermediate in the synthesis of carbohydrates (there seems to be no doubt that sugars and other soluble carbon compounds, produced in photosynthesis may contribute to the increase in the osmotic value of the guard cells, especially at the later stages of stomatal opening).

There is, however, the question of stomatal opening in the dark. Allway & Mans­field (1967) found that stomata on leaves in which photophosphorylation was strongly inhibited, could open widely in response to a low CO₂-concentration, which suggests that ATP formed directly from photophosphorylation is not essential in stomatal opening, everywhere and in all cases.

Evidences also seem to suggest the participation of an active transport mechanism, using ATP derived from oxidative phosphorylation of aerobic respiration. Evidences have also come to light, indicating that when epidermal strips are immersed in a solution containing K+ ions, these accumulate in the guard cells, using respiratory ATP, as stomata open in darkness. (Fujino, 1967; Fischer, 1968).

Guard cells do have an unusually high concentration of K+ when they are open in the light and relatively small quantities when they close in the dark. The influx of K+ ions is accompanied by an efflux of H⁺ ions mediated by a H⁺-K⁺ exchange pump located in the plasma membranes of the guard cells.

Electrical neutrality is maintained probably by anions of malate which are synthesized actively in the guard cells by photosynthetic fixation or respiration in light (or dark CO₂ fixation (?) in Crassulacean plants where stomata open at night), at the same time that K⁺ influx occurs.

It is now known that some of the plant hormones may play important roles in the regulation of transpiration. The most remarkable effect is shown by abscisic acid (ABA). When water stress induces stomatal closure, the stomata remain closed even after the stress is relieved.

It is now known that water stress results in a rapid movement of ABA from mesophyll tissue to epidermal cells and the ionic and metabolic status of guard cells is altered. Natural or applied ABA accumulates in guard cells.

The effect of ABA slowly disappears. ABA probably regulates stomatal opening and closure by affecting the water level in the guard cells. It has been suggested that ABA may in- influence the permeability of cells.

The temperature of the air surrounding the leaf affects the speed with which the stomatal pore opens. Within limits at which protoplasm functions (up to 35-45°C.) the length of the time for opening is reduced to one half for every 10°C. rise in temperature. The explanation is most probably osmotic and also to some extent chemical.

Increase in temperature increases the permeability of the unthickened part of the cell wall of the guard cells, thereby facilitating rapid entry of water into the guard cells. The enzymatic transformation of starch to sugar is also quickened by an increase in temperature.

Rates of photosynthesis increase as also the rate of utilisation of CO₂ of the intercellular spaces due to rise of temperature and all contribute to a rapid opening of the stomata due to increased temperature.

Daily Movement of Stomata:

In general, the stomata tend to show a diurnal periodicity closing at night and opening during the day. This periodicity is clearly related to light. There is great variation in the behaviour of the stomatal pore of the different leaves of the same plant and even on the different parts of the same leaf. The stomata on the upper surface may behave differently from those on the lower.

Some stomata, however, open at night, others close at noon when the light intensity is at a maximum. Wilting may cause most of stomata to close regardless of the light factor.

There are several types of diurnal movement of the stomata:

(1) Stomata of this type under favourable conditions are open all day and closed all night. Under less favourable moisture conditions the stomata close partially for a time during the middle of the day, when the rate of entry of water into the guard cells is incapable of keeping pace with the high rates of transpiration.

The period of partial midday closure increases to complete closure as the moisture conditions in the guard cells become less and less favourable. With the appearance of midday closure, night opening develops which progressively increases with the increase in the day closure, until finally there may be a partial opening of the stomata all night and a closure all day.

This may happen under extreme scarcity of moisture in the leaves. This type of opening and closing is observed mostly in thin leaved mesophytes, e.g., peas, beans, etc.

(2) Movement of stomata of this type is markedly different from the previous type under the same favourable conditions. The stomata of potato are open continuously during day and night except for about 3 hours following sunset.

Midday closure is seldom observed and that only under extreme moisture deficiency conditions. Potato, cabbage, onion, banana, etc., afford examples of this type of stomatal movement.

(3) In the third type, which is typical of all cereals, there is absolutely no opening during the hours of darkness, no matter how slight and how short the day opening may be. There are many closed stomata even during the daytime.

Structural Features of Plant Which Influence the Rate of Transpiration:

Structural differences in leaves also certainly account in part for different rates of transpiration. Many of the supposed effects of structural differences in plants influencing transpiration rates are not, however, verified by experimental evidence.

Our discussion will be limited to the following structural features of plants supposed to influence transpiration rates:

Cutinised and Waxy Thickening of the Epidermal Cells:

Water certainly evaporates more readily from uncutinised epidermal cell walls than from these which are coated with a layer of cutin. It has been observed, that cell walls which are heavily cutinised or covered with wax, show considerably less water loss from them. Cutin as also suberin is cer­tainly very effective in checking loss of water from leaf surface.

The deposition of cutin on the epidermal cell walls, however, has one serious disadvantage; it tends to bring about a closure of stomata, thus severely restricting exchange of gases like CO₂ and O₂ between the plant and the atmosphere—a function absolutely essential for normal metabolic activity of the green plants.

A heavily cutinised leaf surface is usually shiny and reflects much of the solar radiation falling on it. Theoretically, because most of the radiations falling on the cutinised leaf surface are reflected, less is available for absorp­tion. The total effect will be to reduce transpiration rates through reduced heat absorption.

It must also be understood clearly that magnitude of the thickness of cuticle is not a limiting factor in retarding rates of transpiration. After a particular thickness of deposi­tion of cutin on the epidermal cell walls is attained, any further increase in thickness has no appreciable influence on the rates of water loss.

Epidermal Hairs:

Living hairs on the epidermal cells most probably increase the rate of cuticular transpiration by increasing the surface area of the leaves. Dead hairs, on the other hand, act as a mechanical barrier to the effect of light and wind on stomatal trans­piration.

It should be pointed out clearly, however, that many actual experiments have indicated that the efficiency of a coating of hairs dead or living in lowering transpiration rates may be negligible.

Ratio of Internal/External Surface of Leaves:

We know that most of the water vapour lost from leaf surface evaporates from the walls of the mesophyll cells which are bound by intercellular spaces and which constitute the internal evaporating surface of the leaves.

This proportion area of internal evaporating surface to external total leaf surface varies greatly not only in leaves of one species compared to those of the other, but may also vary in the leaves of same species if they have developed under different environmental condition.

In general, the larger the ratio of the internal evaporating surface, i.e., the total area of the intercellular spaces to the total leaf surface, the greater is the rate of transpiration.

Sunken Stomata:

Stomata of many species of plants, adapted to conditions of extreme soil and atmospheric drought, are sunken in pits below the level of the epidermal surface and likewise are well below the level of atmospheric disturbances in the vicinity of the leaf.

Plants with sunken stomata have generally been found capable of significant reduction in transpiration. Diffusion through a small microscopic opening such as stomata is certainly slower if the water vapour must pass through a relatively longer tube (depth of the pit) before reaching the external air than diffusion through a shorter tube (thickness of the stomatal pore only).

Structure and Distribution of the Root System:

Under some environmental conditions, a particular plant may develop a greater total leaf surface in proportion of the extent of its root system. This structural adaptation will certainly favour maintenance of higher transpiration rates.

The distribution of root system in the soil frequently influences the water balance in plants. In a habitat where deep-rooted and shallow-rooted plants are growing side by side, the former may transpire more rapidly during dry periods than the latter.

In the former the roots penetrate to a soil horizon which may still contain available water while the roots of the latter are in a moisture-deficient surface soil due to drying of superficial layers of soil if caused by atmospheric and soil drought.

Environmental Factors and Transpiration:

The rate of transpiration of a plant varies from day to day, from hour to hour and frequently still more rapidly. The difference in the rapidity with which water is lost by plants when the stomatal pores are even half open is primarily due to the effects of environmental conditions.

When the stomatal pores are fully open, the diffusion of water vapour from the intercellular spaces of the leaves to the external air is primarily controlled by the laws of diffusion.

In other words, the larger the difference between the vapour pressure or concentration within the intercellular spaces of the leaf and that of the surrounding external air, the greater is the transpiration rate. So any environmental factor which will tend to steepen this gradient will facilitate exit of water vapour from the plants to the air outside.

The important environmental factors, which help in the development of a difference between the vapour pressure inside and outside the plant and thus facilitating rapid diffusion of water vapour out of the plants, are given below.

In this discussion we assume that the supply of water is abundant in the leaves and absorption keeps pace with trans­piration and the stomatal pores are not less than 1/3 open:

Light:

Though light occupies a position of prime importance among the environ­mental conditions, its effect on the development of a difference in vapour pressure within the plant and the outside air is negligible except in so far as its indirect un­doubted effect in increasing the temperature of the leaf.

The principal effect of light in transpiration, as we have seen in our previous discussions, is predominantly through its influence on the opening and closing of stomata. In the complete absence of light, in most species of plants under normal environmental conditions, the stomatal pores are usually closed.

Humidity:

In general, the greater the vapour pressure of the atmospheric air, the slower is the rate of transpiration, other factors remaining unchanged. We know that the air in the intercellular spaces of the leaf is maintained nearly always at the saturation point if the supply of water to the leaf is uninterrupted and abundant while in the out­side atmosphere, conditions are rarely favourable for attainment of saturation vapour pressure.

It is well known that plants show very high transpiration rates on dry days compared to moist ones and as a result wilting of the aerial parts is a common pheno­menon on hot, dry days if the supply of water to the leaf is not fast enough to keep pace with rapid diffusion of water vapour from the leaf to the external air.

On rare occasions, when the vapour pressure of the air outside approaches saturation point and in extreme cases when the diffusion pressure difference between inside and outside becomes zero, no transpiration occurs even when the stomata are fully open.

Temperature:

Before active transpiration starts leaves exposed to direct sunlight usually have temperatures from 2-5° C. in excess of that of the atmosphere.

When the temperature of the leaf and the surrounding atmosphere is raised by direct sunlight, unless the leaf is markedly deficient in water, the vapour pressure of internal atmosphere, i.e., the intercellular spaces of the leaf maintains essentially a saturation vapour pressure for the prevailing temperature.

As the internal volume is constant in size and is small, the evaporation of water from wet mesophyll cells is sufficient to saturate the comparatively small volume of air inside the leaf In the sur­rounding atmosphere, however, vapour pressure conditions are very different—on clear days, there is frequently little change in the vapour pressure—evaporation into the atmosphere is insufficient to permit a rapid building up of the vapour pressure towards the value for a saturation atmosphere.

Increase in temperature certainly brings about an increase in the movement of the water molecules and if the volume of the external atmosphere remains constant a small increase in vapour pressure would result. But this again is nullified by an expansion of atmospheric volume brought about by an increase in temperature.

Thus, within a certain range of temperature, at which protoplasm remains fully active, the effect of rise in temperature always results in an increase in the difference between vapour pressure within the plant and the outside atmosphere and hence an increase in the rate of transpiration.

Wind:

If all other external conditions are favourable, usually a moderate increase in the velocity of wind, results in an increase in the rate of transpiration. It may be due to the effect of wind in removing the near saturated layer of air in contact with the leaf surface.

The result of this removal of saturated atmosphere from the immediate vicinity of the leaf will be a steepening of the vapour pressure gradient that is in a reduction of the distance across which the vapour pressure difference exists.

This will, however, primarily affect the cuticular transpiration and only relatively slightly, the stomatal transpiration. This is due to the fact, that the removal of saturated layer of air in contact with leaf, will alter only the distance across which vapour pressure difference exists in stomatal transpiration compared to the cuticular transpiration, only a little, where the distance is simply from one side of the cuticle to the other.

In stomatal diffusion of water vapour the said distance is from the wet mesophyll cell walls across the intercellular spaces and the stomatal pore to the external surface of the leaf and this distance is only slightly affected by removal of saturated layer from the vicinity of the leaf surface.

The effect of wind in removing the saturated air from the immediate vicinity of the leaf surface is probably of less importance than its effect in causing swaying of branches and shoots and the bending, twisting and fluttering of the leaves.

It has been shown experimentally that immobile leaves usually transpire much less than leaves which are fluttering or bending in the wind, when both are exposed to winds of equal velocity.

Such bending may increase the rate of water vapour loss from leaves in part, by sudden compressing of the intercellular spaces which actually shakes the water vapour out of the intercellular spaces through the stomata into the external atmosphere.

Winds of high velocity may sometimes actually reduce transpiration rate by causing closure of stomata. If the wind produces a cooling effect on the leaves due to increased heat emission, this may conceivably bring about a reduction in the rate of water loss.

Atmospheric Pressure:

The effect of atmospheric pressure on the transpiration rates is in general only of theoretical importance. It can be shown experimentally that a reduction in atmospheric pressure results in an increase in the rates of water vapour loss from leaves.

The theoretical explanation will be that a reduction in the density of the atmosphere would permit the diffusion of water vapour to occur into it more rapidly. However, for all practical purposes, in any given locality, the variation in atmospheric pressure is too slight to have any significant effect upon rates of transpiration.

The high altitude flora shows a general trend of high transpiration rates compared to the flora of the plains, if other soil and atmospheric factors are not limiting.

Antitranspirants:

There are certain compounds which act as inhibitors of trans­piration rate through their highly specific action on guard cells. The most reliable of such inhibitors found, thus far, is phenyl mercuric acetate. This compound closes stomata in many plants (such as Vinca, tobacco, maize, etc.) when sprayed on leaves, at even as low as concentration as 10^-4 M.

From critical experimentation, this compound was found to inhibit primarily the stomatal diffusive resistance—a single treatment may close the stomata for about 6 weeks—the growth rate of the plants, however, remaining unaffected. Several growth retardants also reduce transpiration rates.

Other compounds have also been successfully tried, such as monomethyl ester of nonenyl succinic acid and decenyl succinic acid (10^-3 M), CO₂ etc.

Exudation of Water in Liquid Form:

Guttation:

We have already touched on the phenomenon of guttation in our pre­vious discussions of root pressure and active water absorption. By far the commonest and conspicuous manifestation of root pressure is the exudation of drops of liquid from the edges and tips of leaves.

This process of escape of water in the liquid form from uninjured plants, particularly from leaves, is called guttation. Guttation has been re­ported from more than 300 genera of flowering plants, although there are many species in which it has not been observed.

Guttation occurs more frequently and abundantly under conditions which favour rapid absorption of water by roots combined with reduced transpiration rates. Thus guttation is most common in plants growing in moist warm soil with their aerial parts surrounded by humid air.

Only when the rate of absorption is sufficiently in excess of transpiration so as to cause development of positive root pressure in the xylem vessels, guttation takes place. Conditions which hinder intake of water by roots such as a cold, dry soil, a high concentration of solutes in the external medium, and also conditions favouring high trans­piration rates, prevent guttation.

Guttation can be stopped by reducing the root pressure, for example, simply by watering the soil with a dilute solution of sugar or KNO₃, thus increasing osmotic pressure of the soil solution, which may greatly reduce water absorption by roots.

Guttation is very commonly and frequently ob­served from the tips and edges of leaves of grasses, Nasturtium, Colocasia, tomato, etc., early in the morn­ing, after a moist and warm night.

Guttation occurs through specialised structures known as hydathodes or water stomata or water pores. The term water stomata is very inappropriate, for hydathodes have no osmotically active guard cells but consist of a large opening, below which there is a rather large air chamber, bordered by a mass of thin-walled, loosely arranged parenchymatous cells (epithem). The xylem of a small vein terminates below each air chamber.

The positive pressure originating in the root and developed in the xylem vessels forces water in the liquid form out of the xylem into the intercellular spaces.

The inter­cellular spaces are thus injected with liquid water (under all normal conditions they are occupied by air, always at near saturation point, by evaporation of water from wet mesophyll cells) which floods the intercellular spaces of the epithem, ultimately causing an overflow through the pore of the hydathodes to the exterior of the leaf.

Guttation may also occur through ordinary stomata as in some grasses and legumes. While most plants exude only a few drops of water during an entire night, young leaves of species of Colocasia have been observed to lose as much as 10-100 ml of liquid water in a single night by guttation.

Although Colocasia antiquorum exudes almost pure water, guttation liquid usually contains small quantities of soluble sugars and salts. Instances of injury to the leaves in the nature of tip-burn have been reported in some species of plants where large concentrations of salt are left on the surface of the leaves in the region of apices and edges, when evaporation removes the guttation water.

The white incrustation which is sometimes seen on the surface of the leaves, is also due to evaporation of guttation water, leaving the dissolved salts as a thin layer on the surface of the leaves.

It is probable that guttation results in a thorough soaking of the leaves, which may conceivably make such leaves more susceptible to the attacks of pathogenic bacteria and fungi.

Glands:

Glands are found on almost all parts of the plant. Many of them are nothing but modified epidermal hairs or cells. Nuclei of gland cells are usually rela­tively large compared to the total cell volume. Certain types of glands secrete a very dilute solution, mainly of sugars and salts.

Sometimes nectar, resins, volatile oils and enzymes are also secreted by glands. This exudation of sap is commonly called secretion and is apparently caused by forces which develop within the gland and not by the hydrostatic pressure developed in the xylem vessels. Glands certainly are not closely connected with xylem elements as hydathodes are. The mechanism of the development of the forces causing secretion by glands, is not clear.

The outermost cell layer of the glands located in the lower third of Nepenthes pitchers is an epithelium with cylindrically shaped typical gland cells, having a dense cytoplasm and characteristic cell wall incrustations.

The digestive fluid in the pitcher lumen is secreted from this inner pitcher wall; pitcher fluid contains an appreciable amount of chloride. Nectar secretion, in leaves and flowers, requires the expenditure of metabolic energy and it is also hormonally controlled.

Most gland cells contain a very large number of mitochondria, irrespective of the gland type or substance transported. In the excretion of salt, the role of these mitochondria may be to supply energy for the working of ion pumps.

The hydropote glands of the lower epidermis of floating water-lily (Nymphaea) leaves contain large nuclei; the cell wall-protuberances and the space left in glands are filled with mito­chondria. A high proportion of activity of the transport of at least SO₄^— seems to be associated with mitochondria.

Bleeding:

If the stem of a herbaceous or woody plant be cut or broken, especially in the spring, a slow exudation of sap often occurs from the cut stump. This phenomenon is called bleeding and under certain favourable conditions large quantities of sap solu­tion, as much as 5-6 litres, may be lost in a single day.

This process differs notably from the phenomenon of bleeding from a wound in animals by the fact that whereas the composition of blood of one species of animal is approximately constant, that of the bleeding sap of a plant may vary within wide limits, according to the season of the year and the place of the wound. Bleeding, however, is not a natural phenomenon.

Some plants yield a bleeding sap that contains a high percentage of sugar, up to about 18-20%. They are nearly all monocotyledonous palms.

It is interesting to note that in plants that exude a bleeding sap containing a high percent of sucrose, the bleeding sap originates in the phloem, whereas in many herbaceous plants (e.g., tomato) the exuding bleeding sap is from the xylem.

In the xylem-bleeding sap, the sugars are largely absent whereas the concentration of inorganic ions, especially calcium, is very high.

In some species of plants, bleeding appears to be due to the development of a positive pressure in the xylem vessels—root pressure; in others to pressures developed in the sieve tubes in phloem instead of xylem, and in still others it may be due to locally developed pressure in the neighbourhood of injury.

Wilting Co-efficient:

Wilting co-efficient is a physiological measure of the water relations of the soils. It is defined as the percentage of water content (expressed as % dry wt.) of a soil when the plant or plants growing in it has just reached the condition of permanent wilting.

A permanently wilted plant is usually considered to be one which will not recover its turgidity unless water is supplied to the soil. Permanent wilting is not necessarily an accurate indicator of the wilting co-efficient.

Thus in the seedlings of bean, a relatively large proportion of water content of the plant is stored in the lower part of the hypocotyl. During loss of water, transpiring leaves draw upon this reserve water in the stem when the supply of water from the soil is cut off. Thus the critical point of soil moisture may be reached previous to permanent wilting.

In order to determine the wilting co-efficient of a sample of soil, the sample is enclosed in a water-proof vessel and the ‘test’ plant is generally allowed to develop from seed in the soil until it has attained a small size.

The soil surface is then sealed over so that all loss of water from the soil occurs through the plant. Since no water is added to the soil the plant eventually shows signs of permanent wilting due to loss of water from leaves by transpiration. As soon as this occurs, a sample of soil is removed and its water content determined by drying in an oven.

Before extensive determinations of wilting percentage for plants growing under different soil and climatic conditions, it was supposed that plants differed markedly in their capacity to reduce the water content of the soil.

It was assumed that xerophytes, which endure drought conditions could deplete the moisture content of a soil to lower value before showing permanent wilting than those species growing in normal supply of water in the soil. Extensive investigations have shown, however, that hydrophytes, xerophytes, mesophytes—all reduce the water content of a given type of soil to about the same value, before showing permanent wilting.

While the wilting point of a given soil shows no appreciable variation when mea­sured by means of different plants growing in it, this value varies greatly with the type of the soil. The percentage of water remaining in a soil when permanent wilting of plants growing in it occurs, ranges from 5-10% for sandy loam, 10-15% for silty loam and 15-20% for clay soil.

Thus we see that different soils have different water-holding capacities. The wilting point at which plants can no longer remove water from the soil whatever the texture of the soil may be, sand, silt or clay corresponds to a force of about 15 atmospheres. No root cells can possibly have osmotic pressure higher than 15 atmos­pheres and water held in the soil at a force of more than 15 atmospheres is thus com­pletely unavailable to the plant.

Thus, the amount of water available to the roots of most plants is held in the soil by forces between 0-3 atm. (i.e., at field capacity) and a maximum of 15 atm. The quantity of available water certainly varies with the type of the soil; it is relatively large in fine textural clay and relatively small in a coarser sandy soil.

The force with which water is held in the soil increases with decreasing water con­tent and hence at permanent wilting point it is independent of the species of plant growing in a soil. In general, wilting point seems to be controlled almost entirely by soil conditions and type of soil—it is only slightly influenced by the species of plant growing in it or by climatic conditions to which the plant is exposed.

The significance of wilting point lies in the fact that it is essentially a measure of that fraction of soil water which is unavailable to the plants and is in turn a measure of the hygroscopic and chemically bound water present in the soil.

The determination of wilting percentage under field conditions, presents many difficulties compared to the more or less easy manipulation which could be obtained in the laboratory. Large dis­crepancies are sometimes found between the values determined by laboratory methods and those determined actually in the field, for the same type of soil.

In the laboratory, small water-proof containers are used and effective penetration by roots in the small soil sample can be taken for granted and a fairly uniform reduction of water content of the small soil sample can be assured.

In field, conditions are very different, however, soil is less effectively penetrated by roots, some portion of the soil in the field may be less com­pletely drained of water than others, soil at the immediate vicinity of the roots may be at wilting point while more remote portions of the soil may still be at field capacity, etc.

Other difficulties which stand in the way of correct determination of wilting point under field conditions are the direct evaporation of water from the soil reducing the water content of the soil, local variation in the soil texture, i.e., some parts of the field may have more clay while others, more silt or sand, lack of uniformity of root deve­lopment, etc.

To surmount the difficulties encountered in the determination of wilting co-efficient of the soil under field conditions and from our knowledge that wilting co-efficient is practically the same for a given soil for any plant under all climatic conditions, several indirect purely physical methods have been proposed for the determination of wilting percentage under field conditions.

These methods are based on the relationship of the wilting point to the moisture retentiveness of the soil as measured by certain physical standards. By determining in the laboratory, some factors on the moisture retentiveness of a sample of soil from a field in which the plants are growing, wilting co-efficients can be calculated quite accurately for all types of soil. In these determinations of wilting co-efficients, the part played by plants is altogether eliminated.

Large scale determinations of wilting co-efficients have been one of the most important routine work in ecological and agronomic ones in recent times. By deter­mining the wilting points of different crop fields, we can obtain some idea about the texture of the soil—suppose, the wilting point of a field is about 15%, we can guess that the soil is mostly clay, whereas a value of about 5-10% will show that the soil is mostly sandy.

Consequently if the soil is clay, more water is needed for normal growth of the plants and water must be added to the soil by irrigation when there is no rainfall than when the soil is sandy where less irrigation-water will be needed.

Of the various physical methods employed for the determination of wilting percentage of soils, the two most commonly used are following:

(1) Relation of the wilting percentage to the moisture equivalent of the soil. The moisture equivalent of a soil is defined as the percentage of water that a soil can retain in oppo­sition to a centrifugal force 1000 times that of gravity.

Wilting co-efficient =moisture equivalent/ (1.84 ± 0.013).

(2) This method depends upon the determination of percentage of coarse, medium and fine particles in several samples of a soil from a field and then multiplying the three with three constant factors and finally adding them up.

Wilting co-efficient =percentage of sand (coarse) particles x 0.001+percentage of silt (medium sized) particles x 0.12+percentage of clay (very fine) particles x 0.57.