Historical Perspective on Plant Physiology!
It is important to know the history of plant physiology in order to understand the contemporary concepts in this subject.
Looking back, we find that the progress of science was for obvious reasons very slow in the eighteenth and the first half of the nineteenth century. However, the importance of photosynthesis concerned only the gas exchange which accompanies the process. Ingenhousz in 1779 demonstrated that plants purified the air only in light, whereas in the dark the same plants made the air impure.
In 1804, de Saussure published his work in which he confirmed the findings of Ingenhousz regarding two types of gas exchange, one in the presence of light and the other in darkness. He also demonstrated that in the presence of light, green tissues alone carried out CO2 absorption and O2 evolution concurrent with water utilization, ultimately leading to a gain in plant weight.
In the latter part of the nineteenth century and the early part of the twentieth century, investigators studied the factors which influenced the rate of photosynthesis and the relative importance of each of these.
In 1943, Liebig’s law of the minimum was proposed which stated that if in a given soil one essential mineral was deficient or absent, while others were abundant, growth would not be possible unless the deficient element was added to the soil.
In 1905, F.F. Blackman published a paper, ‘Optima and Limiting Factors’ in which he explained the influence of several separate factors on a process, the rate of which is determined not by all these factors at once but by one or two which are most deficient and this is known as the ‘principle of limiting factor’.
In 1826, the theory of osmosis was published by Dutrochet and in the middle of the nineteenth century, important contributions to plant physiology were made by Boussingault.
However, a rapidly expanding activity under the leadership of Sachs and his contemporaries was initiated in the second half of the nineteenth century. Thus, by 1900 there was a fairly good understanding of plant structure and of the major physiological processes of plants such as photosynthesis, respiration, plant-water relations, mineral nutrition and translocation.
During the first 30 years of the twentieth century, many important contributions were made, many of which were based on an extension of earlier work, resulting in an increase in the knowledge of plant processes. This was possible mainly because of improvements in experimental techniques.
The work begun by Traube, Pfeffer, de Vries and others in the second half of the nineteenth century paved the way for further work on permeability and salt accumulation. In 1990, ten elements were recognized as essential to plants and more elements have been added to the list with the passage of time.
An entirely new concept was developed by Renner, Ursprung and Blum in the area of water movement which was shown to depend on suction force or water potential gradients rather than on osmotic pressure gradients.
Turning to the problem of mineral nutrition, we find that a number of extensive investigations were undertaken to determine the elements which are essential for higher plants.
The early investigations conducted by the method of solution culture in the later part of the nineteenth century by the German scientists Sachs and Knop led to the development of the classical concept of ten essential elements. This concept was challenged and it was later realized that a few more elements are also essential for plant metabolism.
In 1939, two American plant physiologists, Arnon and Stout, suggested the criteria to determine the essentiality of elements for plant nutrition. Through the research of several workers like Broyer et al., Nicholas, Bollard and Butler, it is now established that sixteen elements are essential for the growth of all higher plants.
The water culture technique has been popular for growing plants either in the laboratory for experimental purpose or for the production of crops on a commercial scale. Such a procedure is termed as hydroponics or soilless growth of plants and the nutrient solution formulated for this purpose by Hoagland and his associates is still used with success.
In addition, foliar nutrition is utilized in modern agriculture to supply fertilizers to crop plants. In this method, mineral nutrients are supplied as foliar sprays in order to get quick effects, besides the usual addition of chemical fertilizers to the soil.
The discovery of photosynthesis is a thrilling chapter in the history of science. This is the basic process of life which harvests the energy of sunlight and converts it into a form that can support the life of organisms, produces organic food material out of inorganic compounds, and maintains the oxygen reservoir of the atmosphere.
It is quite obvious that the conversion of CO2 to carbohydrates with the help of a reducing agent, viz., NADPH provided by the photochemical reaction is the most thoroughly studied area of photosynthesis. Tracer technique using the radioactive carbon isotope 14C was applied to understand in detail the reactions involved in the reduction of CO2 to carbohydrate.
Melvin Calvin of California together with A. A. Benson and J. Bassham and other co-workers proposed in 1954 the path of carbon in photosynthesis and for this unique contribution this pathway is named as the Calvin cycle or the C3 cycle; Calvin was awarded the Novel prize in 1961.
In this pathway the identification of two key intermediates, PGA and RuBP, was an important landmark in the study of metabolic reaction sequences. Research on crop plants, particularly tropical species, is rewarding both in terms of physiology and agronomy.
An example is the discovery of a more efficient mechanism of CO2 uptake through PEP carboxylase by physiologists of the Hawaiian Sugar Planters Association. This led M.D. Hatch and C.R. Slack (1966, 1968) to suggest an alternative pathway of CO2 fixation called the C4 dicarboxylic acid pathway or the C4 pathway.
The process of photorespiration was found to be associated with C3 plants which fix CO2 by RuBP carboxylase which has also been found to behave as an oxygenase producing glycolate, a C2 compound. Glycolate is the source of CO2 in photorespiration in which process considerable quantities of early products of photosynthesis are respired away.
This pathway of glycolic acid (a C2 compound) metabolism has been shown to be a cycle of reactions, termed as photo-respiratory carbon oxidation cycle which has led to the formulation of the C2 cycle of photosynthesis.
In 1937, Robin Hill of Cambridge University was studying the role of H2O in photosynthesis in a cell free system. He observed that isolated chloroplasts when illuminated were able to produce O2 in the presence of a suitable oxidizing agent. This reaction is known as the Hill reaction and the oxidizing agent is known as a Hill reagent.
It was an early speculation that in photosynthesis also, some light energy may be stored in the form of high energy phosphates; photosynthesis was also shown to be associated with ATP synthesis. Photosynthetic phosphorylation was demonstrated in 1954 by D.I. Arnon and his associates in illuminated chloroplast preparations.
The sequence of reactions of biological oxidation which involves the breakdown of respiratory substrate is generally referred to as respiratory metabolism and it involves a series of reactions, each catalyzed by a specific enzyme. The metabolism of pyruvate to CO2 and H2O under aerobic conditions takes place in a cyclic manner.
This cyclic sequence of reactions was described in the 1930s and is known as tricarboxylic acid cycle or citric acid cycle. It is also known as the Krebs cycle after the name of Hans Krebs, the English biochemist, who first recognized its functional role.
ATP which is regarded as the energy currency of living cells is generated from ADP and inorganic phosphate by a process known as oxidative phosphorylation which is dependent on electron transport along the respiratory chain of electron carriers.
In the early 1960s, Peter Mitchell, the English biochemist, proposed the chemiosmotic theory to explain the mechanism of ATP synthesis which is widely accepted. According to this theory, the energy of electron flow in the respiratory chain is partially conserved as a proton motive force which drives the formation of ATP from ADP and Pi.
Biological nitrogen fixation is the major route by which gaseous nitrogen is introduced into the ecosystem and only certain prokaryotic organisms are able to carry out this process. One of the wonders of nature is that some bacteria in association with plants can reduce nitrogen to ammonia by means of an enzyme nitrogenase.
A cluster of genes, known as nif genes are responsible for this phenomenon.
If we can transfer these genes from nodulated legumes to cereals and make them function, the growing crops will directly assimilate elemental nitrogen from the atmosphere and the problem of procuring costly chemical fertilizers can be partly solved. It is known that the desirable traits like nif and Hup genes are borne on plasmids or extra-chromosomal DNA.
Techniques are now available through modern methods of genetic engineering and biotechnology for the incorporation of these genes into both bacteria and the host so that nitrogen fixation can be largely increased. Most important contribution in this area has been made by Postgate (1982), Sprent (1979), Newcomb (1981) and Veeger and Newton (1984).
In the 1 920s, F.A.F.C. Went enunciated the proverb, “no growth without growth substance”. It was realized that cells possess an inherent capacity to grow and for this purpose they need a great variety of non-nutrient substances which promote cell multiplication, cell enlargement and cell maturation. This led to the development of the concept of plant hormones.
In the classical work of Charles and Francis Darwin published in 1880, the responses of grass coleoptile to light and gravity are described and it was suggested that the power of movement of grass coleoptile is controlled by hormones.
In the excellent treatise ‘Phytohormones’ published in 1937, F.W. Went and K.V. Thimann contributed immensely to the knowledge of auxins which were isolated from plant source in 1934 by Kogl and Kostermans and in 1935 by Thimann.
According to some authors, an irreversible increase in volume of the plant is synonymous with growth and auxins are the substance that are responsible for this. For about a quarter of a century after the identification of their existence, auxins reigned supreme as the only growth-regulating substances in plants.
However, the coleoptiles, the experimental material used for auxin research could only grow by cell extension. This placed limitations on learning more on the subject of growth and the active agents that could influence it. Thus attention was turned to agents that promote growth in systems in which cell division is an important part of the response.
The second natural growth regulator, the gibberellin was discovered only by chance. In Japan, Kurosawa was trying to determine as to why rice seedlings infected with the fungus Gibberella fujikuroi become excessively tall as compared to their non-infected counterparts.
In 1962, he showed that the fungus could produce a growth-promoting substance which was later isolated and named gibberellin. Brian et al. (1955) and Cross et al. (1961) worked out its structure. The gibberellins are present in higher plants was demonstrated by Phinney et al. and it became clear that gibberellins play an important role in the control of growth and development.
Equally interesting is the history of discovery of cytokines. In the 1950s, Folke Skoog and his associates at the University of Wisconsin used coconut milk to grow pieces of tobacco tissues while looking for other possible sources of growth factors. They found that such a factor was also present in yeast extract in soluble form the absorption spectrum of which showed that it was a purine.
Since purines are constituents of nucleic acids, one of Skoog’s associates, C.O. Miller used ‘Herring sperm DNA’ from an old bottle instead of yeast extract and this material proved to be capable of inducing tobacco cells to grow and divide. When the old bottle was used up, fresh DNA was brought and used but it failed to show bioactivity.
Then the laboratory shelves were searched for old samples of DNA all of which proved to be active. So Miller took fresh DNA, aged it in an autoclave following which it became active. From this observation, the logical conclusion is that the growth-promoting factor is the breakdown product of nucleic acids.
Skoog, Miller, Strong and others isolated the factor which was a derivative of adenine, one of the purine bases that make up nucleic acids (1955). Because of its specific effect on cytokinesis, the newly isolated product was named kinetin (1956).
Ethylene which is a gaseous plant hormone is unique in its structural simplicity. The growth- regulating properties of ethylene were first recognized in 1901 by the Russian botanist Neljubow who reported that illuminating gas from burning coal contained ethylene which effectively caused growth and gravitropism in dark-grown pea seedlings.
Subsequently, ethylene was shown to be a component of various fumes, smoke and industrial gases which were shown to exert growth-regulatory effects. That ethylene is a natural plant product was positively proved by Gane in England in 1934.
Soon afterwards, Crocker, Hitchcock and Zimmerman (1935) established that ethylene is a fruit-ripening hormone and ripening fruits synthesize high concentrations of ethylene. Adaptation of gas chromatography to ethylene analysis has made possible its measurement in a plant tissue most accurately. This largely stimulated ethylene research in the 1960s giving it a rank of an endogenous hormone.
The history of the discovery of abscisic acid is also very interesting. In the early 1960s, several groups of workers were independently attempting to isolate and purify a growth-regulating substance. One group led first by H.R. Cams and later by F.T. Addicott of the University of California at Davis, was interested in the possibility of isolating a substance from plant tissues which would accelerate leaf abscission.
They obtained from young cotton bolls two partially purified fractions which accelerated leaf abscission in young cotton seedlings. They called these abscisin I and abscisin II.
Further work was concerned with the isolation and identification of abscisin II. At the same time, K. Rothwell and R.W. Wain at Wye College, London University, following the earlier work of R.F.M. van Steveninck in New Zealand, were attempting to identify a substance which accelerated flower-drop in lupins (1964).
Following a quite different approach, P.F. Wareing and his co-workers at Aberystwyth, Wales, U.K. were trying to determine as to what makes trees stop growing in autumn and causes them to form resting buds and thus become dormant.
Wareing et al. obtained from sycamore (Acer pseudoplatanus) leaves an acidic extract which was highly active as a growth inhibitor and which was able to induce the formation of resting buds in sycamore seeding’s when applied to their leaves.
They gave the name ‘dormin’ to this active substance. Further purification of dormin was taken over by J.W. Cornforth at Milstead Laboratory of Shell Research Limited, England and this resulted in its isolation in crystalline form. Abscisin II, dormin and lupin flower abscission factor proved to be one and the same compound.
At the Sixth International Conference of Plant Growth Substances held in Ottawa (1967), the new name abscisic acid which gives an indication of the compound’s chemical nature was approved.
The transition of plants from vegetative growth to flowering is the most essential period in the ontogenesis of plants. It is now widely recognized that morphologically a flower is a transformed leaf shoot. The earliest concept of the physiological nature of plant flowering was based on the hypothesis of Sachs on flower-forming substances which was first published in 1880.
In 1918, Klebs put forward his theory of the role of the ratio of nutrients. A new basic idea about the physiology of flowering appeared at about the same time when Gassner (1918) found a difference in the requirement of temperature for flowering among various plant forms and discovered the phenomenon of thermo-induction in plants.
The term verbalization was introduced by Lysenko (1948) which is in fact the thermo-induction of winter forms caused by low temperature during a certain period.
An important role in the flowering of annual and perennial plants is played by reactions to the day length, i.e., photoperiodism and to low temperature, i.e., vernalization. W.W. Garner and H.A. Allard reported in their papers of 1920 and 1923 the results of experiments performed with the giant tobacco, commonly known as the Maryland mammoth.
They concluded that the time of blossoming of many kinds of plants may be determined by the length of the daylight period which under natural conditions varies from season to season in latitudes north or south of the equator.
The photoperiod that promoted early flowering of the Maryland Mammoth tobacco was termed as short day and this plant was referred to as a short-day plant.
The terms ‘photoperiod’ and ‘photoperiodism’ were coined by them. Regarding the metabolites necessary for flower formation, Chailakhyan (1937) suggested that plant flowering was concerned with substances of hormonal nature formed in leaves known as ‘florigens’, the identity of which is till not clear.
It was further suggested that the flowering hormones or the florigen complex included two groups of substances formed in leaves:
(i) gibberellins which are necessary for growth and elongation of stems, and (ii) substances which are necessary for flower formation called anthesins, the hypothetical hormonal substances.
It was further conjectured that L-D plants do not flower under S-D conditions because of the shortage of gibberellins, while S- D plants do not flower under L-D conditions because of the deficiency of anthesins.
Following the discovery of the vernalization phenomenon. Melchers (1939), concluded that vernalization results in the formation of specific substances of hormonal nature known as ‘vernalins’ which also are hypothetical substances.
Lang (1965) has proposed that there is a direct connection between vernalin and florigen, i.e., the complex of flowering hormones, which is as follows:
low temperature → vernalized condition → vernalin → florigen
Thus the history of plant physiology indicates that rapid advances in knowledge always resulted from improvements in research techniques. These improvements in instrumentation have enabled scientists to measure different physiological processes with great accuracy and rapidity, thus increasing the scope and quantity of research.
A thorough understanding of many important plant processes was impossible till the advent of many sophisticated instruments.
Now instruments can measure biological parameters in seconds, automatically programmed by computers. With the help of isotopes of phosphorus, carbon and sulphur, we can find a single gene in a cluster of chromosomes or a single protein with the help of antibodies specific for that protein.
The invention of thermocouple psychrometers made it much easier to study the effects of water stress on plants, whereas stomatal behaviour can be measured with the help of diffusion porometers. Quantitative measurements of CO2 uptake by many kinds of plants growing in a variety of environments have become possible with infrared gas analysers which measure CO2 accurately.
The availability of radioactive isotopes made it possible to study the uptake and translocation of ions and organic compounds, the carbon pathway in photosynthesis and the successive steps in various other metabolic processes.
Biotechnology or genetic engineering is the present-day trend for the improvements of plants. It is the manipulation of genetic material in various organisms. In principle, genetic engineering refers to the techniques of adding, subtracting or replacing segments of DNA which actually carries the information of life.
This new discipline of biology started at MIT, USA, when Nobel Laureate, H. G. Khurana and his school first reported total synthesis of an artificial gene with the potential for functioning inside a living cell.
Phenotypic expression of an organism takes place through the biochemical processes, that are the result of information encoded within the base sequence of DNA of the organism’s genome as well as its interaction with the environment.
Now, the scientists are able to isolate and produce unlimited copies (cloning) of specific nucleotide sequences (genes). This has enabled plant physiologists to study physiological phenomena by identifying the genetic regulatory mechanisms that control physiology.
Genetic engineering has been utilized in many ways in the field of agriculture, particularly for the protection of crop plants against pests, viruses, and herbicides. About one-sixth of the global food production is lost by insect pests. But the continuous use of pesticides damages the environment. Bacillus thuringiensis that produce BT-toxins is used as alternative biological insecticide.
Non-selective herbicides can be used as selective herbicides by genetically transformed herbicide- resistant plants. Glyphosate-resistant cotton, rapeseed, and soybean have already been produced. Similarly glufosinate-resistant maize and rapeseed have also been licensed for cultivation.
Improvements of yield and quality of crops through the generation of transgenic plants have now been achieved. Transgenic tomato have been raised with altered ripening process and improved quality and storage stability.
Nowadays, transgenic plants are being produced to increase the stress tolerance of cultivated plants through alteration or over expression of gene involved in stress resistance. In conclusion, plant physiology with aid of gene technology has a fundamental role to play in the struggle for food supply in the world.