Proc. Indian Acad. Sci. (Plant Sci.), Vol. 93, No. 3, July 1984, pp. 231-244 �9 Printed in India.

Piant celi physiology (1934-84): Recollections and reflections

F C STEWARD Professor Emeritus, Comell University, Ithaca, New York, USA Mailing address: 1612, Inglewood Drive, Charlottesville, Virginia 22901, USA

Keywords. Plant cell physioiogy;, metabolic machines; osmotic machines; ion accumulation; growth promoting substances; trace elements; solute composition, development.

1. Prologue

This essay relates largely to the past; this prologue sets it in the context of the present. An invitation to cont¡ to the golden jubilee celebrations of the Academy

founded by Sir C. V. Raman, Nobel Laureate in physics---is not to be treated lightly. If the responsibility ig accepted how should it be discharged? A piece of o¡ research dedicated to this end should not be t¡ and merely to recapitulate would also be unworthy. This essay therefore will not stress specific cont¡ with their cited observations in support--to do so would merely invite becoming submerged in a mass of details. A different airo is to view plant physiology more comprehensively and to see how it has fared in a period of great advances in physical science. This is best done through salient topics with which the author was in touch throughout and which invariably distinguish living plants from inanimate systems. Although the account is brief its scope is broad.

As plant physiology emerged into the twentieth century the trend was already set upon applications of a rational system of chemistry on the one hand and on developments from the cell theory on the other. The main focus today is upon causal physicochemical explanations of vital phenomena. The pe¡ in question (1934-1984) was dominated by technical advances in plant biochemistry, intermediary metabolism, enzymology, and the philosophy of genetics and molecular biology. The prevalent trend was toward reductionism, i.e. the attack upon problems and systems reduced to units sucia that they may be separately comprehended. This approach may seem to be vindicated by the wealth of information that it has yielded. But how lar does the summation of that information comprehend the life of the organistas in question?

The reactions of intermediary metabolism sponsored by innumerable enzymes, each seemingly gene-controlled, now seem overwhelming. Any modern text that summarises biochemistry readily overflows with charts that link reactions into schemata and schemata into concepts, whether of photosynthesis and respiration as they involve carbohydrates and organic acid metabolism or the biochemistry of nitrogen com- pounds for the amino acids and protein metabolism synthesis and breakdown. The speci¡ reactions and systems that relate to other broad areas of biochemistry have also proliferated. But most noteworthy is the recognition of phosphate bond energy as the energy currency through which specific energy requiring tmnsactions are negotiated as it has pervaded all of biochemistry and physiology.

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232 F C Steward

The combined resourcr ofradioactive labelling ofsubstrates and ofvarious forms of chromatography (now vr far advanced from the first efforts in tbese directions early in the post World War II period) have produced volumes of data (often described and interpreted by scir art forros as in Scientific American). These now convey an air of certainty to events that are presumed to occur at templates, on membrane surfaces and in organelles.

Membran• phenomena; and what hitherto seemed to be the rdatively simple events of their pr to water and to solutes have been overtaken by concepts matbematical and mechanistic, that at the turn of the century and in the wake of the classical period of cell physiology would have been inconceivable.

But not withstanding the imprr scope of all these and otber events the over- riding considr still involvr the organisations in nature that make them feasible and the forcr and enr relations that integrate them into a coherr working whole.

The cell theory and the ¡ thoughts about protoplasm and the cell nucleus awaited the slow but nevertheless dramatic ris• of submicroscopic morphology. This was later int•rprr by electron microscopy and its satellite techniques. The picture that emerged was of ah internal univers• of complexity in which the vital events occur in cells and their organell•s. So the over-riding questions that have still to be faced concern the means by which all the attributes with which cells ate endowed are motivated to proceed harmoniously in time and spacr and compatibly with ah evolutionary history, on the onr hand, and an inbefited d•velopmental plan, on the other.

But all these questions and interprr at the cr level must also be comprehensible at the level at which cells are organised into tissues and organs and, in higher plants especially into the growing regions from which tbey derive. Morphology and embryology, which in their first descriptiv• phases wer• predominant areas of biology were later overwhelmed by physiology, biochemistry and nutrition etc, have now come again into their own. It is through morphology at all levels, from the molecular to the subeellular, and in the growing regions of plants that milieux are created in which events arr shaped that otherwisr are difficult to comprehend in physical and chr terms.

Tbere is a paradox here that may be illuminated by the following story. In the 1920's, as an undergraduate in cbemistry at Leeds, the writer came into contact with an able laboratory demonstrator, who shall remain anonymous, but who was tben frr from Oxford. His first work had ber in the discipline of crystallography. However he had forsaken this subject becausr as he said, it was on• in which aU was seemingly known, in the sensr that all oftbe feasible crystallographic forms were thr known to occur. So he left crystallography for spectroscopy which he deemed closr to the frontiers of science. But whatr162 the wisdom of otherwise ofthat choice, crystallography was about to take on a new and unexpected scope. The repeating regularity of molecular structures in spac•, whetber in polysaccharides (as in cellulose) or in nitrogen compounds (as in proteins and the dbonucleic acids), essentially in tbeir structures display the features of the crystallographic state. They also extend its interpretations into more dynamic spberes which have brought this type of physical structure of matter close to the very mysteries of life.

But also in the 1920's and •arly 1930's one used to challr students with the numerous reasons why naturr built so much complexity upon the compounds of carbon with carbon and also with nitrogen. But why not silicon? Little did we then know how much nature could harness the storage of usable information and energy,

Plant cell physiolcgy 233

into the geometry of arrangements of carbon with nitrogen and phosphorus, while it left to silicon the basis of the chemistry of minerals. Strangely perhaps, we then knew so little about the near miraculous properties now known to flow from the recoverable storage of useful information on silicon chips.

Thus we should now look beyond the present glut ofinformation about plants, their metabolism and chemical working to seek in their organisation at all levels (from moler to morphology) the clues to tbeir sur without violating the p¡ of matter and energy as they operate in the material universe. The 'message' could stop here, but ir will be pardonable to present some illuminating sequences-7-known to the writer through personal experience--that illustrate how this general philosophy carne about.

One may begin with problems of plant cells as they absorb and hold water and solutes from the ambient media. In plants as indeed in animals water is the most abundant natural constituent. In fact nature imposes restrictions upon the water of the animate world, which distinguish ir from the inanimate world. But plants in the waters of the earth, or in their terrestrial habitats create and maintain distinctive compositions which set them apart from their environments. This is no mean physical achievement for it involves the organisation we call life and, with dissolution and death, uniform physical equilibria return. Problems of membranes at boundary surfaces inevitably arise to know how lar their behaviour is intelligible in terms of familiar physicoche- mir principles. Viewed as chemical working machines the ceUs of plants in their metabolism have developed and use a great and growing array of biochemical substances and systems so that, over the 50 years in question, a body of bior knowledge inconceivable at the beginning of Raman's r (or reine) has emerged. Also, since metabolism works, perforce, according to an inherited plan genetic transmission of the metabolic information and the mystery of its regulatory control has loomed large in the period.

Whereas in the pe¡ physicists and astronomers have presented a truly awe inspiring picture of the material universe, biologists may now add to cosmology their own vision of complexity in an internal biological universe (inner space vs outer space) that must nevertheless also operate according to a physical plan. This plan, however, contrasts with the cosmic one which operates over the vast distante of time and space for it is comprehended within the sub-r and even molecular dimensions within cells and their organelles. But cells are also organised externally into tissues and organs and, in higher plants, they a¡ from growing regions. Thus within the plant body other challenging problems oforganisation and integration arise. Asa higher plant grows and develops it creates a morphology that provides the internal environment in which metabolising cells function in many organ specifir ways. In turn morphology is often modulated by environments. Ir science is viewed as an adventurous journey into the unknown, as indeed it has ber in the physical universe, then the challenges of plant r and plant physiology are as arresting, though perhaps more attainable, as are those of astronomy and cosmology.

Time was when the style and title of natural philosophy could embrar all the enquiries being made by man about his world. As knowledge increased specialisation ensued. Mathematics, astronomy, agriculture, medicine, the natural scienr chem- istry, physics with botany, zoology and bacteriology and later genetics appeared as distinctive disciplines. Thenceforward the process of specialisation and reduction in the scope ofa field of enquiry to be covered proceeded. More and more was expected about

234 F C Steward

less and less. As exciting new developments occurred at the overlapping margins of the more traditional disciplines they gained recognition and support by labels under which they flourished f o r a time. Cytology, general physiology, cell physiology, general cytology, biochemical cytology, dynamic biochemistry, enzymology, biochemical genetics, molecular biology, biological and genetic enginee¡ and so on. But the loss of the erstwhile simplicity ofsubjects was more than ¡ in the terminology of substances and systems, involved in natural processes. Acronyms, often meaningless without a glossary multiplied and the modern terminology of complex organic structures and ofenzymes, is a linguistic nightmare. Though meant to achieve precision ir certainly has complicated communication to the point where one wonders whether truth as properly understood, needs to be so 'unrecognisable'.

Often newly identified areas of specialisation acquire their identity through an advance made in laborato~ technique or in instrumentation. As each new area is pursued to the limit it reaclies the point where further understanding demands that it be seen in a wider context. Thus the first gains achieved by speciaiisation and reductionism need then to be balanced by, or set in the perspective of, an enlightened holism. Some landmarks along this route may now be noted.

2. Towards plant cells as metabolic and osmotic machines

Cell physiology, an off-shoot from the cell theory in the mid 19th century began to pay plant physiological dividends in the late 19th c•ntury as the foundations of the subject were laid and modern plant physiology developed through the first quarter of the 20th cr But, ev•n as early as this, it was evident that physical and botanical sciencr were to be joint beneficiaries of the new knowledge. The names of de Vries, Pfeffer, Hoffmeister, van t' Hoff and Arrhenius, became as familiar in the 1920's to students of physical chemistry and of botany as indeed were the terms osmosis, osmotic pressure, isotonic solutions, plasmolysis etc. The study of the "attraction for water" of solutions, whether they, were bounded by "semi-permeable membranes" supported by porous pots or the membranes of living cclls, gave rise to a theory of solution that brought the gas laws and gas pressure into harmony with what carne to be called osmotic pressure. Thesr relationships were made intelligible through the colligative properties of solutes that operate by reducing the vapour pressure (or the escaping tendency) of the pure solvent. Once seen, these relationships, based on strict thermodynamic proof, are powerful testimony to the role of water in the physiology of plants which nr162 not be obscured by the modern craving for frequently revised terminology and for elaborated symbolism (whereas P for osmotic pressure earlier sut¡ the greek alphabet is now fully exploited from E to ~b!).

Plant cr as osmotic systems, bounded by membranes, invitr studies and speculation upon the nature of these membranes and of their permeability properties but also aroused speculation upon how cells acquire their internal solute concentra- tions, especially where these are heavily composed of inorganic ions that only occur in the waters of the earth at low concentrations and which as charged ions, do not readily enter the cells across their boundary membranes. Concurrently much was being learned about the nature properties and structure of limiting membranes at the surfacr between immiscible phascs (of which the water/air surfacr is a specific case in point). Substancr that reduce the interfacial tension tend to accumulate there and their

Plant cell physioloay 235

behaviour in oriented thin ¡ like "gases in two dimensions" when expanded or like liquids, even solids, when compressed allowed them to recapitulate in films many of the familiar properties of matter in bulk. Al1 this brought a great body of knowledge about molecularly oriented thin films to bear and to generate speculation upon the role of membranes in cells. But questions arose whether such membranes play in cells a passive physical role o ra more active and dynamic one by virtue of the fact that they are an intimate part of a system that is essentially alive.

3. The accumulation of ions: osmotic work by cells

In 1929 a happy circumstance brought together Hoagland (of the Division of Plant Nut¡ at Berkeley, California) and the w¡ (then a Rockefeller Foundation Fellow on leave from Leeds, University in England). Prior work in Leeds and at Cornell University had focussed attention on the ditticulty with which solutes (especially ions) traversed isolated membranes composed of plant cells and the great tenacity with which slices of such tissue retain their solutes, even against water under aseptic conditions. In the mid 1920's, Hoagland, directed to Nitella by Setchell, an algologist, discovered that the Nitella cells could "accumulate" bromide ions from very dilute solutions in pond water in the light but not so in the dark. Hoagland very percr reeognised that this was a case of cells deriving energy from their own metabolism, fuelled by photosynthesis, to drive a non-r movement of ions. Hoagland used Nitetla asa model system but he was ultimately interested in roots and their relations to the very dilute soil solutions from which they also accumulate ions. To find out the conditions under which thin slices of potato tuber tissue, without any direct access to photosynthesis, could also accumulate bromide led to the important discovery that these cells harness their aerobic respiration to the process. To enable the potato cells to pr the act of ion accumulation they responded to the necessary oxygen supply in well aerated solutions and they liberated carbon dioxide from stored starch which was hydrolysed as the cells were activated in response to aeration (This work anticipated by many years the widespread recognition of"active transfers" of ions and solutes driven by metabolism).

From this point on (1929 to 1934) progress was made both in Leeds and in Berkeley along lines that foretold much of the later developments in this field, developments which iUustrated how dependent plant cell physiology was to become upon physical and biochemistry, on the one hand, and upon a perceptive insight into the role of oflls on the other; even ultimately On the behaviour ofcells being stimulated to grow and so to perform cell physiological functions that they otherwise could not achieve.

4. Towards a synthesis of form composition and energy

In retrospect the year 1934 marks a stage in this work and in its philosophy. Aftera further pe¡ of work in Hoagland's laboratory (1933-34) and also periods at the Dry Tortugas to study Valonia in its habitats an opportunity was seized to take stock. This occurred in an interlude on the Sands of Monterey, California.

Using the thin discs of potato tuber methods had br worked out to study the effects of different variables on the salt and water relations of the tissue and the concurrent effects on its metabolism as they were reflected in the respired carbon-

236 F C Steward

dioxide and the conversion of soluble non protein-N to insoluble protein-N. Meanwhile Hoagland had perfected the use ofexcised root systems of barley so grown that they hada vica¡ ability for short periods to absorb large amounts of KBr ions whilr concomittantly, they emitted CO 2 as organic acids declined. The two systems had illuminating points of both similarity and difference.

It was already known that the cells of the potato discs could in moist air alone, retain their viability for long pr and even draw upon their stored organic nitrogen (amino acids) to nourish the cells when they eventually divid•d in the formation of a phellogen. (In fact the potato cells were autotrophic for substances that were later to be rr as necessary for other systems o f mature cells to undergo cell division). In consequence the potato cells would retain their active metabolism and ability to accumulate ions (K and Br) for relatively long pr241 Moreover different conditions which affected the tissue did so in ways that also tended to influence, even to suppress, the salt intake and also the synthesis of alcohol insoluble-N (protein).

But the cells of the barley roots were different. Shorn of their shoots and without any ability for secondary growth the barley roots did not rely upon new growth or protein metabolism to foster salt uptake but replaced organic solutes accumulated by those cells during their earlier development under conditions such that their normal complement ofsalts was trasferred to the shoot in exchange for high sugar and organic acid content sent to the roots. When excised these barley roots therefore enjoyed a brief pr241 as "as high sugar low salt"-roots which accumulate I(. Br- ions in exchange for solutr already accumulated during their attachment to the shoot and without a surge of new protein synthesis of new cells.

Thus, at this pe¡ general schemata were drawn up that related the ability of different systems to accumulate ions using the energy of metabolism to do work and with the duration and extent of the absorption process being related to the ability of the cells for new growth and to the prior nutrition they had received.

Premature attempts were made at Berkeley to see whether the use of metabolites to produce metabolically useful energy could be detr by calorimetric methods to measure energy output and to err balance shr to trace the uses made of the endogenous substrates. Although this work showed how such balance she•ts could be prepared they did not, and could not, identify specifically a measurable moiety of metabolism and energy that could be allocated solely to work done in accumulating salts. The picture was, and is, that the conditions established the status oran on going active metabolism in the cells and, once this was established, the cells took the process of accumulating salts in st¡ along with other concomittant events (e.g. protein synthesis etc.).

It was recognised, however, that further clues to understanding the continuing process ofions absorption in the potato and similar systems were need•d and especially its relation to further growth in the cells and to the part played by the nitrogen compounds. With the methodology then available there was just time before the outbreak of World War II to trace out the effects of different variables on the endogenous nitrogen compounds and the conversion ofstored starch to carbohydrates to CO2 etc and their relations to bromide accumulation. AII of this contributed to what was termed the biochemical background against which ion accumulations occurred. AII this was very much more than a problem related solely to membranes. (The results w•re writt•n-up sent from London to Hoagland who communicated them to Plant Physiology even during the war). But the intimate relations ofcell growth and protein

Plant cell physiology 237

metabolism to ion accumulation had to wait until after the end ofWorld War II. As this story unfolded it showed again how intimately and indissolubly the problems of cell physiology became integrated with cell growth and development and can only be fuUy understood when this is appreciated. Indeed even in Valonias and Nitella their normal sap composition in their habitats had reflected this fact.

5. The af termath of war: a new approach

A fresh start in research provided the stimulus to seek a more versatile system than even the thin discs of potato tuber. The airo was to seek a system amenable to work under completely aseptic conditions, in which the cells could be placed for long periods under conditions conducive to their most rapid growth by cell multiplication and/or enlargement and, alternatively, under conditions of maintained quiescence with minimal growth. AU this required that external media should contain nutrients, organic and inorganic, as well as any special exogenous stimuli. To achieve these ends attention was turned to the so-called tissue culture methods then in use by White and Gautheret, although, in point of fact the modus operandi finaUy adopted for secondary carrot phloem explants was entirely redesigned to fit the needs of these investigations.

At that time the field of growth promoting substances (which has since proliferated enormously) was still dominated by the almost exclusive attention paid to IAA as the universal auxin. In the outcome the events (circa 1950) which transformed the approach to problems of salts and water in plant cells also impinged upon many other areas of enquiry. This was especially true for the following problem areas which were profoundly affected.

The growth promoting substances involved in the induction of growth in otherwise mature or quiescent cells. The nitrogen and protein metabolism of plants, especially the composition of the endogeneous, nitrogen compounds and their mobilisation during growth induction and protein synthesis. The aseptic culture of isolated explants of plant tissue and especially as this is carried out in liquid media. The ability to produce and culture free somatic cells. The totipotency of free ceUs and their ability to behave as somatic embryos and thence to produce clonal populations of plantlets. Finally the interest in the organ specific solute compositions of ceUs during normal ontogeny, was revived and especially as this may be subject to environmental effects upon morphogenesis; effects that originate in the growing regions.

Because so many inter-related topics were affected in these ways the full impact ofall cannot be separately stressed here. It can be seen, however, by reference to other publications. A general point may, however, be made.

Whenever it becomes the vogue, even fashion, to attack problems in a prescribed, acceptable way real progress may become deadlocked. This undoubtedly occurred in the belief that many problems should be attacked without encountering the "compli- cations of growth". But what is plant physiology without "the complications of growth"?

The steadfast disregard over many years of cell multiplication as the first and all-

P - - 4

238 F C Steward

important facet ofgrowth, that it so clearly is, while the role ofauxin 0~) , at stages in the coleoptiles of grasses that involve only cell enlargement, was termed the growth hormone, was clearly unfortunate.

Also the failure to perfect liquid media for the aseptic culture ofangiosperm cells thus confining this technique for too long to random proliferations on relatively large explants on agar was not conducive to rapid progress. Furthermore, to restrict these media to none other than well known constituents and fully "defined" substances discouraged investigators for years from searching for the unknown systems that cause rapid cell multiplication.

6. Cells in eulture and in situ: Their physiology as they grow

But, even so, when a system based on culture of carrot ce|ls that could be placed alternatively under conditions conducive to rapid cell multiplication o r a relative quiescence was clearly in sight, its exploitation in the problems of solutes in cells was deferred. Deferred, in fact until the system and methods for its full analysis were developed. When, in fact this position was reached and its wide implications were realized ir also prompted a return to the problems of growth as they are seen in the growing regions. For this culture of plants (potato and carrot), under the controlled conditions by then available in growth chambers, were exploited so carrying the knowledge gained about cells in culture to their behaviour in situ during normal development. This emphasises a hitherto much neglected area of plant physiology namely the extent to which metabolism, forro and composition of plants with their genetic composition already de¡ are affected by environmental factors as they interact both diurnally and seasonally.

But one should return to the main theme of research into the ability of cells to accumulate solutes. This involves relating the process in question to cells as they grow and develop. The mature understanding of these problems emerged from the use of the system ofcultured cells which consisted ofsmall, uniform, cylindrical tissue explants of carrot root secondary phloem cut lar enough from the cambium so that in situ they would not divide again. Whereas aseptically (whether in water dilute salts of even nutrient solutions) they merely enlarge somewhat their rapid growth in culture by cell multiplication may nevertheless be chemicaUy induced.

The role of plant growth hormones, or growth regulatory substances was long dominated by the analogy with hormones in the animal body and their specific"actions a t a distance". This emerged from the classical tole of I^A as it exerted its effects primarily upon cell enlargement as in the oat coleoptile. But the most powerful "Growth promoting effects" on the cells such as those of carrot root, otherwise unlimited by endogenous or exogenous nutrients ate those that first promote cell multiplication and thereafter ceU enlargement in balance. To promote these effects recourse was made not to already "defined" substances, but to substances from the environments of zygotes as in the contents of the coconut (its liquid endosperm or coconut water or milk), of the immature fruits of Aesculus or even extracts of immature grains of com (Zea mays). Under appropriate conditions that have been described elsewhere these stimuli cause the carrot explants to grow rapidly and, as they do so, they acquire solutes (organic and inorganic) in their cells.

The first solutes to be absorbed and secreted internally into the cultured cells as they

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multiply are organic solutes (sugars and organic acids, the latter electrochemically balanced by I( which is, however, not yet accumulated to high coneentrations or accompanied by CI-). In fact it is later when cell multipli~ttion subsides and enlargement intervenes that the endogenous organic solutes can be replaced with salts (preferentially ofl(although now it may be accompanied by C1- and even some Na). In fact in this active developmental state cells may be induced reversibly to replace organic solutes previously absorbed with inorganic salts exogenously supplied.

In other words the cellular control is p¡ over the activity of water in the cells with enlarging vacuoles and not with ionised salts per se. Thus investigators have been so preoccupied with the electrogenic events at membrane surfaces in pre-formed mature cells and not primarily with the de hOyO accumulations of solutes that the significance of total solutes and their colligative properties upon internal water have been obscured.

Nevertheless, when one mores from cells in aseptic culture to cells as they develop in situ they accumulate solutes in accordance with their morphological setting and the environmental conditions oflength ofday or night or the temperatures by day or night. Potato cells in developing leaves or in tubers acquire solutes according to the environmental conditions that regulate development. AII that need be said here is that the variables that affect metabolism in these developing systems also affect ttie total solutes they contain and their relative composition in terms of organic and inorganic solutes in situ in the intact plant body. This is true of cells of both potato and carrot plant during development.

However, the belated investigation of problems of solute composition in cells under aseptic culture led to other unexpected discoveries.

7. Roles of growth promoting substances and trace elements

The study ofthe naturally occurring agents that induce the growth ofexplants ofcarrot phloem in aseptic culture produced two types of unexpected conclusions. These relate first to the nature of the growth stimulants per se and also to their interactions with trace elements.

The most effective stimuli for growth induction of carrot cells were found in such fluids as coconut milk or water; similar fluids or extracts from immature fruits of Aesculus (particularly A. woerlitzensis) or in immature com (Zea mays) grains. These were divisible into different systems that could be separately assayed. Two such systems differed in that they reacted synergistically in the presence of either tAA or with certain other naturally occur¡ constituents of which myo-inositol was the prime example. These partial growth promoting systems, designated I and II comprised their respect- ive active cell division fractions (AF t or AF z) become effective in presence of their respective synergists (AFt + inositol) or (AFz + IAA) and they may also act best in presence of casein hydrolysate. A'Ft has been best obtained from immature Aesculus fruits and AFe from coconut has been found to be replaceable by zeatin. However, with or without casein hydrolysate these systems only produce their full effects under an appropriately balanced trace element regime. While Fe is paramount in the relation of the trace elements, to the growth stimulus due to the exogenous growth factors the tissue also responds to trace element regimes involving Fe, Mn and Mo. To work out and presenta complete picture ofall these interactions between components of growth

240 F C Steward

factor systems and with trace elements as they affect the growth and composition of carrot cultures proved to be a formidable task. (It has been described as far as possible in two series of papers, the one published in the Annals of Botany, the other in Planta). However, in addition to the varied responses of the tissue to the growth factor systems and to trace elements a most far-reaching and unexpected one on salt composition was observed and described.

The consistent effect is that cultured carrot cells as indeed many other angiosperms accumulate more I~ than N/~mso much so that often I~ very greatly predominates over Na. However, it was found that under the unbalanced stimulus of certain adenyl cytokinins; normally a constituent of carrot growth factor system II, this relationship was so drastically changed that Na even predominated over I~ in the cultured cells. In a study of homologues of zeatin (applied without tAA) it was found that their maximum effect on the Na/K content of the cultured cells occurred when the zeatin side chain was -(CH2)4" H. But in the presente of ~ the number of cells increased and their Na/K ratio was, as usual, low. This is a very far-reaching effect ofa growth factor system on the ionic composition of the cells. It would, however, be tedious to enumerate here all the ways in which external va¡ affect not only the growth but also the composition of the cultured cells. The overall conclusions may be summarized as follows.

8. The final modulation of solute eomposition

After cells are endowed with their unique genetic inheritance their behaviour with respect to metabolism and to their solute contents is mediated during development in various ways. First their morphological environment has an over-riding effect i.e. whether cells develop in one tissue or organ or in another. Second, after the cells develop in a pre-determined location (e.g. in leafor tuber in potato plants, in secondary phloem in storage roots of carrot of elsewhere) they will still respond during development to over-riding effects ofexternal physical (i.e. environmental) variables in which length of day (or night) and temperature by day or night interact. One may extrapolate from the studies on cells in aseptic culture and expect that ifall the possible effects of mineral and trace elements nutrition together with the effects ofenvironments on the composition of cells in storage organs and leaves were fully investigated their range and scope w.ould be very large indeed. Conversely to select arbitrarily contrasted pairs of these variables for separate investigation and publication would produce fragments oftoo dubious value from which to draw over-all conclusions. However, the short answer is that the biochemical composition and the salt and water content ofcells as they develop in situ and respond during ontogeny is a very complex question which is unlikely to be amenable to preconceived mathematical or electrogenic analysis. It must be subject to an essentially obvious conclusion. Plants in their environments develop as physical systems that enable them to respond in their cells to interacting external variables in ways that gire them the physical stability that they so clearly possess and this physical stability seems to be mediated ultimately through the properties of water.

9. Problems: physical and biologicai

Paramount problems still seem to be the following. How do plants endowed with their unique genetic inheritance respond during development so that, in toto (though

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perhaps not necessarily gene by gene) they achieve the remarkable degree of response and stability in their form, composition and behaviour? Also as cell tissue cultures have shown, ontogeny and diversity go hand in hand even while this is still compatible with a maintained totipotency at the somatic cell level as shown by the events of somatic embryogenesis in culture. But the means by which the information carried by genes is recalled for expression in all this remains a physiological mystery.

However, to allow for inherited variations to occur through sexual reproduction, cells at meiosis depart from the diploid state, and in plants, they may even continue to develop in this state as gemetophytes. Although haploidy has been observed to occur and is so documented for carrot cells in culture the precise circumstances that induce in culture what is a normal programmed event in the life cycle is not yet known.

Physicists and physics have current problems which are much publicised. Beset by the multiplicity oftheir sub-atomic particles, they do not know the"glue" that holds the elementary particles together in the structure of matter. Even so the splendour of modern physical instrumentation enables enquiry to range to the limits of the cosmos and speculation to embrace the origin of the universe and the evolution of galaxies and of planets sucia as our own.

But modern biology also has its seemingly insoluble but challenging problems though these may be less dramatically publicised. When Darwin and the origin and evolution oflife on earth are again being called into question one may ¡ ask the very pertinent question. How was matter first so organised that it could independently perform useful work? Also, having been so organized in cells (as in modern plants) how may they without loss of totipotency display the observed range of diversity in forro and function? Genetics tells us that continuity ofestablished traits is maintained in self- reproducing systems and, development shows that within tolerated limits diversity may be established; albeit the means by which genetically transmitted characteristics are regulated is still obscure. Genetics and molecular biology do not tell us how out of matter and energy life emerged. 'Modern' proposals to achieve this de nooo seem to presuppose the prior existence of elaborated biological organisations that it is the ultimate purpose to explain. But has this essay a useful and final message?

10. Epilogue

New information may well continue to expand at its accelerating rate. Minutiae about every topic in plant physiology and botany may continue to overflow even the new journals as rapidly as they appear. Papers may get even shorter and focus only upon single and often trivial events. Readers who may increasingly scan only the tables of contents by title may not even pause to wonder where it all leads. The same furrows may be ploughed even narrower and deeper. But perhaps this celebration volume may induce some to see that it is not now enough to pursue "reductionism" vertically without relating it "ho¡ and "holistically" to see how the whole scheme of things, animate and inanimate, works. The final dilemma, that overrides all the details, is the ultimate mechanism of their control. Hopefully a general synthesis, as between botanists and physicists may emerge. Botanists comprehend and convey the respect for the organisation of plants, their ceUs and organelles. The physicists may be able to speculate upon how the 'breath of life' representing order and reduced entropy, was breathed into an inorganic world of matter and energy. If so, and in consort, they may

242 F C S t e w a r d

be able to comprehend the problems not only o f the origin o f life o f evolution and inheritance but also o f development and diversity.

But the formidable secrets o f nature ate not thosr that can be easily resolved in simplistic terras for, highly improbable and indirect means utilising complex organis- ations and structures have br elaborated to achieve what often seem to be simple objectives.

However detailed is the modern description o f cells and organistas in terms o f their material content, their chemistry, their physical structure, their morphology , their genetics they only opr in life in-as-much as i t is all integrated into a coherent whole which, today, can be respected but not defined. In this sense biology rivals cosmology in its challenge.

The t remendous strides made by molecular biology and genetics in the understand- ing o f unir controls over chemical events have not yet coverr the obvious and long familiar situations in which nature achieves physical controls over large blocks o f very diversified information. Morphological informat ion that controls diversity in the plant body during the life cycle; biochemical informat ion that controls the responsiveness o f composi t ion to forra and environments d u ¡ morphogenesis and physical informa- t ion that ensures that d u ¡ development and in changing environments th› parts o f the plant body comprise a coherent stable whole.

Plant cell physiology should not be complacent till these challenges ate met.

Referenees

Appemlix: Surtes

The following classified sources are iisted in lieu ofa fuil bibliography. The listings will gire access to other rcferences. They have been made so as to support references made in the text to trends in the research dcscribed.

A. Morphogenesis and composition: trends 1963-74

Steward F C 1974 Trends in botanical thought and research: in retrospr and in prospr Curr. Sci. 43 363- 365

Steward F C and Krikorian A D 1975 Mr and its regulation then and now; Biochem. Physiol. Pflanzen 168 375-384

$tr F C 1976 Muitiplr intetactions betwer factors that control cr and development; in Perspectives in experimental biology (�91 N Sunderland (Oxford: Pergamon Press) VoL 2 pp. 9-23

Steward F C and Krikorian A D 1975 The cultu¡ of highr plant celis; in Forro andfunction in plants (eds) H Y Mohan Rara et al and B M Johri Comra. VoL 144-170

Steward F C and Krikorian A D 1979 Problems and potentialities of culturr plant celis in retrospect and prospect; in Plant cell and tissue culture: principle3 and applications (r W R Sharp et al (Columbus Ohio: Ohio State University Press) 221-262

Steward F C 1968 Totipotency of angiosperra cells: its significance for morphology and embryology; Phytomorpholooy 17 499-507

Steward F C and Mohan Raro H Y 1959 Determining factors in cell growth: some implications for morphogenr in plants, in Advances in morphooenesis (eds) Abr and J Brachet (New York: Academic Pr�91 Vol. I pp. 189-265

Steward F C 1963 Totipotency and variation in cultured cr metabolic and morphogenetic manifestatinn p. 1-25, Carrots and coconuts: some investigations on growth p. 178-197 in papr from plant tissue and organ culture--a symposium (ed) by S C Maheshwa¡ and N S Rangaswamy.

B. Solutes in cells in relation to nutrition and growth

Steward F C 1984 Solutes and cr their responses during growth and development: in Plant physiology: a treatise (eds) F C Steward, J F Sutcliffe and J E Dale (New York: Academic Press) (in press)

Plant cell physiology 243

Steward F C, Ulises Moreno and Roca R 1981 Growth and composition of potato planta as affected by environments; Ann. Bot. (Suppl. 2) 48 1-45

Steward F C et al 1962 Growth nut¡ and metabolism of Mentha piperita~ Memoir 379, Cornr University, Agric. Exp. Sta. Parts I-VIII p. 144

Steward F C 1968 Growth and organisation ofplants (Reading, Mass: Addison Wesley) p. 564 Steward F C 1964 Plants at work (Reading Mass: Addison Wesley) p. 184 Steward F C and Sutcliffe J F 1959 Plants in re|ation to inorganic salta, in Plant physiology: a treatise (ed.)

F C Steward (New York: Academic Press) Vol. II p. 253-478 Steward F C and Mott R L 1970 Cells, solutes and growth: salt ar re-examined; Int. Rey. Cytol. 28

275-370 Steward F C and Millar F K 1954 Salt accumulation in plants; a reconsideration of the role of growth and

metabolism; Parts A and B in Symp. Soe. Exptl. Biol. Vol. 8 pp. 367-406 Mott R L and Steward F C 1972 Solute accumulation in plant ceUs V. An aspect of nutrition and

development; Ana. Bot. 36 915-937 Steward F C, Mott R L and Rao K V N 1973 Investigations on the growth and metabolism of cuitured

explanta of Daucus earota V. Effects of trace r and ~owth factors on the solutes accumulated; Planta (Berl) 3 219-243

C. Trends in Plant Physiology (1961-71)

Steward F C 1971 Plant Physiolos~. The changing problems, the continuin 8 quest; Annu, Rey. Plant Physiol. 22 1-22

Steward F C 1970 From cultured cells to whole planta: the induction and control of their 8rowth and morphogenesis; The Croonian Lecture 1969; Proc. R. Soc. (London) B175 1-30

Steward F C 1970 Totipotency variation and clonai development of r cells; Endeavour 29 117-124 Steward F C 1970 How plants grow; Friday evening discourse 6th March~ Proc. R. lnst. Gt. Br. 43 394-426 Steward F C 1961 Vistas in plant physioiogy: problems of organisation, growth and morphogenesis; Duff

Memorial Volume; Can. J. Bot. 39 441-460 Steward F C et al 1961 Growth induction in explanted ceUs and tissues: Metabolic and morphogenetic

manifestations; in 19th Annual Growth symposium: Synthesis of molecular and cellular structure (ed.) D Rudnick (New York: Ronald Press) p. 193-246

D. Trends in nitrogeneous constituents (1947-1983)

Durzan J D and Steward F C 1983 Nitrogen metabolism; in Plant physiology, a treatise (ed) F C Steward and R G S Bidwell (New York: Academic Press) Voi VIII pp 55-265

Steward F C and Street H G 1947 The nitrogencous constituents of plants; Annu. Reo. Biochem. 16 471-502 Steward F C and Pollard J K 1957 Nitrogen metabolism in planta; ten years in retrospect4 Annu. Rey. Plant

Physiol. 8 65-114 Steward F C et al 1959 Nutritional and environmental effecta on the nitrogen metabolism of planta; Soc. Exp.

Biol. 13 148-176 Steward F C and Bidwell R G S 1958 Nitrogen metabolism respiration and growth of plant tissue. IV. The

impact of growth on protein metabolism and respiration and carrot tissue explants: General discussion of resulta~ J. Exp. Bot. 9 285-305

Steward F C and Bidwell R G S 1966 Storage pools and turnover systems in growing and non-growing cells: experimenta with Cl4-sucrose, Cl*-glutamine and C I" Asparagine~ J. Exp. Bot. 17 726-741

Steward F C and Durzan I D 1965 Metabolism of nitrogenous compounds; in Plant physiology: a treatise (ed.) F C Steward, Vol. A4 (New York: Academic Press) p. 379-686

E. Cultured cells and totipotency (see also A) 1958-1970

Steward F C, Mapes M O and Smith J 1958 Growth and organized development ofcultured cells. ! Growth ancl division of free cells~ Aro. J. Bot. 45 693-703

Steward F C 1958 | II lnterpretations of the growth from free celts to carrot plants; Aro. J. Bot. 45 709-713 Mitra J, Mapes M O and Steward F C 1960 IV The behaviour of the nucleus; Aro. J. Bot. 47 357-368 Steward F C, Mapes M O, Kent A E and Holsten R D 1964 Growth and development of p|ant cells; Scienee

143 20-27 Steward F C, Ammirato P V and Mapes M O 1970 Growth and development of totipotent cel|s: Some

prob|ems, procedures and perspectives: Ann. Bot. 34 761-787

244 F C Steward

F. Salt accumulation in cells (1931-1940) (see also B)

Steward F C 1931 The absorption and accumulation of solutes by living plant cells. II a technique for the study of respiration and salt absorption in storage tissue under controlled conditions; Protoplasma 15 497-516

Steward F C 1933 V Observations upon the effects of time, oxygen and salt concentrations upon absorption and respiration by storage tissue; Protoplasma 18 208-242

Steward F C et al 1943 X Time and temperature effects on salt intake by potato discs and the influence of the storage conditions of the tubers on metabofism and other properties, Ann. Bot. N. S. 7 221-260

Steward F C 1935 Mineral nutrition of plants; Annu. Rey. Biochem. 4 519-544 Steward F Cand Prevot P 1936 Salient features of the root system relative to the problems ofsalt absorption;

Plant Physiol. 11 509-534 Steward F C and Harrison J A 1942 Absorption and accumulation of rubidium bromide by barley plants.

Localisation in the root of cation accumulation and of transfer to the shoot; Plant Physiol. 17 411-421 Steward F C 1937 Salt accumulation by plants: the role of growth and metabolism; Trans. Faraday Soc. 33

1006-1016 Steward F C and Martin J B 1937 Valonia at the Dry Tortugas withspecial reference to the problems of salt

accumulation in plants; Papersfrom the Tortugas Laboratory I l 89-170 Steward F C, Stout P R and Preston C 1940 The balance sheet of metabolites for potato discs showing the

effects of salts and dissolved oxygen on metabolism at 23~ Plant Physiol. 15 409--447

G. On orowing points (c.f. also B for Mentha)

Steward F C, Wetmore R H eta11954 A quantitative chromatographic study of nitrogeneous components of shoot apices; Aro. J. Bor 41 123-134

Steward F C, Wetmore R H et al 1955 The nitrogenous components of shoot spices of Adiantura pedatum; Ara. J. Bor 42 946-948

Barker W G and Steward F C 1962 Growth and development of the Banana I The growing regions of the vegetative shoot II. The transition from the vegetative to the floral shoot; Ann. Bor 26 339-423

Barber J T and Steward F C 1968 The proteins ofTulipa and their relations to morphogenesis; Dev. Biol. 17 326-449

Mohan Rana H Y, Monasi Raro Ram and Steward F C III A. The origin of the inflorescence and development of the flowers B. The stem structure and development of the fruit; Ann. Bor 26 657-673

Steward F C and Krikorian A D 1972 Problems of integration and organisation: Control mechanism; in Plant Physiology: a treatise (ed.) F C Steward (Nr York: Academic Press) Vol. VIC, p. 367-419

Steward F C et al 1971 The behaviour ofshoot apices of Tulipa in their relation to floral induction; Dev. Biol. 25 310-335

H. On chemicals active in growth induction of cells

Steward F C and Krikorian A D 1971 Plants chemicals and growth (New York: Academic Press) pp. 232 Steward F C, Mott R L and Shaw G 1973 Effects of adenyl cytokinins on the solutes of cultured cells;

Phytochemistry 12 2335-2339

I. Biology and Cosmology

Taylor R 1968 The biological time-bomb (New York and Cleveland: World Publishing Co.) pp. 140 Taylor R 1983 The great evolution mystery (New York: Harper and Row) pp. 277 Jeremy R 1980 Emropy, A new world view (New York: Viking Press) pp. 305 Paul D 1981 The edfle ofinfinity (New York: Simon and Schuster) pp. 194 Lederman L M 1983 Prospects for progress in particle physics Bull. Aro. Acad. Arts Sci. 37 31-53 Krikorian A D and Steward F C 1979 Is gravity a morphological determinant in plants at the cellular level?

(Cospar) Life Sci. Space Res. XVII 271-284 Krikorian A D and Steward F C 1978 Morphogenetic responses of cultured totipotent cells ofcarrot at zero

gravity Science 200 67-68 Steward F C 1983 Integration ofenergy, forro and composition in Plant Physiol: A Treatise (eds) F C Steward

and R G S Bidwell (New York: Academic Press) Vol VIII pp. 403-405