the hydrion concentration of plant tissues

THE HYDRION CONCENTRATION OF PLANT TISSUES

I. THE METHOD

by JAMES SMALL~ D. Sc.

Received for publication, July 16, 1926

INTRODUCTION

Our knowledge of reaction or hydrion concentration in plants has been confined mainly to the results of determinations of the hydrion concentration of liquids which have been expressed from various parts of the plant and examined either by means of indicators or by electrometric methods. Readers desiring to consult previous literature are referred to the second edition of CLARK's valuable treatise (4). Inter- esting references which do not occur in CLARK'S extensive list are GAUDICHAUD (1848), SACHS (1862); ARRHENIUS, IRWIN, JACOBS (19~); HOAGLAND and DAVIS, PEARSALL and PRIESTLEY, OLSEN (1923); HERKLOTS, PEARSALL and EWING, ROBBINS, SCART~[ (1924); PFEIFFER (19 5); (19 6).

In addition to these determinations we have the observations by HAAS (6), where the coloured substances occurring naturally in the cell were taken as indicators of hydrion concentration. ATKINS (3) seems to have been the first to publish a record of the use of the ordinary synthetic indicators in an attempt to determine the reaction of living tissues.

The method of ATKINS involves a comparison of the tints yielded, thus "Using methyl red, the sclerenchyma and bast fibres give a salmon pink to pink colour, corresponding to pH 5"4 to 5"~; the wood appears a faint pink, not more acid than pH 5'4 to 5"6 and the medullary rays are yellow. The upper limits of acidity are therefore fixed. It remains to determine the acidity of the portions appearing uniformly yellow of

The hydrion concentration of plant tissues. I . 325

the same tint with di-ethyl red. With phenol red the full yellow appeared, showing an acidity of ptI 6"6 or more. With brom thymol blue a yellow with a green tinge was seen, matching the colour with pH 6"9." In order to avoid an error here due to a green tinge in the tissue a drop method with expressed sap was used. This method applied to fresh sections of Salvia Verbenaca gave pH 5"9--5"4 for the sclerenchyma and bast fibres, ptI 5"4--5.6 for the wood walls and pit 6"0 for medullary rays, medullary and cortical parenchyma.

When one c o n s i d e r s - 1. that a carefully prepared standard indicator colonr is generally regarded as necessary for accuracy even to �9 9 in pH values; 9. that the concentration of the indicator in the solution to be tested must not differ materially from that in the standard solution; and 3. that both solutions are usually placed in test-tubes of standard size and viewed in a comparator which permits a view only through the whole thickness of the fluid, one is rather surprised at the publication of this method of using indicators on sections. The possible errors involved are numerous and include 1. variation in depth of colonr due to variation in the thickness of the sections, 9. variation in depth and/or quality of tint from tissue to tissue due to variation in the rate of penetration of the colouring fluid, 3. variation in the quality of tint due to sap which has escaped from cut cells, 4. the 'protein' error, and 5. the 'salt ' error.

The first two sources of error are so obvious to a critical in- vestigator that CLARK (4, p. 118) referring to "the use of unequal depths of solutions through which the colors are viewed" writes that "There are errors of technique . . . . which may be passed over with only a word of reminder". He also refers to certain optical effects (ibid. p. 65), e.g. where a dichromatic purple such as that of brom phenol blue may appear red or blue according to the thickness or thinness of the layer of fluid traversed by the light examined.

With regard to the second source of error we have in fact found tissues which, although giving a stronger red with di-ethyl red than surrounding tissues, give a yellow with methyl red while the surrounding tissues give a red with methyl red, and similar results have been obtained in other cases with other indicators. The depth of the tint observed is, therefore, no certain guide to the exact hydrion concentration of a tissue.

The third source of error can be avoided by suitable washing. The fourth or 'protein' error has its source in the removal, by adsorp-

326 Small

tion or othel~wise, of the indicator from the field of action and must be considered in the case of the living cell. The fifth or 'salt ' error, according to K/tLTttOFF (4, p. 121) is ~-0"95 for brom cresol purple and 0"50N NaC1, --0"10 for methyl red and 0"50NNaC1 and -~0"35 for brom phenol blue and 0"50N KC1. With 0"25N KC1 and brom phenol blue the error is still -~0"15, while for 0"10N KC1 it falls to ~-0"05. According to S~RENSEN and PALITSCH (4, p. 120) this 'salt ' error ranges from a pit change of--0"10 with neutral red and 35 parts of phosphates per 1000 to -~ 0"2'2 with a-naphthol phthalein ~ d 35 parts of borate per 1000 of solution.

In dilute solutions of salts such as are normally encountered in the plant cell, it seems probable that this error would not amount to more than a pH change of ! 0'1, but with less dilute solutions it must be taken as seriously affecting the usefulness of indicators when their use involves a comparison of TINTS.

Notwithstanding the possible sources of error in this method of determining the hydrion concentration of plant tissues, such a method is surely more reasonable and more likely to bring out a closer approxi- mation to the truth concerning the significant variations in reaction in plants than are the electrometric methods so commonly used. I t seems impossible at the present stage to apply electrometric methods to the interior of living plant cells. The nearest approach to an nn- mixed sap used for such determinations is that of Valonia used by C~OZIER (5). But considering the comparatively extensive variation in the pH of neighbouring cells, the determination of the pH of a liquid, which is a crude mixture of cell wall fragments, cytoplasm and vacuolar sap with most of their inclusions, does not seem capable of providing us with very satisfactory data upon which to base conclusions concerning the rSie of hydrion concentration in the life of the plant. The ~tata obtained by C~OZlE~ (5) concerning the reaction of liberated vacuolar sap of Valonia may be more reliable and more significant, but even in that case the very process of liberation may have involved significant changes in the hydrion concentration of the sap. For example, in the case of a very slightly buffered phosphate solution the carbon dioxide content may be the factor governing the pH of the sap and wounding the cell may profoundly alter the carbon dioxide content (cp. LILLIE, 12), and allowing the carbon dioxide of the sap to attain an equilibrium with the air will certainly alter that factor.

The hydrion concentration of plant tissues. I. 327

The usual methods, involving the expression of the sap, seem to the writer to have somewhat the same significance as would determinations of the pH of an average chemical laboratory by a process which involved as its first step the crushing and mixing of all the receptacles and their contents. Quite apart from the inevitable interactions, the resulting data, while more or less true as a kind of average, would furnish no very obvious clues as to the processes included in the normal activities of the laboratory. Nevertheless, many of those who use these methods work their data out to the second decimal place and some even claim a significance for variations in the last figure of such data. It should be clear, from the facts already known concerning the variation of pH from tissue to tissue, that the relative development of the various tissues must be studied and taken into account, before any reliance at all can be placed upon small variations in the hydrion concentration of a mixed liquid derived from a heterogeneous mass of tissues.

THE INDICATORS

Any indicator method of determining hydrion concentration which depends upon a finely developed colour-senSe does not seem to be capable of general application, except in the hands of a trained colour specialist.

The method described below depends upon the fact that in a two-colour indicator series one colour is what may be called a dominant. Thus red or blue is dominant over the yellows given by most of these indicators, i .e . on dilution or thinning of the layer viewed, without any change in pH, the pink or orange or green tints retain the red or blue element of the tint more conspicuously than the yellow element. The actual tint may theoretically and to the trained colour specialist remain the same, but the apparent tint of red diluted becomes pink, pink diluted becomes paler pink, orange diluted becomes doubtful whereas pinkish orange becomes pink, and similarly with blue to pale blue, green to pale green, yellowish green to green; whereas yellow diluted becomes apparently colourless at much earlier stage of dilution or thinning.

In the range of tints given by any one of the indicators used there is a point where the yellow is definitely yellow and another where the colour becomes definitely dominated by the other element, either red or blue, i. e. the dichromatic tint becomes definitely pink or

398 Smal l

green. Between these points is usually a range of 0"4 in the pH; and it is this part of the range and this part only which we use to obtain our results.

The following table shows the scheme used with given indicators, and the method has been extended occasionally to other indicators.

Alk. Col. Acid Col. Indicator Abbrtn. Range pH Range pH

Bromo- ores01 purple . . . .

Di-ethyl red . . . . . . .

Methyl red . . . . . . .

Benzene- azo- ~ naphthylamine

9 Bromo- cresol green . . . .

Bromo- phenol blue . . . .

BCP

DER

MR

BAN

BCG

BPB

pale blue to > 6"2 deep purple

yellow > 6"0

yellow > 5"6

yellow > 4"8

pale green to > 4"4 deep blue

pale green to > 4"0 deep blue

i Introduced into the series in the autumn of 1923. s Introduced into the series in the autumn of 1925.

yellow < 5"8

pale pink to (5"6 deep red

pale pink to <: 5"2 deep red

pale pink to < 4"4 deep red yellow ( 4"0

< 3"4 yellow

In considering this table it should be noted, firstly that no degree of dilution will make yellow appear pink or green, and secondly t h a t the pale blues, greens or pinks do not appear yellow on dilution. The only case where any difficulty has arisen in the interpretation of the results is with BAN. Here the yellow is not the same distinctive colonr as in the other series, but with strict attention to the quality of the yellow as seen in the test-tube alkaline range there has been no further trouble in the classification of the colours found.

TECHNIQUE Hand sections of fresh material are cut of such a thickness that

the section always has the parenchymatous layer in some part just one cell thick. These sections are carefully washed with neutral water to remove the liberated contents of broken cells. This neutral water is prepared by half-filling a bottle or washing flask with distilled water which has invariably been found to be acid to phenol red (cp. 20); introducing a few drops of that indicator which has its point of least colour at pH 7; and then filling up with the normally slightly alkaline


tap water of Belfast, until when mixed the water is practically colourless. A distinct although very pale yellow tint is corrected by the addition of more tap water, while a distinct pale pink tint is corrected by the admixture of more distilled water. The presence of the minute quantity of phenol red does not interfere in any way with subsequent operations, and in the bottle it acts as a constant check on the con- tinued neutrality of the washing water. The latter is a very necessary precaution as will be seen later.

Three washed sections from the same part of the plant are then placed in each watch-glass of diluted indicator solution, left over night, washed again with neutral water, examined under the microscope, and the colours are then recorded. Control sections in each case are used to check the natural colouration which may interfere with the tests, especially in the epidermis. The natural colour is sometimes pink or red and the results with Ml~ and DEI~ are therefore subject to special interpretation.

By carefully controlled experiment it has been found that although the cotour with indicators deepens during the night it has not in any case changed from yellow to pink or green or vice versa. The tint varies but the kind of colour remains the same. The indicator solutions used are those prepared by the British Drug Houses as ready for use, and the usefulness of our method is confirmed by the fact that after an experience of four years we have been able to detect alkali coming from the bottles in one batch of Solutions. The solutions were after- wards obtained in amber glass bottles which do not yield alkali to the solutions. The composition of these indicator solutions is given as follows--

Bromo-cresol purple . . . . 0"04% mouo Na salt in 20% alcohol Di-ethyl red . . . . . . . 0"02% in 6 0 % alcohol M~thyl red . . . . . . . 0"02% in 6 0 % alcohol Benzene azo-a-naphthylamine. 0 '01% hydrochloride in 30% alcohol Bromo-cresol green . . . . 0"04 ~ mono Na salt in 20 % alcohol Bromo-phenol blue . . . . 0"04 % mono Na salt in 90 % alcohol

It will be noted that the indicators are in alcoholic solution. By carefully controlled experiments with aqueous solutions made up from dry B. D. H. indicators Miss S. H. MARTIN, M. Sc., was able to demonstrate conclusively that there was no difference between the kind of colour given by sections with the diluted standard solutions and that

3 3 0 S m a l l

given with fresh aqueous solutions of the same indicators. Aqueous methyl r ed (B. D. H.) used for some time gave no difference in the colour but gave paler tints of the same kind of colour.

In some cases the indicator was taken up by the cells to such a small extent that certain tissues appeared colourless. In many such experiments the sections were mounted in a hanging drop in a gas chamber and when carbon dioxide was passed through the chamber a reddish tint with methyl red or di-ethyl red resulted, showing that the yellow of the alkaline range of these two indicators when very dilute may appear colourless in the sections under the microscope. Red being a more dominant colour shows up when the pH is changed to the acid range of the indicator. Similar experiments with the yellow of the acid range of bromo-cresol purple gave similar results with ammonia vapour or, when the pH appeared to be about 5"9, with carbon dioxide; at the latter point neither yellow nor purple are very distinct.

The six indicators given above enable us to distinguish the following ranges of reaction or pH.

Symbols Colours and Indicators m ptI Range for Notes

p t tRanges

blue to purple BCP . . . . . . . yellow BCP, yellow DER . . . . . yellow BCP, eolourless DER, yellow MR red DER, yellow MR . . . . . . . red DER, eolourless MR, yellow BAN . red MR, yellow BAN . . . . . . . red MR, indet. BAN, green to blue BCG red BAN, green BCG . . . . . . . red BAN, indet. BCG, green to blue BPB yellow BCG, green to blue BPB . yellow BPB . . . . . . . . . .

Wider Ranges Indefinite

yellow BCP, green to blue BPB . red DER, green to blue BPB . . . .

Acid range of first series red MR, green to blue BPB . . . .

> 6"2 <5"8 >6"0 <5"8 > 5 ' 6 <5"6 > 5 ' 6 <5"6 >4"8 < 5 ' 2 > 4 . 8 < 5 ' 2 >4"4 < 4 4 > 4 ' 4 <4"4 > 4 ' 0 <4"0 > 4 ' 0

< 3"4

<5"8 > 4 ' 0 < 5.6 > 4'0

<5"2 >4"0

A a

b e

d e

f

definite higher ranges

are uncommon approx. 5"9 1

approx. 5"6

g approx. 4"4 h i approx. 4"0 k

X = ( a - h) Y = ( d - - h)

Z : ( e - - h)

1 The yellow with BCP is stronger than the indefinite purple about 6"0--5"8.


These symbols have been used throughout the analyses of the results as it is much easier to follow in this way the changes in reaction of the various tissues and from tissue to tissue in a section.

It should be noted that in addition to the indicators determining each range there are others; the colours given by these others act an additional check on the values obtained for the hydrion concentration.

The ranges indicated by X and Y are taken as too wide to be useful and have been eliminated from the records of results which are to follow in this series. Such ranges occur in practice because BAN and BCG were not used in the first series, and also because when used later they and also other indicators sometimes failed to give a definite colouration.

The colour resulting from each experiment in each tissue is noted and from these colours the pH range can readily be determined and translated into a ledger as one or other of the symbols (letters).

From the above account it will be seen that, when a definite kind of colour rather than a tint is taken, the results with fresh sections and series of indicators can be quite definite and precise within the limits imposed by the use of ranges of pH rather than guesses at more exact figures. The use of a series of indicators in this way may reduce the width of the range indicated, e.g. b or 5"8--5"6, or it may bring the range within narrower limits, e. g. c or 5"6--5'6. The other indicators employed in each case not only check higher or lower ranges, but may also differentiate other tissues of the same section within similar narrow limits.

l~ESULTS

J This method of investigating th pI-I of plant tissues has been employed during the past five years in the following researches by Miss M.W. REA, M. Sc., and Miss S. It. MARTIN, M. Sc.--

1. A preliminary survey of the tissue reactions of flowering and other stems; 2. A complete survey of the tissues of the sunflower (Helianthus annuus) from the dry seed to the mature flowering stage; 3. A similar survey of two varieties of the broad bean (Vicia Faba); 4. A survey of the tissue reactions in the stems and leaves of herb- aceous stems throughout the year; 5. A similar survey of the stems and leaves of woody plants for each month in the year; 6. A survey o~ the tissue reactions of selected species growing in markedly different

332 Small

habitats; 7. A survey of tissue reactions in succulent plants; 8. A more extended survey of the tissue reactions in flowering stems.

In addition to these surveys, some investigation of the substances which act as buffers in plant cells has been carried out by Miss S. H. MARTIN, using the hypocotyl of the sunflower seedling. Although confined to this special tissue these results demonstrate the possibility of carbon dioxide content being the factor governing the hydrion concentration of the living cell in at least some plant tissues; and therefore, between pH 6"0 and pH 4"0 all methods of pH determination which allow of the carbon dioxide in the sap reaching equilibrium with that in the air must be suspect unless accompanied by a demonstration that the buffer substances present in the material concerned eliminate any significant action of carbon dioxide. 0LSE~ (13), for example, has shown that the liberation of carbon dioxide from soil water, which is normally much more strongly buffered, may give a shift of as much as 0"6 in the pH.

It isproposed to present all these results as a series under the general title which is used as a heading for this description of the method.

Department of Botany, The Queen's University of Belfast.

REFERENCES

1. ARRHENIUS, 0. Absorption of nutrients and plant growth in relation to hydrogen ion concentration. Journ. Gen. Physiology, Vol. 1, p. 81, 1922.

2. ATKINS , W. R. G. Some factors affecting the hydrogen ion concentration of the soil and its relation to plant distribution. Sci. Proe. Roy. Dublin Soc. (N. S.) 16, XXX, 1922.

3. The hydrogen ion concentration of plant cells, ibid. 16, XXXI, 1922. 4. CLARK, ~r ]~. The determination of hydrogen ions. Williams and Wilkins, Balti-

more, 2nd Edt., 1922. 5. CROZIER, W . J . Intraeellular Acidity in Valonia. Journ. Gem Physiology, I. 6,

p. 5817 1919. 6. GAUDICHAUD, M. Des sues s6veux aeides, et de quelques exer6tions alcalines.

Comptes Rendus, XXVII, 37, 1848. 7. HAAS, A.R. The acidity of plant cells as shown by natural indicators. J. Biol.

Chem., 27, p. 233, 1916. 8 . HERKLOTS, C~. A.C. The effects of an artificially controlled hydrion concentration

upon wound healing in the potato. New Phytologist, XXIII . 5, p. 240, 1924. 9. HOAGLAND, D. R. and DAvis, A.R. The composition of the cell sap of the plant

in relation to the absorption of ions. Journ. Gem Physiology, Yol. 5, p. 629, 1928.


10. IRWIN, M. The permeability of living cells to dyes as affected by hydrogen ion concentration. Journ. Gen. Phys., Vol. 2~ p. 223, 1922.

11. JACOBS, M.H. The influence of ammonium salts on cell reaction. Journ. Gem Phys., Vol. 2, p. 169, 1922.

12. LILLIE, R.S. On the connection between change~ of permeability and stimulation and on the significance of changes in permeability to carbon dioxide. Amer. Journ. Physiol., XXIV, p. 14, 36, 1909.

13. OLSEN, C. Studies on the hydrogen ion concentration of the soil and its significance to the vegetation. Compt. Rend. d. tray. d. Laboratoire Cartsberg, XV. 1, 1923.

14. PEARSALL, W.H. and Ew~G, J. The isoeleetrie points of some plant proteins. Bioehem. Journ., XVIII. 2, p. 39.9, 199.4.

15. PEARSALL, W. H. and PRIESTLEY, J. ]~. Meristematic tissues and protein isoelectric points. New Phytologist, XXII. 4, p. 185, 199.3.

16. PFEIFFER~ H. Uber die Wasserstoffionenkonzentration [H'] als Determinationsfaktor physiologischer Gewebegeschehen in der sekundiiren Rinde der Pflanzen. New Phytologist, XXIV. 2, p. 65, 1925.

17. ROBBINS, W. J. Isoelectrie points for the myeelium of fungi. Journ. Gem Phys., VI. 3, p. 259, 1924.

18. SACHS, J. Ueber saure, alkalische und neutrale Reaktion der S~fte lebender Pflanzen- zetlen. Bot. Zeitung, XX, 9.64, 1862.

19. SCARTH, •. W. Can the hydrogen ion concentration of living protoplasm be determined? Science, LX, p. 431, 1924.

20. The toxic action of distilled water and its antagonism by cations. Trans. Ray. Soc. Canada, XVIII , p. 97, 1924.

21. SMITH, E. PHmm. Acidity of the medium and root production in Coleus. Nature, Vol. 117, p. 339, 1926.

the hydrion concentration of plant tissues

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