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ESTIMATION OF DIFFUSION DEFICIT BY CORRELATION WITH RELATIVE TURGIDITY AND BETA-RADIATION ABSORPTION By P_ C. WHITEMA.c'<* and G. L. WILSON*

[Manuscript received August 9, 1962]

Summary Difficulties of technique prevent extensive measurement of the diffusion pressure deficit (DPD) of plant tissues. The possibility of estimation by correlation with more easily measured indices of water status has been examined for leaf of Sorghum l.,'ulgare. The relative turgidity-DPD correlation provides an accuracy of estimate of' about 10% of DPD values for a given leaf position on the plant at a constant stage of leaf expansion. Differences between leaf position and ,vith varying physiological age of the one leaf reduce the usefulness of the correlation. Beta-radiation absorption, while following the trend of water content, is inadequate for useful estimate of DPD.

1. INTRODUCTION Diffusion pressure deficit (DPD) appears to be the most suitable index of the water status of plant tissue, despite doubts recently raised by Carr and Gaff (1961) as to what precisely is measured when DPD is estimated by various methods. It expresses the total of water absorption in the same units as may be used for water retention by the environment, both and atmosphere, with which moisture exchange occurs. Furthermore, it is sensitive to small changes in the tissue water content. However, estimation ofDPD by the standard flotation methods described by Crafts, Currier, and Stocking (1949) or Slatyer's (1958) vapour equilibra­ tion method, is tedious and time consuming, and for many purposes, precludes its use. This paper examines two methods which have been proposed for estimating DPD by calibration with more easily measured indices of water content.

(a) Relative Turgidity Weatherley (1950) expressed the water content of leaf tissue as a ratio between the actual water content and the content at full turgor, and defined this as the relative turgidity (RT). It is a sensitive indicator of changes in the plant's water balance and Weatherley and Slatyer (1957) provided an experimental demonstration of its relationship with DPD. These authors, and later Kramer (1959) and Brix (1960) suggested that the less laborious procedure for measuring RT might be used as an index of DPD in leaf tissue.

* Department, University of Queensland, Brisbane.

Aust. J. Biol. Sci., Vol. 16, No.1 CORRELATIVE ESTIMATION OF DIFFUSION PRESSURE DEFICIT 141

The usefulness of the RT-DPD correlation has been examined in respect of the uniformity between leaves taken simultaneously from varying positions on the plant, or between leaves of one physiological age, taken from plants differing in developmental stage. (b) Beta-mdiation Absorption Mederski (1961) showed that beta-radiation absorption was highly correlated with RT, sensitive to small RT changes, and could therefore provide a continuous non-destructive record of changes in the plant's water status. He suggested that this technique provides a means of determining changes in RT which could in turn be extended to the prediction of DPD. The variability in the beta-radiation absorption-RT relationship is also examined in this paper.

II. MATERIALS AND ME'rHODS All determinations were made on Sorghum vulgare (Pers.) cv. Saccaline grown in pots in the glasshouse. (a) RT-DPD Relationship The vapour equilibration procedure suggested by Weatherley and Slatyer (1957) was employed. Tissue, brought to full turgor, is placed over a with whose DPD it is presumed to come to equilibrium and the RT thus attained is determined. By selecting a range of of varying DPD an RT-DPD curve for the tissue is constructed. The detailed procedure was based on Slatyer (1958). Plants were sampled in the glasshouse between 6.00 and 7.00 a.m. Ten 1-cm diameter leaf disks were cut with a sharp punch from each leaf sampled, five from each side of the midrib along the entire leaf length. These 10 disks, comprising the sample unit, were transferred to distilled water in Petri -dishes, and held at a constant of 20±0·5°C for 4 hr to bring them to full turgor. After surface drying for 30 sec between sheets of filter paper under a constant of 50 g, the disks were weighed quickly on a torsion balance (±O· 5 mg) and transferred to a microdesiccator similar to that used by Slatyer except that Carr and Gaff's (1961) stopper arrangement was used. lI'Iicrodesiccators were arranged to provide three replicates of 10 DPD's, varying from 9 to 116 atm and held in a water-bath at 23±0·01oC in a constant-temperature room at 20±0·5°C. Sodium chloride solutions were used to provide the DPD's required. The disks, after remaining in the microdesiccators for 24 hr, were removed, weighed immediately, and again after oven drying. RT's were calculated. for the corresponding DPD's. The variability between successive leaves was examined for Sorghum plants between 8 and 9 weeks old. Leaves 4, 5, 7, 9, and 10, numbered from the first leaf produced, were sampled and three replicate RT-DPD relationships determined for each. The effect of increasing plant age was studied by sampling, on successive occa­ sions, leaves 6, 7, and 8 as they became fully expanded. For each leaf position, 142 P. C. WHITEMAN AND G. L. WILSON

10 plants were bulked and the material used for four replicate estimations of the RT-DPD relationship.

(b) Beta·radiation Absorptionr-RT Relationship Changes in radiation absorption with changing water content were followed by placing leaf tissue between a carbon-14 source and a thin-window Geiger tube at a constant height above the source. All operations were carried out in a constant­ temperature room at 20±O· 5°C. Strips of laminae 10 em long and 3 em wide were cut from the central part of the leaf, between the margin and midrib, and floated on distilled water for 4 hr to bring them to full turgor. They were then blotted dry, quickly weighed on a torsion balance (±O·05 mg), and beta-absorption was measured. At short intervals as the tissue strips dried down, weighings and corresponding counts were made, and finally the oven dry weight determined. Preliminary trials revealed a very obvious contribution to variability by the non-aqueous leaf material, and, in an attempt to minimize the effect of this component, measurements were made and counts at sampled state - counts, oven dry X 100 counts at full turgor - counts, oven dry calculated. This quantity, called here relative absorption, may give a better estimate of the influence of varying water content. This required a count on dry tissue, prepared by taping the material to a sheet of paper before the final oven drying. One experiment examined the variability between comparable leaves by following the changes in count rate with decreasing RT for leaf 13 on seven different plants. In another experiment a further source of variability was introduced by using differing leaf positions, from 11 to 14, spread over several plants.

III. RESULTS (a) RT-DPD Relation.ship In the comparison of successive leaves at the one time, the data were not suitable for statistical analysis. However, the regressions of RT on log DPD comprised a series of closely parallel lines (similar to those shown in Fig. 1 for other leaf data), with a progressive decrease in RT at any DPD for successive leaves up the stem. There is therefore a leaf position effect. The comparison of leaves 6, 7, and 8 at the same stage of expansion is shown in Figure 1. The regressions differ significantly between leaves 6 and 7 and between leaves 7 and 8 (P

(b) Beta-radiation Absorptionr-RT Relationship The relationship between absorption and RT for leaf 13 is shown in Figure 2. While absorption increases with increasing water content, there is marked variability, which is, however, reduced when absorption is expressed as relative absorption (Fig. 3). CORRELATIVE ESTIMATION OF DIFFUSION PRESSURE DEFICIT 143

'00 x

> r 0 ,•i3 70 r ~ 60 C < •"W '0

.0

30 "0 "2 ,.. ,., "8 2'0 LOG DIFFUSION PRESSURE DEFICIT

Fig. l.~Relationship between RT and DPD for leaves 6, 7. and S, each at the just fully expanded stage.

'00 XX

'X 90 l

xix

]' z 0.0 J{ • 30 ~ ~ ..o @ 2 ~ •< J , W o >

."" 70 W~ r• 20 ,z • l, S \''\xx ~ x, • x x~x 00 xXxxJ/t"xx,'io: '0 x x ,')c ,­ x xx"" x Xx x~x~~ x x ~~ 'o,I-----"o----c:-----~----~----'" o 20 40 '0 80 000 o 20 40 60 80 100 RELATIVE TURGIDITY (%) RELATIVE TURGIDITY (oro) Fig. 2 Fig. 3

Fig. 2.~Relationship between beta-radiation absorption and RT for leaf 13.

Fig. 3.-Relationship between relative absorption (see text, p. 142) and RT for leaf 13. 144 P. C. WHITEMAN AND G. L. WILSON

When varying leaf positions \vere compared, relative absorption showed a fairly smooth relation with RT for any individual leaf (Fig. 4) except for irregularities at the higher RT's. Compared with the previous experiment, however, variability is greatly increased when leaves of differing position are introduced.

.= • ____ .;:::f5,.Ji'5"'X -/~9 ~/O x /t;/O~

90

z o I-I t "o •m < w > ~. 80 ;/ ~ 18 "

x

70

x/ 50 60 70 eo 90 000 RELATIVE TURGIDITY (~;) Fig. 4.-Relationship between relative absorption (see text, p. 142) and RT for various leaf positions from different plants.

IV. DISCUSSION (a) RT-DPD Relationship One use of the RT-DPD correlation could be its establishment for a particular leaf position on the stem at a constant stage of expansion. While a direct measure of variability between plants has not been undertaken, the data of Figure 1 allow a test of the reliability of the correlation, as influenced by technique and the general variability which existed within the leaf material bulked from the one leaf from 10 plants. By plotting the confidence limits (P ~ 0·05) for the regression lines, probable errors of estimates of DPD may be determined graphically. Because of the logarithmic transformation, errors are greater on the higher side of the means. Assuming Slatyer'B (1957) estimate that RT may be determined with an accuracy of ±l %, the correlative estimates of DPD for a range of RT's, and the corresponding CORRELATIVE ESTIMATION OF DIFFUSION PRESSURE DEFICIT 145 errors, are shown in Table 1. These errors approximate to 10% of the mean values. At low DPD's, these are slightly better than the ±2 atm claimed by Slatyer (1958) for the direct vapour equilibration method, but not as good as the O· 5 atm, for DPD's below 10 atm, stated recently by Brix (1962) for vapour . At higher DPD's errors increasingly exceed those claimed for the other methods. Nevertheless, the 10% reliability may be acceptable for many purposes and merit the use of the correlative method.

TABLE 1 PROBABLE ERRORS (P = 0·05) OF DPD ESTIMATED GRAPHICALLY FR01\{ THE RT-DPD CORRELATION FOR LEAF 6 AT THE JUST FULLY EXPANDED STAGE

Estimated RT(±I) Probable Error MeanDPD (%) (atm) (atm)

90 9·8 +0·94 -0·90

70 22·4 +2·2 -2·0

50 51·3 +5·0 -4·5

40 77·6 +8'7 -7·3

With the introduction of different leaf positions on the plant, variability increases further. This was seen, in the first place, when successive leaves at different stages of growth were simultaneously compared and again when successive leaves were compared as they reached comparable stages of expansion, that is, at the same physiological age. The additional unreliability introduced into the estimation of DPD by correlation with RT determined on leaves selected on the basis of comparable physiological age may be seen from the data of Figure 1. Whereas an RT of 50±1 % would, from the data for the sixth leaf, indicate a DPD of 51·3± (approx.) 4·7 atm, it would, on application to leaf 7, represent a value of 56·2± (approx.) 4·8 atm. If the data for leaves 6 and 7 are combined, the sigriliicant difference between the two regression lines leads to increased variance and therefore a greater error than for either leaf alone.

(b) Beta-mdiation Absorption Beta-radiation absorption was shown to vary widely between the different leaves on one plant and between different plants due perhaps to thickness and density variations. The calculation of relative absorption reduced variability and the data of Figure 3, which is for one leaf position on the stem at a fixed stage of expansion, allows for an RT estimation to ±8·0o;. (P = 0·05). It is apparent from earlier discussion that this magnitude of error precludes the further correlative step to estimate DPD. The radiation absorption-DPD correla- 146 P. c. ,,;'HITEMAN AND G. L. WILSON tion might of course have been examined directly. However, the unreliability of the relationship with RT, which has been shown to follow DPD rather closely, indicated that radiation absorption could provide little more than an approximate index of the water status. Furthermore, the gain in accuracy of the RT estimation by calculating relative absorption depends on destructive sampling of tissue and therefore sacrifices what could have been the particular advantage of the approach.

V. AOKNOWLEDGMENTS Thanks are due to lVIr. H. M. Finucan, Department of Mathematics, University of Queensland, and to Mr. P. Haydock, Division of Tropical Pastures, C.S.I.RO., for assistance ·with statistical aspects of the , and to Dr. K. T. Glasziou of the Commonwealth Sugar Refineries David North Plant Research Centre for the provision of a carbon-14 source. During part of the work, P. C. Whiteman was the holder of a General Motors Holden Post-Graduate Research Fellowship and expresses his gratitude for this assistance.

VI. REFERENCES BRIX, H. (1960).-Determination of viability of loblolly pine seedlings after wilting. Bot. Gaz. 1210 220. Brux, H. (1962).-The effect of water stress on the rates of photosynthesis and in tomato plants and loblolly pine seedlings. Physiol. Plant. 15: 10. CARR, D. J .• and GAFF, D. F. (1961).-The role of the cell wall water in the water relations of leaves. In "Plant-Water Relationships in Arid and Semi-Arid Zones". p. 117. (Proc. U.N.E.S.C.O. Symp., Madrid.) CRAFTS, A. S., CURRIER, H. B., and STOCKING, C. R. (1949).-In "Water in the Physiology of Plants". p. 101. (Chronica Botanica Co.: Waltham, Mass.) KRAII1ER, P. J. (1959).-The role of water in the physiology of plants. Advanc. Agron. 11: 51. l\fEDERSKI, H. J. (1961).-Determination of internal water status of plants by beta-ray gauging. Soil Sci. 92: 143. SLATYER, R. O. (1957).-The influence of progressive increases in total soil moisture stress on , growth, and internal water relationships of plants. Aust. J. Biol.Sci. 10: 320. SLATYER, R. O. (1958).-The measurement of diffusion pressure deficit in plants by a method of vapour equilibration. Aust. J. Biol. Sci. 11: 349. WEATHERLEY, P. E. (1950).-Studies in the water relations of the cotton plant. I. New Phytol. 49: 81. WEATHERLEY, P. E., and SLATYER. R. O. (1957).-Relationship between relative turgidity and diffusion pressure deficit in leaves. Nature 179: 1085.

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