Michigan. Thesoils were fertilized as described previously (3), and after thorough intermixing of the fertilizer with the soil, the respective treatments were placed in two-gallon

PLANT PHYSIOLOGY

special reference to the interchange of energy be-tween the leaf and its surroundings. Proc. Roy.Soc. London, Ser. B. 76: 29-111. 1905.

2. BRUNT, D. Physical Dynamical Meteorology. Cam-bridge University Press, London. 1939.

3. CLUM, H. H. The effect of transpiration and en-vironmental factors on leaf temperatures. Amer.Jour. Bot. 13: 194-230. 1926.

4. CURTIS, 0. F. Leaf temperatures and the cooling ofleaves by radiation. Plant Physiol. 11: 343-364.1936.

5. LAUSCHER, F. Berichte tiber Messunger der nacht-lichen Ausstrahlung auf der Stolzalpe. Met. Zeit.45: 371-375. 1928.

6. MEYER and ANDERSON. Plant Physiology. D. VanNostrand, New York. 1939.

7. SHULL, C. A. Rate of adjustment of leaf tempera-tures to incident energy. Plant Physiol. 11: 181-188. 1936.

8. WAGGONER, PAUL E. and SHAW, R. H. Temperatureof potato and tomato leaves. Plant Physiol. 27:710-724. 1952.

9. WALLACE, R. H. and CLUM, H. H. Leaf tempera-tures. Amer. Jour. Bot. 25: 83-97. 1938.

10. WATSON, A. N. Further studies on the relation be-tween thermal emissivity and plant temperatures.Amer. Jour. Bot. 21: 605-609. 1934.

SOME OBSERVATIONS ON THE REDUCTION OF 2,3,5-TRIPHENYLTETRAZOLIUM\CHLORIDE IN PLANT TISSUE AS INFLUENCED BY MINERAL NUTRITION 1, 2

JOHN C. BROWNPLANT INDUSTRY STATION, U. S. DEPARTMENT OF AGRICULTURE, BELTSVILLE, MARYLAND

Reduction of 2,3,5-triphenyltetrazolium chloride(TTC) to the insoluble red formazan, which was de-veloped by Lakon (9) as a test for seed viability; hasproven of some indicative value, in tissue tests, ofnutritional irregularities. Reduction of the dye ismost pronounced at nodes (5) and in meristematictissues irrespective of their position in plants. Hewittand Agarivala (7) have observed tissues of plantsgrown with low molybdenum to show much less re-duction of TTC than normal plant material. Theyreport the distribution of molybdenum in plants grownwith an adequate molybdenum supply as similar tothe distribution of the precipitated formazan.

In previous experiments in which enzymatic activ-ity was studied as indicative to copper or iron de-ficiencies and their interactions in plants (3, 4), fifteenplant species were grown on five soil treatments ofvarying iron- and copper-supplying capacity. Sevenplant species developed lime-induced chlorosis butdid not develop copper-deficiency symptoms; fourplant species developed severe copper-deficiency symp-toms but did not develop lime-induced chlorosis; twoplant species showed partial symptoms of both typesof chlorosis and two plant species grew well on allfive soil treatments.A marked difference in the ascorbic acid oxidase

activity of these plants was observed between treat-ments. The ascorbic acid oxidase activity was muchgreater in the nodes than in the leaves of both cornand wheat and was found to be indicative of the ex-ternal copper supply. Iron accumulated in the nodesof copper-deficient corn but did not accumulate in thenodes of corn grown on the calcareous soil. The as-corbic acid oxidase activity in the latter nodes wasfifty times greater than it was in copper-deficient cornnodes. This marked difference in activity localized

1 Received April 13, 1953.2 This project was supported in part by the U. S.

Atomic Energy Commission.

in the nodal areas of corn prompted the use of TTCas a further index to the nutritional irregularity.

METHODS AND MATERIALSMinn. Hybrid #800 corn (Zea Maize), Thatcher

spring wheat (Triticum sativum), and cockleburs(Xanthium pensylvanicum) were grown on five dif-ferent soil treatments with various levels of iron andcopper supplying capacity. These soil treatmentswere: (1) Calcareous soil, pH 7.8; (2) 2/3 calcareous1/3 organic soil (by volume), pH 7.5; (3) 1/3 cal-careous 2/3 organic, pH 7.2; (4) organic soil, pH 6;and organic soil plus added CUSO4 5H20. The cal-careous soil was a Millville silty clay loam from Logan,Utah; and the organic soil was from the MichiganState College Experimental Muck Farm, East Lan-sing, Michigan. The soils were fertilized as describedpreviously (3), and after thorough intermixing of thefertilizer with the soil, the respective treatments wereplaced in two-gallon glazed pots, equally moistened,and allowed to stand for one week before planting.The lime-induced chlorotic and copper-deficient

corn plants were harvested before the tassels becamevisible. The wheat plants were harvested just beforethe heads became visible and after severe copper-deficiency symptoms had developed in treatments 3and 4. The cocklebur plants were harvested after theburs had developed.The corn plants were cut off at the surface of the

soil, and the cut ends immediately placed in a brownbottle, in subdued light, containing a 0.5% aqueoussolution of TTC. The upward movement of the dyewas observed by its reduction to the red formazan inthe leaves, especially the veins, of the plant. Someplants were left in the solution for about six hours,and then the stalks were cut open to observe to whatextent and where TTC had been reduced. Otherplants wvere left in the solution for twenty-four hoursbefore observations were made.The entire wheat plant was harvested including as

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much of the root systems as could be effectively re-moved from the soil. The roots of these plants werewashed in water and then placed in the TTC solutionand the upward movement and reduction of the dyedetermined as for corn. In addition, nodes and rootswere cut from wheat plants harvested from the sametreatment and placed in the TTC solution. The bursfrom the cocklebur plants were cut open and placedin this solution. Such experiments have (been carriedout three times for corn; twice for wheat, and oncefor the cockleburs.

RESULTS AND DISCUSSIONThe fruit of corn, wheat and cockleburs was most

affected by copper deficiency. In each case the ears,heads and burs formed, but they did not continue todevelop and mature. Figure 1 shows the nature ofthe copper-deficiency symptoms with the undevelopedburs of the cocklebur plant. Figure 2 shows the ex-tent of the TTC reduction in the nodes o,f corn. Theleast reduction of TTC occurred in plants which weregrown on the copper-deficient organic soil.

In the stalks of corn and wheat which were cutopen, the red formazan precipitate was observed tobe located principally in the nodes. The internodes

FIG. 1. Comparative bur development on cocklebursgrown on soils (left to right): copper-deficient organicsoil and organic soil to which CUSO4 *5H20 was added.Greatest reduction of 2,3,5-triphenyltetrazolium chlorideoccurred in the burs which were grown on the soil towhich CUSO4* 5H20 was added.

FIG. 2. Comparative growth of corn and reduction of2,3,5-triphenyltetrazolium chloride (TTC) in the nodesof corn grown on the five soil treatments (left to right):(1) calcareous soil, (2) J calcareous, A organic soil (byvolume), (3) A calcareous, i organic, (4) organic soil plusCuS04-5H20, (5) organic soil. The greatest reductionof TTC occurred in plants grown on the calcareous soil;the least reduction occurred in the copper-deficientplants, soil 5. The latter plants showed some browningin the nodal areas, but very little of the red formazanoccurred in these plants.

showed no, or very little, reduction of the dye. Thered formazan precipitate was most pronounced in theveins of leaves.

Table I includes data of the ascorbic acid oxidaseand catalase activity taken from previous experiments(3, 4), iron and copper concentrations as determinedspectrographically by Dr. A. W. Specht and J. W.Resnicky, and comparative ratings of TTC reductionin corn and wheat.The comparative ascorbic acid oxidase activity,

copper concentration, and TTC reduction ratings forcorn and wheat were similar when grown on the fivetreatments. The catalase activity and iron concen-

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TABLE ICOMPARATIVE ENZYMATIC ACTIVITIES OF ASCORBIC ACID OXIDASE AND CATALASE, COPPER AND IRON CONCENTRATIONS,

AND THE REDUCTION OF TTC IN TISSUE OF MINN. HYBRID #800 CORN AND THATCHER SPRING WHEAT

SOIL COMPARATIVE Cu * PPM COMPARATIVE COMPARATIVE Fe * PPMTR&AT-AAO ACTVITy b

LEAVES RATINGS oF TTC CATALASE LAEMENTS LEAVESW REDUCTION IN ACTIVITY c LEAVESLEAvEs NoDEs DRY WT NODES t IN LEAVES WT

Corn1 51 371 10 + + + + + 24 814 30 171 9 + + + + 70 742 19 261 11 + + + + 62 1103 19 91 3 + + + 125 935 - 4 7 3 + 193 150

Wheat1 23 163 11 + + + + + 427 854 24 118 25 + + + + 458 1102 15 49 + + + 393 1103 7 32 ..+ + + 310 1105 5 15 2 + 241 95

* Determined spectrographically by Specht and Resnicky.t This is a visual comparison and rating of the TTC reduced in the nodes of corn and wheat. The larger the

number; the greater the reduction of TTC.aSoil treatments: (1) Calcareous soil, (2) i calcareous, A organic (by volume), (3) A, calcareous, i organic,

(4) Organic soil plus CuSO4.5H20, (5) Organic soil.b ul of oxygen taken up/hr x 2 ml aliquot of plant sap extracted from 10 grams fresh weight sample using ascorbic

acid as substrate. Sample pulverized and extracted with 5 ml of demineralized water.cVolume of 02 evolved (ml)/5 min x gram fresh weight of plant material.

tration differed between the two plants in some treat-ments. The catalase activity was comparativelytwenty times greater in wheat than in corn whengrown on the calcareous soil. Corn showed symptomsof lime-induced chlorosis; wheat did not show thesesymptoms. Yields were less and copper-deficiencysymptoms were much greater for wheat than for cornwhen grown on the copper-deficient soils. The ad-dition of copper to the organic soil increased theascorbic acid oxidase activity in both corn and wheat,but it decreased the catalase activity in corn and in-creased it in wheat. The iron concentration decreasedin the corn leaves but did not vary appreciably in thewheat leaves with the addition of copper to the or-ganic soil.The effect of copper on the fruit development in

cockleburs was shown very clearly in the failure ofthe 'burs to develop (fig. 1) when copper was deficient.Plants grown on the calcareous soil, and organic soilto which copper was added, had a higher ascorbic acidoxidase activity and copper concentration than theplants grown on the copper-deficient soils (4). Thegreen weight of burs developed on these plants wasten times greater than that harvested from the twomost copper-deficient treatments. TTC was reducedto a much greater extent in the large developing bursof normal plants than in the undeveloped burs ofcopper-deficient plants.

Reduction of TTC appeared to be greatest in themeristematic regions of the plant. As the corn andwheat became more mature, the older nodes showedless reduction of TTC. Porter et al. (10) have indi-cated that the development of the non-diffusible red

formazan in a specific tissue is presumably an indica-tion of the presence of active respiratory processes inwhich electrons are transferred to TTC. That wheatgerm contains cytochrome oxidase was shown byBrown and Goddard (2) who found that the oxidasewas inhibited by HCN, NaH3 and photoreversibly byCO. Allen and Goddard (1) dbserved that matureleaves of wheat are not inhibited by these samepoisons. In differentiation of the leaves, Goddard(6) concludes there has been a change in metabolismthat either blocks the sensitivity of the cytochromeoxidase to these poisons or a new oxidase system hasbeen developed which replaces the cytochrome oxi-dase. James (8) more recently has found the cyto-chromes, cytochrome oxidase and ascorbic oxidasepresent in barley embryos; polyphenol oxidase wasabsent. The reaction mechanism in young roots wasfound to be quite different from that of the embryos.The cytochrome system and polyphenol oxidase wereboth absent in young roots, and the only oxidase yetidentified was ascorbic oxidase. These data indicatethat so fundamental an attri.bute of the living cell asits transfer of electrons to atmospheric oxygen maysuffer changes of mechanism even at a time when thecell is in full vigour of growth.

In this study, the reduction of TTC is best corre-lated with ascorbic acid oxidase activity and copperconcentration than with catalase activity or the con-centration of iron in these plants.

CONCLUSIONSCopper deficiency retards the development of re-

productive organs of corn, wheat and cockleburs. The

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dye 2,3,5-triphenyltetrazolium chloride was onlyweakly reduced in the copper-deficient plants. Maxi-mum reduction of TTC was obtained in plants grownon a naturally calcareous soil or an organic soil towhich copper was added.

Comparative ascorbic acid oxidase activity, catalaseactivitv and the concentration of copper and iron incorn and wheat showed that the reduction of 2,3,5-triphenyltetrazolium chloride in these plants could bestbe correlated with copper than with iron nutrition.

LITERATURE CITED1. ALLEN, P. J. and GODDARD, D. R. A respiratory

study of powdery mildew of wheat. Amer. Jour.Bot. 25: 613-621. 1938.

2. BROWN, A. H. and GODDARD, D. R. Cytochrome oxi-dase in wheat embryos. Amer. Jour. Bot. 28: 319-324. 1941.

3. BROW.N, J. C. and HENDRICKS, S. B. Enzymaticactivities of copper and iron deficiencies in plants.Plant Physiol. 27: 651-666. 1952.

4. BROWN, J. C. The effect of a dominant iron or

copper requiring metabolic system in the plant onthe development of lime-induced chlorosis. Sub-mitted for publication in Plant Physiol.

5. DUFRENOY, J. and PRATT, R. Histophysiologicallocalization of the site of reducing activity instalks of sugar cane. Amer. Jour. Bot. 35: 211-229.1948.

6. GODDARD, D. R. Metabolism in relation to growth.Growth: Supplement to Vol. XII. 17-45. 1948.

7. HEWITT, E. J. and AGARIVALA, S. C. Reduction oftriphenyltetrazolium chloride by plant tissues andits relation to molybdenum status. Nature 169:545-546. 1952.

8. JAMES, W. 0. The terminal oxidases in the respira-tion of the embryos and young roots of barley.Proc. Roy. Soc. London B 141: 289-299. 1953.

9. LAKON, GEORG. Topographischer Nachiveis der Keim-fahgkeit der Getreidefruchte durch Tetrazolium-salze. Ber. Deutsch. Bot. Ges. 60: 299-305. 1942.

10. PORTER, H. H., DURALL, M. and ROMM, H. J. Theuse of 2,3,5-triphenyltetrazolium chloride as ameasure of seed germinability. Plant Physiol. 22:149-159. 1947.

THE EXTRACTION AND COLORIMETRIC ESTIMATION OF INDOLE-3-ACETIC ACIDAND ITS ESTERS IN DEVELOPING CORN KERNELS 1, 2, 3

0. N. HINSVARK, WM. H. HOUFF, S. H. WITTWER AND H. M. SELLDEPARTMENTS OF HORTICULTURE AND AGRICULTURAL CHEMISTRY,

MICHIGAN STATE COLLEGE, EAST LANSING

The chemical identification of auxin, indole-3-aceticacid (IAA), isolated from the ethanol extracts of im-mature corn kernels by Haagen-Smit, Dandliker,Wittwer and Murneek (3) initiated considerable in-terest in both the quantitative and qualitative aspectsof the auxin economy of corn kernels during theirontogeny. Avery, Berger, and Shalucha (1), Wittwer(8) and Stehsel (6) have studied these changes inauxin content during the development of the cornkernel. Recently Redemann, Wittwer and Sell (5)have reported the isolation of ethylindole-3-acetatefrom corn kernels and found that this substance wasapproximately 100 times more effective than IAA as atomato fruit-setting agent.To supplement biological methods of auxin assay, a

colorimetric method was first described by Mitchelland Brunstetter (4). This procedure was later im-proved by Tang and Bonner (7), and by Gordon andWeber (2). Essentially, the method takes advantageof the red coloration formed through the mild oxida-tion of IAA.4 Although sensitive, it is not alwayssuitable for the determination of the auxin concentra-tion in plant materials because of the presence of otherpigments and the frequent formation of relatively

1 Received April 27, 1953.2 Journal Article No. 1465 from the Michigan Agricul-

tural Experiment Station, East Lansing, Michigan.3 This research was supported by the Horace H. Rack-

ham Research Endowment of Michigan State College.4 This oxidation product will be described in another

publication.

stable colloidal suspensions. Since difficulties are en-countered with extracts of plant materials, it seemedadvisable to describe further refinements of the colori-metric methods developed in this laboratory and espe-cially those procedures adapted for the quantitativedetermination of IAA and its esters in corn kernelsduring the various stages of development.

EXPERIMENTALCOLORIMETRIC MEASUREMENTS: The procedure is

modified from that of Gordon and Weber (2), in that0.05 M ferric chloride in 10% (by volume) perchloricacid was used to develop the red oxidation product,maximum intensity of which was attained in 45 min-utes when held at 30°C. The 10% perchloric acid wasused in preference to 35% to facilitate extraction inthe final separation. The red color was quantitativelyextracted with isobutyl alcohol, and the isobutyl alco-hol layer centrifuged to remove the suspended waterdroplets. Since the red color is unstable in thismedium, the time after extraction to reading wasstandardized at five minutes; the absorbancy was de-termined at 530 m/i on a Beckman D.U. spectropho-tometer. Under these conditions, Beer's law wasobeyed over a concentration range of 0 to 0.8 mg/25ml isobutyl alcohol and an absorbancy index of 2.7 ml(cm mg IAA)-l.APPLICATION OF THE METHOD TO PLANT MATERIAL:

To ascertain the accuracy of the method, weighedamounts of IAA were added to 500 gm samples ofimmature corn kernels at the milk stage and macer-ated with ethyl acetate in a Waring blendor. The

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