SULFUR METABOLISM OF PLANTS Effect of Sulfur Dioxide on Vegetation MOYER D. THOMAS,RUSSEL H. HENDRICKS, AND GEO. R. HILL American Smelting and Refining Company, Salt Lake City, Utah 1
Sulfur b an essential constituent of the plant proteins. Excess sulfur above that needed for organic aompounds is prcuent as sulfate. Organic sulfur values have narrow limit. in the different species of plants but sulfate concentrationm vary over a wide range depending on the sulfur supply to the plant. Sulfur fractionation and distribution have been studied by means of radiosulfur and
paper chromatography. Cystine and methionine account for a large part of organic sulfur in alfalfa leaves. Nitrogen and sulfur nutrition are closely related. Effect of sulfur dioxide absorption in sublethal amounts; mechanism of sulfur dioxide injury; role of environmental facton, in controlling absorption of gas by the plants; and effect of leaf destruction on yield are discussed.
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raised above the level reached by H. barely adequate supplyRather the additional uptake will appear in tho tissue largely as sulfate which in extreme cases can reach five to ten times the organic suSfur content before it becomes injurious to the systmn (91-93). In general the leaves of plants contain a considerably h i g h percentage of sulfur than the stems (in alfalfa about three times as much) and somewhat more than the flowers, though uwally less than the seeds. In view of the wide fluctuations of tho sulfate fraction with sulfur supply, the organic fraction (total sulfur minus sulfate) seems to be the more significant indm to the sulfur needs of the plant. For example, the organic sulfur in the needles of conifers is normally about 0.10% (dry I)a.ris); in alfalfa leaves and the leaves of many field crops, 0.25% (rang(? 0.16 to 0.40%); and in cabbage lerwes and other crucifers, 0.5 to 0.7%.
ULFUR is an essential element for plants because it is an essential constitucnt of most plant proteins and their amino acid fragments, cystine, methionine, and glutathione. There are, of course, other organic sulfur compounds in plants such as thiamine; methyl, allyl, or vinyl sulfides and disulfides; mustard oil; and many other isothiocyanates including their glucocides, sinigrin, and sinalbin (8, 13); and perhaps sulfonates like taurine. Recently a dithiol isobutyric acid has been isolated from asparagus (6). The identified organic sulfur compounds in plants is very limited, and it is not known whether any of these compounds besides the amino acids are essential to the plants.
UPTAKE OF SULFUR The principal source of sulfur for plants is soluble sulfate in the soil. Organic sulfur in the soil is doubtless a potential source, but some organic sulfur compounds may be absorbed directly, whereas others would need to be solubilized and perhaps converted to sulfate. Another source which may be important in industrial areas is sulfur dioxide in the air. The gas is absorbed into the leaves where it can serve aa a nutrient, if the sulfur supply is limited. Absorption of sulfate from the soil and its distribution throughout the plant is rapid. Likewise the reduction of the hexavalent sulfur and its incorporation into organic compounds may be rapid depending on the needs of the plants. For example, radioactive cystine sulfur has been isolated from sulfur deficient tomato leaves which were killed 10 minutes after the addition of radiosulfate to the nutrient solution. Uptake of sulfate from a nutrient solution occurs principally during the daytime and is slight at night. This has been clearly observed in tomatoes by using radiosulfate (18). Samples of tomato leaves were taken in early morning and late afternoon on successive days after adding radiosulfate to the nutrient solution. Usually there was an increme in the total radiosulfur and also the organic fractions during the day, but there was no change or a decrease during the following night. Evidently the uptake is associated with the large daytime transpiration stream because on cloudy days, uptake is reduced. The amount and disposition of sulfate taken up by the plant depends on the supply. If the supply is deficient or barely adequate practically all the sulfur will be incorporated into organio forms, and little or no sulfate will remain, In this range the color of the leaves and the rate of growth of the plant will depend on the sulfur supply because sulfur deficient plants are chlorotic, etiolated, and smaller than normal. If the supply of sulfate is plentiful, additional sulfate will be absorbed, but growth will not be appreciably stimulated, nor will the organic sulfur level of the various plant structures be greatly
FRACTIONATION OF SULFUR Attempts have been mpde to fractionate the sulfur in a nuinher of plants (19, $8). Sulfate can of course be separated readily from organic sulfur compounds. The latter are subjected to an alkaline digestion under hydrogen with magnesium and cadn~ium hydroxide which hydrolyzes the sulfur from cystine and a few other closely related compounds but does not attack methionine or other substances with firmly bound or oxidized sulfur. After removing the hydrolyzed cystine sulfur as hydrogen eul fide. the remainder is recovered partly as soluble organic sulfur and partly as insoluble compounds (largely proteins) along with the insoluble plant residues. These separations have been useful for distinguishing between the labile sulfur compounds close1~-related to cystine and the more stable sulfur compounds. It h w been found ($9) that the labile (cystine) sulfur content of alfalfa and sugar beet leaves, normally about O.lO%, may be about half this amount under sulfur deficiency conditions but concentrations appreciably greater than about 0.13% h a w not been observed even with m abundant sulfur supply. Sulfur deficient sugar beet roots contained practically the same amount of labile sulfur as the normal beets but only about one tenth as much as the leaves. PAPER CHROMATOQRAPHY
Another promising method of fractionation and one that is beginning to yield results is the method of paper chromatography. In cooperation with Steward (18) and his associates at Rochester University, the authors have been able to separate methionine, cystine, and a number of other sulfur compounds from alfalfa leaves. By using radiosulfur, the identification and an estimate of the concentration of cystine and methionine are positive.
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Table I. Radiosulfur Fractions in Tomato Leaves after Addition of Radiosulfate or Radiosulfur Dioxide to Plants at Midnight Time of Treatment Sampling 802 fumigation at 11 P.M. 1-6 hoursb 3-8 day8 SO days NazSO, addition to nutrient 7 houre solution at 12: 30 A.M. 1-8 days 48 days 5 D r basis. b Berore daylight.
Total Sa6 in Leaves, Counts/Seo./G.5 300 330 300 54 400 180
Percentage of '8% As watersoluble Insoluble Sulfate Cystine S organic S organic 9 51 7 29 13 21 21 37 21 26 41 16 42 l7 8 37 13 25 6 22 47 2 17 30 51
Several other unidentified radioactive organic sulfur compounds have also been found in these leaves. Alfalfa leaf fractions containing radiosulfur such as (1) the alcohol-soluble fraction with its free amino acids and amides; (2) water-soluble, heabcoagulable proteins; and (3) water- and alcohol-insoluble proteins have been studied. The proteins have to be hydrolyzed with strong acid. The amino acids and amides are swept in one direction on a filter paper by a capillary stream of a phenol-water mixture and in the other direction by moist collidine-lutidine. The separated compounds are revealed by ninhydrin, and the sulfur compounds are found both by the Geiger counter and by a radioautograph. In making the chromatograms, the sample on the paper is treated with ammonia to neutralize any free acid and with hydrogen peroxide to form cysteic acid from cystine and methicnine sulfoxide or sulfone from methionine. When the activities of the hydrolyzed protein samples were counted before making the chromatogram, 50 to 65% of these counts could be found in the spots derived from cystine and methionine. These indicated percentages of the sulfur amino acids should be raised considerably, because, owing to partial absorption of the weak sulfur radiation by the paper, a given amount of S35shows less activity if spread out over a large area of paper than if concentrated in a smaller area. Only a relatively small amount of radiosulfur could be found in other places as definite spots. One to four such weak spots were found in several autographsfor the most part where ninhydrin color was absent; this suggests the spots were not due to amino acids. Some indefinite streaks were also noted. Methionine predominated over cystine in the water- and alcohol-insolubleprotein, whereas the reverse was true in the water-soluble heat-coagulable protein. In the unhydrolyzed alcohol-soluble fraction nearly all the activity was found where glutathione would be expected. A trace of cystine was revealed but no methionine. After hydrolysis of this fraction more cystine but no methionine was found; this suggested that glutathione was actually present. Evidently methionine is so avidly built into proteins that its concentration in the free state is low. TRACER EXPERIMENTS
Using radiosulfur as a tracer, the relative sulfur concentrations in the organic fractions can be followed conveniently by the Geiger counter, after separating the sulfur as barium sulfate. In an experiment (18) in which a fumigation with radiosulfur dioxide was applied to sulfur deficient tomato plants a t 11 P.M., a leaf sample taken before daylight showed that only about half the absorbed sulfur remained as sulfate; the other half was converted into organic forms with the soluble noncystine organic fraction predominating. Table I gives the sulfur distribution in this sample and in subsequent samples from the same plot. Evidently light is not necessary for the reduction of sulfate. The table also gives similar data for a plot treated with radio sulfrtte. The sulfate rapidly and nearly completely disappeared from the sulfate treated leaves, but an appreciable amount persisted in the sulfur dioxide treated leaves throughout this
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period. The insoluble organic fraction increased uniformly until, after several weeks, it predominated in the lesvw. RADIOAUTOGRAPHS
Because the organic sulfur is associated principally with the proteins, those cells of the plants are richest in sulfur which are also richest in proteins. Using radiosulfur it has been shown by radioautographs that in the roots and stems of tomatoes and alfalfa, the principal sulfur accumulation occurs in the inner and outer phloem and cambium areas whereas the pith and xylem areas are relatively low in sulfur. In sugar beets (18), autographs correspond closely with the ring pattern of the beet, but more detailed examination shows that the accumulation is once more principally in the phloem and cambium though the latter are so intimately associated with the xylem that it is difficult to differentiate between the various areas. The storage cells are low in sulfur. Small lateral roots of all plants studied show higher concentrations than the main fleshy root. This is to be expected because the small young roots are developing rapidly and are composed primarily of meristematic tissue with sulfur-rich protoplasm. The older roots have a considerable amount of storage tissue. In tomato stem's (18) the inner and outer phloem and outer layers of the xylem adjacent to the phloem have strong activity when the radiosulfur is added as sulfate to the roots, but the inner two thirds of the xylem cylinder is low in sulfur. On the other hand the entire xylem is lacking in activity when the radiosulfur is added to the leaves as sulfur dioxide. Evidently in the former case the radiosulfur moves upward in the xylem as well as laterally and downward in the phloem, whereas in the latter case it moves only downward in the phloem. In wheat (S,80) as much as 60 to 80% of the radiosulfate taken up by the plant is found in the kernels a t maturity. Here it is accumulated more strongly in the embryo than in the endosperm, though the aleuron layer around the endosperm shows large accumulations also. This sulfur appears to be largely bound in the proteins. Similarly, tomato fruits (18) show maximum concentrations in the seed, particularly in the embryo, but appreciable amounts are also found in the vascular strands, the locular surfaces, and the skin.
SULF'UR AND NITROGEN In view of the essential presence of sulfur in the plant proteins, the relationships of nitrogen and sulfur are of interest. Nightingale ( 1 1 ) and Eaton (8) have shown that nitrogen-rich sulfurpoor plants, particularly the stems, carry considerable amounts of nitrate, amide and free amino nitrogen, and also more than normal amounts of some carbohydrates. They suggest that this condition is first, due to a lowered rate of nitrate reduction; secondly, to less than normal protein synthesis because of an inadequate supply of the sulfur-containing amino acids; and thirdly, to considerable proteolysis in the stems. Evidently sulfur deficiency causes abnormal nitrogen metabolism. These observations have been confirmed with alfalfa (2'4.4)using high nitrogen, low sulfur nutrient solutions. But when the supplies of both nitrogen and sulfur are low, the abnormalities disappear suggesting that there is enough sulfur present to take care of all the nitrogen that is available.
SULFUR DIOXIDE The action of sulfur dioxide on vegetation has been extensively studied for over 76 years. Most of these studies have been
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INDUSTRIAL AND ENGINEERING CHEMISTRY
carried out to determine the effects of injurious concentrations of the gas on plants (4, 9, 14, 16, 17, 96-99),but more recently sublethal concentrations have been investigated also (9, 14, 91, 27). Kata has recently reviewed the literature on the relations of sulfur dioxide in the atmosphere to plant life (6). The mechanism of sulfur dioxide injury is still not fully understood, but the following discussion summarizes present knowledge of the subject. Sulfur dioxide is a very soluble gas which is readily absorbed by the leaves of plants. Only a limited absorption occurs on external s u r f a m unlem they are wet. Most of the absorbed gas enters the leaves through the stomata and dissolves on the moist surfaces of the mesophyll cells. Here its behavior depends on the rate at which it enters the system. If the rate is slow the gas may be oxidized, or to a small extent reduced, as rapidly as it is absorbed. It is also rapidly neutralized by the organic bases in the leaf. Absorption, oxidation, and neutralization may thus continue for a long time until relatively large amounts of sulfate have accumulated. The uptake of the acid which the sulfate represents reduces the buffer capacity of the leaf appreciably but does not change the pH of the tissues (99). There is only a slight tendency for this sulfate to be translocated out of the leaves. Alfalfa leaves hold the sulfate with particular tenacity; in one series of experiments 97% of the gas absorbed was accounted for in the leaves as sulfate (99). Presumably the organic sulfate in alfalfa has very little mobility. I n tomato leaves, the sulfate is not BO firmly k e d . Build-up of sulfate can continue until the buffering capacity of the leaves is reached. There appears to be little or no effect on the appearance of the leaf or on its fundtioning as measured by photosynbhesis until saturation of the buffer capacity is approached. Then the leaf becomes chlorotic, ceases to function, and is shed. If the rate of this proceea does not exceed appreciably the rate at which the plant can grow new leaves to replace the old, there is little or no deleterious effect of the gas on the growth and development of the plant. However, this process may be significant in plants like the conifers which cannot grow new leaves quickly to replace the old. If the plant happens to be suffering from sulfur deficiency, sulfur dioxide added in this way will stimulate growth, though not quite so much as will an adequate amount of sulfate supplied to the roots (21). If the sulfur dioxide is added to the leaves a t a greater rate than it can be oxidized and neutralized immediately upon entry, a build-up of sulfite occurs which can cause injury to the cells at a much lower level of concentration than the saturation sulfate level (9,%’), Intercostal or marginal areas collapse and dry out, leaving regions that are ivory-colored in most plants, but brown or brownish-red in some. Occasionally when insufficient sulfur dioxide is added to cause acute injury, chlorotic markings appear after 3 to 6 days. Sulfur dioxide can be recovered from the leaves of these plants by distillation with phosphoric acid both before and after collapse of the cells. The persistence of sulfite after a fumigation has been studied by Nielson (10) and his results are fully confirmed by the authors. The amount of sulfur dioxide recoverable after a fumigation falls off with time. In bright sunlight the sulfur dioxide usually disappears in 1 to 2 hours, though fully collapsed leaves retain the sulfite for a longer time; a t nigh+ it may persist for 10 to 12 hours. The initial stages of leaf collapse are reversible. When a short Fumigation with a high concentration is applied, the leaf may exhibit by transmitted light a water-soaked appearance in the interveinal areas. If these areas are not subjected to too strong a drying influence, either of wind or sunshine, the liquid may be reabsorbed into the cells and little or no injury may result. For example, sugar beet plots have been treated with this type fumigation. A concentration, 10 to 16 p.p.m., was applied for 10 minutes. After about 15 minutes, water-soaked areas began to appear. The extent of these areas increased until after I hour,
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when they involved about half the leaf area on the plot. They were most readily apparent by transmitted light. This condition was noticeably less marked after 2 hours and was nearly cleared up in 3 hours. Permanent leaf destruction amounted to only 1 to 2%. It is likely that if the fumigation had been prolonged somewhat, the plasmolysis would not have been reversible and leaf destruction would have been extensive. The experiment suggests that acute sulfur dioxide injury is, at least in part, due to a high local concentration of sulfurous acid on the mesophyll cells which causes plasmolysis. The high local concentration may be reached suddenly if the sulfur dioxide content of the air is sufficiently high, or it may be reached gradually with a milder fumigation. I n the latter case part of the absorbed sulfur dioxide will be oxidized and neutralized before the lethal concentration is reached, thus requiring the absorption of more gas than a shorter Pigher concentration fumigation to cause the same amount of leaf destruction. I n other words, the toxicity of a given dose of the gas will depend on its absorption rate. However, acute sulfur dioxide injury is doubtless not entirely a plasmolytic effect, otherwise sulfite and sulfate would have similar toxicity unless the cells are much less permeable to the sulfite than to sulfate. Evidently the sulfite exerts a toxic action probably due to its reducing properties. This would explain also the delayed chlorotic injury when the fumigation was not severe enough to cause acute markings. The toxicity of the sulfite may be manifested in several other ways: 1. Photosynthertis (6, 9, 27) may be inhibited temporarily if the amount of sulfur dioxide absorbed is somewhat less than that required to cause acute injury. 2. Respiration ap ears to be less definitely affected by moderate fumigations but g a t z et al. (9, page 393) have observed both increase and decrease in respiration rate under certain conditions. 3. The gas can influence some of the enzyme systems in the plants. Unpublished studies at this laboratory indicate that catalase activity of alfalfa leaves could be reduced radically by a morning fumigation that caused acute injury, but i t would be only slightly reduced by a similar fumigation accompanied by acute injury in the late afternoon. Diastase activity appeared to be increased by the fumigation treatments, but experiments on the oxidase and peroxidase systems were inconclusive. 4. Noack (1%)sug ested that sulfur dioxide injury to vegetation was characterizetby inactivation of the iron in the chloroplasts, interfering with its catalytic properties in assimilation. Secondar photochemical oxidative processes then caused bleaching and L a t h of the cells.
ACTION OF SULFITE ON WATER PLANTS Brooks ( 1 ) found in a study of the water plants, nilella, elodea, spirogyra, and hydrodictyon, that the toxicity of a sulfite solution was greater, the lower the pH. For example, in 0.0053N sulfite solution equivalent to 170 p.p.m. sulfur dioxide in the solution, elodea was uninjured by 4 hours’ exposure at pH 8.3, but was killed in 4 hours at pH 5.3 or 4.2. In 0.001 N sulfite, etodea withstood 72 hours at 7.9, but was killed in 12 hours at pH 4.3. Nitella was a little more resistant than elodea, but spirogyra and h y d r e dictyon were definitely more sensitive, in that order. The authors confirmed Brooks’ p H observations and have found that acidity increased toxicity because more sulfite was absorbed a t the lower pH values. Brooks did not study sulfate solutions, but the pond water he used contained 0.00154 N sulfate and 0.00108 N chloride which were within the range of toxic sulfite concentrations. All the plants except hydrodictyon grew luxuriantly in this water a t pH 8.0, and none of them except hydrodictyon was injured when the water was acidified by hydrochloric acid to pH 4.0. All were injured at pH 3.0. Evidently sulfate and acid sulfate are relatively nontoxic as compared with sulfite. Brooks suggested that there is really a close parallel between the treatment of water plants with sulfite solutions and the fumigation of land plants with sulfur dioxide, in spite of the fact that his sulfite concentrations were apparently 25 to 100 times greater than corresponding toxic concentrations of sulfur diovide in the
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Table 11. Relative Resistance of Plants t o Sulfur Dioxide* (Plants in order of resistance, with alfalfa, as standard, = 1.00) 2.19 1.00 Hydrangea Alfalfa 2.2R 1.00 Cockleburr Barley 2.29 1.00 Peach Prickly let,tuce 2.29 1.06 Linden Four o’clock 2.34 1.06 Nasturtium Rhubarb 2.41 1.07 Elm Cosmos 2.41 1.10 Birch Sweet pea 2.44 1.20 Iris Ragweed 2.45 1.22 Plum Radish 2.46 1.24 Poplar Dock 2.57 1.25 Horseradish Bouncing bet 1.25 2.59 Sweet cherry Mignonette 2.60 1.26 Canna Beet 1.30 Gladiolus 2.60 Oats 3.00 1.38 Potato (Irish) Bachelor button 3.25 1.44 Castor bean Squash 1,50 3.28 Maple Bean 1.52 3.28 Boxelder Carrot 3.33 1,52 Wisteria Wheat 1.59 Hibiscw 3.68 Aster 1.59 3.81 Virginia creeper Dandelion 3.96 1.62 Lilac Sweet William 3.96 1.64 Corn Parsley 5.22 1.65 Gourd Black mustard 5.84 1.71 Snowball Tomato 6.42 1.74 Celery Parsnip 7.67 1.79 Muskmelon Apple (foliage) 7.86 1.81 Arbor vitae Sweet clover 1.94 Currant (blossoms) 11.88 Catalpa 2.03 15.48 Privet Cabbage 20.99 2.06 Corn (silks and tassels) Hollyhock 2.14 25.50 Apple (blossoms) Gooseberry 2.14 Apple (buds) 87.29 Marigold 5 Table prepared by O’Gara about 1920.
air. From absorption data based on sulfite uptake by dead and living plants in the dark he estimated that the living cells of elodea absorbed about 0.50 gram of sulfur dioxide per hour per kg. of fresh plants from a solution of about 0.004 N sulfite at pH 8.0. This solution caused injury in about 6 hours, representing a lethal dose of 3.0 grams of sulfur dioxide per kg. of active plant cells. Thomas and Hill (E6)estimated that the minimum lethal dose of sulfur dioxide for the mesophyll cells of alfalfa is about 0.54gram per kg. of fresh mesophyll tissue if added instantaneously to the cells. If added over a period of 6 hours the lethal dose is estimated to be about 2.8 grams per kg. of active cells. Because of the close agreement of these dosage figures, some of Brooks’ experiments were repeated. Difficulty was encountered because of oxidation of the sulfite by the air. This was overcome by treating the plants in stoppered flasks connected to a supply of hydrogen. The,flasks were filled to the brim with sulfite solution and when samples of the latter were removed for sulfite titration hydrogen was admitted to the flasks instead of air. Some experiments were carried out similarly in the sulfide digestion apparatus (19). Sulfite remaining in the plant tissue could then be determined by washing the plants with water, acidifying with phosphoric acid, and removing the sulfur dioxide with hydrogen while boiling under reflux. The evolved gas was determined conductometrically after absorption in hydrogen peroxide solution. Data obtained with several water plants, such as elodea and uaucheriu, indicate that the plants absorb the sulfite in the dark only one half to one tenth as rapidly as was found by Brooks, but in the light at pH 6.0, the rate of sulfite loss from the solution was approximately the same as that quoted by Brooks, though in one experiment with elodea using a sulfite solution of initial concentration 0.0028 N the rate was two to three times as great. Recovery of the absorbed sulfite from the plants accounted for only 10 to 50% of the sulfite loss from the solution. Evidently the sulfite w m partly oxidized after absorption. In every case, less sulfite was removed from the solution and recovered from the plants a t pH 8 than at pH 6. This would explain the greater toxicity of the latter solutions. The limited number of experiments that have thus far been carried out tend to support the suggestions of Brooks that the living absorptive cells of such different species as alfalfa and elodea or vaucheria growing in different media have about the
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same sensitivity to the toxic action of sulfite. This suggests a fundamental protoplasmic effect. If such is the cme, it would be expected that plant cells in general would have similar sensitivity to sulfur dioxide, if they could absorb the gas in equivalent amount.
INFLUENCE OF ENVIRONMENT Although it is possible that the absorptive cells of different plants may be equally susceptible to injury by sulfur dioxide, it is certainly not true that different plants have the same susceptibility in the field, nor is any particular plant equally susceptible under different environmental conditions. Different plants vary widely in their susceptibility to injury by sulfur dioxide. Alfalfa and barley are usually the most sensitive of the crop plants, but some of the common weeds such as Guuru purwijlora, dock, malva, and prickly lettuce are about as sensitive. The latter are useful indicators in diagnosing a sulfur dioxide fumigation. O’Gara has rated other plants by comparison with alfalfa, using a resistance factor. For example, he found that oats and wheat will usually withstand about 1.3 to 1.5 times the gas concentration or exposure time that will cause injury to alfalfa under similar environmental conditions. Corresponding resistance factors for other common plants are given in Table TI, which was prepared by O’Gara on the basis of experiments carried out near Salt Lake City, Utah. Experience has shown that O’Gara’s factors are essentially correct so far as the authors have had occasion to check them, though plants grown in humid climates will usually show greater sensitivity than those grown in arid places. This is possibly due to morphological differences in theJeaves which have a more impervious epidermis with fewer stomata, if they were exposed to a dry rather than a moist atmosphere during their formative period. Environmental conditions during a fumigation are also important in modifying the sensitivity of the plant to the gas. In general the different investigators ( 4 , 6 , 9 , 1 6 , l 7 , % 6 , 2 8 , %agree 9) that sensitivity is highest when the soil is moist and the relative humidity and the light intensity are high. Another important consideration is the time of day when the fumigation occurs. With all environmental factors held nearly constant throughout the day, many plants are most sensitive to sulfur dioxide between midmorning and noon or early afternoon on a well-lighted day. Sensitivity is much lower in the early morning and falls off decidedly in the late afternoon (6). At night the sensitivity of plants is usually low though some plants such as potato are about as sensitive at night as during the day (10). If a plant has low light intensity in the morning and high light in the afternoon, the time of maximum sensitivity will be midafternoon. Evidently several hours are required, after a period of darkness or low light, to establish maximum sensitivity to the gas. These environmental conditions operate to influence the sensitivity of plants to sulfur dioxide primarily by controlling the stomatal apertures which in turn control the rate of absorption of the gas. High resistance in the late afternoon, at night, in the early morning, and under conditions of low moisture supply, appears to be due to closed or partially closed stomata. The potato does not normally close its stomata at night and is therefore susceptible in the dark. Conversely, alfalfa normally closes its stomata a t night and is resistant, but it has been noted that alfalfa leaves with open stomata are very susceptible at night. The time-of-day effects, already referred to, are associated with an opening and closing of the stomata and parallel change in the amount of gas absorbed. Loftfield (7) has illustrated beautifully, by photomicrographs, the normal diurnal trends of stomatal openings for the leaves of a number of plants. However, the decrease in sensitivity observed in the afternoon may in part be due to build-up in carbohydrates in the leaf which may reduce the toxicity of the absorbed sulfur dioxide. Kat2 and Ledingham (9, page 298) have discussed stomatal effects, arriving at similar conclusions.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
EFT’ECT ON YIELD Sulfur dioxide injury to vegetation is essentially leaf injury. Time-concentration-absorption curves have been worked out for the destruction of alfalfa leaves ($6, $6). As indicated in Table I1 much higher concentrations are required to injure other plant structures. There is no evidence to suggest that the gas is a systemic poison. Rather the effects are confined to the areas visibly injured, and no support has been found for the theory of “invisible injury” (4, 6, 9, 16,27). It has been shown (6, $6)that reduction of yield is a function of the percentage of leaf area destroyed and that the effect of fumigation can be simulated by clipping off an equivalent amount of leaf tissue from similar unfumigated plants. In vegetative plants like alfalfa, simple linear relations relate leaf destruction and yield. The yield of grain ($6),on the other hand, is reduced by a given amount of leaf destruction to a variable extent depending on the stage of growth. It is greatest at the blossoming stage and falls off both with younger and more mature plants. LITERATURE CITED (1) Brooks, P. M., dissertation, Stanford University (March 1943). (2) Eaton, 9.V., Botan. Gaz., 102,53G56 (1941). (3) Harrison, B. F., Thornas, M. D., and Hill, Geo. R., Plant Phyaiol., 19,245-57 (1944). (4) Holmes, J. A., Franklin, E. C., and Gould, R. A., U. 8. Bur. Mines, Bull. 98 (1915). (6) Jansen, E. F., J . Biol. Chm., 176,657-64 (1948). (6)Kata, Morris, IND. ENG.CHEM.,41, 2450-65 (1949). (7)Loftfield, J. V. G., Camegie Inst., Wash. Pub. 314 (1921). (8) McNair, J. B., Am. J. Botany, 28, 179-84 (1941). (9) National Research Council of Canada, Ottawa, “Effeot of Sulfur Dioxide on Vegetation,” 1939.
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Nielsen, J. P., dissertation, Stanford University (November 1938).
Nightingale, 0.T., Schermerhorn, L. G., and Robbins, W. R., Plant Physiol., 7, 665-95 (1932). Noack, Kurt, 2.Angew. Chem., 42, 123-6 (1929). Peterson, W. H., J. Am. Chem. Soc., 36, 1290-1300 (1914). Setterstrom, C., IND.ENO.CHEM.,32,473-9 (1940). Setterstrom, C., and Zimmerman, P. W., Contrib. Boyce Thomp son Inst., 10,155-81 (1939). Steward, F. C., Thompson, J. F., Miller, F. K., Thomas, M. D., and Hendricks, R. H.. Plant Physiol., in press (1950). Swain, R. E., IND. ENQ.CHEM.,15,296-301(1923). Thomas, M. D., Proc. Auburn Conference on Use of Radioactive Isotopes in Agricultural Research, pp. 103-17,Alabama Polytechnic Institute (December 1947). Thomas, M. D., and Hendricks, R. H., J. Biol. Chem., 153,31325 (1944).
Thomas, M.D.,Hendricks, R. H., Bryner, L. C., and Hill, Geo. R., Plant Physiol., 19,227-44 (1944). Thomas, M. D., Hendricks, R.H., Collier, T. R., and Hill, Geo. R., Ibid., 18,345-71 (1943). Thomas, M. D., Hendricks, R. H., and Hill, Geo. R., Ibid., 19, 212-26 (1944). (23)Thomas, M. D.,Hendricks, R. H., and Hill, Geo. R., Soil Sci., 70,9-18 (1960). (24)Ibid., pp. 19-26. (25)Thomas, M. D.,Hendricks, R. H., and Hill, Geo. R., Stanford
Research Inst. Pub., Air Pollution Symposium, Pasadena, Calif., pp. 143-7 (1949). (26) Thomas, M. D., and Hill, Geo. R., Plant Physiol., 10, 291-307
(1935). (27)Ibid., 12,309-83 (1937). (28)Zimmerman, P. W.,Proc. Stanford Research Inst. Pub., Air Pollution Symposium, Pasadena, Calif., pp. 135-41 (1949). (29)Zimmerman, P. W.,and Crocker, W., Contrib. Boyce Thompson Inst., 6,455-70 (1934). R E C E ~ VJuly E D 1, 1950.
SOURCES OF HYDROGEN SULFIDE IN WYOMING RALPH H. ESPACH U. 8. Bureau of Mines, Laramie, Wyo. Within the past several years, there have become available in the central and north central areaa of Wyoming and at the petroleum-refiningcenter at or near Billings, Mont., a large preuent supply and future reserve of hydrogen sulfide. The reserve is estimated at over 6,500,000 tons, from which almost 500 tons of elemental sulfur could be produced daily for a period of at least 23 years. The raw material for a heavy chemical industry-sulfuric acid-is
available. The preparation of superphosphates from Idaho, Wyoming, Montana, and Utah phosphate rock can proceed with the amsuranqe of ample acid supplies from within the area. Marginal chrome ores in southern Montana can be processed into usable products of industry, and other ores, the processing of which depends on the use of sulfuric acid, can become valuable resources. Other uses of sulfur or sulfuric acid within the area are indicated.
A
sandstone in the Elk Basin field, that large volumes of hydrogen sulfide were discovered. The hydrogen sulfide was associated with hydrocarbon gases in solution in the oil of the subsurface Tensleep reservoir. Following this, other oil and gas fields yielding quantities of hydrogen sulfide were developed. Worland and Big Sand Draw, in 1946; Neiber Dome, in 1947; Silver Tip, in 1948; and Beaver Creek and Riverton, in 1949, were the largest fields. Figure 1 is a map of Wyoming and adjoining areas showing the location of these fields. By January 1950 approximately 162,000,000,000 cubic feet of hydrogen sulfide had been proved to exist in association with both free gas and solution gas (principally hydrocarbons in solution in petroleum under existing conditions of pressure and temperature in the subsurface oil reservoirs). Table I summarizes information concerning the more important sources of hydrogen sulfide in
T SEVERAL localities in Wyoming the presence of hydrogen sulfide associated with other gases has been known for many years. The Indians named the river Shoshone because to them it waa “stinking water” owing to the hydrogen sulfide in the gases liberated from the hot springs emptying into the river. The hot springs a t Thermopolis also yield hydrogen sulfide. HYDROGEN SULFIDE FROM PETROLEUM AND NATURAL GAS FIELDS The production of gas in the South Baxter Basin field in 1922 and in the Garland field in 1927, and of petroleum in the Oregon Basin field in 1927 and in the Frannie field in 1928 resulted in fmding some quantities of hydrogen sulfide. However, it waa not until November 1942, with the discovery of oil in the Tensleep
