Mineral Deficiency & Organic Constituents in Tobacco Plants

Transcription

1 Mineral Deficiency & Organic Constituents in Tobacco Plants III. Plant Growth & Alkaloid Contents Related to Gradual Development of Calcium or Boron Deficiency Symptoms' T. C. Tso, J. E. McMurtrey, Jr., & R. N. Jeffrey United States Department of Agriculture, Agricultural Research Service, Crops Research Division, Beltsville, Maryland In the preceding papers (7, 8) of this series we presented the effects of certain mineral deficiency on the organic constituents of tobacco plants. Changes in the organic composition were associated with the development of typical symptoms resulting from an abnormal supply of a specific mineral. Mineral deficiency symptoms, in general, appear initially in a localized area and are very mild. They gradually extend to other portions of the plant and cause further characteristic effects. The severity of the symptoms increases with age and development of plants. In the case of calcium or boron deficiency, initial symptoms appear at the tip of a tobacco plant. Further abnormalities or symptoms gradually advance toward the midd(le portion and finally to the whole plant if the specific nutrient is withdrawn at an early stage of the plant growth. In tobacco plants, calcium or boron deficiency causes, in addition to other symptoms, natural decapitation which is physically similar but physiologically different from manual topping in tobacco cultivation. The present and following reports (in preparation) provide data on the corresponding changes in organic constituents including alkaloids, sugars, organic acids, andl amino acids relating to the gradual development of Ca- or B-deficiency svmptoms. In this paper results are reported on the plant growth and the alkaloid content in the at various degrees of severity of deficiency symptoms and at different stalk positions. Materials & Methods Plants of Nicotiana tabacumii L. cv. Connecticut Broadleaf were studied. Nutrient solutions of full strength with reduced level of Ca or B or without Ca or B were prepared according to McMurtrey's formula (2) in which the levels of Ca and B are 245 and 1 ppm, respectively. All experiments were carried out in the greenhouse. Tobacco seeds were germinated in sterilized sand and seedlings were sprayed occasionally with one-tenth strength complete nutrient. When seedlings were about five inches tall and had five or six Received May 17, 1962., they were transferred first to a starter solution of half-strength complete nutrient. Three weeks later when the plants were fully established, eaclh plant was transferred to the experimental solution in one of sixty 10-liter jars. These jars, their covers, and aerators are made of pyrex glass, and water used in this experiment was obtained from double (listillation with an all-glass still. Two separate crops were used for this investigation. The first, designated as winter crop. was transferred on Dec. 29, 1959, to different solutions including complete nutrient and nutrient solution complete for all other elements, but with only 10 % of the usual level of Ca or B. or without addition of Ca or B. The secon(d crop, designated as spring crop, was transferred on April 11, 1960, to solutions similar to those of the winter crop, except that or 1 % B was used instead of 10 % Ca or 10 % B. Calcium and boron deficiencies caused natural decapitation of tobacco plants as the apical buds ceased to grow. As soon as this synmptom appeared, some of the corresponding control plants growing in complete nutrient were topped (decapitated) and suckers were removed thereafter to serve as an additional control designated as the "topped control." Plants were harvested when (leficiencv symptoms had reached various stages. The last sampling occurred when the entire plants of and -Ca treatmlents showed severe symptoms whiclh ould have resulted in the loss of if the plants had not been harvested. Three plants of each treatment were harvested at each timle indlicatel, and the corresponding plant parts were combined to obtain enough mlaterials for chemical investigation. Only w,ere analyzed, but the fresh weights of stems and roots are reported. Leaves were divided into three groups on the basis of their stalk position-top. miid(dle, and bottom. Extractions were made at roomii temperature (22 + 2) using a Waring Blendor. For alkaloid deternmination each 50 g of fresh plant material wvere extracted for 7 minutes xvith 50 % acetone, taking account of the water present in the plant material. Individual alkaloids were separated and semi-quantitatively evaluated by paper chromatography (6). Three or more chemical analyses were nmade on each sanmple. Since Connecticut Broadleaf tobacco con- 804

2 TSO ET AL.-MINERAL DEFICIENCY & CONSTITUENTS OF TOBACCO tains nicotine principally and no marked change in the composition of minor alkaloids was observed, only total alkaloid is reported. 'able I Plant Appearance & Development of 1960 Winter Crop 805 Results & Discussion Seasonal variations appeared to cause marked differences between winter and spring crops. Although there was little temperature change under greenhouse conditions between the winter crop (Dec. 8-Feb. 4) and the spring crop (March -May 3), the difference in daylength was considered to contribute to the variations between these two crops (1). The spring plants grew much faster; the deficiency symptoms appeared earlier and developed more rapidly (tables I & II). In general, however, the symptoms which developed in the two crops were rather similar. The fresh weights of the top and the roots from the topped controls were greater than those from other treatments, but the weights of the middle and bottom in the two controls (topped & untopped) differed less. The fresh weight of the - Ca plants was always smaller than that of plants given other treatments. Plants supplied with 10 % of the usual amount of Ca or B nutrient level developed as well as untopped control plants until they approached the fourth sampling when mild deficiency symptoms were evident. Plants supplied with only 1 % of the usual amoult of Ca or B nutrient level, however, had initial deficiency symptoms within 4 days of the appearance of such symptoms in -Ca or plants. Ca-deficiency symptoms generally appeared earlier than symptoms. The alkaloid contents of different plant parts associated with various stages of deficiency symptom developed in the two crops are shown in tables III and IV. The order of alkaloid content per plant of different treatments agrees with that reported previously (8); i.e., topped controls had the highest total alkaloid content and, -Ca, and untopped control followed in decreasing order. DaysafterFrw gpat start (Dec. Plant treatments & Fr wt (g/plant) 29) of deficiency symptoms Top Middle Bottom treatments Sample Root Stem Total 0 Normal plants put into different solutions First sampling (a) -Ca symptom started to show a peculiar hooking downward - Ca of tips of the young making up the bud. (Mild stage) (b) Some control plants topped 15 Second sampling (a) -Ca symptom intensified by death of tips & margins of young -Ca Remaining dark green and thickened (Moderate stage) (b) symptom started to show. Tips of young ceased to grow and ashy (Mild stage) 27 Third sampling (a) symptoms intensified by death of terminal bud. Leaves Topped thickened & brittle (Moderate control stage) Fourth sampling (a) 10 % Ca & 10% B plants showed early deficiency symp- Topped toms (Mild stage) control (b) -Ca plants-all terminal 10 % Ca growth stopped. Some necrotic 10 % B spots & chlorosis in older -Ca ( Severe stage) (c) plants-upper & some middle of plant tend to roll in half circle from top toward base, light green in color. Lateral buds developed in the axils (Severe stage)

3 806 Days after start (Apr. 11) of treatments Plant treatments & deficiency symptoms* PLANT PHYSIOLOGY Table IT Plant Appearance & Development of 0 Normal plants put into different solutions 3 -Ca symptom started to show (Mild stage) 5 (a) symptom started to show (Mild stage) (b) Some of control plants topped 7 First sampling (a) plants started to show symptoms (Mild stage) (b) plants started to show symptoms (Mild stage) 15 Second sampling (a) Both -Ca and symptoms reached moderate stage (b) and symptoms reached advanced mild stage 22 Third sampling (a) -Ca and symptoms reached severe stage (b) and symptoms reached moderate stage * As described in table I 1960 Spring Crop Fr wt (g/plant) Sample Root Stem Top Middle Bottom Total Ca Topped control -Ca Topped control - Ca Days after start of treatments Table III Total Alkaloid Content in Leaves Associated With Various Degrees of Deficiency Symptoms in 1960 Winter Crop (mg/plant part) Sample & symptoms Top Middle Bottom Ca (mild) Ca (moderate) (mild) Topped control (moderate) Topped control % Ca (mild) % B (mild) Ca (severe) (severe) Total Alk. X 104 alkaloids Root

4 Days after TSO ET AL.-MINERAL DEFICIENCY & CONSTITUENTS OF TOBACCO Table IV Total Alkaloid Content in Leaves Associated With Various Degrees of Symptoms in 1960 Spring Crop (mg/plant part) Deficiency Sample Top Middle Bottom Total Alk. X 104 treatments symptoms alkaloids Root % Ca % B Ca (mild) (mild) Topped (mild) % B (mild) Ca (moderate) (moderate) Topped control (moderate) (moderate) Ca (severe) (severe) %7 In the winter crop the alkaloid content in the plants supplied with 10 % of the usual level of Ca or B was lower than in the untopped controls. In the spring crop the alkaloid contents of the or B plants were between those of the corresponding -Ca and plants and the untopped controls. Since it has been claimed that nicotine synthesis is proportional to the root weight (3), the last columns of tables III and IV show the ratio of alkaloid content of to fresh weight of roots. The alkaloid content of other plant parts is not included; however, since always contain the majority of the alkaloid in plants grown under these conditions, the ratios could not be much more uniform between treatments if the alkaloid content of the remaining parts had been determined. The ratios, expressed as alk. X 104, vary from 1 root to 6 for untopped control plants with no clear evidence of a difference related to age at harvest or season when grown. The ratios for topped control plants fall in the region from 13 to 23. Those with mild calcium-deficiency symptoms fall in an intermediate range and those with moderate or severe calciumdeficiency symptoms are more nearly equal to those of manually topped plants. The plants with mild boron-deficiency symptoms had ratios at or slightly above those for untopped control plants, but the values rose much more rapidly with this type of physiological topping to 39 and 69 for the plants with severe boron-deficiency symptoms. Since the amount of root branching affects the number of root tips in which most nicotine synthesis is generally believed to occur, the roots in the different treatments were carefully examined. The borondeficient plants showed slight increase in root branching (5), but not as much an increase as in the ratio of alkaloid to root weight of about ten times. No difference in proportion of root branching was observed in the plants of the other treatments. To examine further the relationship between alkaloid content and root weight, a comparative study was made (as shown in table V) on the change of fresh weight of roots and the corresponding change in alkaloids. Since no systematic relationship between these two sets of data was found, it appears that the alkaloid production is not in direct proportion to total root weight. The distribution of alkaloids at last sampling in from different stalk positions varied from dif- Table V Proportion of Root Weights & Corresponding Alkaloid Contents of Different Treatments at Last Harvest Winter crop Spring crop Ratio Root Leaf Roo Leaf wt wt alkaloid ot alkaloid /plant wt /plant Topped control Untopped control % B (winter) or (spring) Untopped control % Ca (winter) or (spring) Untopped control No boron Untopped control No calcium Untopped control

5 808 ferent treatments. All untopped control plants and plants with 10 % or 1 % of the level of regular Ca or B supply had an alkaloid distribution with higher total alkaloid content in the bottom than in the upper (9). Manually topped plants in both crops had higher total alkaloid content in the top than in the middle and bottom. It wvas principally due to the better development of top and consequently greater fresh weight. Physiologically topped plants resulting from -Ca or - B had the highest total alkaloid content in either middle or bottom. The top of these Ca- or B-deficient plants could no longer function as normal due to severe dlamage from their respective deficiencies; therefore, their normal metabolic activities, including alkaloid formation and accumulation which usually took place in the top, were shifted to middle or bottom, depending upon the extent of the leaf damage or the severity of the symptoms. Scholz (4) used a different approach to study the effect of boron on alkaloid production in N. rustica. Leaf cuttings appeared to develop more root and produce more alkaloicl when grown in a solution containing 0.5 ppm boron than in one to which no boron was added. This result showed the complexity of physiological action of boron in Nicotiana plants. Summary Nicotiana tabacutmt L. cv. Connecticut Broadleaf plants were used to study the change in plant growth and alkaloid content related to the gradual development of Ca- or B-deficiency symptoms. Consequently, seasonal variations, winter or spring, caused differences in rate of plant growth and the rate of symptom development; however, the symptoms produced, the relative plant growth, and alkaloid synthesis following different treatments in these two crops were similar. When calcium or boron was applied at 10 % or 1 % of the usual level, symptom development was slower and the modification of the alkaloid content was similar to that observed at the same stage of symptom development in the plants from which calcium or boron was completely withheld. Decapitation, or manual topping, increased root growth, but such increase was not found in plants I'LANT PHYSIOLOGY physiologically topped (lue to Ca or B (leficiency. The ratio of alkaloid found in the to root weight increased with nmanual topping and Nith physiological topping. The weight of the root itself appears not to be in direct proportion of alkaloid production. The patterns of leaf alkaloid (listribution regarding stalk positions varied from different treatments andl were affected by the severity of the (leficiency symptoms. Acknowledgment We thank Mrs. Tamara P. Sorokin for the determination of tobacco alkaloids. Literature Cited 1. GAUCH, H. G. & W. M. DUGGER, JR The physiological action of boron in higher plants: A review & interpretation. Univ. of Maryland Agr. Exp. Sta. Tech. Bul. A MCMURTREY, J. E., JR Distinctive effects of the deficiency of certain essential elements on the growth of tobacco plants in solution cultures. USDA Tech. Bul SOLT, MARIE L., R. F. DAWSON, & D. R. CHRISTMAN Biosynthesis of anabasine & of nicotine by excised root cultures of Nicotiana glauica. Plant Physiol. 35: SCHOLZ, G tyber die Bedeutung des Bors fur die alkaloid produktion. Z. Pflanzenernahr. Dung. Bodenk. 80: STEINBERG, R. A Effect of boron deficiency on nicotine formation in tobacco. Plant Physiol. 30: Tso, T. C., R. N. JEFFREY, & T. P. SOROKIN Biochemical studies on tobacco alkaloids. II. The relative absorption & assimilation of C14 from C14,O gas & from NaHC1403 solution. Arch. Biochem. Biophys. 92: Tso, T. C. & J. E. MCMURTREY, JR Mineral deficiency & organic constituents in tobacco plants. II. Amino acids. Plant Physiol. 35: Tso, T. C., J. E. MCMURTREY, JR. & TAMARA SORO- KIN Mineral deficiency & organic constitutents in tobacco plants. I. Alkaloids, sugars, & organic acids. Plant Physiol. 35: VICKERY, H. B Chemical investigations of the tobacco plant. XI. Composition of the green leaf in relation to position on the stalk. Conn. Agric. Exp. Sta. Bul. 640.