Abscission: Quantitative Measurement With A ... - Plant Physiology

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1. E) Explooded view of abscissor. An explant holding vise consisted of 2 pieces of. Plexiglas 0.9 cm wide by 5.1 cm high by 51 cm long held together at their 5.1.
Plant Physiol.

(1969) 44, 1139-1143

Abscission: Quantitative Measurement With A Recording Abscissor L. E. Graker and

F.

B. Abeles

Plant Sciences Laboratories, Fort Detrick, Frederick, Maryland 21701 Received March 31, 1969. Abstract. The construction, operation, and effectiveness of an abscission measuring instrument called an abscissor is described. The device measured the force required for a springopposed plunger to shear abscission zone explants and was capable of automatically recording break strength data. Examples of data obtained with the abscissor are presented to demonstrate its capability of rapidly measuring signifioant changes in explant brcak strength.

Two approaches have been used to obtain quantitative data on abscission. The first is the use of pressure applicators that tell the operator the number of abscission zone explants that will separate at some force required to separate plant parts as a result of predetermined force. The second is to measure the a pulling or shearing force. Examples of the first group of abscissors are the pres-ure applicators described by Mi:chell and Livingston (10) and Addicott et al. (5). The second group includes the simple but effective use of a laboratory balance (6), the use of a modified Chatillion force measuring device for measuring fruit abscission (7,8), and the use of an Instron Linear Stress-Strain Analyzer (11). This paper describes the construction and operation of a simple self-recording abscissor that measures the force required for a spring-opposed plunger to shear abscission zone explants.

Materials and Methods Plant Materials. The methods used to grow bean (Phaseolus vulgaris L. cv. Red Kidney) and cotton (Gossypium hirsutum L. cv. Acala 4-42) plants and to prepare and store explants have been described earlier (1, 2, 3). In the experiment in which the effect of ethylene on the break strength of explants from 18 varieties of P. vulgaris was tested, the plants were grown in the greenhouse during January and February 1968. Supplementary lights (150-watt incandescent bulbs spaced 4 meters apart and 2 meters above the bench) were used to give a 16-hr photoperiod. Seeds were planted in 10-cm diameter pots filled with soil and grown for 14 days before explants were excised. Cellulase Determinations. Bean explants were inserted in 1.5 % agar in petri plates and placed in a desiccator containing 10 ppm ethylene. Details of the techniques utilized to add ethylene to desiccators has been described earlier (4). At the time indicated in Fig. 4, explants were withdrawn and frozen until subsequent analysis for cellulase. Cellulase was extracted- by homogenizing 10 separation layers with 4 ml of 0.05 M potassium phosphate buffer, pH 7, in

a Ten Broeck homogenizer. The homogenate was filtered thru Mira-cloth (Calbiochem Corporation) and centrifuged at 10,000g for 10 min. A 1 ml por.ion of the cupernatant was analyzed for cellullase by adding it to 1 ml of 1.5 % sodium carboxymethyl cellulose (CMC) and measuring the viscosity of I ml of the mixture after a 16-hr incubation at 40Q. A model LVT Wells- Brookfield microviscometer (Brookfield Engineering Laboratories, Stougliton, Massachtisetts) was used to measure the viscosity of the CMC. Details of tllis method have been presen.ed earlier (4). Construction and Opcration of the Abscissor. A Plexiglas abscissor was used to measure the break strength of abscission zone explants. Details of its construction and a wiring schematic are shown in Fig. 1. The resistance of a decade resistance box was set so that the output across the ends of a wire wound resistor (Olhnmite Dividolim vitreous enameled adjustable resistor) was equal to 250 my. Other values can be used depending on the input of the recorder. The potential across the resistor was measured between 1 end of the resistor and the brush by a recording potentiometer. The brush was attached to a spring-opposed plunger and the amount of force required to push the plunger a fixed distance was regulated by the resistance of the spring. By inserting springs of different strengths between the plunger and cap of the abscissor, various force ranges were obtained. Fig. 2 slhows calibration data indicating that there was a straight line relationship between the force against the plunger and the output of the recorder. This curve was obtained by placing a known force on the face of the plunger held in a vertical position and reading the deflection on the recorder. The curve intercepts the abscissa at 25 grams in this example because a finite amount of force is required to overcome the force of the spring holding the plunger against the front adjusting screw. The distance traveled by the steel brush can be set by adjusting screws at each end of the slot in which the brass brush hiolder moves. Electrical contact to the brush was made by means of taut copper slide wires attached to these adjusting screws.

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BRUSH HOLDER SLIDE WIRE

IKQ RESISTOR SPRING

BARREL

CAP

PLUNGER

,

1 cm

1 cm

SIDE

PLU NGER

FRONT

1 cm SLIDE WIRE BRUSH HOLDER FIG. 1. Construction details and schematic of the recording abscissor. The 1-cm line indicates the scale of the drawing. A) (top left) Overall view of the abscissor. B) (top right) Front view of the abscissor. C) (bottom left) Top view of the abscissor. D) (battom right) Schematic of the abscissor. 350

300/ 250

/ -

200/ 0

150. 100 / 50A

FIG. 1. E) Explooded view of abscissor.

An explant holding vise consisted of 2 pieces of Plexiglas 0.9 cm wide by 5.1 cm high by 51 cm long cm faces by bolts fitted held together at their with wing nuts. Steel guide pins set in the 5.1 cm side of 1 piece of Plexiglas were inserted into holes in the other piece of P.lexiglas to insure proper alignment. Holes centered on the crack between the 2 pieces of Plexiglas were drilled along the length of the vise. In order to accommodate explants of 5.1

tI

0

10.

20

30 40 50 60 Recorder Reading

I _

70

80

_

90 100

FiG. 2. Calibration curve showing the relationship between force and displacement of the abscissor plunger as measured by a recording potentiometer. Dashed lines indicate 95 % confidence limits.

different sizes, 2.5 mm, 3.5 mm, or 4 mm holes were used. The explant vise was held with the long axis horizontal by a clamp fastened to the laboratory bench.

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APP-AMINOACYL-tRNA TRANSFER FACTORS

FrG. 3. Methods used to apply force with the abscissor plun-cr against A) bean and B) c-tton explants held in the explaint vise. The break strength of the explants was measured by placing the abscissor plunger against. the explants a. shown in Fig. 3. The abscissor plunger was pushed against the explants in a slow steady motion un.il the explants separated. The speed at which the plunger spring was compressed was limited by the speed at which the recorder pen can move across the chart paper wilhout appreciable lag. The plunger

pushed perpendicularly against the adaxial sides of the pulvinus of bean explan:s and the cotyledonary pe.iole of co:ton explants (Fig. 3). During a break strength determination the recorder pen moved across the chart paper as the spring was compressed and then returned to the zero base line when the tension was released by separation of .he explant. The recorder reading at

was

Table I. Break Strength Force for Separati:n of Explasnts Frotmi Different Bean Varieties After 30 Hours Wf ith or Without Ethylente Each value represents the average of 4 experiments consisting of 50 explants. Explants were isolated from greenhouse-grown plants. Control

Variety Perry Marrow Lows Chanipion Stringless Green Refugee Dark Red Kidney Tennessee Green Pod Red Kidney Red Mexican 34 Black Valentine Pinto Univ. of Idaho III Sanilac Top Crop Plentiful Saginaw Oregon State Univ. 2065 Tendercrop Earliwax Michelite Slenderwhite

276 253 228 2 9 216 2'0 199 183 183 175 175 175 174 140 137 134 117 114

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Break strength force 10 ppm C2H4

47 35 38 44 35 38 47 38 38 21 32 41 53 27 41 38 24 35

(g)

Difference

± S.D.

189 178 137 100 140 97 160 79 152 94 68 126 148

47 68 91 94

97

± 50 ± 38 ± 27 + 35 ± 56 ± 32 + 29 ± 44 ± 44 - 27 ± 21 ± 50 + 50 ± 21 + 44 ± 27 ± 21 ± 38

87 75 91 119 76 113 39 104 31 81 107 49 26 93 69 43 23

17

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the peak of this curve was converted to grams by use of graphs similar to Fig. 2 and was considered the break strength of the explant.

Results and Discussion The break strength required for separation of bean explants from 18 different varieties of Plhaseoliis vulgaris L. after 30 hr with or witlhout ethvlene treatment is shown in table I. As observed earlier by Morre (11), the diameter of the explant plays a role in determining the force required for separation, i.e., larger explants required more force for separation than small ones. However, the varietal differences shown in table I were onlv partially due to differences in diameter. The linear correlation between the diameter of the explants fromi different varieties and the force required for separation was small (0.4). The data in table I slhow that the abscissor was able to measure varietal differences in break strength and that 10 ppm ethylene reduced the break strength of all varieties. It is interesting to note that the second largest reduction in break strengtlh occurred in Red Kidney, the variety used for most of the experimental work on abscission from this and other laboratories. Fig. 4 presents data showing a loss in break strength of Red Kidney explants after 14 hlr of exposure to 10 ppm etlhvlene. Cellulase has been proposed as one of the enzymes responsible for cell separation (4, 9). To clheck this idea and take advantage of the ability of the abscissor to record the onset of abscission, a set of explants were analyzed for thleir cellulase content over the same period of time alloted for break strengtlh measure-

ments. In agreeiment with the idea that cellulase is one of the enzymes responsible for cell separation, we found tllat an increase in cellulase activity pre-

ceded the loss of break strength. A similar experiment hias been reported earlier by Morre (11). He found that pectinase (as measured by a cucumber pericarp assay) activity rose prior to thle separation of bean explants and that increased separation of bean explants was correlated witlh a decrease in break strengthi as measured by the Instron. He found that 200 g were required to pull freshly excised explants. whichi is similar to the lateral shear forces measured by the abscissor.

Conclusions The results obtained with the abscissor indicate that it combines some of the better features of pressure applicators and strain gauges. It is rapid. A skilled operator can measure the break strength of 100 explants in 20 min. This time includes loading explants in the explant vise as well as breaking the explants witlh the abscissor. The time required to break an explant is about 1 sec. Analogous with the Instron, the recording feature of the abscissor means that data can be examined at the operator's convenience. Hlowever. care must be taken that the rate of pressure application does not exceed the span speed of the recorder. The abscissor also shares with other techniques (6, 7, 8, 11) the ability to measure a range of break strength forces. Ihowever, no force less than 10 g can be recorded witlh the present instrument because tllis force is required to keep the plunger at the zero position of the abscissor. Because the abscissor measures a range of break strength forces it is able to indicate the onset of change in break strength, wlhich in turn reflects the initial action of cell wall degrading enzymes. In general practice we have found that a difference of 20 g in break strength was enough to indicate a significant difference between 2 samples of 10 explants excised from plants grown under controlled conditions. As indicated in table I, explants from greenhouse plants were more variable and larger differences in break strength were required for significance.

Acknowledgments HOURS

FIG. 4. Change in break strength and cellulase

con-

tent of bean abscission zone explants. Explants were analyzed for break strength and cellulase content as a

function of time after excision from the plant. All explants were exposed to 10 ppm ethylene during the course of the experiment. Each break strength determination is the average of 90 observations and each cellulase determination is the average of 2 cellulase assays. Increasing cellulase activity is characterized by increased ability of the enzyme to reduce the viscosity of a 1.5 % solution of CMC.

The authors thank Gerald R. Leather and Leonard Forrence for assistance during the performance of these experiments. The bean seeds were a gift from the following: Dr. W. J. Zaumeyer, USDA, Crops Division, Beltsville, Maryland; Ferry Morse Seed Company Incorporated, Mountain View, California; Mfr. R. Jorden, Agwvay Incorporated, Buffalo, New York; Mr. M. LeBaron, Branch Experiment Station, University of Idaho, Kimberly, Idaho; Mr. T. Kiely, Charter Seed Company, Twin Falls, Idaho; and Mr. W. R. Fancher, Agway Incorporated, Canandaigua, New York.

CRACKER AND ABELES-ABSCISSION: QUANTITATIVE MEASUREMENT

Literature Cited 1. ABELES, F. B. 1967. Mechanism of action of abscission accelerators. Physiol. Plantarum 20: 442-54. 2. ABELES, F. B. AND R. E. HOLM. 1966. Enhancement of RNA synthesis, protein synthesis, and abscission by ethylene. Plant Physiol. 41: 133242. 3. ABELES, F. B. AND R. E. HOLM. 1967. Abscission: The role of protein synthesis. Proc. N. Y. Acad. Sci. 144: 367-73. 4. ABELES, F. B. 1969. Abscission: role of cellulase. Plant Physiol. 44: 447-52. 5. ADDICOTT, F. T., H. R. CARNS, J. L. LYON, 0. E. SMITH, AND J. L. MCMEANS. 1963. On the physiology of abscisins. Regulateurs Naturels de la Croissance Vegetale 123: 687-703.

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6. DE LA FUENTES, R. K. AND A. C. LEOPOLD. 1968. Senescence processes in leaf abscission. Plant Physiol. 43: 1496-1502. 7. HARTMANN, H. T., M. FADL, AND J. WHISLER. 1967. Inducing abscission of olive fruits by spraying with ascorbic acid and iodoacetic acid. Calif. Agr. 21: 5-7. 8. HENDERSHOTT, C. H. 1964. The effect of various chemicals on fruit abscission on pineapple oranges. Proc Am. Soc. Hort. Sci. 85: 201-09. 9. HORTON, R. F. AND D. J. OSBORN. 1967. Senescence, abscission and cellulase in Phaseolus vulgaris. Nature 214: 1086-88. 10. MITCHELL, J. W. AND G. A. LIVINGSTON. 1968. Methods of studying plant hormones and growthregulating substances. Agriculture Handbook No. 336. U. S. Govt. Printing Office, Washington, D. C. 11. MORRE, D. J. 1968. Cell wall dissolution and enzyme secretion during leaf abscission. Plant Physiol. 43: 1545-59.