Effect of Boron on Cell Elongation and Division in Squash · elongation index in which cell length...
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Plant Physiol. (1977) 59, 884-887 Effect of Boron on Cell Elongation and Division in Squash Roots Received for publication August 26, 1976 and in revised form January 3, 1977 MARTIN S. COHEN Department of Biology, University of Hartford, West Hartford, Connecticut 06117 ROBERT LEPPER, JR. Department of Botany, University of Rhode Island, Kingston, Rhode Island 02881 ABSTRACT This work establishes that cessation of root elongation of intact squash (Cucurbita pepo L.) plants is an early result of boron deficiency. Root elongation is slowed by 6 hours and is virtually stopped as early as 24 hours after boron is first withheld from the nutrient solution. As root elongation ceased, cell elongation progressed distally into the region normally occupied by the apical meristem and eventually the meristem became indistinguishable. Differentiation was determined by use of an elongation index in which cell length was compared to cell width. This index ranged from a low of 0.8 in boron-sufficient root meristems to a high of 3 in root meristems grown in a boron-deficient nutrient solution for 98 hours. It is concluded that a continuous supply of boron is not essential for cell elongation but is required for maintenance of meriste- matic activity. Boron may act as a regulator of cell division in this tissue. treated and were grown in modified Shive nutrient solution containing boron (+B) as described by Albert and Wilson (1). On the 5th day, roots were washed and placed into either freshly prepared modified Shive nutrient solution to which boron was added (+B) or into nutrient solution without added boron (-B). Water for both types of solutions was obtained by passing dis- tilled H2O through a Millipore Milli-Q2 system. Just before placing the plants into +B or -B nutrient solutions, two roots/ plant were marked by placing small India ink marks 10 mm from the root tip. Subsequent root elongation was determined by measuring the distance from the root tip to the mark. Net root elongation was determined after periods of 0, 6, 12, 24, 48, 72, and 98 hr of +B and -B treatment and root tips were harvested for histological examination at these times. The terminal 1 cm of each root was excised, fixed in formaldehyde-acetic-acid-alcohol (6), dehydrated, embedded in paraffin, sectioned, and stained with Sharman stain (11). Alexander (2) studied the onset of boron deficiency in squash plants grown in solution culture in which boron was withheld. He examined the roots of boron-deficient plants after periods of 48, 72, and 98 hr of boron deficiency to observe histological effects of the treatment. After 48 hr of boron deficiency, he reported a decrease in the size of the root cap; collapsed, disintegrated, and enlarged cells or groups of cells in the peripheral portions of the stele; and differentiation close to the root meristem. Examina- tion of roots after 98 hr of boron deficiency commonly revealed death of the extremities of the tip. In the region of elongation, individual cells could no longer be recognized. Varying degrees of response were observed in any given experiment. Other workers have questioned whether boron is necessary for either cell elongation or cell division. Alexander (2), Neales (7), and Sommer and Sorokin (8) suggested that the primary influ- ence of boron is not on cell division since abortive lateral primor- dia are formed in boron-deficient roots. Normal mitoses oc- curred as lateral meristems began development but cell division ceased soon thereafter. On the other hand, several investigators have suggested that boron is required for cell division (5, 9). For example, in boron-deficient field beans, very low mitotic indices are observed (10). Recently, we have reported that intact boron- deficient squash roots quickly lose their capacity for DNA syn- thesis and cell division (4). There have been no previous investigations which quantify early morphological effects of boron deficiency. The present work was undertaken in order to define specifically such changes in the intact root tips of squash plants. MATERIALS AND METHODS Squash (Cucurbita pepo L., cultivar Early Prolific Straight- neck) seedlings were grown from seed supplied by Joseph Harris Company, Rochester, N.Y. All seedlings were 5 days old when RESULTS Elongation Measurements. Root elongation measurements were made during the treatment period from 0 to 98 hr (e.g. beginning from the 5th day onward). Measurements made dur- ing the first 24 hr of boron deficiency are presented in Figure 1. All points represent means of at least 100 roots. At all times, the mean total elongation of the intact + B roots exceeded that of the intact -B roots. Even more apparent is the difference in elonga- tion rates between +B and -B intact roots. After 6 hr of treatment, the +B roots had elongated at a rate of 1.5 mm/hr while the -B roots had elongated at a rate of only 0.5 mm/hr. After 12 hr, the elongation rate of the +B roots (1.5 mm/hr) greatly exceeded the elongation rate of the -B roots (0.2 mm/ hr). This difference became even more exaggerated after the third and fourth 6-hr periods as the elongation rate of the -B roots decreased to essentially zero while the elongation rate of the +B roots increased to 1.8 and 2.6 mm/hr, respectively. The growth rate of the +B roots increased during the period from 0 to 24 hr of treatment while that of the -B roots decreased to zero during the same period. Qualitative Histology. As viewed in median longitudinal sec- tion (Fig. 2), the root tips from seedlings grown in +B nutrient solution displayed an intact epidermis, well developed cortical region, central cylinder, and root cap. The meristematic region was composed of a group of initials which are the source of the central cylinder, cortex, and column of the root cap. The cells were filled with cytoplasm and there was a large, centralized nucleus containing a large nucleolus. In root tips from seedlings grown in -B nutrient solution for a period of 24 hr (Fig. 3), cells of the root cap appeared to have less cytoplasm than the controls based upon a decrease in the intensity of staining of the cells. In all regions, the cell walls appeared thicker than in the controls and the outlines of the cells had lost their regular rectangular appearance, becoming irregu- 884 www.plantphysiol.org on July 15, 2018 - Published by Downloaded from Copyright © 1977 American Society of Plant Biologists. All rights reserved.
Transcript of Effect of Boron on Cell Elongation and Division in Squash · elongation index in which cell length...
Plant Physiol. (1977) 59, 884-887
Effect of Boron on Cell Elongation and Division in Squash RootsReceived for publication August 26, 1976 and in revised form January 3, 1977
MARTIN S. COHENDepartment of Biology, University of Hartford, West Hartford, Connecticut 06117ROBERT LEPPER, JR.Department of Botany, University of Rhode Island, Kingston, Rhode Island 02881
ABSTRACT
This work establishes that cessation of root elongation of intact squash(Cucurbita pepo L.) plants is an early result of boron deficiency. Rootelongation is slowed by 6 hours and is virtually stopped as early as 24hours after boron is first withheld from the nutrient solution. As rootelongation ceased, cell elongation progressed distally into the regionnormally occupied by the apical meristem and eventually the meristembecame indistinguishable. Differentiation was determined by use of anelongation index in which cell length was compared to cell width. Thisindex ranged from a low of 0.8 in boron-sufficient root meristems to ahigh of 3 in root meristems grown in a boron-deficient nutrient solutionfor 98 hours. It is concluded that a continuous supply of boron is notessential for cell elongation but is required for maintenance of meriste-matic activity. Boron may act as a regulator of cell division in this tissue.
treated and were grown in modified Shive nutrient solutioncontaining boron (+B) as described by Albert and Wilson (1).On the 5th day, roots were washed and placed into either freshlyprepared modified Shive nutrient solution to which boron wasadded (+B) or into nutrient solution without added boron (-B).Water for both types of solutions was obtained by passing dis-tilled H2O through a Millipore Milli-Q2 system. Just beforeplacing the plants into +B or -B nutrient solutions, two roots/plant were marked by placing small India ink marks 10 mm fromthe root tip. Subsequent root elongation was determined bymeasuring the distance from the root tip to the mark. Net rootelongation was determined after periods of 0, 6, 12, 24, 48, 72,and 98 hr of +B and -B treatment and root tips were harvestedfor histological examination at these times. The terminal 1 cm ofeach root was excised, fixed in formaldehyde-acetic-acid-alcohol(6), dehydrated, embedded in paraffin, sectioned, and stainedwith Sharman stain (11).
Alexander (2) studied the onset of boron deficiency in squashplants grown in solution culture in which boron was withheld. Heexamined the roots of boron-deficient plants after periods of 48,72, and 98 hr of boron deficiency to observe histological effectsof the treatment. After 48 hr of boron deficiency, he reported adecrease in the size of the root cap; collapsed, disintegrated, andenlarged cells or groups of cells in the peripheral portions of thestele; and differentiation close to the root meristem. Examina-tion of roots after 98 hr of boron deficiency commonly revealeddeath of the extremities of the tip. In the region of elongation,individual cells could no longer be recognized. Varying degreesof response were observed in any given experiment.
Other workers have questioned whether boron is necessary foreither cell elongation or cell division. Alexander (2), Neales (7),and Sommer and Sorokin (8) suggested that the primary influ-ence of boron is not on cell division since abortive lateral primor-dia are formed in boron-deficient roots. Normal mitoses oc-curred as lateral meristems began development but cell divisionceased soon thereafter. On the other hand, several investigatorshave suggested that boron is required for cell division (5, 9). Forexample, in boron-deficient field beans, very low mitotic indicesare observed (10). Recently, we have reported that intact boron-deficient squash roots quickly lose their capacity for DNA syn-thesis and cell division (4).There have been no previous investigations which quantify
early morphological effects of boron deficiency. The presentwork was undertaken in order to define specifically such changesin the intact root tips of squash plants.
MATERIALS AND METHODS
Squash (Cucurbita pepo L., cultivar Early Prolific Straight-neck) seedlings were grown from seed supplied by Joseph HarrisCompany, Rochester, N.Y. All seedlings were 5 days old when
RESULTS
Elongation Measurements. Root elongation measurementswere made during the treatment period from 0 to 98 hr (e.g.beginning from the 5th day onward). Measurements made dur-ing the first 24 hr of boron deficiency are presented in Figure 1.All points represent means of at least 100 roots. At all times, themean total elongation of the intact + B roots exceeded that of theintact -B roots. Even more apparent is the difference in elonga-tion rates between +B and -B intact roots. After 6 hr oftreatment, the +B roots had elongated at a rate of 1.5 mm/hrwhile the -B roots had elongated at a rate of only 0.5 mm/hr.After 12 hr, the elongation rate of the +B roots (1.5 mm/hr)greatly exceeded the elongation rate of the -B roots (0.2 mm/hr). This difference became even more exaggerated after thethird and fourth 6-hr periods as the elongation rate of the -Broots decreased to essentially zero while the elongation rate ofthe +B roots increased to 1.8 and 2.6 mm/hr, respectively. Thegrowth rate of the +B roots increased during the period from 0to 24 hr of treatment while that of the -B roots decreased tozero during the same period.
Qualitative Histology. As viewed in median longitudinal sec-tion (Fig. 2), the root tips from seedlings grown in +B nutrientsolution displayed an intact epidermis, well developed corticalregion, central cylinder, and root cap. The meristematic regionwas composed of a group of initials which are the source of thecentral cylinder, cortex, and column of the root cap. The cellswere filled with cytoplasm and there was a large, centralizednucleus containing a large nucleolus.
In root tips from seedlings grown in -B nutrient solution for a
period of 24 hr (Fig. 3), cells of the root cap appeared to haveless cytoplasm than the controls based upon a decrease in theintensity of staining of the cells. In all regions, the cell wallsappeared thicker than in the controls and the outlines of the cellshad lost their regular rectangular appearance, becoming irregu-
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BORON DEFICIENCY IN SQUASH ROOTS
sO1
45-44,
Z36
z 25-26ois-
0 is ;g _---_ _ _O10~~LS * 12 18 24
H O U R SFIG. 1. Total root elongation of +B (solid line) and -B (dotted line)
roots during the first 24 hr of treatment.
larly shaped. In root tips from seedlings grown in -B nutrientsolution beyond 24 hr (Figs. 4 and 5), the differences describedabove were present and became progressively more pronounced.In the root tips from seedlings grown in -B nutrient solution fora period of 98 hr (Figs. 6 and 7), all of the boron deficiencyeffects were seen in the extreme. Many cells appeared empty,the boundary between the stele and root cap was generallyabsent, and thus the meristematic region could not be distin-guished with certainty. These descriptive results were inter-preted to mean that elongation of the cells of the central cylinderhad proceeded distally into the region normally occupied by themeristem, which was thus no longer identifiable.
Quantitative Histology. In order to support this interpretationquantitatively, measurements of cell dimensions were made todetermine the extent to which differentiation had occurred in thecells of the central cylinder in boron sufficient and boron defi-
FIG. 2-7. Median longitudinal sections of squash root tips at the end of the treatment period. 2: +B 24 hr (x 480); 3: -B 24 hr (x 480); 4: -B48 hr (x 300); 5: -B 72 hr (x 300); 6: -B 98 hr (x 300); 7: -B 98 hr (x 495).
Plant Physiol. Vol. 59, 1977 885
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Plant Physiol. Vol. 59, 1977
Table I. Effect of baron on elongation in squash roots.Cell Length, Cell Width, and Elongation Indices (El) in Two Regions Proximal to theMeristem at the End of the Treatment Period for +B and -B root tips. Means ± SaE arePooled Values for Ten or More Root Tips. Means of Total Root Elongation are given foreach Treatment Period.
Proximal Di.stance frorm Merister'.
0-160 unn 160-320 pM
Length of Cell Cel1 Cell Cell AverageTreatment Treatment Length Width E.I. Lengtih Width E.I. Total Root
(Ilours) (pu) (p) (jim) (m) Elongation(mm)
+B 24, 48, 72 10.2 + 0.8 13.0 + 0.6 0.8 + 0.1 13.8 + 1.1 11.5 + 0.5 1.2 + 0.5 45.4-B 24 11.1 + 0.8 8.4 + 0.7 1.4 + 0.2 14.6 + 0.6 6.7 + 0.1 2.2 + 0.1 7.6-B 48 13.7 + 0.8 7.6 4- 0.7 1.9 + 0.2 21.1 + 0.4 7.1 + 0.3 3.0 + 0.1 0.3-B 72 17.3 + 0.7 7.0 + 0.8 2.5 + 0.1 20.8 + 1.0 8.1 + 0.0 2.6 + 0.1 0.2-B 98 17.9 + 0.8 6.2 + 0.4 3.0 + 0.2 ... I ... 1 ...1 0.0
Data not recorded
cient roots. Since cellular elongation is a significant, easily iden-tifiable aspect of differentiation in the cells of the central cylin-der of squash roots, this parameter was chosen for study. Table 1presents quantitative data on cell elongation occurring in +Band -B root tips. The root tip was arbitrarily divided into tworegions for study. One region (0-160 ,um proximal to the meris-tem) contains cells recently produced by the meristem while thesecond region (160-320 ,um proximal to the meristem) containscells produced earlier in time by the meristem. Measurementswere made of cell length and width of 10 cells in each of theseregions in 10 roots for each treatment. Each value in Table I isbased upon measurements of 100 cells.Measurements of cell length were compared to cell width in
order to calculate an El' which served as a measure of celldifferentiation. At each time interval of both +B and -B treat-ment, cells farther from the meristem have a higher El thanthose closer to the meristem. When comparing the El of cells in+B roots throughout all times of the treatment period, nosignificant changes occur as would be expected in an activelydeveloping root. However, when comparing the El of cells in-B roots, the El significantly increases during the treatmentperiods. This establishes that cell elongation is increased underconditions of boron deficiency (Table I). This increase, however,does not continue beyond 72 hr of treatment.
DISCUSSION
Comparison of -B to control root tips revealed that theprincipal, observable effects of boron deficiency are: (a) cessa-tion of total root elongation, and (b) progressive elongation ofcells of the central cylinder. This was established both by qualita-tive observations and supporting quantitative measurements.
Calculation of the ratios of cell length to cell width (El) (Table1) produced a very useful measure of cellular elongation in thestele. Comparison of cellular elongation in the stele to total rootelongation revealed that these values were correlated to eachother and also to the total time of boron deficiency. As theperiod of boron deficiency increased, the elongation rate of theintact roots decreased and the extent of cell elongation in-creased. The latter was revealed by increases in the values of theelongation indices calculated for boron sufficient and deficientroots.
I Abbreviation: El: elongation index.
Cells located in the region 160 to 320 ,um proximal to themeristem were always more elongated than those located in theregion 0 to 160 ,um proximal to the meristem. Cells locatedfarther from the meristem are produced earlier in time thanthose located closer to the meristem and thus they would beexpected to be longer since they had been developing into tissuesof the stele for a longer time than had cells produced morerecently. All of the observed cells began their differentiation bycell elongation in the presence of boron. Their continued andprogressive elongation during periods of 24, 48, and 72 hr ofboron deficiency indicates that the process of cell elongation inthese roots is not dependent on a continuing supply of boron.Birnbaum et al. (3) reported that boron appears to be involved
in elongation of fibers developing on excised, unfertilized, hor-monally stimulated, cotton ovules grown in vitro. It is presentlyunclear what relationship these tissues and this system have tothose employed in the present study.
Total root elongation, which is a result of two processes, celldivision and cell elongation, was observed to decrease essentiallyto zero after a period of 24 hr of boron deficiency. This lack ofroot elongation cannot be attributed to a cessation of cellularelongation as this continued up to 72 hr of boron deficiency(Table I). Thus, cessation of total root elongation was caused bya failure of cell division in meristematic cells. The meristem istherefore no longer able to progress ahead of the region of cellelongation and ultimately, elongation progresses into the regionnormally occupied by the meristem which then is no longerdistinguishable or functional.
This conclusion is further supported by autoradiographic evi-dence (4) which revealed that the decrease in rate of total rootelongation during boron deficiency is caused by a cessation ofDNA synthesis and cell division during the first 20 hr of borondeficiency. It seems clear that continuing cell divisions in meris-tematic cells of squash roots are dependent on a continuoussupply of boron and an early effect of boron deficiency is thecessation of cell division in this tissue. It can further be specu-lated that boron may be part of an intracellular regulatorymechanism for cell division in meristematic tissue.
LITERATURE CITED
1. ALBERT LS, CM WILSON 1961 Effect of boron on elongation of tomato root tips. Plant
Phvsiol 36: 244-2512. ALEXANDER TR 1942 Anatomical and physiological responses of squash to various levels of
boron supply. Bot Gaz 103: 475-491
886 COHEN AND LEPPER
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3. BIRNBAUM EH, CA BEASLEY, WM DUGGER 1974 Boron deficiency in unfertilized cotton
(Gossypium hirsutum) ovules grown in vitro. Plant Physiol 54: 931-9354. COHEN MS, LS ALBERT 1974 Autoradiographic examination of intact boron-deficient
squash roots treated with tritiated thymidine. Plant Physiol 54: 766-7685. HAAs AR, LV KWTz 1931 Nutrition and composition of the Deglet Noor palm in relation
to the decline disease. Hilgardia 5: 511-5306. JENSEN WA 1962 Botanical Histochemistry. WH Freeman & Co, San Francisco pp 55-837. NEALES TF 1960. Some effects of boron on root growth. Aust J Biol Sci 13: 232-2488. SOMMER AL, H SOROKIN 1928 Effects of the absence of boron and some other essential
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elements on the cell and tissue structure of the root tips of Pisum sativum. Plant Physiol 3:237-260
9. WHnInNGTON WJ 1957 The role of boron in plant growth. L. The effect on general growth,
seed production, and cytological behaviour. J Exp Bot 8: 353-36710. WHrMnNGTON WJ 1959 The role of boron in root growth. II. The effect on growth of the
radicle. J Exp Bot 10: 93-10311. VENNING FD 1954 Manual of Advanced Plant Microtechnique. WC Brown Co, Dubuque
Iowa pp 19-20
Plant Physiol. Vol. 59, 1977 BORON DEFICIENCY IN SQUASH ROOTS
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