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Biology Department, Brookhaven National Laboratory, Upton, New York 11973
Received for publication July 27, 1998. Accepted for publication October 22, 1998.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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24% after 2 d postanthesis (dpa). The amount of nuclear DNA at 2 dpa is 5.4 ± 0.27 pg. At 3–4 dpa it increases to 6.7 ± 0.24 pg and by 5 dpa it is 6.8 ± 0.70 pg. These values were obtained by nuclear fluorescence after staining with Hoechst 33258. Human oral squamous cell nuclei were used as a DNA standard. Nuclear DNA content increases in fibers growing on either fertilized or unfertilized ovules. The increase also is detectable in Feulgen stained nuclei using two-wavelength cytospectrophotometry. All measurements were made on isolated fiber cell nuclei using a newly developed method tailored to cotton fiber cells. The results imply that during the early stages of development fiber cell nuclei either selectively amplify certain sequences or enter S-phase replicating a portion of their genome.
Key Words: cotton • development • fibers • Gossypium hirsutum • Malvaceae • nuclear DNA
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Van't Hof, 1998
). Treatment with 
-amanitin indicates that during the first 2 d postanthesis (2 dpa), nuclei synthesize poly (A)+ RNA and continue to do so several days thereafter (Triplett, 1998
). Also, during the first 4 dpa genes involved with the cell cycle can be active, since up to 30% of the fiber cells divide on ovules cultured at this time (Van't Hof and Saha, 1997
). The cell cycle genes are silent after 4 dpa, but other specific genes are active. For example, from 5 to 24 dpa the E6 gene is expressed (John and Crow, 1992
), at 12–15 dpa a series of ATPase and proton-translocating pyrophosphatase genes function (Smart et al., 1998
), and at 17 dpa the cellulose synthase gene, celA1, is active (Pear et al., 1996
). All this transcriptional activity occurs against a background of secondary wall deposition, which begins at 10 dpa (Seagull, 1990
).
Because the nucleus is the center of much of the developmental activity in young fiber cells, one questions the nature of the nuclei themselves. Do they, during all the developmental activity, maintain a constant amount of DNA? The question was motivated by the fact that other plant cells are known to increase nuclear DNA content either before or during differentiation (Innocenti and Avanzi, 1971
; Avanzi, Maggini, and Innocenti, 1973
; Nagl, 1976
; Grisvard and Tuffet-Anghileri, 1980
; D'Amato, 1989
; Schweizer et al., 1995
). Though well grounded and developmentally important the answer to this question required new techniques tailored to cotton fiber cells. In our hands, nuclei in fiber cells are difficult to stain by the Feulgen method, a method commonly used for DNA measurements. Also, the cell wall of fibers interferes with nuclear fluorescence thereby making fluorometric DNA measurements questionable.
The findings presented in this paper are free of these difficulties. They result from using a newly developed technique that gives clean isolated nuclei useful for measurements of DNA content. The measurements show fiber cell nuclei at 2 dpa have 5.4 pg DNA, the 2C amount reported for Gossypium hirsutum (Firoozabady, 1986
; Michaelson et al., 1991
), and that the nuclei increased their DNA content 24% at 3–5 dpa. The implication of this finding is that cotton fiber cells during the early stages of development either selectively amplify certain sequences or enter S-phase replicating a portion of their genome.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Emasculation
The morning a flower opened the style and anthers were removed to prevent fertilization and the emasculated flowers remained attached to the plant until the bolls were harvested.
Nuclear isolation
Bolls were removed from the plant, opened, and the ovules dislodged using a smooth-edged microspatula. The freed ovules were immediately suspended in 2–3 mL of 0.5% Sparkleen (w/v) for 5 min under vacuum at room temperature. Sparkleen is a biodegradable commercial detergent sold by Fisher Scientific Corp., Pittsburgh, Pennsylvania. It contains Calgon (Calgon Corp.), a water-softening agent, and has a phosphate content of 14% expressed as P, 72% expressed Na3PO4, and 31% expressed as P2O5. After removal of the Sparkleen, the ovules were rinsed once or twice with 2–3 mL distilled water and fixed in ice-cold 2% formaldehyde in phosphate buffer (0.066 mol/L Na2HPO4, 0.066 mol/L NaH2PO4, pH 6.8) for 12 min. Following fixation the ovules were rinsed once or twice with ice-cold buffer free of formaldehyde and remained suspended in cold buffer awaiting nuclear removal (Kraszewska et al., 1985
).
Ovules aged 2 dpa were placed on a dry freshly subbed microscope [5 g gelatin/L, 0.5 g CrK(SO4)2 - 12H2O/L] and viewed at 12 or 25x under a dissecting microscope. Holding the ovule with a fine-tipped forceps we cut the outermost tips of the fibers once with a surgical micro shears (Laschal Surgical, Inc., Laschal Micropoint, LA-5, GBC Scientifics, Gaithersburg, Maryland). The ovule was rotated so that the cut fibers faced the surface of the microscope slide and gently blotted 3–4 times forming a circle on the dry surface of the slide. The total amount of liquid from blotting one ovule is 2–4 µL, and 2–3 cut ovules may be used per slide. Excess buffer reduces the number of free nuclei, and crushing the ovule on the microscope surface produces excess debris. After blotting the slide is air dried at room temperature.
For ovules aged 3 dpa and older there are minor procedural differences. Because the fibers are longer, more liquid adheres to the ovular surface. This liquid is removed by gently touching the ovule to absorbent tissue and adding 1–2 µL to the surface where the cut fibers will be blotted. Also, it is useful to remember to clean the shears after each cut. This reduces the number of free fibers in the blotted area.
Nuclear staining and DNA measurement
The nuclei can be stained by the Feulgen method. This is not the preferred method, but it was used to confirm the results obtained by nuclear fluorescence. Feulgen staining was accomplished by 30 min hydrolysis with 5 mol/L HCl at room temperature, a distilled water rinse, and covering the blotted nuclei with 50–100 µL of Schiff's reagent and allowing them to stain in the dark at room temperature for 1–2 h. The stained nuclei were rinsed with distilled water, twice bleached for 10 min with a solution of 22.5 mmol/L K2S3O5, and 50 mmol/L HCl, and air dried. The dried preparations were mounted in immersion oil type B, refractive index 1.5150, and covered with a coverslip. The DNA content of individual nuclei was measured by the two-wavelength method using a Leitz cytospectrophotometer. A total of 100 nuclei each were measured for fibers with a mean length of 0.7 and 4 mm.
To measure DNA amounts by fluorescence 8–12 µL of aqueous Hoechst 33258 (Galbraith, Mauch, and Shields, 1981
), 200 ng/mL, was added to blotted dried nuclei, covered with a coverslip, and sealed with rubber cement. Individual nuclear fluorescence, excited by epi-illuminated UV irradiation, was measured using a Zeiss microspectrophotometer equipped with 365 nm excitation and 420 nm barrier filters. For each dpa sampled a total of 300 to >600 nuclei were measured. Human oral squamous cells spread on the same slide containing isolated fiber cell nuclei served as a DNA standard and as a control. The 2C value for the squamous nuclei is 5.6 pg, a value representing the mean published for human skin (5.6 pg) and twice that of human sperm (Altman and Katz, 1976
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Patau, Das, and Skoog, 1957
; Mendelsohn, 1958
; Patau and Srinivasachar, 1960
). The nuclei measured were from 0.7-mm fibers on young ovules and from 4-mm fibers growing on older ovules. Since fibers elongate as they develop, their length is one criterion of developmental stage. The results are given in the form of histograms reflecting the relative amounts of nuclear DNA, in arbitrary units, expressed as a percentage of the total number of nuclei measured (Fig. 1). The histograms show that the distributions differ. The nuclei from 0.7-mm fibers had a mean ±1 SD of 0.89 ± 0.20 and those from 4-mm fibers had a mean of 1.26 ± 0.33. Clearly, the longer fibers have nuclei with more DNA. How much more is unknown as no standard was included with these measurements.
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Besides using a different method of measuring nuclear DNA content, the criterion for fiber cell age was changed from length to dpa. The fluorescence measurements also include nuclei isolated from fibers on unfertilized ovules and a nuclear DNA standard. A total of 610 measurements was made of nuclei of fibers aged 2 dpa. The nuclei came from a minimum of six different ovules, 100 per ovule. The measurements from fibers aged 3 + 4 dpa were combined to give a total of 522, a minimum of 5 ovules, 100 nuclei each. Finally, 411 nuclei were measured from fibers aged 5 dpa. These came from at least four different ovules, 100 nuclei each. The results, expressed as a percentage of the total number of nuclei measured, are presented as a histogram. The statistics of the measurements are in arbitrary units of the mean nuclear fluorescence and 1 SE of the means from samples consisting of 100 nuclei per sample.
The histograms in Fig. 2 show the fluorescence, in arbitrary units, of nuclei isolated at 2, 3 + 4, and 5 dpa. The fluorescence of nuclei aged 2 dpa is less than that of nuclei isolated at either 3 + 4 or 5 dpa. The mean fluorescence at 2 dpa is 1.53 ± 0.07, at 3 + 4 dpa it is 1.90 ± 0.06, and at 5 dpa the mean is 1.92 ± 0.20. These statistics are evidence that fiber cell nuclei increase their DNA content between 2 and 3 dpa and that no further increase occurs up to 5 dpa.
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), indicates that the fiber cell nucleus at 2 dpa has 5.4 pg of DNA. This amount falls at the upper end of a 4.3–5.6 pg range, the 2C range reported for Gossypium hirsutum (Firoozabady, 1986
; Michaelson et al., 1991
). Using the same standard and a mean fluorescence of 1.9–1.92 arbitrary units for nuclei of fibers aged 3 dpa and older shows these older nuclei have 6.7–6.8 pg of DNA, 1.3–1.4 pg more than nuclei at 2 dpa. Thus, the fiber cell nucleus begins development with a 2C amount of DNA and adds 1.3–1.4 pg of DNA between 3 and 5 dpa.
Increased nuclear DNA content is independent of fertilization. Nuclei isolated at 4 dpa from fibers on unfertilized ovules have an increased DNA content when compared to nuclei at 2 dpa from fertilized ovules (Fig. 3). The mean fluorescence of 413 measurements, 100 each from at least four ovules, is 2.03 ± 0.20 arbitrary units. This level of fluorescence is equivalent to 7.1 ± 0.80 pg of DNA, an amount that is higher but not statistically different from the 6.7 pg measured for nuclei at 3 + 4 dpa from fertilized ovules. Finally, after 4 dpa no significant increase in the mean nuclear DNA content is detected in nuclei from fibers on unfertilized ovules aged 6 dpa. These nuclei have 7.2 pg DNA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The nuclear DNA values are based on nuclear fluorescence using Hoechst 33258. They are not due to RNA as Hoechst 33258 does not fluoresce in the presence of RNA (Brown et al., 1991
). Also, RNA is not a factor when using Feulgen stain and measurements using this stain show an increased nuclear DNA content in older fibers.
The standard used to calculate the amount of additional DNA was human squamous cells (the authors). These cells have a 2C nuclear DNA content of 5.6 pg (Altman and Katz, 1976
), an amount identical to the 2C value reported for cotton (Michaelson et al., 1991
). In this respect, the human cells meet the requirement that a cytophotometric standard have nearly the same 2C value as the cells of the experimental material (Johnson et al., 1999
). Further, since human squamous cells have cytoplasm and their nuclei lack prominent nucleoli, they are easily distinguished from an isolated fiber cell nucleus with its enlarged nucleolus. The oral squamous cells can be smeared on the slide beside fiber cell nuclei at the first preparatory step so that both nuclei, human and cotton, are subject to the same treatment. Finally, the use of human squamous cells as a standard is validated by the fact that the 2C value, 5.4 pg, determined in these experiments using cv. MD51ne differs by <4% from 5.6 pg reported for cv. Tamcot CAMD-E using laser flow cytometry (Michaelson et al., 1991
).
It is significant that the 2C value determined in this work using Hoechst 33258 closely agrees with that obtained with unfixed nuclei stained with the fluorochrome propidium iodide (Michaelson et al., 1991
). The close agreement rules out the possibility that the increased nuclear DNA content is due to either the fluorochrome, Hoechst 33258, or the formaldehyde fixation protocol used to isolate fiber cell nuclei. Besides this close agreement, there are two other reasons why neither Hoechst 33258 nor the fixation protocol is responsible for the increased nuclear DNA content seen in fiber cell nuclei. First, it is known that a 3% solution of formaldehyde at 35°C denatures duplex DNA (Feldman, 1973
), and it is also known that single-stranded DNA reduces the fluorescence intensity of Hoechst 33258 (Simola et al., 1975
). Since the fiber cell nuclei were fixed at 4°C for 12 min it is unlikely that DNA denaturation occurred. Further, if denaturation did occur the result would be a reduction in fluorescence and a lower estimate of nuclear DNA content rather than the observed increase. The second reason is that a short fixation time minimizes crosslinking of protein to DNA. To form such crosslinks, formaldehyde treatment requires either 100 h at 4°C (Solomon, Larson, and Varshavsky, 1988
) or a shorter time at temperatures in the 30°C range (Feldman, 1973
). Since the fiber cell nuclei were fixed for 12 min at 4°C, there is a low probability that the increased DNA content is due to nucleoprotein crosslinking.
The finding that fiber cell nuclei increase their nuclear DNA content soon after anthesis may be viewed in two ways. The first is that a portion of the genome is amplified. Which portion is unknown, but likely candidates are sequences encoding genes whose products are required for the extensive growth that occurs after 5 dpa. The added DNA also may be associated with the micronucleolus that appears nearly simultaneously with the increase in nuclear DNA. Finally, the added DNA may represent aggregated chromatin that surrounds the enlarged macronucleolus (data not shown). Chromatin aggregation is one way the allotetraploid cotton nucleus could rearrange selected sequences from each diploid parent in a manner that enhances fiber cell expression.
A second view is that the fiber cell, after 2 dpa, enters S-phase and stops after replicating a portion of its genome. In this case, the DNA replicated could be either sequences that normally replicate early in S-phase, selected genes associated with fiber cell development, or a combination of both. The latter would occur, if fiber specific genes normally replicate early in S-phase.
Further work at the molecular level using a variety of methods, including in situ hybridization of selected probes, is needed to determine whether the added nuclear DNA represents selectively amplified genes or sequences replicated in early S-phase.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Avanzi, S., F. Maggini, and M. Innocenti. 1973 Amplification of ribosomal cistrons during maturation of metaxylem in the root of Allium cepa. Protoplasma 76: 197–210.
Brown, S. C., C. Bergounioux, S. Tallet, and D. Marie. 1991 Flow cytometry of nuclei for ploidy and cell cycle analysis. In I. Negrutiu and G. Gharti-Chhetri [eds.], A laboratory guide for cellular and molecular plant biology, 1–11. Birkhauser, Basel.
D'Amato, F. 1989 Polyploidy in cell differentiation. Caryologia 42: 183–211.[ISI]
Feldman, M. Y. 1973 Reactions of nucleic acids and nucleoproteins with formaldehyde. In J. N. Davidson and W. E. Cohn [eds.], Progress in nucleic acid research and molecular biology, vol. 13, 1–49. Academic Press, New York, NY.
Firoozabady, E. 1986 The effects of cell cycle parameters on cell wall regeneration and cell division of cotton protoplasts (Gossypium hirsutum L.). Journal of Experimental Botany 37: 1211–1217.
Galbraith, D. W., T. J. Mauch, and B. A. Shields. 1981 Analysis of the initial stages of plant protoplast development using 33258 Hoechst: reactivation of the cell cycle. Physiologia Plantarum 51: 380–386.[CrossRef]
Grisvard, J., and A. Tuffet-Anghileri. 1980 Variations in the satellite DNA content of Cucumis melo in relation to dedifferentiation and hormone concentration. Nucleic Acid Research 8: 2843–2857.
Innocenti, A. M., and S. Avanzi. 1971 Some cytological aspects of the differentiation of metaxylem in the root of Allium cepa. Caryologia 4: 283–292.
John, M. E., and L. J. Crow. 1992 Gene expression in cotton (Gossypium hirsutum L.) fiber: cloning of the mRNAs. Proceedings of the National Academy of Sciences, USA 80: 5769–5773.
Johnson, J. S., M. D. Bennett, A. L. Rayburn, D. W. Galbraith, and H. J. Price. 1999 Reference standards for determination of DNA content of plant nuclei. American Journal of Botany. 86: 609–613.
Kraszewska, E. K., C. A. Bjerknes, S. S. Lamm, and J. Van't Hof. 1985 Extrachromosomal DNA of pea-root (Pisum sativum) has repeated sequences and ribosomal genes. Plant Molecular Biology 5: 353–361.[CrossRef][ISI]
Mendelsohn, M. L. 1958 The two-wavelength method of microspectrophotometry. I. A. microspectrophotometer and tests on model systems. Biophysical and Biochemical Cytology 4: 407–414.
Michaelson, M. J., H. J. Price, J. R. Ellison, and J. S. Johnson. 1991 Comparison of plant DNA contents determined by Feulgen microspectrophotometry and laser flow cytometry. American Journal of Botany 78: 183–188.[CrossRef][ISI]
Nagl, W. 1976 DNA endoreduplication and polyteny understood as evolutionary strategies. Nature 261: 614–615.[CrossRef][Medline]
Patau, K. 1952 Absorption microphotometry of irregular-shaped objects. Chromosoma 5: 341–362.
———, N. K. Das, and F. Skoog. 1957 Induction of DNA synthesis by kinetin and indoleacetic acid in excised tobacco pith tissue. Physiologia Plantarum 10: 949–966.[CrossRef]
——— and D. Srinivasachar. 1960 A microspectrophotometer for measuring the DNA-content of nuclei by the two wave length method. Cytologia 25: 145–151.
Pear, J. R., Y. Kawagoe, W. E. Schreckengost, D. P. Delmer, and D. M. Stalker. 1996 Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase. Proceeding of the National Academy of Sciences, USA 93: 12637–12642.
Ramsey, J. C., and J. D. Berlin. 1976a Ultrastructural aspects of early stages in cotton fiber elongation. American Journal of Botany 63: 868–876.[CrossRef][ISI]
———, and ———. 1976b Ultrastructure of early stages of cotton fiber differentiation. Botanical Gazette 137: 11–19.[CrossRef]
Schweizer, L., G. L. Yerkdavis, R. L. Phillips, F. Srienc, and R. J. Jones. 1995 Dynamics of maize endosperm development and DNA endoreduplication. Proceedings of the National Academy of Sciences, USA 92: 7070–7074.
Seagull, R. W. 1990 Tip growth and transition to secondary wall synthesis in developing cotton hairs. In I. Heath [ed.], Tip growth in plant and fungal cells, 261–284. Academic Press, New York, NY.
Simola, K., R. K. Selander, A. De La Chapelle, G. Corneo, and E. Ginelli. 1975 Molecular basis of chromosome banding I. The effect of mouse DNA fractions on two fluorescent dyes in vitro. Chromosoma 51: 199–205.[ISI][Medline]
Smart, L. B., F. Vajdani, M. Maeshima, and T. A. Wilkins. 1998 Genes involved in osmoregulation during turgor-driven cell expansion of developing cotton fibers are differentially regulated. Plant Physiology 116: 1539–1549.
Solomon, M. J., P. L. Larson, and A. Varshavsky. 1988 Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell 53: 937–947.[CrossRef][ISI][Medline]
Triplett, B. A. 1998 Stage-specific inhibition of cotton fiber development by adding -amanitin to ovule cultures. In Vitro Cell and Developmental Biology 34: 27–33.
Van't Hof, J. 1998 Production of micronucleoli and onset of cotton fiber growth. Planta 205: 561–566.[CrossRef][ISI]
———, and S. Saha. 1997 Cotton fibers can undergo cell division. American Journal of Botany 84: 1231–1235.[Abstract]
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