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Developmental Biology and Genetics |
2USDA/ARS, Crop Genetics and Production Research Unit, 141 Experiment Station Road, Stoneville, Mississippi 38776 USA; 3University of Arkansas, CSES Department, University of Arkansas, Fayetteville, Arkansas 72701 USA
Received for publication January 21, 2005. Accepted for publication September 13, 2005.
ABSTRACT
The cell cycle in cotton (Gossypium hirsutum) fibers is poorly understood. The objective of this study was to evaluate the cell cycle status and DNA content in developing cotton fibers. The DNA content and cell cycle distribution in fiber and hypocotyl cells were determined by flow cytometry. Expression levels of minichrosomal maintenance protein (mcm), cyclin B, and a retinoblastoma-like protein (rb) genes were determined with real-time PCR in fibers and dividing and nondividing tissues. No endoreduplication occurred, nor did genome size or percentage of G1-phase nuclei differ between hypocotyls and fibers. Approximately 13 and 17% of fiber nuclei were in the S phase in 14 days after anthesis (d) fibers and 25 d fibers, respectively. The mcm and cyclin B were expressed at higher levels in fibers than in mature leaves, but expression levels in fibers were less than 15% of meristematic tissues. Rb was expressed in fibers at levels less than 50% of mature leaves or meristematic tissues. Based on an apparent increase in S-phase cells as fibers mature and the low level of expression of genes associated with cell cycle progression, we conclude that S-phase arrest occurs in developing cotton fiber.
Key Words: • cyclin B • fiber DNA content • flow cytometry • Gossypium hirsutum • minichromsomal maintenance protein • Naked Seed mutant • retinoblastoma protein
Short and long seed hairs are characteristic of several members of the genus Gossypium. These trichomes are single-celled projections that, once matured, will have elongated up to 3000 times their initial length. In cultivated cotton (G. hirsutum L.), about one in every four seed epidermal cells differentiates into a fiber (Stewart, 1975
) via four distinct stages of development (Basra and Malik, 1984
). On the day of anthesis, fiber initials appear first near the chalazal end of the ovule, followed by progressive appearance toward the micropylar end (Stewart, 1975
; Basra and Malik, 1984
; Graves and Stewart, 1988
). Rapid elongation occurs from 3 to 21 d, overlapping with a stage of massive cellulose deposition and synthesis of secondary cell walls beginning about 16 d and continuing to 42–45 d. Fibers then undergo a poorly defined maturation stage until the carpel walls open about 45–50 d, at which time the fibers dehydrate. The typical cotton seed will have 14 000–18 000 fibers at maturity, but as many 30 000 have been reported (Basra and Saha, 1999
). All fibers, however, are not equal. The relatively long fibers used for spinning yarn are referred to simply as "fibers" or "lint fibers." The shorter fibers that remain on the seed coat after ginning are called "fuzz fiber" or "linters."
A 25% increase in DNA content in 3- and 5-d fibers relative to 0 d fibers has been reported (Van't Hof, 1999
), an increase interpreted as selective amplification of the genome or entry into the S phase of the cell cycle. In contrast, Basra and Saha (1999)
reported that 98% of fiber cells are arrested in the G1 phase of the cell cycle early in fiber development, leaving only 2% of fiber cells to account for the variation in DNA content previously reported (Van't Hof, 1999
). To further complicate the matter, in vitro studies of fiber elongation provide evidence that some semblance of a cell cycle exists in cotton fiber. If young ovules are cultured in media with appropriate phytohormones, they continue to expand as single cells. However, if the ovules are placed in media lacking phytohormones, up to 25% of fibers are induced to divide (Van't Hof and Saha, 1997
). This phenomenon, however, is unique to in vitro conditions as multicellular fibers are almost never observed in field grown plants. These reports suggest that regulation of the cell cycle is unusual in cotton fibers.
The cell cycle is regulated by cyclin proteins that interact with cyclin-dependent kinases. Cyclin D expression is associated with nonreplicating or G1 cells (Meijer and Murray, 2000
; Dewitte and Murray, 2003
). Cyclin A and cyclin B are mitotic cyclins associated with DNA synthesis (S), G2, and mitosis (M) (Ito et al., 1997
; Chaubet-Gigot, 2000
). Endoreduplication, reported in both plant and animals, is an aberration of normal cell cycling in which multiple S phases occur without M (Joubès and Chevalier, 2000
) and may result from low cyclin-dependent kinase activity during mitosis (Inzé, 2005
). The retinoblastoma protein (Rb) controls commitment to DNA synthesis and entry into mitosis in animal cells. Rb homologs that interact with cyclin D exist in plants, but their function has not been clearly established (Ach et al., 1997
; Durfee et al., 2000
). The minichromosomal maintenance (MCM) protein family are DNA synthesis-related proteins required for chromosome maintenance and replication in yeast, Drosophila, and other organisms (Gibson et al., 1990
; Feger, 1999
). In yeast, MCM protein complexes are first associated with origins of replication in the G1 phase of the cell cycle followed by progressive movement away from the origins, suggesting a helicase-like role for the proteins (Kelly and Brown, 2000
). The anaphase promoting complex (APC) monitors mitosis and prevents premature passage into anaphase by controlling stability of mitotic proteins (Ach et al., 1997
; Kominami et al., 1998
; Harper et al., 2002
). Several observations suggest that the APC functions differently in animals than in plants. For example, in yeast and animals, agents that disrupt mitotic spindle formation trigger the APC to stop mitosis in metaphase (Harper et al., 2002
). In plants, agents that disrupt microtubules, such as colchicine and oryzalin, are routinely used to induce polyploidy indicating that cell cycling continues in the absence of chromosomal separation (Grandjean et al., 2004
). Additionally, Arabidopsis with a mutated APC gene can undergo normal mitosis, though some aspects of meiosis are impaired (Capron et al., 2003
).
The objectives of this study were to evaluate the DNA content, cell cycle regulation, and expression of three genes involved in DNA synthesis and mitosis in several dividing and nondividing cotton tissues and in developing fibers. The DNA content of fiber was identical to that of hypocotyls. Additionally, no endoreduplication was observed in cotton fiber, but relative to hypocotyls, a higher fraction of cells were in the S phase. The expressions of the cell cycle regulatory genes were consistent with the observations of S-phase arrest in cotton fiber cells.
MATERIALS AND METHODS
Plant material
Flowers of field grown plants of Gossypium hirsutum cv. DES119 (or other cultivar, as specified) were tagged on the day of anthesis, and at the specified day after anthesis (13, 14, or 25), the developing boll was harvested. Fibers were pulled from the ovule and immediately used for nuclear isolation. Frozen fibers often failed to yield intact nuclei. Hypocotyls were harvested from seed soaked overnight in water and germinated for 3–5 days at 30°C in the dark on a paper towel soaked in 10 mM CaSO4.
Nuclei isolation and staining
Fibers were carefully separated from the ovule to avoid contamination of the fiber with other ovule cells. All subsequent steps were performed on ice. One gram of fiber or 0.4 g of hypocotyl tissue were placed in a petri dish containing 10 mL of ice-cold nuclear isolation buffer (45 mM MgCl2, 20 mM MOPS [pH 7], 30 mM sodium citrate, 0.1% Triton-X-100, 2% polyvinylpyrolidine 40, 0.1% diethyldithiocarbamic acid) and chopped with a new double-edge razor blade for 2 min. The samples were gently shaken on a rotary shaker for 5 min. The homogenate was poured through Miracloth (Calbiochem, San Diego, California, USA), and the petri dish and Miracloth were rinsed with 5 mL of nuclei isolation buffer. Nuclei were further purified by filtration through a syringe-mounted nylon filter with a 20-µm exclusion limit. After all samples had been filtered, the nuclei were pelleted in a swinging-bucket-rotor centrifuge (Eppendorf, Westbury, New York, USA) at 228 x g. The supernatant solution was discarded and the pellet of nuclei was resuspended in 300 µL nuclear isolation buffer. Three µL of DNases-free RNase A (1 mg · mL–1, 10 µg · mL–1 final concentration) and 30 µL propidium iodide (PI; 1 mg · mL–1, 100 ppm final concentration) were added and the suspension incubated at 4°C for at least 30 min prior to analysis by flow cytometry.
DNA content measurement and cell cycle analysis
Propidium-iodide-stained nuclei from 14 and 25 d fibers and hypocotyls were analyzed for DNA content with a FACsort flow cytometer (Becton Dickinson Immunocytometry System, San Jose, California, USA). Three standards recommended by Price and Johnston (1996)
were used to estimate DNA content for the tissues: Oryza sativa cv. IR36 (2C = 1.01 pg), Zea mays cv. W64A (2C = 5.47 pg), and Hordeum vulgare cv. Sultan (2C = 11.12 pg). However, external rather than internal standards were used (Hendrix and Stewart, 2005
). The mean fluorescence of the G0/1 peak for each standard was determined after analysis of the data collected by the CellQuest software. These values were plotted, and a regression equation was calculated each day for use in estimation of the DNA content of the experimental samples (Hendrix and Stewart, 2005
). Two samples of each standard were measured randomly during analysis as a control for instrument drift.
For DNA content of cotton tissue, G0/1- and G2/M-phase nuclei were identified by their symmetric peaks at expected fluorescence values, and S-phase nuclei were identified as those with fluorescence values intermediate to the G1 and G2 peaks. Such delineation enabled the number and mean fluorescence of nuclei in each cell cycle phase to be quantified.
Statistical analysis of data
Four replicates of at least 500 G1-phase nuclei were measured for each tissue type in the study. For DNA content, data are reported as the mean 2C DNA mass in picograms (±SE). For cell cycle analysis, data are reported as the mean percentage (±SE) of total nuclei measured in a particular cell cycle phase delineated as described. Means were separated with a Dunnet's test (
= 0.05) with hypocotyls as the control group in JMP 5.0.1 (academic version, SAS Institute, Cary, North Carolina, USA).
RNA isolation and cloning of cell cycle regulatory genes from cotton fibers
Total polyribosomal RNA was isolated from the indicated tissues of Gossypium hirsutum cv. DES119 as previously described (Taliercio and Ray, 2001
). Semiquantitative RT-PCR was done as described elsewhere (Taliercio and Kloth, 2004
). The genes, primers, and accession numbers relevant to the semiquantitative PCR are shown in Table 1. The PCR products were separated by electrophoresis through a 1% agarose gel in TAE and visualized with ethidium bromide staining. Selected bands were cloned and sequenced to confirm their identity. All sequencing was done at the USDA, ARS MidSouth Genomics Core Facility. The genes were annotated by comparison to the Arabidopsis genome with the tBLASTx algorithm. Clones are designated with Gh (for Gossypium hirsutum) preceding the name.
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). Because of its utility across tissues, 18S GhrRNA was selected to normalize sample loading. All primers were used at a final concentration of 250 nM except the rRNA primers, which were used at 100 nM. The cDNA was denatured for 3 min at 95°C followed by 40 cycles of 30 s at 95°C, 30 s at 54°C, and 30 s at 72°C. The amplicon's homogeneity was confirmed by analyzing melting temperatures using the standard protocol recommended by BioRad. No DNA contamination was observed in RNA template controls amplified with any primer with the exception of the 18S rRNA controls. Undiluted RNA of many samples amplified a product as early as cycle 30, including the "no template" control. To avoid this apparent contamination, no data from the rRNA primers were used after cycle 26. Relative expression values were calculated by the method of Pfaffl (2001)
. The amplification efficiency of each primer pair used in subsequent calculations was determined with shoot RNA (Table 1). The amplification efficiency of selected primer pairs was confirmed for a variety of cDNAs by amplifying cDNA derived from an equal portion mixture of shoot, leaf, stem, and fiber RNAs. These values are shown in parentheses on Table 1. The efficiency of the rRNA primers was determined on five 0.1 dilutions. The efficiency of all other primers was determined on four or five 0.25 dilutions to accommodate the low level of expression of these genes. All statistical analysis was done on EXCEL (Microsoft, Seattle, Washington, USA). Expression values were normalized to the 18S rRNA gene using a 0.01 dilution of the cDNA and compared with the expression levels in apical shoot RNA. The three samples include two replicates of material harvested in 2004 and one replicate of material harvested in 2003. The trends of gene expression were consistent between the samples harvested in 2004 and 2003. RESULTS
The DNA content of the G1-phase of nuclei from G. hirsutum cv. DES119 fibers at 14 and 25 d and from hypocotyls is reported in Table 2. No significant differences in DNA content were observed among tissue types, indicating that 14 and 25 d fibers behaved very much like somatic cells in this regard. Additionally, no endoreduplication up to 
20 C was observed in either fibers or hypocotyl nuclei (data not shown).
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Analysis of the DNA content and cell cycle distribution of fiber cells further refines our understanding of cell-cycle regulation in developing cotton fibers. The trend observed during the period of fiber development between 14 and 25 days is for a progressively higher portion of nuclei to be present in the S phase (Fig. 1). Apparently, this trend begins early in fibers development because Basra and Saha (1999)
reported 2% of nuclei in young fibers had entered the S phase. The high percentage of fiber cells with arrested cycling early in fiber development reported by Basra and Saha (1999)
appears to dissipate as fibers age and, in the time frame we examined, was not significantly different from hypocotyls cells (79%). Additionally, we found no evidence of endoreduplication in cotton fibers. Thus, the previously reported (Van't Hof, 1998
) 25% increase in fiber DNA content early in fiber development does not represent the beginning of successive genome replications. More likely, the reported early increase in DNA content was due to enlarged micronucleoli in these metabolically active cells (Peeters et al., 1988
; Van't Hof, 1998
).
Little is known about the short, developmentally delayed "fuzz" fibers on the cotton seed coat. To address the possibility that fuzz fibers were contributing S-phase nuclei, the nuclei from Naked seed (fuzzless) mutants were examined. The S-phase arrest was shown to be independent of fuzz fibers. It is also unlikely that the S-phase arrest phenomenon was an artifact due to nucleoli, because development of these organelles peaks at 6–10 d and declines to a constant value around 20 d (Peeters et al., 1988
). In the absence of endoreduplication, and given the low incidence of multicellular fibers in field-grown plants, one of two possibilities might explain the presence of S-phase and G2-phase nuclei in fibers. First, there could be multinuclear fibers that become more numerous as fibers age. While multinuclear fibers have not been reported, the examination of fiber nuclei becomes difficult after the secondary cell wall develops. More likely, however, is a system of cell cycle regulation in which a minority of cells escapes to enter into S phase and arrest or move slowly on to the G2/ M phase. Additionally, variable observations (e.g., absence of the peak, G1-shoulder, or symmetric peaks) in the 14 d samples suggest that the phenomenon begins to be rapidly manifested concurrent with secondary cell wall synthesis (around 16 d). While the percentages of nuclei in S phase are not significantly different between 14 and 25 d fibers, there may be a trend toward a higher percentage of nuclei in S phase as the fibers mature. Increases in S-phase nuclei in the 25 d fibers can be explained by accumulation of S-phase-arrested nuclei because these nuclei probably have no way of reducing their DNA content in the absence of mitosis.
Expression of Ghrb suggested that at least part of the apparatus for moving nuclei into the S phase of DNA replication is present in fibers, though the low level of Ghmcm expression in fibers relative to stems suggested the potential for DNA synthesis was limited. Clearly, the level of Ghcycb expression was much higher in 13 d fibers than mature leaves and was slightly higher in 13 d fibers than stems. Elevated levels of cyclin B are associated with cells in G2/M phases of the cell cycle (Ito, 2000
). The relative increase in expression of Ghcycb compared to stems and leaves is consistent with a portion of fibers arresting in S phase. In fact, if the increase in expression of Ghcycb is confined to the approximately 20% of fiber cells arrested in S or G2/M phase, the potential level of cyclin B in these cells may be underestimated. There appears to be a decrease in expression of cyclin B between 13 d and 25 d of fiber development, but no clear trend toward lower expression was observed among five fiber samples harvested at 3-day intervals between 10 d and 22 d (data not shown), though it was evident by 25 d. It is difficult to speculate about the role these genes may play based on mRNA levels alone because cyclin B and Rb are posttranscriptionally regulated (Ach et al., 1997
; Durfee et al., 2000
; Ito, 2000
). However, the presence of the mRNAs and the patterns of expression of Ghrb, Ghcycb, and Ghmcm are consistent with a minority of fiber cells arresting in S- or G2/M-phases of the cell cycle (Reichheld et al., 1996
, 1999
).
Cotton fibers provide a useful system to investigate control of the cell cycle in a population of synchronously differentiating cells. It is particularly interesting that a significant number of fibers appear to arrest in S/G2/M phase and are also associated with an elevated level of cyclin B transcripts. In animal and yeast cells, checkpoints have been described that prevent entry into the S phase before the cell is ready to synthesize DNA and prevent the entry into anaphase before the appropriate time. The gatekeeper for entry into the S phase is the retinoblastoma protein, and the gatekeeper for completion of mitosis is the APC. Even though plants have many genes similar to the apparatus of cell cycle checkpoints, there are differences in the way these checkpoints operate in plants. For example, in Arabidopsis thaliana meristems, DNA synthesis continues in the nucleus in the presence of agents that disrupt the microtubules (Grandjean et al., 2004
). Fibers may represent a case where the cell cycle is arrested during the S or G2/ M phase, making it a valuable system to identify components of a cell cycle checkpoint in plants and the role these genes play in differentiation.
FOOTNOTES
The authors thank P. Johnson for technical assistance and R. Turley and B. Triplett for review of this manuscript. This work was supported by the USDA-ARS CRIS No. 6402-21000-029-00 (E.T.) and by Cotton, Inc. Grant No. 02– 258 (J.M.S.).
Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. 
4 Author for correspondence (ETaliercio{at}msa-stoneville.ars.usda.gov
)
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