Chromosome instabilities and programmed cell death in tapetal cells of maize with B chromosomes and effects on pollen viability

B chromosomes (B's), knobbed chromosomes, and chromosome 6 (NOR) of maize undergo nondisjunction and micronucleus formation in binucleate tapetal cells. These chromosome instabilities are regular events in the program of tapetal cell death, but the B's strongly increase A chromosome instability. We studied 1B and 0B plants belonging to selected lines for high or low B transmission rate and their F1 hybrids. These lines are characterized by meiotic conservation or loss of B chromosomes, respectively. The female B transmission (fBtl) allele(s) for low B transmission is dominant, inducing micronucleus formation and B nondisjunction. We hypothesize that the fBtl allele(s) induces knob instability. This instability would be sufficient to produce B loss in both meiocytes and binucleate tapetal cells. B instability could, in turn, produce instabilities in all chromosomes of maize complement. To establish whether the chromosomal instabilities are related to the tapetal programmed cell death (PCD) process, we applied the TUNEL technique. PCD, estimated as the frequency of binucleate tapetal cells with TUNEL label, was significantly correlated with the formation of micronuclei and the frequency of pollen abortion. It can be concluded that the observed chromosome instabilities are important to the PCD process and to the development of microspores to form viable pollen grains.     

On the mechanism of chromatin loss induced by the B chromosome of maize

Knobbed regions of the regular maize complement frequently are eliminated at the second microspore division in spores which have two or more B chromosomes. Evidence is presented that no or little loss occurs in spores with one B and that the rate is not increased in spores with more than two B's.—The B chromosomes from an unrelated strain proved as effective in inducing loss as did the B's of the original high loss stock.—Chromatin loss induced by B's is restricted to knobbed A chromosomes and occurs only at the second microspore division. Knobbed chromosomes 3, 5, and 9 have been tested and all interact with B's to give loss. Chromosomes with large knobs are more frequently broken than are those with smaller knobs and knobless chromosomes show negligible loss.—Although knobs and B's are essential for chromatin elimination, modifying genes can markedly affect the rate of loss.——Two knobbed heterologous chromosomes undergo simultaneous loss more frequently than expected from independent events. The data indicate that joint loss occurs in competent cells and that preferential assortment of the two deficient chromosomes to specific poles is unlikely.—B chromosomes and deficient chromosomes assort independently at the second microspore anaphase.—Genetic data from crosses with marker genes in both arms of chromosome 3 show that breakage of the postulated dicentric bridge does not occur solely at the centric region since a variety of deficient chromosomes were recovered.—Nondisjunction of B chromosomes and elimination of knobbed chromatin take place during the second microspore mitosis. The argument is advanced that the two phenomena result from faulty replication of heterochromatic segments. The position of the nonreplicating segment in the two kinds of chromosomes determines whether nondisjunction or breakage takes place.—Finally, it is suggested that all of the reported effects of the B chromosome can be accounted for if the B is a parasitic entity having no genetic function other than controlling the replication of its proximal heterochromatic knob and increasing the ability of B-containing sperm cells to compete successfully for fertilization of the egg.     

Sporophytic nondisjunction of the maize B chromosome at high copy numbers

It has been known for decades that the maize B chromosome undergoes nondisjunction at the second pollen mitosis.Fluorescence in-situ hybridization(FISH)was used to undertake a quantitative study of maize plants with differing numbers of B chromosomes to observe if instability increases by increasing B dosage in root tip tissue.B chromosome nondisjunction was basically absent at low copy number,but increased at higher B numbers.Thus,B nondisjunction rates are dependent on the dosage of B's in the sporophyte.Differences in nondisjunction were also documented between odd and even doses of the B.In plants that have inherited odd humbered doses of the B chromosome,B loss is nearly twice as likely as B gain in a somatic division.When comparing plants with even doses of B's to plants with odd doses of B's,plants with even numbers had a significantly higher chance to increase in number.Therefore,the B's nondisjunctive capacity,previously thought to be primarily restricted to the gametophyte,is present in sporophytic cells.     

Sporophytic nondisjunction of the maize B chromosome at high copy numbers

It has been known for decades that the maize B chromosome undergoes nondisjunction at the second pollen mitosis.Fluorescence in-situ hybridization (FISH) was used to undertake a quantitative study of maize plants with differing numbers of B chromosomes to observe if instability increases by increasing B dosage in root tip tissue.B chromosome nondisjunction was basically absent at low copy number,but increased at higher B numbers.Thus,B nondisjunction rates are dependent on the dosage of B's in the sporophyt...     

The supernumerary B chromosome of maize: drive and genomic conflict

The supernumerary B chromosome of maize is dispensable, containing no vital genes, and thus is variable in number and presence in lines of maize. In order to be maintained in populations, it has a drive mechanism consisting of nondisjunction at the pollen mitosis that produces the two sperm cells, and then the sperm with the two B chromosomes has a preference for fertilizing the egg as opposed to the central cell in the process of double fertilization. The sequence of the B chromosome coupled with B chromosomal aberrations has localized features involved with nondisjunction and preferential fertilization, which are present at the centromeric region. The predicted genes from the sequence have paralogues dispersed across all A chromosomes and have widely different divergence times suggesting that they have transposed to the B chromosome over evolutionary time followed by degradation or have been co-opted for the selfish functions of the supernumerary chromosome.     

Sperm Identification in Maize by Fluorescence in Situ Hybridization

The two sperm cells of common origin within the pollen tube of flowering plants are each involved in a fertilization event. It has long been recognized that preferential fusion of one sperm with the egg can occur in B chromosome-containing lines of maize. If the second pollen mitosis begins with a single B chromosome, nondisjunction will result in one sperm possessing two B chromosomes and the other containing no B chromosomes. The B chromosome-containing sperm most often fertilizes the egg, whereas the sperm nucleus with no B chromosomes fuses with the polar nuclei. Despite the obvious advantages of being able to recognize and then track, separate, and analyze one sperm type from the other, it has not been possible because of the lack of sufficient detectable differences between the two types of sperms. In this study, we used a B chromosome-specific DNA sequence (pZmBs) and in situ hybridization to identify and track the B chromosome-containing sperm cell within mature pollen and pollen tubes. Our results are consistent with conclusions from previous genetic studies related to B chromosome behavior during pollen formation. Within pollen tubes, the position in which the B chromosome-containing sperm travels (leading or trailing) in relation to the sperm cell lacking B chromosomes appears to be random.     

Involvement of knob heterochromatin in mitotic abnormalities in germinating aged seeds of maize.

Seeds of three inbred lines of maize, with contrasting numbers of heterochromatic knobs and stored under two different ageing treatments, were analyzed for the occurrence of abnormalities at mitotic anaphase in root meristems 3, 7, 21,42, and 56 days after germination, and in root meristems of their freshly harvested selfed progeny. The largest category of detectable aberrations involved breakage of knobbed chromosome arms. We have obtained evidence that knob heterochromatin plays a central role in the origin of primary chromosome bridges. The initial event responsible for the occurrence of breakages and lagging chromosomes was characterized by the nondisjunction of newly replicated sister chromatids, which was observed to occur preferentially at the knob level. Such configurations, and all the other types of abnormalities (as for example, lagging chromosomes, typical chromosome bridges, fragments, and micronuclei), were observed at decreasing frequencies throughout root growth. Nevertheless, we have detected the occurrence of breakage-fusion-bridge cycles that were initiated by broken chromosomes. The relationship between late-replicating DNA in maize knob heterochromatin and the vulnerability of such regions to breakage is discussed. Our observations suggest a similarity between the mechanisms involved or associated with the origin of the described abnormalities and those reported to occur in cultured maize cells.     

Centromere function and nondisjunction are independent components of the maize B chromosome accumulation mechanism

Supernumerary or B chromosomes are selfish entities that maintain themselves in populations by accumulation mechanisms. The accumulation mechanism of the B chromosome of maize (Zea mays) involves nondisjunction at the second pollen mitosis, placing two copies of the B chromosome into one of the two sperm. The B chromosome long arm must be present in the same nucleus for the centromere to undergo nondisjunction. A centromere, containing all of the normal DNA elements, translocated from the B chromosome to the short arm of chromosome 9 was recently found to be epigenetically silenced for centromeric function. When intact B chromosomes were added to this genotype, thus supplying the long arm, the inactive centromere regained the property of nondisjunction causing the translocation chromosome 9 to be differentially distributed to the two sperm or resulted in chromosome breaks in 9S, occasionally producing new translocations. Translocation of the inactive B centromere to chromosome 7 transferred the nondisjunction property to this chromosome. The results provide insight into the molecular and evolutionary basis of this B chromosome accumulation mechanism by demonstrating that nondisjunction is caused by a process that does not depend on normal centromere function but that the region of the chromosome required for nondisjunction resides in the centromeric region.     

Accumulation of multiple copies of maize minichromosomes

Multiple copies of B chromosomes in maize (Zea mays) can accumulate in the genome using the B chromosome's accumulation mechanism, specifically nondisjunction at the second pollen mitosis and preferential fertilization of the egg. Using this mechanism, we accumulated 4 different-sized minichromosomes derived from the B chromosome to test the chromosome limits of the cell. The accumulation of normal B chromosomes is associated with multiple phenotypes including white stripes and asymmetric leaf blades, but when minichromosomes are accumulated these symptoms are absent. We also found that multiple B chromosome-derived minichromosomes can coexist with A chromosome-derived minichromosomes. During the years that these experiments were conducted, we found many B chromosome rearrangements and fragments, 2 recoverable A chromosome fragments, and observed a minichromosome breakage-fusion-bridge cycle in roots.     

Laser microirradiation of kinetochores in mitotic PtK2 cells: chromatid separation and micronucleus formation

Irradiation of the kinetochore region of PtK2 chromosomes by laser light of 532 nm was used to study the function of the kinetochore region in chromosome movement and to create an artificial micronuclei in cells. When the sister kinetochores of a chromosome were irradiated at prometaphase, the affected chromosome detached from the spindle and exhibited no further directed movements for the duration of mitosis. The chromatids of the chromosome remained attached to one another until anaphase, at which point they separated. No poleward movement of the chromatids was observed, and at telophase they passively moved to one of the daughter cells and were enclosed in a micronucleus. The daughter cell containing the micronucleus was then isolated by micromanipulation and followed through subsequent mitoses. At the next mitosis, two chromosomes, each with two chromatids, condensed in the micronucleus. These chromosomes did not attach to the spindle and showed chromatid separation, but no poleward movements at anaphase. They were again enclosed in micronuclei at telophase. The third generation mitosis was similar to the second. Occasionally, both the irradiation-produced and naturally occurring micronuclei exhibited no chromosome condensation at mitosis. Feulgen-stained monolayers of PtK2 cells with naturally occurring micronuclei showed that some micronuclei stain positive for DNA and others do not. This finding raises questions about the fate of chromosomes in a micronucleus.     

Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay

Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.     

Laser microirradiation of kinetochores in mitotic PtK2 cells

Irradiation of the kinetochore region of PtK₂ chromosomes by laser light of 532 nm was used to study the function of the kinetochore region in chromosome movement and to create artificial micronuclei in cells. When the sister kinetochores of a chromosome were irradiated at prometaphase, the affected chromosome detached from the spindle and exhibited no further directed movements for the duration of mitosis. The chromatids of the chromosome remained attached to one another until anaphase, at which point they separated. No poleward movement of the chromatids was observed, and at telophase they passively moved to one of the daughter cells and were enclosed in a micronucleus. The daughter cell containing the micronucleus was then isolated by micromanipulation and followed through subsequent mitoses. At the next mitosis, two chromosomes, each with two chromatids, condensed in the micronucleus. These chromosomes did not attach to the spindle and showed chromatid separation, but no poleward movements at anaphase. They were again enclosed in micronuclei at telophase. The third generation mitosis was similar to the second. Occasionally, both the irradiation-produced and naturally occurring micronuclei exhibited no chromosome condensation at mitosis. Feulgenstained monolayers of PtK₂ cells with naturally occurring micronuclei showed that some micronuclei stain positive for DNA and others do not. This finding raises questions about the fate of chromosomes in a micronucleus.     

A procedure for localizing genetic factors controlling mitotic nondisjunction in the B chromosome of maize

The B chromosome of maize undergoes nondisjunction at the second pollen mitosis at rates as high as 98% (Roman, 1948; Carlson, 1969a). Nondis-junction is controlled by at least two separable regions on the B chromosome (Roman, 1949; Longley, 1956; Carlson, 1969b; Ward, 1972). A procedure for identifying and localizing the chromosomal sites required for nondisjunction is reported here. A translocation between the B and chromosome 9 (TB-9b) was utilized. Plants carrying TB-9b were screened for mutants of nondisjunction, i.e. translocations in which nondisjunction does not occur. Two such translocations were identified in a small screening. While the mutant translocations have not been analyzed in pachytene, they are most likely deletions or rearrangements of regions on the B chromosome vital to nondisjunction. Diminutive and rearranged B chromosomes are known to arise spontaneously in small populations (Randolph, 1941; Longley, 1956). — Also reported here are the nondisjunctional properties of the B⁹ isochromosome (Carlson, 1970) and several telocentric (or subtelocentric) derivatives of this chromosome. Some derivatives of the isochromosome are virtually incapable of nondisjunction, and should provide information on the role of the centromere in nondisjunction.     

B Chromosome Behavior in Maize Pollen as Determined by a Molecular Probe

The B chromosomes of maize typically undergo nondisjunction during the second microspore division (generative cell division). When the microspore nucleus contains only one B chromosome, two kinds of sperm result, one with two B chromosomes and one with no B chromosomes. The sperm with the B chromosomes preferentially fertilizes the egg cell. Previous studies of these phenomena have been limited to genetic analysis and chromosome spreads. In this study we show that a B chromosome-specific probe can be used with fluorescence in situ hybridization (FISH) analysis to detect the presence, location, and frequency of B chromosomes in intact interphase nuclei within mature pollen of maize. Using genetic line TB-10L18, our results indicate that nondisjunction of the B centromere occurs at an average frequency of 56.6%, based on four plants and 1306 pollen grains analyzed. This is consistent with the results of genetic studies using the same B-A translocation. In addition, our results suggest that B chromosome nondisjunction can occur during the first microspore division. Spatial distribution of the B chromosome-specific probe appears to be largely confined to one tip of the sperm nucleus, and a DNA fragment found outside the pollen nuclei often hybridizes to the B chromosome-specific probe.     

Locating a site on the maize B chromosome that controls preferential fertilization.

In maize, the B chromosome can undergo nondisjunction at the second pollen mitosis, producing sperm with two B chromosomes and sperm with zero B chromosomes. Preferential fertilization is the ability of the sperm carrying two B chromosomes to transmit more frequently to the embryo of a kernel than the sperm lacking the B chromosome. A translocation involving the B chromosome and chromosome 9, TB-9Sb, has been used to study preferential fertilization. The B-9 chromosome has the same properties of nondisjunction and preferential fertilization as the standard B chromosome. Deletion derivatives of B-9, which lack the centric heterochromatin and possibly some adjacent euchromatin, were tested for their ability to induce preferential fertilization. They were found to lack the capacity for preferential fertilization.     

A test of homology between the B chromosome of maize and abnormal chromosome 10, involving the control of nondisjunction in B's

Summary Nondisjunction of B and B-translocation chromosomes occurs regularly in maize at the second pollen mitosis (Roman, 1947; Blackwood, 1956). The mechanism of nondisjunction was studied using the A-B interchange, TB-9b. The B⁹ chromosome of the interchange undergoes nondisjunction at the second pollen mitosis, while the 9^B chromosome does not (Roman, 1947). It was shown that the 9^B chromosome must be present in a plant for nondisjunction of the B⁹ to occur. This is consistent with the reports of Roman on TB-4a (1949) and Longley on TB-10a (1956). It was also demonstrated that the influence of the 9^B chromosome is limited to pollen grains containing it, and does not extend to all the pollen of a plant.A test of homology between the B chromosome and abnormal chromosome 10 was also made. The ability of abnormal 10 to substitute for the 9^B chromosome and induce nondisjunction of the B⁹ was tested. Nondisjunction did not occur at a detectable rate in the presence of abnormal 10, and the results failed to support Ting's proposal (1958) concerning the origin of abnormal 10.     

Analysis of Nondisjunction Induced by the R-X1 Deficiency during Microsporogenesis in Zea Mays L

The r-X1 deficiency in maize induces nondisjunction at the second mitotic division during embryo sac formation. However, it was not known if this deficiency also induces nondisjunction during the microspore divisions. Microsporogenesis in plants lacking or containing this deficiency was compared using two approaches. First, chromosome numbers were determined in generative nuclei. Many (8.3%) of the generative nuclei in r-X1-containing plants were aneuploid; however, those from control plants were all haploid. Thus, this deficiency induces nondisjunction during the first microspore division. Second, nucleoli were analyzed in microspores. The only nucleolar organizing region in maize is on chromosome 6. If chromosome 6 underwent nondisjunction during the first microspore division, one nucleus in binucleate microspores would contain no nucleolus and the other would contain two nucleoli (or one nucleolus if the nucleoli fused). Only one (0.03%) microspore of this type was observed in control plants while 1.12% were found in r-X1-containing plants. Thus, the r-X1 deficiency induces nondisjunction of chromosome 6 during the first microspore division. However, both of the sperm nuclei in trinucleate microspores contained one nucleolus in r-X1-containing and control plants; thus, this deficiency does not induce nondisjunction of chromosome 6 (and presumably other chromosomes) during the second microspore division.     

A New Property of the Maize B Chromosome

TB-9Sb is a translocation between the B chromosome and chromosome 9 in maize. Certain deletions of B chromatin from the translocation cause a sharp decrease in B-9 transmission compared to the rate for standard TB-9Sb. The deletions remove components of a B chromosome genetic system that serves to suppress meiotic loss in the female. At least two distinct B-chromosome regions suppress meiotic loss: one on the B-9 and one on 9-B. The system operates by stabilizing univalent B-type chromosomes. It allows the univalents to migrate to one pole in meiosis, despite the absence of a pairing partner. The findings reported here are the first evidence for genetic control of meiotic loss by a B chromosome. However, it is proposed that the practice of suppressing meiotic loss is common to the B chromosomes of all species. The need to suppress meiotic loss results from the fact that B chromosomes are frequently unpaired in meiosis and subject to very high frequencies of loss. B chromosomes may utilize one or more of the following methods to suppress meiotic loss: (a) regular migration of univalent B's to one pole in meiosis, (b) enhanced recombination between B chromosomes and (c) mitotic nondisjunction.     

Nondisjunction and isochromosome formation in the B chromosome of maize

Bianchi et al. (1961) found that sectored losses of B-translocation chromosomes occur at a significant rate during early development of the endosperm and sporophyte. The losses were attributed to nondisjunction of the chromomosome, since B type chromosomes are known to undergo nondisjunction at the second pollen mitosis. Sector formation was further analyzed in the present paper, using the translocation, TB-9b. It was found that losses of the B⁹ chromosome during early endosperm mitoses occur only if the 9^B chromosome is present. In addition, sectors are produced in the sporophyte only if the 9^B and B⁹ chromosomes are inherited from the male parent. Both of these findings suggest that nondisjunction is indeed responsible for the B⁹ losses (see text). However, cytological observation of sectored plants demonstrates that isochromosome formation, rather than nondisjunction, produces most B⁹ losses in the sporophyte. The conflicting results can be reconciled by assuming that the same basic event, perhaps stickiness of the B⁹ chromosome, produces nondisjunction at the second pollen mitosis and isochromosome formation in the developing sporophyte. Observation of the isochromosome in pachytene reveals that a heterochromatic region corresponding to the short arm of the normal B⁹ is missing. The normal B⁹ chromosome is, therefore, an acrocentric chromosome.     

Chromosome spatial order in human cells: evidence for early origin and faithful propagation

We have investigated the origin and nature of chromosome spatial order in human cells by analyzing and comparing chromosome distribution patterns of normal cells with cells showing specific chromosome numerical anomalies known to arise early in development. Results show that all chromosomes in normal diploid cells, triploid cells and in cells exhibiting nondisjunction trisomy 21 are incorporated into a single, radial array (rosette) throughout mitosis. Analysis of cells using fluorescence in situ hybridization, digital imaging and computer-assisted image analysis suggests that chromosomes within rosettes are segregated into tandemly linked “haploid sets” containing 23 chromosomes each. In cells exhibiting nondisjunction trisomy 21, the distribution of chromosome 21 homologs in rosettes was such that two of the three homologs were closely juxtaposed, a pattern consistent with our current understanding of the mechanism of chromosomal nondisjunction. Rosettes of cells derived from triploid individuals contained chromosomes segregated into three, tandemly linked haploid sets in which chromosome spatial order was preserved, but with chromosome positional order in one haploid set inverted with respect to the other two sets. The spatial separation of homologs in triploid cells was chromosome specific, providing evidence that chromosomes occupy preferred positions within the haploid sets. Since both triploidy and nondisjunction trisomy 21 are chromosome numerical anomalies that arise extremely early in development (e.g., during meiosis or during the first few mitoses), our results support the idea that normal and abnormal chromosome distribution patterns in mitotic human cells are established early in development, and are propagated faithfully by mitosis throughout development and into adult life. Furthermore, our observations suggest that segregation of chromosome homologs into two haploid sets in normal diploid cells is a remnant of fertilization and, in normal diploid cells, reflects segregation of maternal and paternal chromosomes. Received: 19 January 1998; in revised form: 28 May 1998 / Accepted: 30 June 1998