Chromosome nondisjunction and instabilities in tapetal cells are affected by B chromosomes in maize

Abnormal mitosis occurs in maize tapetum, producing binucleate cells that later disintegrate, following a pattern of programmed cell death. FISH allowed us to observe chromosome nondisjunction and micronucleus formation in binucleate cells, using DNA probes specific to B chromosomes (B's), knobbed chromosomes, and the chromosome 6 (NOR) of maize. All chromosome types seem to be involved in micronucleus formation, but the B's form more micronuclei than do knobbed chromosomes and knobbed chromosomes form more than do chromosomes without knobs. Micronuclei were more frequent in 1B plants and in a genotype selected for low B transmission rate. Nondisjunction was observed in all types of FISH-labeled chromosomes. In addition, unlabeled bridges and delayed chromatids were observed in the last telophase before binucleate cell formation, suggesting that nondisjunction might occur in all chromosomes of the maize complement. B nondisjunction is known to occur in the second pollen mitosis and in the endosperm, but it was not previously reported in other tissues. This is also a new report of nondisjunction of chromosomes of the normal set (A's) in tapetal cells. Our results support the conclusion that nondisjunction and micronucleus formation are regular events in the process of the tapetal cell death program, but B's strongly increase A chromosome instability.     

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...     

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.     

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.     

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.     

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.     

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.     

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.     

Abnormal Development of Tapetum and Microspores Induced by Chemical Hybridization Agent SQ-1 in Wheat

Chemical hybridization agent (CHA)-induced male sterility is an important tool in crop heterosis. To demonstrate that CHA-SQ-1-induced male sterility is associated with abnormal tapetal and microspore development, the cytology of CHA-SQ-1-treated plant anthers at various developmental stages was studied by light microscopy, scanning and transmission electron microscopy, in situ terminal deoxynucleotidyl transferasemediated dUTP nick end-labelling (TUNEL) assay and DAPI staining. The results indicated that the SQ-1-treated plants underwent premature tapetal programmed cell death (PCD), which was initiated at the early-uninucleate stage of microspore development and continued until the tapetal cells were completely degraded; the process of microspore development was then blocked. Microspores with low-viability (fluorescein diacetate staining) were aborted. The study suggests that premature tapetal PCD is the main cause of pollen abortion. Furthermore, it determines the starting period and a key factor in CHA-SQ-1-induced male sterility at the cell level, and provides cytological evidence to further study the mechanism between PCD and male sterility.     

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.     

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.     

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.     

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.     

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.     

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.     

Ultrastructural aspects and programmed cell death in the tapetal cells of Lathyrus undulatus Boiss

Programmed cell death (PCD) in the tapetum of Lathyrus undulatus L. was analyzed based on light, fluorescence and electron microscopy to characterize its spatial and temporal occurrence. Development and processes of PCD in secretory tapetal cells of Lathyrus undulatus L. were correlated with the sporogenous cells and pollen grains. At early stages of development the tapetal cells appeared similar to pollen mother cells, structurally. Concurrent with meiosis, tapetum expanded both tangentially and radially as vacuoles increased in size. Tapetal cells most fully developed at young microspore stage. However, tapetum underwent substantial changes in cell organization including nucleus morphology monitored by DAPI. The TUNEL staining confirmed the occurrence of intra-nucleosomal DNA cleavage. In addition to nuclear degeneration which is the first hallmark of PCD other diagnostic features were observed at vacuolated microspore stage intensely; such as chromatin condensation at the periphery of the nucleus, nuclear membrane degeneration, chromatin release to the cytoplasm, vacuole collapse according to tonoplast rupture, shrinkage of the cytoplasm, the increase and enlargement of the endoplasmic reticulum cisternae and disruption of the plasma membrane. After vacuole collapse due to possible release of hydrolytic enzymes the cell components degraded. Tapetal cells completely degenerated at bicellular pollen stage.     

Microsporogenesis in Brachiaria dictyoneura (Fig. & De Not.) Stapf (Poaceae: Paniceae)

Microsporogenesis was analyzed in five accessions of Brachiaria dictyoneura presenting x = 6 as the basic chromosome number. All accessions were tetraploid (2n = 4x = 24) with chromosome pairing in bi-, tri-, and quadrivalents. The recorded meiotic abnormalities were those typical of polyploids, including precocious chromosome migration to the poles, laggard chromosomes, and micronucleus formation. The frequency of these abnormalities, however, was lower than those reported for other polyploid accessions previously analyzed for other Brachiaria species. Cell fusion and absence of cytokinesis were also recorded in some accessions, leading to restitutional nucleus formation in some cells. Genetically unbalanced microspores, binucleate, and 2n microspores were found among normal meiotic products as results from these abnormalities. The limitation in using these accessions as pollen donor in interspecific crosses with sexual species with x = 7 or x = 9 in breeding programs is discussed.     

Male-sterility of thermosensitive genic male-sterile rice is associated with premature programmed cell death of the tapetum

The tapetum plays a crucial role in pollen development. This secretory tissue produces numerous nutritive proteins necessary for pollen maturation. The tapetum, whose cells undergo programmed cell death (PCD), is completely diminished by the time the pollen is fully mature. Our previous studies on a thermosensitive genic male-sterile (TGMS) rice (Oryza sativa L.) suggested that male-sterility was due to failure in pollen development. In this paper we describe how further analysis of the TGMS rice revealed that male-sterility is associated with premature PCD of the tapetum. Cytological observations of TGMS rice anthers at various developmental stages indicated that PCD initiates at an early stage of pollen development and continues until the tapetal cells are completely degraded, resulting in pollen collapse. Transmission electron microscopy showed the morphologically distinct hallmarks of apoptosis, including cytoplasmic shrinkage, membrane blebbing, and vacuolation. Identification of DNA fragmentation using the TUNEL assay supports the hypothesis that premature PCD is associated with male-sterility in the rice. The tissue-specific feature of the thermosensitive genic male-sterile phenotype is discussed with regard to PCD during anther development.     

Double fertilization in maize: the two male gametes from a pollen grain have the ability to fuse with egg cells

In flowering plants, two male gametes from a single pollen grain fuse with two female gametes, the egg and central cells, to form the embryo and endosperm, respectively. The question then arises whether the two male gametes fuse randomly with the egg and central cells. We investigated this question using two nearly isogenic maize lines with supernumerary B chromosomes (TB10L18) or without (r-tester). B chromosomes regularly undergo non-disjunction at the second pollen mitosis, producing one sperm cell with zero B chromosomes and one with two. We first confirmed earlier studies showing an excess of transmission of the B chromosomes to the embryo rather than to the endosperm. We then tested the possibility of a directed fertilization. For TB10L18 pollen, we could demonstrate the existence of a size dimorphism between the two sperm cells, correlated to the content in B chromosomes, as detected by fluorescence in situ hybridization (FISH). However, no directed fusion of B chromosome containing sperm to egg cells could be detected when using in vitro fertilization. The absence of directed fusion in vitro could also be demonstrated for control lines. We conclude that both male gametes have the capacity to fuse with the egg cell in maize, although sexual reproduction results in a preferential transmission of supernumerary B chromosomes.