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The Plant Cell 19:3835-3837 (2007) © 2007 American Society of Plant Biologists
Naughty Behavior of Maize Minichromosomes in Meiosis
Department of Plant Breeding and wp45{at}cornell.edu
Imagine a plant genetics lab in the year 2020. After the genomics era, the function of nearly every plant gene is known. Inexpensive resequencing methods allow easy access to natural genetic variation and creation of multiple mutant alleles for each locus. Geneticists can pursue what has been a life-long dream for many of us: studying interactions between large numbers of genes. To do this, they use engineered minichromosomes that carry very large numbers of genes in a single genetic transformation vector.
When we think about the above scenario, it is hard not to get excited about the recent advances in the development of plant artificial chromosomes. Although development of plant minichromosomes was at first slower than in other organisms, the progress in the past few years has been amazing, and in early 2007, the group of Jim Birchler at the University of Missouri–Columbia reported creating minichromosomes in maize (Yu et al., 2007
There are essentially two ways to create minichromosomes: bottom-up and top-down (Houben and Schubert, 2007  ). In the first approach, chromosomes, including centromeres and often telomeres, as well as selectable markers, and, most importantly, genes of interest are combined in vitro and then delivered into the cell. The second approach, pursued by Birchler and colleagues (Yu et al., 2007), is to create a minichromosome by truncating a regularly sized chromosome. However, instead of using one of the 10 normal maize chromosomes, they used the B chromosome. B chromosomes are supernumerary, nonessential chromosomes that are found in many species of plants, animals, and fungi (Jones et al., 2007). Maize B chromosomes are heavily heterochromatic and do not seem to carry any genes. The fact that B chromosomes are similar to normal chromosomes but devoid of genes makes them ideal as gene delivery vehicles because (1) transgenes can be inserted into them without disrupting endogenous genes, which is often the case on normal gene-carrying chromosomes, and (2) they can be added and removed without any consequences for the organism. B chromosomes have an interesting propensity to accumulate in large copy numbers. They often undergo nondisjunction in the second pollen mitosis, which is followed by a preferential fertilization of the B chromosome containing sperm (Jones et al., 2007).
How can the B chromosome be truncated to make a minichromosome? One way to do it is by using a genetic trick that relies on the breakage-fusion-bridge cycle phenomenon first described by Barbara McClintock in 1939 (McClintock, 1939
The key to proper segregation of chromosomes in meiosis are processes that take place in early meiotic prophase I. The first of these processes is establishing meiosis-specific sister chromatid cohesion. After premitotic or premeiotic DNA replication, each chromosome consists of two identical chromatids that are connected by the cohesin complex. In meiosis, this complex consists of four main proteins, SMC1, SMC3, SCC3, and REC8 (Ronceret et al., 2007 ). The maize homolog of REC8 is AFD1 (Golubovskaya et al., 2006). AFD1 is installed along the chromosome axis during premeiotic interphase and leptotene. Later on, before anaphase I, the cohesins are removed, except for the chromosome regions surrounding the centromeres (Figure 1A
), by the action of proteinase called separase and other separase-independent mechanisms (Liu and Makaroff, 2006). In the centromeric regions, sister chromatid cohesion is protected by the SHUGOSHIN1 (SGO1) protein and persists until anaphase II (Hamant et al., 2005). This intricate system allows homologous chromosomes to exchange parts of their chromatids but still retain both chromatids in the first meiotic division. The complete removal of the cohesin complex in the second meiotic division allows sister chromatids to separate in anaphase II. In the absence of REC8, or in the case of a premature cohesin removal in the absence of SGO1, sister chromatids can segregate away precociously in meiosis I instead of meiosis II (Hamant et al., 2005; Golubovskaya et al., 2006).
Another meiotic process critical for proper chromosome segregation is homologous chromosome pairing. However, in contrast with most other meiotic processes, little is known about the mechanisms controlling chromosome pairing in plants. In most species, including plants, homologous pairing is dependent on the progression of meiotic recombination (Pawlowski and Cande, 2005 ). Several lines of evidence suggest that a process called single-end invasion is at the heart of chromosome homology recognition (Bozza and Pawlowski, 2008). In this process, single-stranded DNA overhangs, created by resection of meiotic double-strand breaks, probe double-stranded DNA searching for homology. However, how this microscale recognition relates to faithful pairing of entire chromosomes is less clear. Genomes of most plants, including maize, contain a large percentage of repetitive sequences that could obscure overall chromosome homology. In maize, there are  500 single-end invasion events per cell (Franklin et al., 1999), but it is not known how many of them, if any, occur in repetitive DNA regions.
Our limited understanding of meiotic mechanisms prevents us from predicting the meiotic behavior of chromosomes based on their structural features. Consequently, the only way to know how minichromosomes will behave during meiosis is to test their behavior directly. This is what Han et al. have done, using fluorescence in situ hybridization probes specific to B chromosomes, which allowed them to follow the minichromosomes among all chromosomes in the cell. First, they looked at chromosome pairing in pachytene. They found that minichromosomes had lost the ability to pair with both their B and chromosome 9 progenitors. However, most B chromosomes ( 64%) could still pair with each other when present in two copies per cell. Generally, larger minichromosomes were better at pairing. However, this was not always the case, one of the tiniest minichromosomes (#9) paired more frequently than some of the larger minichromosomes. Consequently, minichromosome size is not the only factor governing pairing.
Han et al. also looked at the disjunction behavior of minichromosomes in a situation with only one minichromosome present in a diploid cell. Two classes of minichromosomes could be distinguished on this basis. The first class showed regular disjunction behavior typical for univalent chromosomes (normal or B). The univalent moved to one pole during meiosis I, and sister chromatids separated at anaphase II. The second class showed an abnormal disjunction in which sister chromatids separated at meiosis I (Figure 1B). Interestingly, minichromosomes lost their autonomous ability for postmeiotic nondisjunction at the second pollen mitosis found in original B chromosomes. This mechanism of accumulation is known to require the tip of B chromosome long arm, which was absent from the minichromosomes (Han et al., 2007
Faced with the unusual behavior of minichromosomes, Han et al. initiated a study to understand its basis. In several lines showing premature sister chromatid separation, they followed the localization pattern of SGO1, which is thought to protect pericentromeric cohesion in meiosis I. In maize afd1 mutants, when sister chromatids separate prematurely during anaphase I, SGO1 is not detected (Hamant et al., 2005
The Han et al. study shows that minichromosomes often do not behave in the same way as normal chromosomes: they do not always pair and frequently undergo premature sister chromatid separation. However, in their abnormal way, they are still meiotically stable, and equational segregation of sister chromatids in meiosis I can alleviate the problem of reduced pairing. The fact that minichromosomes often do not pair may not be such a bad thing after all. If chromosomes do not pair, they also do not recombine, so two similar but not identical minichromosomes might be placed in one cell with no danger that their contents will eventually shuffle.
Although the immediate goal of Han et al. was to analyze the meiotic behavior of minichromosomes, several questions and answers raised by their study are brilliantly relevant for the quest to understand meiotic behavior of chromosomes in general. Because of their small size, minichromosomes could be more easily dissected to identify chromosome features that allow some of them to pair in meiosis while others do not. It will be also interesting to learn why the minichromosomes lost their ability to pair with their chromosome 9 and B chromosome progenitors. Examining whether the minichromosomes initiate recombination in the same way as normal chromosomes should be the first step of this analysis. Equally interesting will be further analysis of the authors' observation that the smallest minichromosomes are defective in meiotic sister chromatid cohesion, while the large chromosomes are not. The work of Han et al. provides a significant advancement for future efforts to design better minichromosomes and also contributes to the understanding of the behavior of normal chromosomes in meiosis.
www.plantcell.org/cgi/doi/10.1105/tpc.108.057968
Bozza, C.G., and Pawlowski, W.P. (2008). The cytogenetics of homologous chromosome pairing in meiosis in plants. Cytogenet. Genome Res., in press. Franklin, A.E., McElver, J., Sunjevaric, I., Rothstein, R., Bowen, B., and Cande, W.Z. (1999). Three-dimensional microscopy of the Rad51 recombination protein during meiotic prophase. Plant Cell 11: 809–824. Golubovskaya, I.N., Hamant, O., Timofejeva, L., Wang, C.J., Braun, D., Meeley, R., and Cande, W.Z. (2006). Alleles of afd1 dissect REC8 functions during meiotic prophase I. J. Cell Sci. 119: 3306–3315. Hamant, O., Golubovskaya, I., Meeley, R., Fiume, E., Timofejeva, L., Schleiffer, A., Nasmyth, K., and Cande, W.Z. (2005). A REC8-dependent plant Shugoshin is required for maintenance of centromeric cohesion during meiosis and has no mitotic functions. Curr. Biol. 15: 948–954.[CrossRef][ISI][Medline] Han, F., Gao, Z., Yu, W., and Birchler, J.A. (2007). Minichromosome analysis of chromosome pairing, disjunction, and sister chromatid cohesion in maize. Plant Cell 19: 3853–3863. Han, F., Lamb, J.C., and Birchler, J.A. (2006). High frequency of centromere inactivation resulting in stable dicentric chromosomes of maize. Proc. Natl. Acad. Sci. USA 103: 3238–3243. Houben, A., and Schubert, I. (2007). Engineered plant minichromosomes: A resurrection of B chromosomes? Plant Cell 19: 2323–2327. Jones, R.N., Viegas, W., and Houben, A. (August 17, 2007). A Century of B chromosomes in plants: So what? Ann. Bot. (Lond.) http://dx.doi.org/10.1093/aob/mcm167. Kaszas, E., and Cande, W.Z. (2000). Phosphorylation of histone H3 is correlated with changes in the maintenance of sister chromatid cohesion during meiosis in maize, rather than the condensation of the chromatin. J. Cell Sci. 113: 3217–3226.[Abstract] Liu, Z., and Makaroff, C.A. (2006). Arabidopsis separase AESP is essential for embryo development and the release of cohesin during meiosis. Plant Cell 18: 1213–1225. McClintock, B. (1939). The behavior in successive nuclear divisions of a chromosome broken at meiosis. Proc. Natl. Acad. Sci. USA 25: 405–416. Pawlowski, W.P., and Cande, W.Z. (2005). Coordinating the events of the meiotic prophase. Trends Cell Biol. 15: 674–681.[CrossRef][ISI][Medline] Ronceret, A., Sheehan, M.J., and Pawlowski, W.P. (2007). Chromosome dynamics in meiosis. In Cell Division Control in Plants, D.P.S. Verma and Z. Hong, eds (Heidelberg, Germany: Springer-Verlag), pp. 103–124. Yu, W., Han, F., Gao, Z., Vega, J.M., and Birchler, J.A. (2007). Construction and behavior of engineered minichromosomes in maize. Proc. Natl. Acad. Sci. USA 104: 8924–8929. Related articles in Plant Cell:
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