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The effects of polyploidy on gene regulation are of interest in plant systems because of the widespread occurrence of polyploid species and populations and the mounting evidence for ancient polyploidization events in the ancestry of apparent diploids (Cui et al., 2006). Plants display higher tolerance to the consequences of genome doubling than vertebrates (Otto & Whitton, 2000; Doyle et al., 2008). Two fundamentally different types of polyploids exist: allopolyploids, which are derived from interspecies hybrids in which two or more parental genomes are maintained in duplicated forms; and autopolyploids, which are derived by multiplication of the same chromosome set (Comai et al., 2000; Comai, 2005). These two types of polyploids are different at the molecular and genetic levels. For example, in autopolyploids, recombination occurs between the multiple homologous chromosomes while in allopolyploids recombination is limited to the pair of homologous chromosomes derived from a single ancestral genome. Because diverged parental genomes are combined in allopolyploids, multiple regulatory changes are expected from the interaction of two disparate systems (Osborn et al., 2003), an expectation validated in multiple studies (Adams & Wendel, 2005; Chen, 2007; Doyle et al., 2008). Predictions are not as obvious for autopolyploidy, where changes to the nuclear environment are expected to be less profound than in allopolyploids (Comai, 2005). Accordingly, evidence for gene regulatory changes in autopolyploids is not clear. A study examining the expression of 18 maize (Zea mays) genes in haploid, diploid, triploid and tetraploid plants showed that for most genes the transcript level per cell was directly proportional to the structural gene dosage. A few genes, however, displayed negative or positive (greater than expected) correlation with ploidy (Guo et al., 1996). The molecular basis for this response is not yet understood. Interestingly, inbred maize autotetraploids display stronger inbreeding depression than diploids (Riddle et al., 2006). Recently, analysis of the transcriptome in an inbred maize ploidy series found frequent but low-level changes (Riddle et al., 2010). Comparison of diploid to triploid and tetraploid maize revealed that the most common trend was a decrease in expression, mirroring the decrease in size observed in maize autopolyploids. In Arabidopsis thaliana autotetraploids, however, no changes in vigor are visible. Depending perhaps on accession and on experimental conditions, phenotypic changes have been described as limited to trichome structure. (Mittelsten Scheid et al., 2003; Yu et al., 2009). Autotetraploids of multiple accessions displayed an increase in flower size and stomatal size, a likely reflection of increased nuclear content and cell size. To a casual observer, however, autotetraploids are nearly undistinguishable from the corresponding diploids (D. Pignatta et al., unpublished). Autopolyploids of multiple species display frequent aneuploidy and this may induce phenotypic alterations that should vary with the type of chromosome imbalance (Comai, 2005). Compelling evidence for tetraploidy-induced changes in epigenetic gene regulation exists for a transgenic locus of A. thaliana, which demonstrates striking ploidy responses, such as the ability to transfer its silencing state from one allele to the other (Mittelsten Scheid et al., 1996, 2003). This phenomenon is reminiscent of paramutation, where changes in chromatin states are mediated by an RNA signal (Chandler, 2007). We examined the possibility that another RNA-mediated phenomenon, transgene-induced post-transcriptional silencing, could also respond to ploidy (Pignatta et al., 2008). Comparing the efficiency of silencing of the endogenous gene CHALCONE SYNTHASE between tetraploids and their progenitor diploids, we found that a change in ploidy neither enhanced nor suppressed the silencing action of dsRNA-generating hairpin transgenes (Pignatta et al., 2008). Therefore, no evidence was found that the small RNA pathway responsible for post-transcriptional transgene silencing was affected by ploidy changes.
Whole-transcriptome expression analysis might discover cases of endogenous genes that respond to ploidy. In potato (Solanum phureja), 10% of the c. 9000 genes analyzed using microarray displayed changes in expression level within a twofold range, with no ‘ploidy-regulated’ gene identified (Stupar et al., 2007). In Saccharomyces cerevisiae (budding yeast), there is conflicting evidence about whether ploidy-regulated genes exist or not. Indeed, the first genome-wide transcriptome analysis in S. cerevisiae appeared to support the hypothesis of ploidy-regulated genes (Galitski et al., 1999). However, a more recent whole-genome transcriptome analysis of haploids and isogenic tetraploids resulted in very similar transcription profiles, although tetraploids failed to up- or down-regulate some genes important for stationary-phase survival (Andalis et al., 2004). The similarity of the gene expression profiles of diploid and tetraploid budding yeast was again confirmed in another study, and further supported by the steady-state protein levels for selected proteins (Storchova et al., 2006).
In A. thaliana, the effects of genome doubling on nonadditive gene expression remain elusive. As part of a study by Wang et al. on nonadditive gene regulation in allopolyploids (Wang et al., 2006), a transcriptome analysis of diploid vs autotetraploid A. thaliana identified only a small percentage of genes significantly differently expressed between the two. Importantly, the study focused on only one lineage of A. thaliana ecotype Landsberg erecta (Ler) (Wang et al., 2006).
At least two problems could complicate the analysis of regulatory changes during autopolyploidy. The first is the pervasive presence of aneuploids in autopolyploid populations (Bond et al., 2004; Henry et al., 2005, 2006). Depending on the species and background, polyploid populations can include 25–50% of aneuploid individuals. For example, in a tetraploid population the observed aneuploids may include pentasomics or trisomics for one to a few chromosomes, with hyperploidy being more common. These chromosomal variants could introduce regulatory variations that depend on dosage alterations rather than on ploidy (Henry et al., 2006). Also, the microarray analyses reported thus far may have lacked the statistical power to detect relatively small changes, because they were too subtle, masked by noise, or tissue-specific.
Here, we addressed this problem using a twofold approach. First, we carried out a microarray analysis in three presumably equivalent lineages of matched diploids and tetraploids of A. thaliana accession Columbia (Col-0). Independent tetraploid events derived from the same starting genotype should allow resolution of lineage-specific changes, such as those attributable to aneuploidy or to the colchicine treatment, from changes that derived from the diploid to tetraploid transition. Secondly, we used transgenic A. thaliana lines engineered with an enhancer gene trap T-DNA (GAL4-VP16- GFP; Fig. 1a). We reasoned that a tool that more efficiently displays changes in the spatial distribution of gene expression based on the sensitive green fluorescent protein (GFP) marker would be more likely to identify subtle changes or changes that may be more visible at specific developmental stages. The A. thaliana transgenic lines (accessions Col-0 and C24) used here carried the enhancer trap in different genomic locations. Expression of the chimeric transcriptional activator GAL4-VP16 gene was activated by an enhancer element present in the plant genome, which was different for each independent transgenic line. This strategy thus separated the effect of the structural part of the gene from that of the genomic positions. Our microarray analysis revealed few changes in gene expression between diploid and tetraploid plants. By contrast, the enhancer-trap analysis revealed that multiple loci in the genome are potentially subject to ploidy regulation. The inconsistency of the answers provided by the two methods is discussed.
Figure 1. Enhancer trap scheme and genotype of transgenic Arabidopsis thaliana lines. (a) T-DNA elements: the chimeric transcriptional activator GAL4-VP16; the neomycin phospho-transferase resistance II (NptII) gene; the 5× upstream activator sequence (UAS); the mGFP-ER reporter gene encoding the green fluorescent protein targeted to the endoplasmic reticulum. RB and LB, right and left borders, respectively; (p), the 35S CaMV minimal promoter; p, the nos promoter; E, endogenous A. thaliana enhancer in flanking genomic region. (b) GFP expression was compared between diploid individuals (2×), which carry two copies of the trap, and autotetraploid individuals (4×), which carry four copies. For simplicity, only one chromosome type is depicted. (c) Single-locus T-DNA enhancer trap lines of accession Columbia (Col-0) with linked genes. For each line, a schematic representation of the T-DNA insertion locus and the copy number are given. The length of linked genes (in black) and the distance from the T-DNA insertion locus (in green) are expressed in nucleotides (nt). The trap sequence is represented as a black rectangle. Arrows indicate the direction of the transcription.
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Changes between diploids and tetraploids of the same species have been reported previously (Galitski et al., 1999; Andalis et al., 2004; Storchova et al., 2006; Wang et al., 2006; Stupar et al., 2007). What is not clear is whether these are true responses to genome doubling, because independent replication of the polyploidization process was not employed. True responses to genome doubling should be reproducible. We applied this criterion by analyzing the transcriptome in three independent lineages of autotetraploid A. thaliana Col-0 and their matched diploids. We also tested the response of transcriptional enhancers by examining the regulation of GFP enhancer traps upon genome doubling. Our results are seemingly paradoxical: microarray analysis of ploidy-induced changes failed to provide a set of robustly regulated genes, yet ploidy-induced changes could be easily demonstrated by the use of enhancer traps. If enhancer traps reproduce the regulation of ploidy-sensitive endogenous genes, why did we not find them in the microarray study? One possibility is that the microarray assay may be biased or not sufficiently sensitive. Inconsistent with this possibility, this is the same microarray system that was used to detect common changes in independently produced synthetic allopolyploids of A. thaliana and Arabidopsis arenosa (Wang et al., 2006). Another possibility is that ploidy-regulated genes are expressed at very low level, or in specific tissues, and thus escape detection (i.e. are in the range of the experimental noise). This is unlikely as GFP activation and suppression were found in multiple tissues of the four responding trap lines and the whole aerial part of 4-wk-old plants was sampled for the microarray experiment (see Materials and Methods section). Moreover, differences in NptII, GFP and GAL4-VP16 were readily found by RT-PCR, which utilizes whole tissue preparations of RNA, but none of the eight endogenous genes compared between diploid and tetraploid wild types exhibited expression changes in response to ploidy. If one assumes that each regulated enhancer trap represents a gene, the total number of ploidy-regulated genes would lie between 2800 and 17 000. This 1% confidence interval (CI) was derived from the observation that four of 16 enhancer traps tested responded positively or negatively to autopolyploidy (CI 0.07 to 0.57 with α = 0.01, or 2800 to 17 000 genes out of c. 30 000). If traps reproduce perfectly the regulation of genes, thousands of genes would be expected to be ploidy sensitive. Moreover, the observed changes in expression levels are of sufficient magnitude (×4 to over ×600) that at least some of the thousands of these ploidy-sensitive genes should be identified in a microarray analysis. We believe, therefore, that our microarray analysis was sufficiently sensitive, but, consistently with previous investigations, few endogenous genes displayed ploidy-dependent regulation.
Our analysis is inconsistent with the induction of widespread, reproducible and large changes in the expression of genes by a change in ploidy. Nonetheless, the frequent and reproducible response of enhancer traps to ploidy changes suggests that autopolyploidy induces changes in the regulatory environment of the cell. These diverging observations would be reconciled if enhancer traps exhibit unique sensitivity to ploidy while endogenous genes are buffered against it. Hypersensitivity of enhancer traps to ploidy may result from reduced complexity of their regulation. Enhancer trap activation requires the capture of positive regulatory signals from genomic sites. Several cases where traps were analyzed in depth (Springer, 2000; Fridborg et al., 2004; Baroux et al., 2005; Leon-Kloosterziel et al., 2005; Slotkin et al., 2009) indicate that their regulation is lower in developmental and probably biochemical complexity than the complete cis-regulatory complement acting on genes, which are subject to complex regulation involving multiple pathways. If multiple positive and negative signals converge on the promoter of a gene, the other pathways may compensate changes in one pathway.
Alternatively, establishment of an altered regulatory environment in response to ploidy changes may affect many genes, but with low penetrance. One explanation of our observations could be that upon polyploidization a large set of genes are potentially affected, but only a smaller and random number become altered, perhaps with unpredictable direction. Indeed, this may explain why only two of the seven genes that were detected to be differentially expressed under the common variance assumption (Fig. 2, Table 1) were regulated in the same direction.
The dependence of enhancer traps on the formation of a T-DNA transgenic locus may also affect another layer of gene regulation: the formation of insulated domains (Ong & Corces, 2009). We observed that in two of four cases the regulation of the NptII gene varied in harmony with GFP. It is possible that the T-DNA disrupts existing domains, resulting in poor insulation and increased susceptibility to perturbation, perhaps because of the spreading of neighboring chromatin states. ‘Position effects’ have been previously described (Singh et al., 2008) and different positions in the genome may be differently sensitive to a change in ploidy. Consistent with this possibility, two different transgenes driven by the same 35S-CaMV promoter, but located in different regions of chromosome 5, reacted differently upon triplication of the chromosome (Huettel et al., 2008). One transgene was strongly down-regulated in the Chr5 trisomics as compared with diploids, while the average expression of the other transgene remained approximately the same. The suppressed transgene was inserted near a cluster of silent transposon-related sequences and tRNA genes, giving rise to numerous small RNAs. The enhancer trap that displays ploidy-dependent suppression, CS70016 (Fig. 4), is inserted inside the putative transposon SADHU2.1 (Fig. 1c), which is in turn located in the 3′ nonterminal inverted repeats of a Mutator-like element (non-TIR MULE) named KAONASHI (Hoen et al., 2006; Rangwala et al., 2006). Interestingly, SADHU2.1 becomes sensitive to ploidy in the enhancer trap line, providing additional evidence for domain-wide regulatory changes. The negative regulation of this enhancer trap line by ploidy may underlie an interaction with suppressive heterochromatin. However, in lines cs70267, cs70091 and cs70007 the trap was inserted into protein-coding genes, which are moderately or highly expressed (Schmid et al., 2005). Thus, the formation of poorly insulated domains could result in susceptibility to multiple perturbations including ploidy.
It is possible that ploidy-regulated changes may exhibit a genome-specific threshold of sensitivity, or that they may be sensitive to the cause of polyploidization. Maize, for example, may be sensitive to autotetraploidy (Riddle et al., 2006, 2010) because of its genomic characteristics – perhaps as a result of the widespread interspersion of genes and transposons. The A. thaliana genome, in contrast, may be more resilient when tetraploid, but be subject to regulatory problems at higher ploidy with connected phenotypic depression of growth. By colchicine treatment of diploids we have produced hundreds of polyploids of A. thaliana (B. P. Dilkes et al., unpublished). Using flow cytometry, in all cases we found tetraploids. The absence of hexaploids and octaploids may result from impaired proliferation of hexaploid and octaploid cells in the meristem of colchicine-treated plants or from failure of gametes with ploidy higher than 2N. Indeed, octaploid A. thaliana are unstable (Yu et al., 2009) and severely compromised in growth (Tsukaya, 2008) and therefore A. thaliana resembles maize in its response to ploidy. Perhaps a higher ploidy threshold is required to trigger deleterious ploidy-related effects in A. thaliana.
Autopolyploidization may occur through different mechanisms. This study used a spindle poison, colchicine. The maize study (Riddle et al., 2006) employed tetraploids produced by nitrous oxide treatment of diploids, which also interferes with spindle function. In nature, tetraploids can arise from spontaneously tetraploidized sectors or from rare 2n gametes and in this latter case pass through a triploid bridge, which may entail an aneuploid stage (Henry et al., 2005). Therefore, different stresses may be imposed on cells and result in different regulatory outcomes.
In summary, using microarray analysis it was not possible to find regulatory changes that could be clearly attributable to changes in ploidy. This was in contrast to the readily demonstrable, large and frequent changes we observed in an analysis of enhancer trap expression. The response of enhancer traps indicates that regulatory pathways acting on transcription can be affected but no effects of ploidy were seen on neighboring endogenous genes in comparisons of diploid and tetraploid wild-type plants. While it is possible that the difference in the results obtained with these two methods might lie in the insufficient sensitivity of the microarray system compared with the exquisite sensitivity of the GFP reporter, we argue that a better explanation is higher complexity of regulatory pathways affecting genes compared with those acting on enhancer traps, or better insulation of chromosome domains from perturbing effects compared with enhancer traps. Buffered regulation of genes with respect to genomic ploidy may have selective advantages and might help explain the pervasive occurrence of polyploidy in the plant kingdom.