Expression partitioning between genes duplicated by polyploidy under abiotic stress and during organ development

Allopolyploidy has been a prominent mode of speciation and a recurrent process during plant evolution and has contributed greatly to the large number of duplicated genes in plant genomes [1-4]. Polyploidy often leads to changes in genome organization and gene expression [5-9]. The expression of genes that are duplicated by polyploidy (termed homeologs) can be partitioned between the duplicates so that one copy is expressed and functions only in some organs and the other copy is expressed only in other organs, indicative of subfunctionalization [10]. To determine how homeologous-gene expression patterns change during organ development and in response to abiotic stress conditions, we have examined expression of the alcohol dehydrogenase gene AdhA in allopolyploid cotton (Gossypium hirsutum). Expression ratios of the two homeologs vary considerably during the development of organs from seedlings and fruits. Abiotic stress treatments, including cold, dark, and water submersion, altered homeologous-gene expression. Most notably, only one copy is expressed in hypocotyls during a water-submersion treatment, and only the other copy is expressed during cold stress. These results imply that subfunctionalization of genes duplicated by polyploidy has occurred in response to abiotic stress conditions. Partitioning of duplicate gene expression in response to environmental stress may lead to duplicate gene retention during subsequent evolution.     

Novel patterns of gene expression in polyploid plants

Genome doubling, or polyploidy, is a major factor accounting for duplicate genes found in most eukaryotic genomes. Polyploidy has considerable effects on duplicate gene expression, including silencing and up- or downregulation of one of the duplicated genes. These changes can arise with the onset of polyploidization or within several generations after polyploid formation and they can have epigenetic causal factors. Many expression alterations are organ-specific. Specific genes can be independently and repeatedly silenced during polyploidization, whereas patterns for other genes appear to be more stochastic. Three recent reports have provided intriguing new insights into the patterns, timing and mechanisms of gene expression changes that accompany polyploidy in plants.     

Evolution of Stress-Regulated Gene Expression in Duplicate Genes of Arabidopsis thaliana

Due to the selection pressure imposed by highly variable environmental conditions, stress sensing and regulatory response mechanisms in plants are expected to evolve rapidly. One potential source of innovation in plant stress response mechanisms is gene duplication. In this study, we examined the evolution of stress-regulated gene expression among duplicated genes in the model plant Arabidopsis thaliana. Key to this analysis was reconstructing the putative ancestral stress regulation pattern. By comparing the expression patterns of duplicated genes with the patterns of their ancestors, duplicated genes likely lost and gained stress responses at a rapid rate initially, but the rate is close to zero when the synonymous substitution rate (a proxy for time) is >~0.8. When considering duplicated gene pairs, we found that partitioning of putative ancestral stress responses occurred more frequently compared to cases of parallel retention and loss. Furthermore, the pattern of stress response partitioning was extremely asymmetric. An analysis of putative cis-acting DNA regulatory elements in the promoters of the duplicated stress-regulated genes indicated that the asymmetric partitioning of ancestral stress responses are likely due, at least in part, to differential loss of DNA regulatory elements; the duplicated genes losing most of their stress responses were those that had lost more of the putative cis-acting elements. Finally, duplicate genes that lost most or all of the ancestral responses are more likely to have gained responses to other stresses. Therefore, the retention of duplicates that inherit few or no functions seems to be coupled to neofunctionalization. Taken together, our findings provide new insight into the patterns of evolutionary changes in gene stress responses after duplication and lay the foundation for testing the adaptive significance of stress regulatory changes under highly variable biotic and abiotic environments.     

Evolution of duplicate gene expression in polyploid and hybrid plants

Allopolyploidy is a prominent mode of speciation in flowering plants. On allopolyploidy, genomic changes can take place, including chromosomal rearrangement and changes in gene expression; these processes continue over evolutionary time. Recent studies of gene expression in polyploid and hybrid plants, reviewed here, have examined expression in natural polyploids and synthetic neopolyploids as well as in diploid and F(1) hybrids. Considerable changes in gene expression have been observed in allopolyploids, including up- or downregulation of expression in the polyploids compared with their parents, unequal expression of duplicated genes, and silencing of one copy. Genes in a variety of functional categories show altered expression, and the patterns vary considerably by gene. Some changes seem to be stochastic, whereas others are repeatable. Gene expression changes can be organ specific. Reciprocal silencing of duplicates in different organs has been observed, suggesting subfunctionalization and long-term retention of duplicates. It has become clear that hybridization has a much greater effect than chromosome doubling on gene expression in allopolyploids. Diploid and triploid F(1) hybrids can show alterations of expression levels compared with their parents. Parent-of-origin effects on gene expression have been examined, and loss of gene imprinting has been shown. Some gene expression changes in polyploids and hybrids can be correlated with phenotypic effects. Demonstrated mechanisms of gene expression changes include DNA methylation, histone modifications, and antisense RNA. Several hypotheses have been proposed for why gene expression is altered in allopolyploids and hybrids.     

Polyploidy and genome evolution in plants

Genome doubling (polyploidy) has been and continues to be a pervasive force in plant evolution. Modern plant genomes harbor evidence of multiple rounds of past polyploidization events, often followed by massive silencing and elimination of duplicated genes. Recent studies have refined our inferences of the number and timing of polyploidy events and the impact of these events on genome structure. Many polyploids experience extensive and rapid genomic alterations, some arising with the onset of polyploidy. Survivorship of duplicated genes are differential across gene classes, with some duplicate genes more prone to retention than others. Recent theory is now supported by evidence showing that genes that are retained in duplicate typically diversify in function or undergo subfunctionalization. Polyploidy has extensive effects on gene expression, with gene silencing accompanying polyploid formation and continuing over evolutionary time.     

植物多倍体研究的回顾与展望

多倍化是促进植物进化的重要力量。多倍体主要是通过未减数配子融合,体细胞染色体加倍以及多精受精三种方式起源的。其中,不减数配子是多倍体形成的主要机制。三倍体可能在四倍体的进化中起了重要作用。过去认为多倍体只能是进化的死胡同,现在发现很多多倍体类群都是多元起源的而不是单元起源的。当多倍体形成后,基因组中的重复基因大部分保持原有的功能,也有相当比例的基因发生基因沉默。多倍体通常表现出不存在于二倍体祖先的表型,并且超出了其祖先的分布范围,因为在多倍体中发生了很多基因表达的变化。主要从多倍体的起源、影响多倍体发生的因素及多倍体基因组的进化等方面回顾并展望多倍体的研究。     

Genomic, regulatory and epigenetic mechanisms underlying duplicated gene evolution in the natural allotetraploid Oryza minuta

Background

Polyploid species contribute to Oryza diversity. However, the mechanisms underlying gene and genome evolution in Oryza polyploids remain largely unknown. The allotetraploid Oryza minuta, which is estimated to have formed less than one million years ago, along with its putative diploid progenitors (O. punctata and O. officinalis), are quite suitable for the study of polyploid genome evolution using a comparative genomics approach.

Results

Here, we performed a comparative study of a large genomic region surrounding the Shattering4 locus in O. minuta, as well as in O. punctata and O. officinalis. Duplicated genomes in O. minuta have maintained the diploid genome organization, except for several structural variations mediated by transposon movement. Tandem duplicated gene clusters are prevalent in the Sh4 region, and segmental duplication followed by random deletion is illustrated to explain the gene gain-and-loss process. Both copies of most duplicated genes still persist in O. minuta. Molecular evolution analysis suggested that these duplicated genes are equally evolved and mostly manipulated by purifying selection. However, cDNA-SSCP analysis revealed that the expression patterns were dramatically altered between duplicated genes: nine of 29 duplicated genes exhibited expression divergence in O. minuta. We further detected one gene silencing event that was attributed to gene structural variation, but most gene silencing could not be related to sequence changes. We identified one case in which DNA methylation differences within promoter regions that were associated with the insertion of one hAT element were probably responsible for gene silencing, suggesting a potential epigenetic gene silencing pathway triggered by TE movement.

Conclusions

Our study revealed both genetic and epigenetic mechanisms involved in duplicated gene silencing in the allotetraploid O. minuta.     

Stochastic and epigenetic changes of gene expression in Arabidopsis polyploids

Polyploidization is an abrupt speciation mechanism for eukaryotes and is especially common in plants. However, little is known about patterns and mechanisms of gene regulation during early stages of polyploid formation. Here we analyzed differential expression patterns of the progenitors' genes among successive selfing generations and independent lineages. The synthetic Arabidopsis allotetraploid lines were produced by a genetic cross between A. thaliana and A. arenosa autotetraploids. We found that some progenitors' genes are differentially expressed in early generations, whereas other genes are silenced in late generations or among different siblings within a selfing generation, suggesting that the silencing of progenitors' genes is rapidly and/or stochastically established. Moreover, a subset of genes is affected in autotetraploid and multiple independent allotetraploid lines and in A. suecica, a natural allotetraploid derived from A. thaliana and A. arenosa, indicating locus-specific susceptibility to ploidy-dependent gene regulation. The role of DNA methylation in silencing progenitors' genes is tested in DNA-hypomethylation transgenic lines of A. suecica using RNA interference (RNAi). Two silenced genes are reactivated in both ddm1- and met1-RNAi lines, consistent with the demethylation of centromeric repeats and gene-specific regions in the genome. A rapid and stochastic process of differential gene expression is reinforced by epigenetic regulation during polyploid formation and evolution.     

Infraspecific DNA methylation polymorphism in cotton (Gossypium hirsutum L.)

Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.     

Genome evolution in polyploids

Polyploidy is a prominent process in plants and has been significant in the evolutionary history of vertebrates and other eukaryotes. In plants, interdisciplinary approaches combining phylogenetic and molecular genetic perspectives have enhanced our awareness of the myriad genetic interactions made possible by polyploidy. Here, processes and mechanisms of gene and genome evolution in polyploids are reviewed. Genes duplicated by polyploidy may retain their original or similar function, undergo diversification in protein function or regulation, or one copy may become silenced through mutational or epigenetic means. Duplicated genes also may interact through inter-locus recombination, gene conversion, or concerted evolution. Recent experiments have illuminated important processes in polyploids that operate above the organizational level of duplicated genes. These include inter-genomic chromosomal exchanges, saltational, non-Mendelian genomic evolution in nascent polyploids, inter-genomic invasion, and cytonuclear stabilization. Notwithstanding many recent insights, much remains to be learned about many aspects of polyploid evolution, including: the role of transposable elements in structural and regulatory gene evolution; processes and significance of epigenetic silencing; underlying controls of chromosome pairing; mechanisms and functional significance of rapid genome changes; cytonuclear accommodation; and coordination of regulatory factors contributed by two, sometimes divergent progenitor genomes. Continued application of molecular genetic approaches to questions of polyploid genome evolution holds promise for producing lasting insight into processes by which novel genotypes are generated and ultimately into how polyploidy facilitates evolution and adaptation.     

Responses of Populus trichocarpa galactinol synthase genes to abiotic stresses

Galactinol synthase (GolS; EC 2.4.1.123) is a member of the glycosyltransferase eight family that catalyzes the first step in the biosynthesis pathway of the raffinose family of oligosaccharides (RFOs). The accumulation of RFOs in response to abiotic stress indicates a role for RFOs in stress adaptation. To obtain information on the roles of RFOs in abiotic stress adaptation in trees, we investigated the expression patterns of nine Populus trichocarpa GolS (PtrGolS) genes with special reference to stress responses. PtrGolS genes were differentially expressed in different organs, and the expressions of PtrGolS4 and PtrGolS6 were relatively high in all tested organs. The expression levels of all PtrGolS genes, except PtrGolS9, changed in response to abiotic stress in gene- and stress-type-specific manners. Moreover, short- and long-term stress treatments revealed that induction of PtrGolS by salt stress is obvious only in the early period of treatment (within 24 h), whereas water-deficit stress treatments continued to upregulate PtrGolS gene expression after two days of treatment, in addition to induction within 24 h of treatment. Consistent with these expression patterns, the galactinol content in leaves increased after four days of drought stress, but not under salt stress. Our findings suggest divergent roles for PtrGolS genes in abiotic stress responses in poplars.     

Differential expression of duplicated peroxidase genes in the allotetraploid Brassica napus

Gene redundancy due to polyploidization provides a selective advantage for plant adaptation. We examined the expression patterns of two peroxidase genes (BnPOX1 and BnPOX2) in the natural allotetraploid Brassica napus and the model diploid progenitors Brassica rapa (Br) and Brassica oleracea (Bo) in response to the fungal pathogen Sclerotinia sclerotiorum. We demonstrated that the Bo homeolog of BnPOX1 was up-regulated after infection, while both BnPOX2 homeologs were down-regulated. A bias toward reciprocal expression of the homeologs of BnPOX1 in different organs in the natural allotetraploid of B. napus was also observed. These results suggest that subfunctionalization of the duplicated BnPOX genes after B. napus polyploidization as well as subneofunctionalization of the homeologs in response to this specific biotic stress has occurred. Retention of expression patterns in the diploid progenitors and the natural allotetraploid in some organs indicates that the function of peroxidase genes has been conserved during evolution.     

Comprehensive sequence and expression profile analysis of PEX11 gene family in rice

PEX11 gene family has been shown to be involved in peroxisome biogenesis but very little is known about this gene family in rice. Here we show that five putative PEX11 genes (OsPEX11-1-5) present in rice genome and each contain three conserved motifs. The PEX11 sequences from rice and other species can be classified into three major groups. Among the five rice PEX11 genes, OsPEX11-2 and -3 are most likely duplicated. Expression profile and RT-PCR analysis suggested that the members of PEX11 family in rice had differential expression patterns: OsPEX11-1 and OsPEX11-4 had higher expression levels in leaf tissues than in the other tissues, OsPEX11-2 was detected only in germinated seeds, OsPEX11-3 was expressed predominantly in endosperm and germinated seeds, and OsPEX11-5 was expressed in all the tissues investigated. We also observed that the rice PEX11 genes had differential expression patterns under different abiotic stresses. OsPEX11-1 and OsPEX11-4 were induced by abscisic acid (ABA), hydrogen peroxide (H2O2), salt and low nitrogen stress conditions. OsPEX11-3 was responsive to ABA and H2O2 treatments, and OsPEX11-5 was responsive to ABA, H2O2, and salt treatments. However, OsPEX11-2 had no response to any of the stresses. Our results suggest that the rice PEX11 genes have diversification not only in sequences but also in expression patterns under normal and various stress conditions.     

Genome-wide quantification of homeolog expression ratio revealed nonstochastic gene regulation in synthetic allopolyploid Arabidopsis

Genome duplication with hybridization, or allopolyploidization, occurs commonly in plants, and is considered to be a strong force for generating new species. However, genome-wide quantification of homeolog expression ratios was technically hindered because of the high homology between homeologous gene pairs. To quantify the homeolog expression ratio using RNA-seq obtained from polyploids, a new method named HomeoRoq was developed, in which the genomic origin of sequencing reads was estimated using mismatches between the read and each parental genome. To verify this method, we first assembled the two diploid parental genomes of Arabidopsis halleri subsp. gemmifera and Arabidopsis lyrata subsp. petraea (Arabidopsis petraea subsp. umbrosa), then generated a synthetic allotetraploid, mimicking the natural allopolyploid Arabidopsis kamchatica. The quantified ratios corresponded well to those obtained by Pyrosequencing. We found that the ratios of homeologs before and after cold stress treatment were highly correlated (r = 0.870). This highlights the presence of nonstochastic polyploid gene regulation despite previous research identifying stochastic variation in expression. Moreover, our new statistical test incorporating overdispersion identified 226 homeologs (1.11% of 20 369 expressed homeologs) with significant ratio changes, many of which were related to stress responses. HomeoRoq would contribute to the study of the genes responsible for polyploid-specific environmental responses.