Jin Koh

Evolution and Expression of Homeologous Loci in Tragopogon miscellus (Asteraceae), a Recent and Reciprocally Formed Allopolyploid

On both recent and ancient time scales, polyploidy (genome doubling) has been a significant evolutionary force in plants. Here, we examined multiple individuals from reciprocally formed populations of Tragopogon miscellus, an allotetraploid that formed repeatedly within the last 80 years from the diploids T. dubius and T. pratensis. Using cDNA–AFLPs followed by genomic and cDNA cleaved amplified polymorphic sequence (CAPS) analyses, we found differences in the evolution and expression of homeologous loci in T. miscellus. Fragment variation within T. miscellus, possibly attributable to reciprocal formation, comprised 0.6% of the cDNA–AFLP bands. Genomic and cDNA CAPS analyses of 10 candidate genes revealed that only one “transcript-derived fragment” (TDF44) showed differential expression of parental homeologs in T. miscellus; the T. pratensis homeolog was preferentially expressed by most polyploids in both populations. Most of the cDNA–AFLP polymorphisms apparently resulted from loss of parental fragments in the polyploids. Importantly, changes at the genomic level have occurred stochastically among individuals within the independently formed populations. Synthetic F1 hybrids between putative diploid progenitors are additive of their parental genomes, suggesting that polyploidization rather than hybridization induces genomic changes in Tragopogon.

Gene loss and silencing in Tragopogon miscellus (Asteraceae): comparison of natural and synthetic allotetraploids.

Whole-genome duplication (polyploidisation) is a widespread mechanism of speciation in plants. Over time, polyploid genomes tend towards a more diploid-like state, through downsizing and loss of duplicated genes (homoeologues), but relatively little is known about the timing of gene loss during polyploid formation and stabilisation. Several studies have also shown gene transcription to be affected by polyploidisation. Here, we examine patterns of gene loss in 10 sets of homoeologues in five natural populations of the allotetraploid Tragopogon miscellus that arose within the past 80 years following independent whole-genome duplication events. We also examine 44 first-generation synthetic allopolyploids of the same species. No cases of homoeologue loss arose in the first allopolyploid generation, but after 80 years, 1.6% of homoeologues were lost in natural populations. For seven homoeologue sets we also examined transcription, finding that 3.4% of retained homoeologues had been silenced in the natural populations, but none in the synthetic plants. The homoeologue losses and silencing events found were not fixed within natural populations and did not form a predictable pattern among populations. We therefore show haphazard loss and silencing of homoeologues, occurring within decades of polyploid formation in T. miscellus, but not in the initial generation.

Tissue-specific silencing of homoeologs in natural populations of the recent allopolyploid Tragopogon mirus

Recent years have seen rapid advances in our knowledge of the transcriptomic consequences of allopolyploidy, primarily through the study of polyploid crops and model systems. However, few studies have distinguished between homoeologs and between tissues, and still fewer have examined young natural allopolyploid populations of independent origin, whose parental species are still present in the same location. Here, we examined the expression of 13 homoeolog pairs in seven tissues of 10 plants of allotetraploid Tragopogon mirus from two natural populations formed by independent polyploidizations between Tragopogon dubius and Tragopogon porrifolius c. 40 generations ago. We compare these with patterns of expression in the diploid parental species from the same locality. Of the 910 assays in T. mirus, 576 (63%) showed expression of both homoeologs, 63 (7%) showed no expression of either homoeolog, 186 (20%) showed nonexpression of one homoeolog across all tissues of a plant, and 72 (8%) showed non-expression of a homoeolog in a particular tissue within a plant. We found two cases of reciprocal tissue-specific expression between homoeologs, potentially indicative of subfunctionalization. Our study shows that tissue-specific silencing, and even apparent subfunctionalization, can arise rapidly in the early generations of natural allopolyploidy.

Homeolog loss and expression changes in natural populations of the recently and repeatedly formed allotetraploid Tragopogon mirus (Asteraceae)

BackgroundAlthough polyploidy has long been recognized as a major force in the evolution of plants, most of what we know about the genetic consequences of polyploidy comes from the study of crops and model systems. Furthermore, although many polyploid species have formed repeatedly, patterns of genome evolution and gene expression are largely unknown for natural polyploid populations of independent origin. We therefore examined patterns of loss and expression in duplicate gene pairs (homeologs) in multiple individuals from seven natural populations of independent origin of Tragopogon mirus (Asteraceae), an allopolyploid that formed repeatedly within the last 80 years from the diploids T. dubius and T. porrifolius.ResultsUsing cDNA-AFLPs, we found differential band patterns that could be attributable to gene silencing, novel expression, and/or maternal/paternal effects between T. mirus and its diploid parents. Subsequent cleaved amplified polymorphic sequence (CAPS) analyses of genomic DNA and cDNA revealed that 20 of the 30 genes identified through cDNA-AFLP analysis showed additivity, whereas nine of the 30 exhibited the loss of one parental homeolog in at least one individual. Homeolog loss (versus loss of a restriction site) was confirmed via sequencing. The remaining gene (ADENINE-DNA GLYCOSYLASE) showed ambiguous patterns in T. mirus because of polymorphism in the diploid parent T. dubius. Most (63.6%) of the homeolog loss events were of the T. dubius parental copy. Two genes, NUCLEAR RIBOSOMAL DNA and GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE, showed differential expression of the parental homeologs, with the T. dubius copy silenced in some individuals of T. mirus.ConclusionsGenomic and cDNA CAPS analyses indicated that plants representing multiple populations of this young natural allopolyploid have experienced frequent and preferential elimination of homeologous loci. Comparable analyses of synthetic F1 hybrids showed only additivity. These results suggest that loss of homeologs and changes in gene expression are not the immediate result of hybridization, but are processes that occur following polyploidization, occurring during the early (<40) generations of the young polyploid. Both T. mirus and a second recently formed allopolyploid, T. miscellus, exhibit more homeolog losses than gene silencing events. Furthermore, both allotetraploids undergo biased loss of homeologs contributed by their shared diploid parent, T. dubius. Further studies are required to assess whether the results for the 30 genes so far examined are representative of the entire genome.

Concerted evolution of rDNA in recently formed Tragopogon allotetraploids is typically associated with an inverse correlation between gene copy number and expression

We analyzed nuclear ribosomal DNA (rDNA) transcription and chromatin condensation in individuals from several populations of Tragopogon mirus and T. miscellus, allotetraploids that have formed repeatedly within only the last 80 years from T. dubius and T. porrifolius and T. dubius and T. pratensis, respectively. We identified populations with no (2), partial (2), and complete (4) nucleolar dominance. It is probable that epigenetic regulation following allopolyploidization varies between populations, with a tendency toward nucleolar dominance by one parental homeologue. Dominant rDNA loci are largely decondensed at interphase while silent loci formed condensed heterochromatic regions excluded from nucleoli. Those populations where nucleolar dominance is fixed are epigenetically more stable than those with partial or incomplete dominance. Previous studies indicated that concerted evolution has partially homogenized thousands of parental rDNA units typically reducing the copy numbers of those derived from the T. dubius diploid parent. Paradoxically, despite their low copy number, repeats of T. dubius origin dominate rDNA transcription in most populations studied, i.e., rDNA units that are genetic losers (copy numbers) are epigenetic winners (high expression).

Comparative proteomics of the recently and recurrently formed natural allopolyploid Tragopogon mirus (Asteraceae) and its parents

• We examined the proteomes of the recently formed natural allopolyploid Tragopogon mirus and its diploid parents (T. dubius, T. porrifolius), as well as a diploid F(1) hybrid and synthetic T. mirus. • Analyses using iTRAQ LC-MS/MS technology identified 476 proteins produced by all three species. Of these, 408 proteins showed quantitative additivity of the two parental profiles in T. mirus (both natural and synthetic); 68 proteins were quantitatively differentially expressed. • Comparison of F(1) hybrid, and synthetic and natural polyploid T. mirus with the parental diploid species revealed 32 protein expression changes associated with hybridization, 22 with genome doubling and 14 that had occurred since the origin of T. mirus c. 80 yr ago. We found six proteins with novel expression; this phenomenon appears to start in the F(1) hybrid and results from post-translational modifications. • Our results indicate that the impact of hybridization on the proteome is more important than is polyploidization. Furthermore, two cases of homeolog-specific expression in T. mirus suggest that silencing in T. mirus was not associated with hybridization itself, but occurred subsequent to both hybridization and polyploidization. This study has shown the utility of proteomics in the analysis of the evolutionary consequences of polyploidy.

Sequence and Expression Studies of A‐, B‐, and E‐Class MADS‐Box Homologues in Eupomatia (Eupomatiaceae): Support for the Bracteate Origin of the Calyptra

Eupomatia (Magnoliales, Eupomatiaceae) has flowers that bear a calyptra, an unusual organ that encloses the floral bud. The structural homology and evolutionary derivation of the calyptra are unknown, although some have proposed that it is a bract, while others favor a derivation from the perianth. To address the evolutionary origin of the calyptra, we isolated, sequenced, and characterized the expression of A‐, B‐, and E‐class MADS‐box homologues from Eupomatia bennettii and a close relative, Magnolia grandiflora (Magnoliaceae). The expression patterns of organ identity genes in floral organs of Eupomatia and Magnolia were very similar. However, the expression patterns of these MADS‐box genes indicated that the ABC model is not strictly applicable to either Eupomatia or Magnolia. For example, A‐class homologues were expressed in carpels and leaves of both Eupomatia and Magnolia. In the calyptra, expression levels of B‐ and E‐class homologues were low and almost identical to those observed in leaf tissue. In contrast, high levels of expression for B‐ and E‐class homologues were observed in the stamens, staminodes, and carpels. These gene expression data agree with recent developmental data and the interpretation of the calyptra as a bract. We also report the presence of various forms of alternatively spliced mRNAs in the cDNA pool from floral organs, and the implications of these mRNAs are discussed.

Comparative Proteomic Analysis of Brassica napus in Response to Drought Stress

Drought is one of the most widespread stresses leading to retardation of plant growth and development. We examined proteome changes of an important oil seed crop, canola (Brassica napus L.), under drought stress over a 14-day period. Using iTRAQ LC-MS/MS, we identified 1976 proteins expressed during drought stress. Among them, 417 proteins showed significant changes in abundance, and 136, 244, 286, and 213 proteins were differentially expressed in the third, seventh, 10th, and 14th day of stress, respectively. Functional analysis indicated that the number of proteins associated with metabolism, protein folding and degradation, and signaling decreased, while those related to energy (photosynthesis), protein synthesis, and stress and defense increased in response to drought stress. The seventh and 10th-day profiles were similar to each other but with more post-translational modifications (PTMs) at day 10. Interestingly, 181 proteins underwent PTMs; 49 of them were differentially changed in drought-stressed plants, and 33 were observed at the 10th day. Comparison of protein expression changes with those of gene transcription showed a positive correlation in B. napus, although different patterns between transcripts and proteins were observed at each time point. Under drought stress, most protein abundance changes may be attributed to gene transcription, and PTMs clearly contribute to protein diversity and functions.

Proteomic analysis of salt tolerance in sugar beet monosomic addition line M14.

Understanding the mechanisms of plant salinity tolerance can facilitate plant engineering for enhanced salt stress tolerance. Sugar beet monosomic addition line M14 obtained from the intercross between Beta vulgaris L. and Beta corolliflora Zoss exhibits tolerance to salt stress. Here we report the salt-responsive characteristics of the M14 plants under 0, 200, and 400 mM NaCl conditions using quantitative proteomics approaches. Proteins from control and the salt treated M14 plants were separated using 2D-DIGE. Eighty-six protein spots representing 67 unique proteins in leaves and 22 protein spots representing 22 unique proteins in roots were identified. In addition, iTRAQ LC-MS/MS was employed to identify and quantify differentially expressed proteins under salt stress. Seventy-five differentially expressed proteins in leaves and 43 differentially expressed proteins in roots were identified. The proteins were mainly involved in photosynthesis, energy, metabolism, protein folding and degradation, and stress and defense. Moreover, gene transcription data obtained from the same samples were compared to the corresponding protein data. Thirteen proteins in leaves and 12 in roots showed significant correlation in gene expression and protein levels. These results suggest the important processes for the M14 tolerance to salt stress include enhancement of photosynthesis and energy metabolism, accumulation of osmolyte and antioxidant enzymes, and regulation of methionine metabolism and ion uptake/exclusion.

Quantitative proteomics of tomato defense against Pseudomonas syringae infection

Genetic and microarray analyses have provided useful information in the area of plant and pathogen interactions. Pseudomonas syringae pv. tomato DC3000 (Pst) causes bacterial speck disease in tomato. Previous studies have shown that changes in response to pathogen infection at transcript level are variable at different time points. This study provides information not only on proteomic changes between a resistant and a susceptible genotype, but also information on changes between an early and a late time point. Using the iTRAQ quantitative proteomics approach, we have identified 2369 proteins in tomato leaves, and 477 of them were determined to be responsive to Pst inoculation. Unique and differential proteins after each comparison were further analyzed to provide information about protein changes and the potential functions they play in the pathogen response. This information is applicable not only to tomato proteomics, but also adds to the repertoire of proteins now available for crop proteomic analysis and how they change in response to pathogen infection.