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Keywords:

  • abiotic stress;
  • cotton;
  • duplicated genes;
  • gene expression;
  • polyploidy

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Polyploidy has occurred throughout plant evolution and can result in considerable changes to gene expression when it takes place and over evolutionary time. Little is known about the effects of abiotic stress conditions on duplicate gene expression patterns in polyploid plants.
  • We examined the expression patterns of 60 duplicated genes in leaves, roots and cotyledons of allotetraploid Gossypium hirsutum in response to five abiotic stress treatments (heat, cold, drought, high salt and water submersion) using single-strand conformation polymorphism assays, and 20 genes in a synthetic allotetraploid.
  • Over 70% of the genes showed stress-induced changes in the relative expression levels of the duplicates under one or more stress treatments with frequent variability among treatments. Twelve pairs showed opposite changes in expression levels in response to different abiotic stress treatments. Stress-induced expression changes occurred in the synthetic allopolyploid, but there was little correspondence in patterns between the natural and synthetic polyploids.
  • Our results indicate that abiotic stress conditions can have considerable effects on duplicate gene expression in a polyploid, with the effects varying by gene, stress and organ type. Differential expression in response to environmental stresses may be a factor in the preservation of some duplicated genes in polyploids.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Polyploidy has been a prominent and ongoing process during plant evolution. The process can contribute to the rapid emergence of new species and it has been estimated that c. 15% of speciation events in angiosperms are accompanied by polyploidy (Wood et al., 2009). Many important agricultural crops are polyploid, such as wheat, canola, potato, sugarcane and cotton (Wendel, 2000). Polyploidy can lead to novel phenotypes, such as changes in flowering time (Schranz & Osborn, 2000; Pires et al., 2004; Wang et al., 2006a) and self-incompatibility (Brennan & Hiscock, 2010). After polyploidy there can be extensive changes to genome structure, probably caused by rearrangements and recombination (e.g. Liu et al., 1998; Gaeta et al., 2007; Lim et al., 2008), but not all polyploids show such responses (Liu et al., 2001; Mestiri et al., 2010). Gene losses have been inferred in natural polyploid populations of recent origin (Tate et al., 2006; Buggs et al., 2009). Many previous studies have revealed frequent epigenetic changes, including DNA methylation and histone modifications, in newly synthesized allopolyploids of Arabidopsis, Brassica and wheat (e.g. Shaked et al., 2001; Kashkush et al., 2002; Madlung et al., 2002; Wang et al., 2004, 2006a,b; Lukens et al., 2006; Gaeta et al., 2007; Beaulieu et al., 2009), but cytosine methylation changes were not found in cotton (Liu et al., 2001). Recently formed natural populations of allopolyploids and hybrids can also show alterations in DNA methylation (Parisod et al., 2009).

Polyploidy can result in changes to duplicate gene expression, both on allopolyploidy and over evolutionary time. Numerous studies of the expression of duplicated protein coding genes in newly created synthetic allopolyploids and their progenitors of Brassica napus, wheat, cotton, Arabidopsis suecica, Citrus and Senecio have shown expression levels that deviate from mid-parent values, biased expression of homeologs and silencing of homeologs (e.g. Comai et al., 2000; Kashkush et al., 2002; Wang et al., 2004, 2006a,b; Adams et al., 2004; Albertin et al., 2006; Hegarty et al., 2006; Gaeta et al., 2007; Pumphrey et al., 2009; Rapp et al., 2009; Bassene et al., 2010). Expression changes can be organ and tissue specific, and can vary by generation in newly synthesized polyploids. Longer term evolutionary effects on duplicate gene expression in natural polyploid plants have been examined in several natural polyploids, especially cotton, wheat and A. suecica (Lee & Chen, 2001; Adams et al., 2003; Bottley et al., 2006; Hovav et al., 2008; Chaudhary et al., 2009; Flagel & Wendel, 2010), as well as in recently formed natural polyploid populations of Senecio, Tragopogon and Spartina (Hegarty et al., 2005; Buggs et al., 2010a,b; Chelaifa et al., 2010). The above studies have indicated that many genes show biased expression of homeologs or homeolog silencing that varies by organ and tissue type and by developmental stage. For example, Adams et al. (2003) showed that there was reciprocal, organ-specific silencing of alcohol dehydrogenase gene A homeologs in Gossypium hirsutum, and Hovav et al. (2008) used homeolog-specific microarrays to demonstrate that c. 30% of homeologs showed biased expression in natural allopolyploid cotton.

There can be changes in homeologous gene expression patterns in polyploid plants in response to abiotic stresses. Liu & Adams (2007) assayed the expression of alcohol dehydrogenase A gene homeologs under multiple stress treatments and in different organs at multiple developmental stages in polyploid G. hirsutum. Variation in homeolog expression patterns in response to stresses was found to be organ and developmental stage specific. Of particular interest was the finding of reciprocal silencing of homeologs in response to different stress treatments (under water submersion treatment one homeolog of the AdhA gene was silenced in hypocotyls, and under cold stress treatment the other copy was silenced in hypocotyls) that indicated subfunctionalization in response to abiotic stress conditions. This is the first and only study so far of homeologous gene expression in response to abiotic stresses in polyploid plants. Thus, the extent of homeologous gene expression changes in response to abiotic stresses remains unknown.

In this study, we investigated the expression of homeologs in response to abiotic stresses in natural allotetraploid G. hirsutum and a synthetic cotton allotetraploid. Cotton (Gossypium) is a useful group for studying polyploidy. There are c. 40 diploid and five natural polyploid Gossypium species recognized (reviewed in Wendel & Cronn, 2003). All five polyploids are allotetraploids (AADD; 2n = 4x = 52) which originated by hybridization between an A genome diploid (genome AA; 2n = 26) and a D genome diploid (genome DD; 2n = 26) c. 1.5 million yr ago (reviewed in Wendel & Cronn, 2003). Homeologous genes in polyploid cotton are designated At and Dt for the genes derived from the A-genome progenitor and the D-genome progenitor, respectively. We designed a series of experiments to determine the following: whether changes in the expression levels of duplicated genes, relative to each other, under abiotic stresses are common phenomena in G. hirsutum; what kinds of stress-specific and organ-specific patterns are present; and whether changes in the expression levels of homeologs under abiotic stresses can occur in a newly synthesized allotetraploid.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials and stress treatments

Gossypium hirsutum L. cv TM1 (Texas Marker-1), which is a highly inbred line, and a synthetic allopolyploid created by doubling the chromosomes of a G. arboreum × G. davidsonii F1 hybrid (synthesized by J. A. Lee, North Carolina State University, and studied in Adams et al., 2004 and Chaudhary et al., 2009) were used for expression experiments. A reciprocal hybrid of the synthetic allopolyploid has not been successfully synthesized to our knowledge. For G. hirsutum cv TM1, the stress treatments of seedlings were separated into two sets. The first set included water submersion (flood simulation) stress, cold stress and control plants that were grown in Liu & Adams (2007). The second set included heat stress, salt stress, drought stress and control plants that were grown for this study. There were two sets because we decided to include more stresses after the experiments with the first two stresses were complete to increase the size of the dataset and to give a broader view of the abiotic stress responses of the duplicated genes. For each stress treatment and control, there were three biological replicates. Six seedlings were pooled for RNA extraction in each replication so that the effects of plant-to-plant variation and slightly different germination and growth rates were minimized. The seedlings were grown in a peat–vermiculite–soil mixture under fixed day : night (16 h day : 8 h night) and temperatures of 24°C in a growth chamber.

For water submersion stress, the seedlings were completely submerged under distilled water for 2 d to simulate flooding; for cold stress treatment, the seedlings were placed in a cold room (4°C) for 2 d; both stress treatments were performed in Liu & Adams (2007). For heat stress treatment, the seedlings were placed in a growth chamber at 43°C for 2 d, which is an intermediate temperature between 41°C (Law et al., 2001) and 45°C (Fender & O’Connell, 1989). For drought stress treatment, the seedlings were removed from the pots and placed on dry filter paper for 2 d, as performed in Jin & Liu (2008). For salt stress treatment, the roots of the seedlings were submerged in an aqueous soil extract solution with 400 mM NaCl for 2 d, as performed in Jin & Liu (2008). All of the above stresses were performed from 6 to 8 d post-planting (dpp). For the stress treatments, all other conditions of plant growth were the same as those of the untreated controls. For the synthetic Gossypium allopolyploids, plant growth, cold stress, heat stress and salt stress were performed from 6 to 8 dpp as above.

Gene choices, sequence alignments, phylogenetic analyses and primer design

We focused on genes from Gossypium species whose total expression levels of both homeologs varied in response to one or more abiotic stresses in previous studies, and genes with a homolog in Arabidopsis thaliana that were stress regulated. Twelve of the genes have been shown to be up- or down-regulated by one or more abiotic stresses in polyploid cotton (Zhao et al., 2003; Qin et al., 2004; Wang et al., 2006c; Ni et al., 2008; Guo et al., 2009). However the expression levels of each homeolog have not been assayed and compared with each other. In addition, we used cotton homologs of 18 genes known to be up- or down-regulated in A. thaliana in response to abiotic stresses, obtained from microarray studies (Kilian et al., 2007). The cotton homologs were identified using tBLASTx searches of expressed sequence tags (ESTs) from GenBank with the gene from Arabidopsis as a query.

Sequences were aligned in ClustalX and analyzed by parsimony analysis using PAUP 4.0 with default settings. Paralogs and homeologs of each gene within Gossypium were identified by constructing phylogenetic trees from sequence alignments. Paralogs were identified by separate and distinct clades in the trees. Homeologs, designated At for that derived from the A-genome parent and Dt for that derived from the D-genome parent, were identified by finding G. hirsutum sequences that branch with sequences from the diploids G. raimondii and G. arboreum, indicating Dt and At homeologs, respectively. Examples of phylogenetic trees are shown in Supporting Information Fig. S1.

Primers were designed to amplify products of c. 200 bp that contained several single nucleotide polymorphisms (SNPs) that could be used to distinguish homeologs with single-strand conformation polymorphism (SSCP); examples of alignments showing SNPs are given in Fig. S1. There were no introns in the amplified regions, so that amplification from genomic DNA could be used as a guide for band locations on the gels.

Nucleic acid extraction, reverse transcription and polymerase chain reaction

Genomic DNA was extracted with the Qiagen DNeasy Plant Mini Kit according to the manufacturer’s instructions, and RNA was extracted from tissues with a hot borate method (used in Adams et al., 2003). DNA and RNA concentrations and purities were measured with a spectrophotometer. RNA quality was checked on agarose gels to identify strong ribosomal RNA bands.

RNAs were treated with RNAse-free DNaseI to remove genomic DNA before reverse transcription, and single-stranded cDNA was synthesized by M-MLV reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. Negative controls were made with diethylpyrocarbonate (DEPC)-treated water instead of reverse transcriptase to check for any remaining genomic DNA. PCR conditions were optimized with genomic DNA for each primer. PCRs were performed in a reaction mixture (10 μl) containing 1 μl of template (genomic DNA or cDNA), 1 μl of 10 × PCR buffer, 0.1 μl of 2.5 mM deoxynucleoside triphosphates (dNTPs), 0.5 μl of each 0.4 μM forward and reverse primer, 0.1 μl of Paq5000 DNA polymerase (Stratagene, Mississauga, Ontario) and 6.8 μl of double-distilled H2O. Thirty cycles of PCR were carried out, and the conditions for each cycle were as follows: 30 s at 94°C, 30 s at the optimized annealing temperature, 1 min at 72°C. PCR products were run on 2% agarose gels (0.5 × tris-borate-ethylenediaminetetraacetic acid (TBE) buffer) for band separation and stained with ethidium bromide for visualization.

SSCP analysis

After PCR amplification, Qiagen quick spin columns were used to clean PCR products to remove primers, and 2–4 ng of clean, double-stranded PCR product was used for SSCP labeling reactions. The labeling reactions were performed in a reaction mixture (10 μl) containing 4 ng of double-stranded PCR product (diluted to 6 μl with water), 0.5 μl of 10 μM primer, 1.0 μl of 10 × PCR buffer, 0.8 μl of 2.5 mM dNTPs, 0.5 μl of 50 mM MgCl2, 1.04 μl of distilled H2O, 0.08 μl of Paq 5000 DNA polymerase (Stratagene) and 0.08 μl 32P-dCTP (3000 Ci mmol−1 stock). The labeling conditions were as follows: 2 min at 94°C; 25 cycles of 30 s at 94°C, 30 s at 54–57°C (depending on predicted primer Tm), 1 min at 72°C; a final extension for 5 min at 72°C.

After labeling, the samples were denatured by mixing 2 μl of PCR product with 8 μl of denaturation solution (0.05% bromophenol blue, 0.05% xylene cyanol, 95% formamide, 20 mM EDTA), heated to 95°C for 5 min, and snap cooled on ice until loading on the polyacrylamide gel (25% MDE® gel solution from Lonza, http://www.lonza.com, 0.1 × TBE, 0.033% g/v ammonium persulfate and 0.05% v/v tetramethylethylenediamine (TEMED)). Electrophoresis was performed in 0.5 × TBE at 8 W for c. 20 h at room temperature. Following electrophoresis, the gels were dried and placed on phosphorimaging screens for 2 d, and the images were visualized on a Bio-Rad PhosphorImager. Band intensities of homeologs were measured using Quantity One software (Bio-Rad) with background corrections. For each gene, the SSCP gel conditions were optimized by adjusting the running time and urea concentration (Table S1) so that two bands were visible for each gene, one for each homeolog, when a single labeling primer was used (or four bands, corresponding to both strands if two labeling primers were used).

DNA sequencing to identify homeologs

After PCR amplification, the products were purified by Qiagen columns, SSCP loading buffer was added and the samples were loaded onto a polyacrylamide gel. After electrophoresis was finished, the gel was fixed in 10% acetic acid for 30 min, washed with distilled water three times, incubated in silver staining solution (0.1% silver nitrate and 0.15% formaldehyde) for 30 min, briefly washed with distilled water and incubated in developing solution (3% Na2CO3, 0.15% formaldehyde, 0.024% Na2SO3). When clear bands were seen, the developing solution was poured out and 10% acetic acid was added to stop development. For DNA sequencing, a single silver-stained band was excised directly from the gel and vigorously rinsed with 500 μl of extractant (2.5% sodium thiosulfate, 1% potassium hexacyanoferrate) for 5 min to remove the silver from the gel. The rinsing process was repeated five times until the gel became transparent. After another rinse with distilled water, the DNA was extracted from the gel with 30 μl of distilled water at 80°C for 15 min. The DNA was re-amplified with a 20-cycle PCR and subjected to Sanger sequencing on an ABI 3730 with ABI Big Dye Version 3.1 (Applied Biosystems, http://www.appliedbiosystems.com) at the Nucleic Acid Protein Service Unit of the University of British Columbia.

Data analysis

SSCP-cDNA data from three replicates or, in some cases, two replicates were analyzed for each stress treatment and organ type. Paired t-tests, performed with R software, were used to determine significant differences in the level of At expression compared with Dt expression (the percentage of total transcripts derived from the At homeolog vs the Dt homeolog) between abiotic stress-treated plants and untreated plants. A 5% false discovery rate (FDR) was used to correct the multiple testing results.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Duplicate gene expression in response to cold and water submersion stresses in G. hirsutum

We assayed duplicate gene expression in response to abiotic stress conditions using 30 pairs of duplicates (60 total genes) in polyploid G. hirsutum. The genes and their functions or putative functions are listed in Table 1. We first assayed expression in G. hirsutum seedlings in response to cold stress and water submersion (flood simulation) stress, as in Liu & Adams (2007) for the AdhA gene, but with more genes. We used the SSCP method to separate genes with very similar sequences (Cronn & Adams, 2003). Fig. 1 shows examples of SSCP-cDNA gels. Among the 30 gene pairs, 15 showed significant changes in the contribution levels of the At and Dt homeologs to the total transcript pool in response to stress treatment in at least one organ (Fig. 2). Twelve gene pairs showed changes in the level of At expression compared with Dt expression, hereafter designated At : Dt, under cold stress treatment, and another eight gene pairs showed such expression changes after water submersion stress treatment. Some genes showed changes under both stresses and other genes only under one stress. Five genes showed significant expression level changes under both cold and water submersion stresses. There was variability among the three different organ types examined: 12 gene pairs showed changes in the At : Dt expression levels in hypocotyls under at least one stress treatment, whereas six gene pairs were affected in cotyledons, and six gene pairs in roots (Fig. 2).

Table 1. Gossypium genes assayed for duplicate gene expression
Gene pairFunction or putative functionAccession numbers
  1. Accession numbers refer to GenBank accession IDs. One accession number is provided for each duplicate gene and thus two for each gene pair. * indicates stress-regulated genes from polyploid Gossypium (Millar et al., 1994; Zhao et al., 2003; Qin et al., 2004; Wang et al., 2006a,b; Ni et al., 2008; Guo et al., 2009). The other genes are homologs of stress-regulated genes from Arabidopsis thaliana (Kilian et al., 2007).

 1Ultraviolet-B-repressible proteinDV850210, CD485666
 2Photosystem II subunit XES839555, DW225871
 3Photosystem II subunit XDN780680, DN780680
 4ATP synthase delta chainAI728201, AI728742
 5ATP synthase delta chainES811921, ES837314
 6AminomethyltransferaseES804546, ES827416
 7AminomethyltransferaseDW242796, AI725406
 8GDSL-motif lipaseES845029, DR459231
 9Coenzyme bindingDN780968, DN804813
10Alternative respiratory pathwayDW228107, DW235082
11*Peptide methionine sulfoxide reductaseDW227959, DW487847
12*Ethylene responsive element binding proteinES809854, DT550565
13*Ethylene responsive element binding proteinES837957, DT459748
14*MAP kinaseDT558090, AI726863
15*MAP kinaseEX167377, DT554369
16*Alcohol dehydrogenase BAF226633, DT553703
17*Alcohol dehydrogenase CAF036575, AF036569
18*Alcohol dehydrogenase DAF250204, AF250205
19Zinc finger family proteinDT571829, DR452483
20Group 4 late embryogenesis-abundant proteinDR463629, DW516643
21Unknown functionDW505446, DN803743
22Cold-regulated plasma membraneDW504511, EX165054
23Unknown functionAW187060, DW505445
24*Ethylene responsive element binding proteinDT459748, DW233937
25*Peptide methionine sulfoxide reductaseDW487846, DW227959
26Manganese ion bindingDR454806, DW504108
27AminomethyltransferaseDT545047, DN781137
28Transferase, transferring hexosyl groupsDW482030, ES841864
29*Zinc finger transcription factorAY887895, DN779306
30*Homeobox proteinEF151309, ES831550
image

Figure 1. Single-strand conformation polymorphism (SSCP)-cDNA gels. A few examples of SSCP-cDNA gels are shown. Gene pair numbers are given beside the gels. ‘At’ and ‘Dt’ indicate two homeologous genes from the A or D genome of the natural allopolyploid Gossypium hirsutum. The numbers under the lanes indicate transcript percentages as determined by the PhosphorImager. The gDNA lane is the genomic DNA control. C, cold stress; Co, cotyledons; D, drought stress; Hy, hypocotyls; S, salt stress; U, untreated plants at 8 d; W, water submersion stress. The numbers 1, 2 and 3 after the organ indicate replicates 1, 2 and 3, respectively. Bands were cut from the gel and sequenced to determine their identity.

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image

Figure 2. Duplicate gene expression levels vary under cold stress and water submersion stress in Gossypium hirsutum. Graphs showing the percentage of transcripts derived from the At homeolog (y axis) in seedling organs of G. hirsutum. C, cold stress; U, untreated control plants; W, water submersion. Error bars represent standard deviations among replicates. The treatments designated by ‘*’ indicate a statistically significant difference compared with untreated plants, detected by t-tests (P < 0.05). Gene pair numbers are indicated under the graphs. Graphs of expression data from all assayed genes are given in Supporting Information Fig. S2; At : Dt data are given in Table S2.

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Among the 15 gene pairs that showed changes in relative expression levels of the homoelogs in response to cold and water submersion stresses, five showed changes in which the difference between stress and control was > 20% (e.g. 60 : 40 vs 80 : 20) after abiotic stress treatments (Fig. 2). The 20% threshold was arbitrary for separating larger changes from smaller changes. Among the largest changes, the two duplicates of an aminomethyltransferase (gene pair 7) showed equal expression of both copies in hypocotyls under untreated conditions, but the Dt copy was almost silenced (At : Dt of 90 : 10) under cold stress treatment (Figs 1, 2). The two duplicates of a gene with coenzyme binding function (gene pair 9) showed a stong bias towards the Dt homeolog (At : Dt of 14 : 86) in hypocotyls under cold stress treatment, whereas there was preferential expression of the At copy under untreated conditions (At : Dt of 65 : 35); Figs 1 and 2. A third example involves a pair of genes for late embryogenesis-abundant proteins (gene pair 20) that showed a considerable increase in the relative expression level of At to Dt in both cotyledons and roots under cold stress (c. 80 : 20 from 45 : 55; Fig. 2). One gene pair (11), a peptide methionine sulfoxide reductase, showed reciprocal changes in the levels of At and Dt expression in response to different stresses: there was an increase in relative expression of At to Dt after cold treatment in hypocotyls (83 : 17, from 66 : 34 under untreated conditions) and a decrease after water submersion treatment in hypocotyls (to 48 : 52).

Duplicate gene expression in response to heat, salt and drought stresses in G. hirsutum

After documenting many genes with changes in the contribution levels of the At and Dt homeologs to the total transcript pool in response to cold and water submersion stresses, we decided to examine three other abiotic stresses to further characterize the duplicate gene expression patterns in response to abiotic stresses. These included three treatments, heat, high salt and drought stress, plus control untreated plants. We examined the expression patterns of 30 homeologous gene pairs in three organ types of G. hirsutum seedlings. Twenty gene pairs showed changes in the relative contributions of the At and Dt homeologs to the transcript pool in at least one organ after one or more of the abiotic stress treatments (Fig. 3). Among the 20 gene pairs, 14 showed expression changes in at least one organ under heat stress treatment, 16 under salt stress treatment and eight under drought stress treatment. Only three gene pairs (4, 14 and 15) showed significant changes in response to all three treatments in the same organ type. The expression patterns of the duplicated genes in response to heat, salt and drought stresses were compared in different organ types. Eleven gene pairs showed changes in the At : Dt expression levels in hypocotyls under at least one stress treatment, 14 gene pairs in cotyledons and nine gene pairs in roots.

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Figure 3. Duplicate gene expression levels vary under heat stress, salt stress and drought stress in Gossypium hirsutum. Graphs showing the percentage of transcripts derived from the At homeolog (y axis) in seedling organs of G. hirsutum. D, drought stress; H, heat stress; S, salt stress; U, untreated control plants. Error bars represent standard deviations among replicates. The treatments designated by ‘*’ indicate a statistically significant difference compared with untreated plants, detected by t-tests (P < 0.05). Gene pair numbers are indicated under the graphs. Graphs of expression data from all assayed genes are given in Supporting Information Fig. S3; At : Dt data are given in Table S3.

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Of the 20 gene pairs that showed significant changes in the At : Dt expression level in response to one or more of the three abiotic stresses, eight gene pairs showed changes in which the difference between stress and control was > 20% (e.g. 60 : 40 vs 80 : 20) after abiotic stress treatments. For example, the expression pattern of gene pair 20 (late embryogenesis-abundant proteins) changed from biased expression of the Dt copy (At : Dt of 28 : 72 to 30 : 70) to preferential expression of the At copy (At : Dt of 61 : 39 to 69 : 31) in all three organs after heat stress treatment and in two organs after salt stress treatment (Fig. 3). Two gene pairs showed reciprocal expression changes in response to different stress treatments, that is, an increase in the At : Dt expression level under one stress treatment and a decrease under another stress treatment. Gene pair 15, mitogen-activated protein kinases, showed reciprocal expression changes in cotyledons: there was an increase in the transcript contribution of At after heat stress treatment (At : Dt of 75 : 25, from 45 : 55 under untreated conditions), and decreases after salt stress treatment (At : Dt of 32 : 68) and drought stress treatment (At : Dt of 38 : 62) (Figs 1 and 3). Gene pair 19, in the zinc finger family, showed a change in homeolog expression bias from Dt to At in cotyledons after heat stress treatment (At : Dt of 69 : 31 compared with 40 : 60 under untreated conditions), whereas there was a greater Dt bias after drought stress treatment (At : Dt of 17 : 83; Fig. 3).

Comparisons of duplicate gene expression in response to five abiotic stresses

We next compared the results from all five abiotic stress treatments. For each stress, we compared the At : Dt values of the stressed and control plants to determine whether they increased, decreased or remained unchanged in response to stress. Then, we compared the effects of all five stresses for each gene. Among the 30 duplicate gene pairs examined, 23 showed significant changes in the contributions of the At and Dt homeologs to the transcript pool after one or more of the five abiotic stress treatments in at least one organ type compared with the untreated control plants. Cold, water submersion, heat, salt and drought stresses induced changes in the expression levels of twelve, eight, fourteen, sixteen and eight gene pairs, respectively. Thus, heat and salt stresses had the largest effects on duplicate gene expression levels. Only one gene pair, an ATP synthase delta chain (4), showed changes in the At : Dt levels in response to all five stress treatments, whereas four gene pairs (3, 15, 16 and 19) showed such expression changes in response to four of the five stresses. Five gene pairs (4, 11, 15, 16 and 19) showed reciprocal At : Dt expression level changes (the level increased in response to one stress and decreased in response to another stress) in a single organ type after different abiotic stress treatments, indicated by ‘+’ and ‘−’ in the same row, and 12 gene pairs showed reciprocal At : Dt expression level changes among different stresses when comparing all three organ types (Fig. 4). Among the three different organ types examined, 17 gene pairs showed changes in At : Dt expression levels after at least one stress treatment in hypocotyls, compared with 15 gene pairs in cotyledons and 13 gene pairs in roots. In addition to comparisons among the expression responses of the homeologs to different stresses, we also compared the organ-specific responses to the same stress. We found a significant change in At : Dt expression levels in only one or two of the organ types in response to cold, water submersion, heat, salt and drought for many of the gene pairs: nine, eight, thirteen, sixteen and eight, respectively. Four gene pairs (3, 8, 11 and 14) showed an increase in At : Dt expression levels in one organ type and a decrease in another organ type in response to the same stress (Fig. 4). For example, gene pair 3 showed an increase in the At : Dt expression levels in cotyledons and roots in response to water submersion, but a decrease in hypocotyls. Overall, there were 45 gene pair/stress/organ combinations in which the level of At transcripts relative to Dt transcripts increased in response to a stress treatment, and 32 gene pair/stress/organ combinations in which the level of At transcripts decreased relative to Dt transcripts. Thus, there appears to be a general bias towards expression of the At homeolog after abiotic stress treatments.

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Figure 4. Summary of duplicate gene expression in response to five abiotic stresses. Changes in At : Dt expression levels between each stress treatment and untreated plants in Gossypium hirsutum. ‘+’ indicates an increase in the At : Dt expression level after stress treatment; ‘−’ indicates a decrease in the At : Dt expression level after stress treatment; ‘0’ indicates that there is no statistically significant difference between the stress treatment and untreated plants. Horizontal shading indicates gene pairs and organ types for which the At : Dt expression level increased after one or more stresses and decreased after other stresses. Vertical shading indicates an increase in the At : Dt expression level in one organ type and a decrease in another organ type after the same stress. The word ‘Genes’ followed by a number indicates the gene pair number. CS, cold stress; DS, drought stress; HS, heat stress; nd, expression not determined; SS, salt stress; WS, water submersion.

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Duplicate gene expression in response to abiotic stresses in a synthetic Gossypium allotetraploid

Synthetic allopolyploids are useful for examining genetic changes that take place immediately on and within a few generations after allopolyploidy. We examined the expression patterns in a synthetic Gossypium allotetraploid of 10 duplicate gene pairs that showed significant changes in the relative expression level of At and Dt after one or more abiotic stress treatment in the natural allotetraploid G. hirsutum. High salt, cold and heat stress treatments were used. Eight gene pairs showed changes in At : Dt expression levels in at least one organ after one or more abiotic stress treatment (Fig. 5). Among the eight gene pairs, seven showed expression changes under salt stress treatment in at least one organ, three were affected by cold stress treatment and three were affected by heat stress treatment. In hypocotyls and cotyledons, six and five gene pairs, respectively, showed changes in response to at least one abiotic stress treatment, but there were no gene pairs in roots with such responses. Among the eight gene pairs that showed At : Dt expression level changes, two showed changes of 20% or greater (e.g. 60 : 40 to 80 : 20) between stressed and unstressed plants. Gene pair 8, GDSL-motif lipases, showed a considerable decrease in At : Dt levels in hypocotyls after heat stress treatment (At : Dt of 27 : 73) compared with untreated conditions (At : Dt of 55 : 45) in the synthetic allopolyploid. Gene pair 20 (for late embryogenesis-abundant proteins) showed biased expression of At under salt stress treatment in hypocotyls (At : Dt of 74 : 26) and cotyledons (At : Dt of 68 : 32) compared with about equal expression under untreated conditions (At : Dt of 48 : 52).

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Figure 5. Duplicate gene expression levels vary in response to abiotic stresses in a synthetic Gossypium allopolyploid. Graphs showing the percentage of transcripts derived from the At homeolog (y axis) in seedling organs of the synthetic Gossypium allopolyploid. C, cold stress; H, heat stress; S, salt stress; U, untreated control plants. Error bars indicate standard deviations among replicates. The treatments with ‘*’ indicate a significant difference compared with control plants, detected by t-tests (P < 0.05). Gene pair numbers are indicated under the graphs. At : Dt data are listed in Supporting Information Table S4.

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Comparison of the expression patterns of the 10 duplicate gene pairs between the natural and synthetic Gossypium allopolyploids showed only two gene pairs with parallel expression changes under particular stress treatments: Gene pair 20 (for late embryogenesis-abundant proteins) showed increases in At : Dt expression levels under salt stress in hypocotyls and cotyledons in both natural and synthetic Gossypium. Gene pair 12, an ethylene responsive element binding protein, showed a small decrease in At : Dt expression level under salt stress in cotyledons in both the natural and synthetic polyploid Gossypium, but expression changes in other organs and stress treatments were not in parallel. All the other genes showed different expression patterns between natural and synthetic Gossypium allopolyploids in response to abiotic stress treatments. For example, gene pair 4 (ATP synthase delta chain) showed mostly the presence of Dt transcripts in all organs under all stresses in the synthetic allopolyploid, compared with almost equal expression of both copies in G. hirsutum. Gene pair 12 showed a large bias in expression towards Dt in the synthetic polyploid under all conditions, but only a slight At bias in G. hirsutum. Overall, there was little correspondence between the natural and synthetic allopolyploid genotypes in their duplicate gene expression levels in response to abiotic stress treatments among the assayed genes.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Abiotic stress-induced changes in duplicate gene expression in a natural and synthetic allopolyploid

In this study, we assayed the expression of 30 gene pairs under five abiotic stress treatments and in three organ types for a total of c. 450 gene pair/stress/organ combinations, plus untreated controls, in G. hirsutum. We found that a large majority of the duplicate gene pairs assayed (23 of 30 pairs; 77%) showed changes in the relative contributions of the homeologs to the transcript pool under one or more abiotic stress treatment and in one or more organ type. Among the 23 gene pairs that showed expression changes under abiotic stresses, 11 had biased expression level differences of > 20% (e.g. 60 : 40 to 80 : 20) compared with untreated plants. There were five gene pairs in which the relative contribution of the At homeolog increased after one stress, compared with untreated plants, but decreased after another stress. This phenomenon is analogous to duplicate gene pairs in which one copy is more highly expressed in some organ types and the other copy is more highly expressed in other organ types, referred to as ‘complementary expression’ (Ganko et al., 2007) or ‘quantitative subfunctionalization’ (Force et al., 1999; Duarte et al., 2006). Thus, this study indicates that stress-specific complementary expression (or quantitative subfunctionalization) has occurred for some duplicate gene pairs in polyploid cotton. (Quantitative subfunctionalization contrasts with qualitative subfunctionalization in that qualitative refers to no expression of one copy in one organ type (or tissue, cell type, condition, etc.) and no expression of the other copy in another organ type (Force, 1999). We think that the term ‘complementary expression’ provides a clearer description of the phenomenon reported here.)

In some cases, the effects of the abiotic stress treatments on duplicate gene expression varied by organ type. Many of the duplicate gene pairs examined in this study showed organ-specific responses to stress treatments (that is, At : Dt expression levels changed after stress treatment in only one or two of the examined organ types). Among these genes, four pairs showed reciprocal changes in At : Dt expression levels among different organs under the same stress treatment, that is, in one organ there was an increase in At : Dt expression and there was a decrease in another organ after the same abiotic stress treatment. Organ-specific responses to stress treatments were not unexpected because homeologous gene expression patterns in polyploids often vary by organ type (reviewed in Adams, 2007).

Overall, it appears that changes in the contributions of homeologous genes to the total transcript pool (transcriptome) in response to abiotic stresses is a common phenomenon in polyploid cotton. Considering that a large majority of the genes examined were abiotic stress regulated in cotton, or homologs of genes regulated by at least one abiotic stress in A. thaliana, 77% of duplicate gene pairs showing changes might not be generally applicable when applied to the whole G. hirsutum transcriptome, but our findings are probably applicable to stress-regulated genes. In the only previous study of the effects of abiotic stress treatments on duplicate gene expression in a polyploid plant, qualitative subfunctionalization in response to abiotic stress conditions was discovered (Liu & Adams, 2007). In that study, only one homeolog of the alcohol dehydrogenase gene AdhA was expressed in hypocotyls under water submersion treatment and only the other copy was expressed in hypocotyls under cold stress treatment (Liu & Adams, 2007). In this study, we did not find any cases of qualitative subfunctionalization, but we did find many cases of changes in the amount of expression of one duplicate relative to the other in response to abiotic stress treatments.

Our findings of altered duplicate gene expression levels and complementary expression in response to abiotic stress treatments demonstrate that abiotic stresses can have a major influence on the expression of duplicated genes, which may aid in the preservation of duplicated genes over evolutionary time. All of the duplicate gene pairs examined in G. hirsutum have multiple amino acid sequence differences (Table S5), although it is not known which of these amino acid differences affect function. Nevertheless, it is possible in a few cases that the product of one of the two homeologs performs better under abiotic stress conditions, and thus there has been selection for a higher expression level of that homeolog relative to the other homeolog, caused by mutations in cis-regulatory elements. The alteration of the expression patterns of some types of gene, such as transcription factors, in response to one or more abiotic stress conditions could affect the expression levels of downstream genes that they regulate. In that regard, the altered gene expression patterns of some genes in response to stress conditions could be the result of the process just mentioned, and not a result of selection on the genes themselves. Alternatively, there may not be any selection acting on some of the genes or their regulators and the expression level changes in response to stress conditions may be neutral, or of no functional consequence, rather than being adaptive.

In addition to finding gene pairs in G. hirsutum whose comparative expression levels change in response to abiotic stresses, we also found that high salt, cold and heat stress treatments had effects on At : Dt expression levels in a synthetic cotton allopolyploid. This finding shows that the phenomenon is not limited to natural allopolyploid species. There are no previous studies of homeologous gene expression changes in response to abiotic stress treatments in synthetic polyploids. However, the results from the synthetic allopolyploid are analogous to those of a previous study of allelic expression in diploid maize hybrids, which showed changes in allelic expression ratios of four genes in response to drought stress (Guo et al., 2004).

There was little correspondence between stress-induced changes in duplicate gene expression in synthetic and natural allopolyploids. One possibility is that there were similar stress-induced expression changes on allopolyploidy, followed by considerable divergence in the expression patterns in the natural polyploid G. hirsutum, perhaps caused by selection in response to environmental stresses. Another possibility is that the phenomenon is not caused by merger of the A and D genomes in a common nucleus of the allopolyploid cotton per se. Instead, the process may be largely random in terms of which genes are affected in the synthetic polyploidy, and probably caused by the various molecular processes that take place on allopolyploidization; these processes are discussed below. Previous studies have shown stochastic changes in duplicate gene expression levels in newly synthesized polyploids, albeit not in response to abiotic stresses (Wang et al., 2004). Even if stress-induced expression changes are random after allopolyploidy, there could be subsequent divergence caused by selection in response to environmental stresses or other factors.

The mechanisms and causes of gene silencing and expression level changes of homeologs in polyploids are starting to become known, but are still relatively poorly understood. They include cis- and trans-regulatory variation, changes in regulatory hierarchies and epigenetic changes such as cytosine methylation, histone modifications and small RNAs (reviewed in Osborn et al., 2003; Riddle & Birchler, 2003; Chen & Ni, 2006). Nothing is currently known about the causes and mechanisms of expression changes in duplicated genes in response to abiotic stresses in polyploids.

Mutations in cis-regulatory elements in which stress-induced transcription factors bind, sometimes referred to as stress-responsive elements, may account for homeolog expression changes in natural polyploids. Changes in the expression of trans-factors that regulate gene expression, particularly in response to abiotic stresses, may also contribute to the expression changes shown in this study. An often-discussed cause of gene expression changes in polyploids is the reuniting of diverged regulatory hierarchies on allopolyploidy from the two parental species, which may alter levels and patterns of gene expression (Osborn et al., 2003; Riddle & Birchler, 2003). In the case of expression changes in response to abiotic stress conditions, it is possible that regulator–sequence interactions in stress-regulated pathways may be altered in a synthetic allopolyploid, or that duplicated regulatory pathways diverge from each other to result in altered gene expression in response to stress in a natural allopolyploid.

There are several possible epigenetic mechanisms for the altered expression of duplicated genes in response to abiotic stress. Changes in cytosine methylation have been shown in several allopolyploid systems, as reviewed in the Introduction section. DNA methylation pattern alterations can occur in response to abiotic stresses (reviewed in Urano et al., 2010) and the patterns of cytosine methylation can affect gene expression (reviewed in Liu et al., 2010). Thus, it is possible that cytosine methylation pattern changes between the two duplicates in a pair may alter the expression levels of one or both genes in response to abiotic stress conditions. In the case of the cotton allopolyploids, divergent methylation patterns between the homeologs could be a factor affecting abiotic stress-responsive expression in G. hirsutum, but it is unlikely to be a factor in the synthetic allopolyploid because synthetic cotton allopolyploids have been shown to experience few cytosine methylation changes (Liu et al., 2001). Other epigenetic changes have been shown to occur in allopolyploids and affect gene expression, including histone methylation and histone acetylation (Wang et al., 2004; Ni et al., 2009). Alterations in histone modifications can occur in response to abiotic stresses (reviewed in Kim et al., 2010), and thus it is a probable candidate mechanism for stress-regulated changes in allopolyploids. Another possibility is the nonadditive expression of small RNAs, which has been shown in Arabidopsis allopolyploids (Ha et al., 2009), that could trigger changes in the amount of microRNA (miRNA) degradation of the homeologs. Abiotic stresses can result in changes in small RNA expression patterns and can affect gene regulation by miRNAs (reviewed in Sunkar et al., 2007).

Duplicate gene expression and responses to abiotic stresses

This study adds to the literature on the expression responses of duplicated genes to abiotic stresses. Expression patterns of genes duplicated by an ancient polyploidy event during the evolution of the Arabidopsis lineage, in response to abiotic stresses, were examined in three relatively recent studies. Kim et al. (2005) found, in a microarray study, that 117 gene pairs showed significant expression responses to oxidative stress by both genes in the duplicate pair, with some of the genes having distinct and sometimes opposite expression patterns. Ha et al. (2007) showed many examples of divergence in expression patterns between the duplicated genes in response to nine abiotic stress conditions in their analysis of the microarray dataset of Kilian et al. (2007). Zou et al. (2009) examined the expression of duplicates derived from the ancient polyploidy event and tandem duplicates with the nine abiotic stress microarray dataset (Kilian et al., 2007) using an ancestral state estimation approach. Numerous putative cases of partitioning of stress responsiveness between the duplicates were inferred. The above studies indicate the long-term evolutionary changes in responses to abiotic stresses by duplicated genes, whereas the present study examined expression in response to abiotic stresses on a shorter term evolutionary time scale of c. 1.5 million yr for G. hirsutum (Senchina et al., 2003) and in a synthetic allopolyploid.

Not only can abiotic stresses affect the expression patterns of duplicated genes, but biotic stresses can also have such effects. For example, expression assays of two peroxidase homeologs in Brassica napus showed differential expression responses of the homeologs to pathogen infection (Zhao et al., 2009). In addition, the studies of anciently duplicated genes in Arabidopsis, mentioned above (Ha et al., 2007; Zou et al., 2009), included microarray data from biotic stresses in addition to abiotic stresses, with data from the two types of stresses analyzed together.

Concluding remarks

This study reveals complex patterns of stress- and organ-specific expression of duplicated genes in a polyploid plant that are variable by gene, stress and organ type. It provides a new perspective on homeologous gene expression patterns in allopolyploids as the first study to examine expression patterns of multiple gene pairs in response to several abiotic stress conditions. It is possible that the expression changes shown here are involved in stress responses. Differential expression in response to environmental stresses may be a factor in the preservation of some duplicated genes in polyploids. Future studies will be needed to determine whether the phenomena shown here apply to other polyploid plants, and also to characterize the genomic extent of stress-induced changes in homeologous gene expression.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank the K. Adams laboratory and anonymous reviewers for helpful comments on the manuscript. This study was funded by the United States Department of Agriculture.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1 Phylogenetic trees and homeologous gene alignments

Figs S2 and S3 Graphs of At and Dt expression levels for all assayed genes in Gossypium hirsutum.

Table S1 Primers used for gene amplification and urea concentration for single-strand conformation polymorphism (SSCP)

Table S2 Single-strand conformation polymorphism (SSCP)-cDNA data for Gossypium hirsutum (cold and water submersion)

Table S3 Single-strand conformation polymorphism (SSCP)-cDNA data for Gossypium hirsutum (heat, salt, drought)

Table S4 Single-strand conformation polymorphism (SSCP)-cDNA data for the synthetic Gossypium allopolyploid

Table S5 Amino acid sequence comparisons between homeologs in Gossypium hirsutum

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