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Combined haploinsufficiency and purifying selection drive retention of RPL36a paralogs in Arabidopsis.

Casanova-Sáez R, Candela H, Micol JL - Sci Rep (2014)

Bottom Line: RPL36aA was found expressed at a higher level than API2 in the wild type.Their double mutant combinations with asymmetric leaves2-1 (as2-1) caused leaf polarity defects that were stronger in rpl36aa as2-1 than in api2 as2-1.Our results highlight the role of combined haploinsufficiency and purifying selection in the retention of these paralogous genes in the Arabidopsis genome.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Alicante, Spain.

ABSTRACT
Whole-genome duplication events have driven to a large degree the evolution of angiosperm genomes. Although the majority of redundant gene copies after a genome duplication are lost, subfunctionalization or gene balance account for the retention of gene copies. The Arabidopsis 80S ribosome represents an excellent model to test the gene balance hypothesis as it consists of 80 ribosomal proteins, all of them encoded by genes belonging to small gene families. Here, we present the isolation of mutant alleles of the APICULATA2 (API2) and RPL36aA paralogous genes, which encode identical ribosomal proteins but share a similarity of 89% in their coding sequences. RPL36aA was found expressed at a higher level than API2 in the wild type. The loss-of-function api2 and rpl36aa mutations are recessive and affect leaf development in a similar way. Their double mutant combinations with asymmetric leaves2-1 (as2-1) caused leaf polarity defects that were stronger in rpl36aa as2-1 than in api2 as2-1. Our results highlight the role of combined haploinsufficiency and purifying selection in the retention of these paralogous genes in the Arabidopsis genome.

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Visualization of API2pro:GUS and RPL36aApro:GUS transgene activity in the Col-0 background.(a), (m) Seedlings and (b), (n) cotyledons. (c–f), (o–r) Vegetative leaves from the (c), (o) first, (d), (p) third (e), (q) fifth and (f), (r) seventh nodes. (g), (s) Inflorescences. (h), (t) Immature and (i), (u) mature flowers. (j), (v) Immature siliques, (k), (w) root tips and (l), (x) mature siliques. Pictures were taken (a), (m) 6, (b–f), (k), (n–r), (w) 14 and (g–j), (l), (s–v), (x) 35 das. Scale bars indicate (a), (c–e), (g), (j), (l), (m), (o–q), (s), (v), (x) 1 mm, (b), (f), (i), (n), (r), (u) 500 μm, (h), (t) 200 μm and (k), (w) 100 μm.
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f5: Visualization of API2pro:GUS and RPL36aApro:GUS transgene activity in the Col-0 background.(a), (m) Seedlings and (b), (n) cotyledons. (c–f), (o–r) Vegetative leaves from the (c), (o) first, (d), (p) third (e), (q) fifth and (f), (r) seventh nodes. (g), (s) Inflorescences. (h), (t) Immature and (i), (u) mature flowers. (j), (v) Immature siliques, (k), (w) root tips and (l), (x) mature siliques. Pictures were taken (a), (m) 6, (b–f), (k), (n–r), (w) 14 and (g–j), (l), (s–v), (x) 35 das. Scale bars indicate (a), (c–e), (g), (j), (l), (m), (o–q), (s), (v), (x) 1 mm, (b), (f), (i), (n), (r), (u) 500 μm, (h), (t) 200 μm and (k), (w) 100 μm.

Mentions: As mentioned above, the non-allelic non-complementation between API2 and RPL36aA is best understood when there is at least some overlap of expression in the same cells. To experimentally demonstrate this, we fused the promoter regions of both genes to the GUS reporter gene. GUS staining of 5 API2pro:GUS and 5 RPL36aApro:GUS independent transgenic lines in the Col-0 background uncovered an identical expression pattern for both genes. Both constructs were ubiquitously expressed, with particularly intense GUS staining in young and actively proliferating tissues, such as those of developing leaves, floral buds and root apices (Figure 5). Interestingly, the relative intensities of the GUS staining matched our qRT-PCR results (Figure 3), with the RPL36aA promoter conferring higher expression levels in wild-type vegetative leaves than the API2 promoter.


Combined haploinsufficiency and purifying selection drive retention of RPL36a paralogs in Arabidopsis.

Casanova-Sáez R, Candela H, Micol JL - Sci Rep (2014)

Visualization of API2pro:GUS and RPL36aApro:GUS transgene activity in the Col-0 background.(a), (m) Seedlings and (b), (n) cotyledons. (c–f), (o–r) Vegetative leaves from the (c), (o) first, (d), (p) third (e), (q) fifth and (f), (r) seventh nodes. (g), (s) Inflorescences. (h), (t) Immature and (i), (u) mature flowers. (j), (v) Immature siliques, (k), (w) root tips and (l), (x) mature siliques. Pictures were taken (a), (m) 6, (b–f), (k), (n–r), (w) 14 and (g–j), (l), (s–v), (x) 35 das. Scale bars indicate (a), (c–e), (g), (j), (l), (m), (o–q), (s), (v), (x) 1 mm, (b), (f), (i), (n), (r), (u) 500 μm, (h), (t) 200 μm and (k), (w) 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927210&req=5

f5: Visualization of API2pro:GUS and RPL36aApro:GUS transgene activity in the Col-0 background.(a), (m) Seedlings and (b), (n) cotyledons. (c–f), (o–r) Vegetative leaves from the (c), (o) first, (d), (p) third (e), (q) fifth and (f), (r) seventh nodes. (g), (s) Inflorescences. (h), (t) Immature and (i), (u) mature flowers. (j), (v) Immature siliques, (k), (w) root tips and (l), (x) mature siliques. Pictures were taken (a), (m) 6, (b–f), (k), (n–r), (w) 14 and (g–j), (l), (s–v), (x) 35 das. Scale bars indicate (a), (c–e), (g), (j), (l), (m), (o–q), (s), (v), (x) 1 mm, (b), (f), (i), (n), (r), (u) 500 μm, (h), (t) 200 μm and (k), (w) 100 μm.
Mentions: As mentioned above, the non-allelic non-complementation between API2 and RPL36aA is best understood when there is at least some overlap of expression in the same cells. To experimentally demonstrate this, we fused the promoter regions of both genes to the GUS reporter gene. GUS staining of 5 API2pro:GUS and 5 RPL36aApro:GUS independent transgenic lines in the Col-0 background uncovered an identical expression pattern for both genes. Both constructs were ubiquitously expressed, with particularly intense GUS staining in young and actively proliferating tissues, such as those of developing leaves, floral buds and root apices (Figure 5). Interestingly, the relative intensities of the GUS staining matched our qRT-PCR results (Figure 3), with the RPL36aA promoter conferring higher expression levels in wild-type vegetative leaves than the API2 promoter.

Bottom Line: RPL36aA was found expressed at a higher level than API2 in the wild type.Their double mutant combinations with asymmetric leaves2-1 (as2-1) caused leaf polarity defects that were stronger in rpl36aa as2-1 than in api2 as2-1.Our results highlight the role of combined haploinsufficiency and purifying selection in the retention of these paralogous genes in the Arabidopsis genome.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Alicante, Spain.

ABSTRACT
Whole-genome duplication events have driven to a large degree the evolution of angiosperm genomes. Although the majority of redundant gene copies after a genome duplication are lost, subfunctionalization or gene balance account for the retention of gene copies. The Arabidopsis 80S ribosome represents an excellent model to test the gene balance hypothesis as it consists of 80 ribosomal proteins, all of them encoded by genes belonging to small gene families. Here, we present the isolation of mutant alleles of the APICULATA2 (API2) and RPL36aA paralogous genes, which encode identical ribosomal proteins but share a similarity of 89% in their coding sequences. RPL36aA was found expressed at a higher level than API2 in the wild type. The loss-of-function api2 and rpl36aa mutations are recessive and affect leaf development in a similar way. Their double mutant combinations with asymmetric leaves2-1 (as2-1) caused leaf polarity defects that were stronger in rpl36aa as2-1 than in api2 as2-1. Our results highlight the role of combined haploinsufficiency and purifying selection in the retention of these paralogous genes in the Arabidopsis genome.

Show MeSH