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Genome-wide analysis of leafbladeless1-regulated and phased small RNAs underscores the importance of the TAS3 ta-siRNA pathway to maize development.

Dotto MC, Petsch KA, Aukerman MJ, Beatty M, Hammell M, Timmermans MC - PLoS Genet. (2014)

Bottom Line: Interestingly, in contrast to data from other plant species, we found no evidence for the existence of phased siRNAs generated via the one-hit model.The phenotypes of Arabidopsis and Medicago ta-siRNA mutants, while strikingly different, likewise result from misexpression of the tasiR-ARF target ARF3.Instead, we propose that divergence in the gene networks downstream of the ARF3 transcription factors or the spatiotemporal pattern during leaf development in which these proteins act constitute key factors underlying the distinct contributions of the ta-siRNA pathway to development in maize, Arabidopsis, and possibly other plant species as well.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.

ABSTRACT
Maize leafbladeless1 (lbl1) encodes a key component in the trans-acting short-interfering RNA (ta-siRNA) biogenesis pathway. Correlated with a great diversity in ta-siRNAs and the targets they regulate, the phenotypes conditioned by mutants perturbing this small RNA pathway vary extensively across species. Mutations in lbl1 result in severe developmental defects, giving rise to plants with radial, abaxialized leaves. To investigate the basis for this phenotype, we compared the small RNA content between wild-type and lbl1 seedling apices. We show that LBL1 affects the accumulation of small RNAs in all major classes, and reveal unexpected crosstalk between ta-siRNA biogenesis and other small RNA pathways regulating transposons. Interestingly, in contrast to data from other plant species, we found no evidence for the existence of phased siRNAs generated via the one-hit model. Our analysis identified nine TAS loci, all belonging to the conserved TAS3 family. Information from RNA deep sequencing and PARE analyses identified the tasiR-ARFs as the major functional ta-siRNAs in the maize vegetative apex where they regulate expression of AUXIN RESPONSE FACTOR3 (ARF3) homologs. Plants expressing a tasiR-ARF insensitive arf3a transgene recapitulate the phenotype of lbl1, providing direct evidence that deregulation of ARF3 transcription factors underlies the developmental defects of maize ta-siRNA biogenesis mutants. The phenotypes of Arabidopsis and Medicago ta-siRNA mutants, while strikingly different, likewise result from misexpression of the tasiR-ARF target ARF3. Our data indicate that diversity in TAS pathways and their targets cannot fully account for the phenotypic differences conditioned by ta-siRNA biogenesis mutants across plant species. Instead, we propose that divergence in the gene networks downstream of the ARF3 transcription factors or the spatiotemporal pattern during leaf development in which these proteins act constitute key factors underlying the distinct contributions of the ta-siRNA pathway to development in maize, Arabidopsis, and possibly other plant species as well.

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Expression of a tasiR-ARF insensitive arf3a transgene recapitulates the lbl1 phenotype.(A) Mutations were introduced in both arf3a tasiR-ARF binding sites that reduce complementarity without affecting the amino acid sequence. Sequences of the tasiR-ARF target sites in arf3a and arf3a-m with base pairing to the tas3d D7(+) ta-siRNA, are shown. (B-E) Expression of arf3a-m from the arf3a regulatory regions produces a seedling phenotype with similarities to weak alleles of lbl1[55]. arf3a:arf3a-m seedlings develop thread-like abaxialized leaves (arrowhead in B) and half leaves (C), as well as leaves with ectopic blade outgrowths surrounding abaxialized sectors on the upper leaf surface radial (D, E). (F) Analogous to the half leaves present in weak lbl1 mutants, half leaves on arf3a:arf3a-m transgenic plants show a partial radial arrangement of vascular bundles around the midvein (arrow). (G) Compared to the near wild-type phenotype of arf3a:arf3a transgenic plants (left) the stature of arf3a:arf3a-m plants (right) is severely reduced. (H-I) Such plants display defects in vegetative and reproductive development. Arrows in (H) mark half leaves, and in (I), a strongly reduced and sterile male inflorescence. Ad, Adaxial; Ab, Abaxial; MV, midvein. Scale bar = 100 µm.
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pgen-1004826-g006: Expression of a tasiR-ARF insensitive arf3a transgene recapitulates the lbl1 phenotype.(A) Mutations were introduced in both arf3a tasiR-ARF binding sites that reduce complementarity without affecting the amino acid sequence. Sequences of the tasiR-ARF target sites in arf3a and arf3a-m with base pairing to the tas3d D7(+) ta-siRNA, are shown. (B-E) Expression of arf3a-m from the arf3a regulatory regions produces a seedling phenotype with similarities to weak alleles of lbl1[55]. arf3a:arf3a-m seedlings develop thread-like abaxialized leaves (arrowhead in B) and half leaves (C), as well as leaves with ectopic blade outgrowths surrounding abaxialized sectors on the upper leaf surface radial (D, E). (F) Analogous to the half leaves present in weak lbl1 mutants, half leaves on arf3a:arf3a-m transgenic plants show a partial radial arrangement of vascular bundles around the midvein (arrow). (G) Compared to the near wild-type phenotype of arf3a:arf3a transgenic plants (left) the stature of arf3a:arf3a-m plants (right) is severely reduced. (H-I) Such plants display defects in vegetative and reproductive development. Arrows in (H) mark half leaves, and in (I), a strongly reduced and sterile male inflorescence. Ad, Adaxial; Ab, Abaxial; MV, midvein. Scale bar = 100 µm.

Mentions: The fact that mutations in lbl1 and rgd2 condition comparable adaxial-abaxial leaf polarity defects supports the finding that LBL1 contributes to development through the biogenesis of TAS3-derived ta-siRNAs. To confirm that this requirement lies specifically in the production of tasiR-ARFs and the downstream regulation of the ARF3 abaxial determinants, we generated transgenic lines that express either a native or tasiR-ARF-resistant version of arf3a. The latter (arf3a-m) harbors silent mutations in each of the two tasiR-ARF target sites (Fig. 6A). Most plants expressing the native arf3a cDNA from the endogenous arf3a regulatory regions (arf3a:arf3a) are phenotypically normal. Only occasionally do such plants develop slightly narrower leaves and these become less evident as the plant matures. In contrast, transgenic plants expressing the tasiR-ARF insensitive arf3a:arf3a-m transgene displayed pronounced vegetative and reproductive abnormalities (Fig. 6B-I). arf3a:arf3a-m seedlings resemble seedlings homozygous for the weak lbl1-ref allele, and develop half leaves and thread-like abaxialized leaves, as well as leaves with ectopic blade outgrowths surrounding abaxialized sectors on the upper leaf surface [55]; (Fig. 6B-F). However, as such plants matured, their phenotypes became progressively more severe and resembled the phenotypes of lbl1-rgd1. Mature arf3a:arf3a-m plants have a dramatically reduced stature (Fig. 6G-H), and exhibit developmental abnormalities in both male and female inflorescences that result in complete sterility (Fig. 6I). Thus, misregulation of arf3a alone is sufficient to recapitulate phenotypic defects seen in lbl1 and rgd2 mutants [17], [18], [55]. The initial milder phenotypes of arf3a:arf3a-m plants are potentially explained by the fact that only arf3a expression is affected in these plants, as ARF3 has been shown to condition dose-dependent phenotypes in Arabidopsis[10]. These data demonstrate a conserved role for the ARF3 transcription factors in promoting abaxial fate, and confirm our findings from genome-wide analysis that LBL1 contributes to development through the biogenesis of TAS3-derived tasiR-ARFs and the regulation of their ARF3 targets. Moreover, the severe defects conditioned by mutations in lbl1 and rgd2 thus underscore the importance of the tasiR-ARF ARF3 regulatory module to maize development.


Genome-wide analysis of leafbladeless1-regulated and phased small RNAs underscores the importance of the TAS3 ta-siRNA pathway to maize development.

Dotto MC, Petsch KA, Aukerman MJ, Beatty M, Hammell M, Timmermans MC - PLoS Genet. (2014)

Expression of a tasiR-ARF insensitive arf3a transgene recapitulates the lbl1 phenotype.(A) Mutations were introduced in both arf3a tasiR-ARF binding sites that reduce complementarity without affecting the amino acid sequence. Sequences of the tasiR-ARF target sites in arf3a and arf3a-m with base pairing to the tas3d D7(+) ta-siRNA, are shown. (B-E) Expression of arf3a-m from the arf3a regulatory regions produces a seedling phenotype with similarities to weak alleles of lbl1[55]. arf3a:arf3a-m seedlings develop thread-like abaxialized leaves (arrowhead in B) and half leaves (C), as well as leaves with ectopic blade outgrowths surrounding abaxialized sectors on the upper leaf surface radial (D, E). (F) Analogous to the half leaves present in weak lbl1 mutants, half leaves on arf3a:arf3a-m transgenic plants show a partial radial arrangement of vascular bundles around the midvein (arrow). (G) Compared to the near wild-type phenotype of arf3a:arf3a transgenic plants (left) the stature of arf3a:arf3a-m plants (right) is severely reduced. (H-I) Such plants display defects in vegetative and reproductive development. Arrows in (H) mark half leaves, and in (I), a strongly reduced and sterile male inflorescence. Ad, Adaxial; Ab, Abaxial; MV, midvein. Scale bar = 100 µm.
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pgen-1004826-g006: Expression of a tasiR-ARF insensitive arf3a transgene recapitulates the lbl1 phenotype.(A) Mutations were introduced in both arf3a tasiR-ARF binding sites that reduce complementarity without affecting the amino acid sequence. Sequences of the tasiR-ARF target sites in arf3a and arf3a-m with base pairing to the tas3d D7(+) ta-siRNA, are shown. (B-E) Expression of arf3a-m from the arf3a regulatory regions produces a seedling phenotype with similarities to weak alleles of lbl1[55]. arf3a:arf3a-m seedlings develop thread-like abaxialized leaves (arrowhead in B) and half leaves (C), as well as leaves with ectopic blade outgrowths surrounding abaxialized sectors on the upper leaf surface radial (D, E). (F) Analogous to the half leaves present in weak lbl1 mutants, half leaves on arf3a:arf3a-m transgenic plants show a partial radial arrangement of vascular bundles around the midvein (arrow). (G) Compared to the near wild-type phenotype of arf3a:arf3a transgenic plants (left) the stature of arf3a:arf3a-m plants (right) is severely reduced. (H-I) Such plants display defects in vegetative and reproductive development. Arrows in (H) mark half leaves, and in (I), a strongly reduced and sterile male inflorescence. Ad, Adaxial; Ab, Abaxial; MV, midvein. Scale bar = 100 µm.
Mentions: The fact that mutations in lbl1 and rgd2 condition comparable adaxial-abaxial leaf polarity defects supports the finding that LBL1 contributes to development through the biogenesis of TAS3-derived ta-siRNAs. To confirm that this requirement lies specifically in the production of tasiR-ARFs and the downstream regulation of the ARF3 abaxial determinants, we generated transgenic lines that express either a native or tasiR-ARF-resistant version of arf3a. The latter (arf3a-m) harbors silent mutations in each of the two tasiR-ARF target sites (Fig. 6A). Most plants expressing the native arf3a cDNA from the endogenous arf3a regulatory regions (arf3a:arf3a) are phenotypically normal. Only occasionally do such plants develop slightly narrower leaves and these become less evident as the plant matures. In contrast, transgenic plants expressing the tasiR-ARF insensitive arf3a:arf3a-m transgene displayed pronounced vegetative and reproductive abnormalities (Fig. 6B-I). arf3a:arf3a-m seedlings resemble seedlings homozygous for the weak lbl1-ref allele, and develop half leaves and thread-like abaxialized leaves, as well as leaves with ectopic blade outgrowths surrounding abaxialized sectors on the upper leaf surface [55]; (Fig. 6B-F). However, as such plants matured, their phenotypes became progressively more severe and resembled the phenotypes of lbl1-rgd1. Mature arf3a:arf3a-m plants have a dramatically reduced stature (Fig. 6G-H), and exhibit developmental abnormalities in both male and female inflorescences that result in complete sterility (Fig. 6I). Thus, misregulation of arf3a alone is sufficient to recapitulate phenotypic defects seen in lbl1 and rgd2 mutants [17], [18], [55]. The initial milder phenotypes of arf3a:arf3a-m plants are potentially explained by the fact that only arf3a expression is affected in these plants, as ARF3 has been shown to condition dose-dependent phenotypes in Arabidopsis[10]. These data demonstrate a conserved role for the ARF3 transcription factors in promoting abaxial fate, and confirm our findings from genome-wide analysis that LBL1 contributes to development through the biogenesis of TAS3-derived tasiR-ARFs and the regulation of their ARF3 targets. Moreover, the severe defects conditioned by mutations in lbl1 and rgd2 thus underscore the importance of the tasiR-ARF ARF3 regulatory module to maize development.

Bottom Line: Interestingly, in contrast to data from other plant species, we found no evidence for the existence of phased siRNAs generated via the one-hit model.The phenotypes of Arabidopsis and Medicago ta-siRNA mutants, while strikingly different, likewise result from misexpression of the tasiR-ARF target ARF3.Instead, we propose that divergence in the gene networks downstream of the ARF3 transcription factors or the spatiotemporal pattern during leaf development in which these proteins act constitute key factors underlying the distinct contributions of the ta-siRNA pathway to development in maize, Arabidopsis, and possibly other plant species as well.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.

ABSTRACT
Maize leafbladeless1 (lbl1) encodes a key component in the trans-acting short-interfering RNA (ta-siRNA) biogenesis pathway. Correlated with a great diversity in ta-siRNAs and the targets they regulate, the phenotypes conditioned by mutants perturbing this small RNA pathway vary extensively across species. Mutations in lbl1 result in severe developmental defects, giving rise to plants with radial, abaxialized leaves. To investigate the basis for this phenotype, we compared the small RNA content between wild-type and lbl1 seedling apices. We show that LBL1 affects the accumulation of small RNAs in all major classes, and reveal unexpected crosstalk between ta-siRNA biogenesis and other small RNA pathways regulating transposons. Interestingly, in contrast to data from other plant species, we found no evidence for the existence of phased siRNAs generated via the one-hit model. Our analysis identified nine TAS loci, all belonging to the conserved TAS3 family. Information from RNA deep sequencing and PARE analyses identified the tasiR-ARFs as the major functional ta-siRNAs in the maize vegetative apex where they regulate expression of AUXIN RESPONSE FACTOR3 (ARF3) homologs. Plants expressing a tasiR-ARF insensitive arf3a transgene recapitulate the phenotype of lbl1, providing direct evidence that deregulation of ARF3 transcription factors underlies the developmental defects of maize ta-siRNA biogenesis mutants. The phenotypes of Arabidopsis and Medicago ta-siRNA mutants, while strikingly different, likewise result from misexpression of the tasiR-ARF target ARF3. Our data indicate that diversity in TAS pathways and their targets cannot fully account for the phenotypic differences conditioned by ta-siRNA biogenesis mutants across plant species. Instead, we propose that divergence in the gene networks downstream of the ARF3 transcription factors or the spatiotemporal pattern during leaf development in which these proteins act constitute key factors underlying the distinct contributions of the ta-siRNA pathway to development in maize, Arabidopsis, and possibly other plant species as well.

Show MeSH
Related in: MedlinePlus