Genome-Wide Analysis of leafbladeless1-Regulated and Phased Small RNAs Underscores the Importance of the TAS3 ta-siRNA Pathway to Maize Development

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.

The Sussex signal: insights into leaf dorsiventrality

“…For instance, Antirrhinum phan mutants show clear adaxial-abaxial polarity phenotypes, as do tobacco and tomato plants harboring mutations in phan orthologs, but rough sheath2 from maize and as1 from Arabidopsis do not (Timmermans et al, 1999;Tsiantis et al, 1999;Byrne et al, 2000;Kim et al, 2003a;McHale and Koning, 2004). Likewise, mutations affecting tasiARF biogenesis condition a strong abaxializing phenotype in maize, rice and tomato, but cause only subtle polarity defects in Arabidopsis (Nogueira et al, 2007;Nagasaki et al, 2007;Chitwood et al, 2009;Douglas et al, 2010;Yifhar et al, 2012;Dotto et al, 2014). The latter difference may be explained, in part, by variation in the spatiotemporal expression of pathway components across species; tasiARF, for example, acts in the incipient primordium in maize and rice but during later stages in Arabidopsis leaf development (see Husbands et al, 2009).…”

Novel DICER-LIKE1 siRNAs Bypass the Requirement for DICER-LIKE4 in Maize Development

“…Even in a single plant, the phenotype of individual leaves varies widely, ranging from having a near wild-type morphology, to occasionally appearing thread-like (Supplemental Figures 3A to 3E). As seen for lbl1 and rgd2 (Timmermans et al, 1998;Dotto et al, 2014), the threadlike dcl4 leaves lack adaxial features, such as macrohairs and bulliform cells, and show a radial symmetry in cross sections with photosynthetic and epidermal cells surrounding a central vascular bundle (Supplemental Figures 3F and 3I). However, this retains adaxial-abaxial polarity, as xylem forms at the adaxial pole and phloem at the abaxial pole.…”

“…A recent study revealed that the five maize ARF3 homologs (arf3a-e) are the only ta-siRNA targets with a role in shoot development (Dotto et al, 2014). A positive correlation between ARF3 dosage and phenotype severity has been observed in Arabidopsis and tomato (Fahlgren et al, 2006;Hunter et al, 2006;Yifhar et al, 2012), further supporting the idea that in comparison to other ta-siRNA biogenesis mutants, dcl4 affects expression of the arf3 targets to a lesser extent.…”

“…Such mutants are also severely stunted and fail to flower. The polarity phenotype conditioned by the rgd2-Ds1 allele, which carries a Ds transposon insertion in the essential PIWI domain, is even more severe and leads to early seedling lethality or occasionally a shootless phenotype (Figures 1C and 1E;Dotto et al, 2014). The less severe phenotype of lbl1-rgd1 mutants may be attributed to the fact that this allele still retains some activity.…”

“…To test this possibility directly, we examined the transcript levels for arf3a-e in dcl4 shoot apices. Considering that the arf3 targets are cleaved at one or both of their tasiR-ARF target sites (Dotto et al, 2014), their expression is predicted to increase upon loss of tasiR-ARF function. Indeed, arf3 transcript levels are increased in lbl1-rgd1 and rgd2-Ds1 shoot apices; likewise, arf3b, arf3c, and arf3e transcripts are upregulated approximately 2-fold in dcl4-3 mutants (Figure 2A).…”