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.

Figure 1. lbl1 affects small RNA biogenesis at select loci.

(A) Compared to wild-type, lbl1-rgd1 mutant seedlings show a reduced stature and develop radial, abaxialized leaves. (B) lbl1 has a relatively subtle effect on the overall small RNA population; in three independent biological replicates 79, 172, and 209 loci generate 21-, 22-, or 24-nt small RNAs, respectively, that are significantly changed (q-value<0.05) at least 2-fold between wild-type and lbl1. Grey dots mark the relative normalized abundance of small RNAs not significantly changed between wild-type and lbl1. Colored dots correspond to significantly changed small RNAs: red, 21-nt; blue, 22-nt; green, 24-nt. (C) Low copy genic regions accumulating significantly fewer 21-nt (left), 22-nt (middle) and/or 24-nt (right) siRNAs in lbl1. Genes marked in bold and underlined show reduced levels for all three siRNA classes. Genes marked in bold show reduced 21- and 22-nt siRNAs. Note that the abundance of 21-nt siRNAs at these loci is generally substantially higher. Values are reported on a log scale and represent the mean normalized read counts (RPM) and standard deviation across three independent biological replicates. * adjusted p-value<0.05; ** adjusted p-value<0.01.

Figure 2. Organization of TAS3 loci generating phased 21-nt ta-siRNAs.

(A, B) Normalized read counts (RPM) for ta-siRNAs in phase with the 3′ miR390 cleavage site (top graphs) and for out of phase small RNAs (bottom graphs) are shown for tas3c (A) and tas3e (B). Red bars, miR390 binding sites; vertical dashed lines, the 21-nt register. Annotations of the TAS3 precursors are shown beneath, with alignments of miR390 to the tas3c and tas3e precursors and the number of PARE signatures in the small window (W_S) over the large window (W_L) at the 3′ miR390 target sites indicated. Black brackets; ta-siRNAs detected in the vegetative apex libraries; dashed lines, predicted ta-siRNAs not detected in our libraries; red brackets, miR390 binding sites; green bars, tasiR-ARFs. (C) Size distribution profiles for small RNAs derived from tas3c and tas3e showing most are 21-nt long. Values shown are the mean normalized read counts (RPM) and SD from three independent biological replicates. (D) Alignment of the eleven tasiR-ARFs processed from the maize TAS3 loci. Asterisks indicate 100% identity.

Figure 3. Regulation of ARF3 genes by ta-siRNAs in the maize shoot apex.

(A) Transcripts of the five maize ARF3 genes each contain two tasiR-ARF target sites spaced approximately 200 bp apart. PARE analysis indicates that both sites in arf3a, arf3d and arf3e transcripts are cleaved, whereas only one target site was validated for the arf3b and arf3c transcripts (asterisks). (B) Expression for four of the five ARF3 genes is significantly changed between wild-type and lbl1 in RNAseq and qRT-PCR analyses. Transcript levels determined by qRT-PCR (mean ± SD; n = 3) normalized to wild-type are shown (* p<0.05). The RPKM values for the ARF3 genes in wild-type and mutant samples are reported in S3 Dataset. (C) The distribution of PARE signatures at arf3d, showing the presence of high abundance signatures at the predicted tasiR-ARF target sites (arrows). Grey dots, PARE signatures and their abundance in reads per 15 million reads; red boxes, exons; white boxes, introns; light red boxes, UTRs.

Figure 4. LBL1 affects the accumulation of 21-, 22-, and 24-nt small RNAs.

(A) Annotation of loci that accumulate significantly fewer small RNAs in lbl1 than wild-type. (B) Annotation of loci that accumulate significantly more small RNAs in lbl1 compared to wild-type. Tables list the number of loci in each category and bar graphs show their relative frequencies.

Figure 5. rgd2-Ds1 mutants resemble the phenotype of lbl1-rgd1 and develop radial abaxialized leaves.

(A) rgd2-Ds1 results from a Ds insertion into the essential PIWI domain of AGO7. (B, C) Compared to wild-type (B), rgd2-Ds1 mutants (C) are severely stunted and develop very narrow and radial leaves (arrows). (D) Transverse section through a wild-type leaf blade. Bulliform cells mark the adaxial epidermis and within the vascular bundles, xylem differentiates adaxially to phloem. (E) Transverse section through a radial rgd2-Ds1 mutant leaf. The mutant leaf consists of an irregular vascular cylinder surrounded by concentric rings of bundle sheath, mesophyll cells and abaxial epidermis, resembling those observed in lbl1-rdg1 mutants . (F) Expression levels for the ARF3 genes are significantly increased in rgd2-Ds1. Transcript levels determined by qRT-PCR (mean ± SD; n = 3) normalized to wild-type are shown (* p<0.05, ** p-value<0.01). Ad, Adaxial; Ab, Abaxial; V, vein; BC, bulliform cells; BS, bundle sheath; M, mesophyll.

Figure 6. 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 . 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.