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

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.

Genic and nongenic contributions to natural variation of quantitative traits in maize.

The complex genomes of many economically important crops present tremendous challenges to understand the genetic control of many quantitative traits with great importance in crop production, adaptation, and evolution. Advances in genomic technology need to be integrated with strategic genetic design and novel perspectives to break new ground. Complementary to individual-gene-targeted research, which remains challenging, a global assessment of the genomic distribution of trait-associated SNPs (TASs) discovered from genome scans of quantitative traits can provide insights into the genetic architecture and contribute to the design of future studies. Here we report the first systematic tabulation of the relative contribution of different genomic regions to quantitative trait variation in maize. We found that TASs were enriched in the nongenic regions, particularly within a 5-kb window upstream of genes, which highlights the importance of polymorphisms regulating gene expression in shaping the natural variation. Consistent with these findings, TASs collectively explained 44%-59% of the total phenotypic variation across maize quantitative traits, and on average, 79% of the explained variation could be attributed to TASs located in genes or within 5 kb upstream of genes, which together comprise only 13% of the genome. Our findings suggest that efficient, cost-effective genome-wide association studies (GWAS) in species with complex genomes can focus on genic and promoter regions.

Targeted forward mutagenesis by transitive RNAi.

A novel technique is described that targets specific populations of transcripts for homology-based gene silencing using transitive RNAi. This approach is designed to target a subset of the transcriptome in order to identify genes involved in a particular localized process, such as photosynthesis. As a proof-of-concept approach, mesophyll cells from Arabidopsis thaliana were laser-microdissected from whole leaves to generate a focused cDNA library that was bi-directionally cloned into a transitive RNAi vector that had been designed to induce silencing of homologous, endogenous genes. Approximately 15% of the transformant plants identified from both sense and antisense libraries exhibited visible phenotypes indicative of photosynthetic defects. Amplification from the genome and sequencing of cDNA inserts identified candidate genes underlying the phenotypes. For 10 of 11 such mutants, re-transformation with an RNAi construct corresponding to the candidate gene recapitulated the original mutant phenotype, and reduction of corresponding endogene transcripts was confirmed. In addition, one of the re-transformed transgenes also silenced transcripts of closely related family members, thereby demonstrating the utility of this approach for mutagenesis of redundant gene functions. Preliminary results using tissue-specific transitive RNAi forward mutagenesis of the Arabidopsis vegetative shoot apical meristem demonstrate the broad applicability of this forward mutagenesis technique for a variety of plant cell types.