Dissecting Leaf Dorsiventral Polarity In Maize

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Leaves are produced from the flank of the shoot apical meristem and are asymmetric from inception. Mutations perturbing dorsiventral cell-fate acquisition may cause the formation of radially symmetric leaves that lack adaxial/abaxial (upper/lower) polarity. However, mutations in the maize (Zea mays) gene ragged seedling2 (rgd2) condition radial leaves that maintain dorsiventral polarity. A positional cloning approach revealed that rgd2 encodes an ARGONAUTE7 (AGO7)like protein. In Arabidopsis, AGO7 forms a complex with microRNA390 (miR390) to cleave the non-protein coding tas3 transcript. The cleavage product is stabilized and converted to double-stranded RNA (dsRNA). The dsRNA is then cleaved into 21nucleotide long fragments termed ta-siARFs. The ta-siARFs then target transcripts of the putative abaxial identify factor auxin response factor3 (arf3) for degradation by AGO7. Previous studies with the abaxialized maize mutant leafbladeless1 (lbl1) implicated a role for ta-siARF function in establishing dorsiventral polarity. Although RGD2 and LBL1 function are required for reduced accumulation of arf3a, arf3a transcripts are spatially restricted to the abaxial regions of leaf primordia independently of ta-siARF biogenesis. Additionally, RGD2 function is required to regulate the accumulation and localization the abaxial identity factor miR166 in shoot apices. These data suggest combined over-accumulation of arf3a and miR166 are insufficient to confer abaxialized leaf phenotypes in maize. A revised model for the role of small RNAs during dorsiventral patterning of maize leaves is discussed. A maize gene of unknown function (AC211276.4_FG008; punctate vascular expression1 (pve1)) was previously identified as being markedly down-regulated in rgd2 mutants. The coding region and putative gene structure of pve1-R is described. Quantitative reverse transcriptase-PCR confirmed that pve1 accumulates to lower levels in rgd2 apices and showed its accumulation is diminished in lbl1 mutants, suggesting pve1 regulation is downstream of ta-siARF biogenesis. Finally, to investigate potential downstream effects of ta-siARF biogenesis on a genomic scale, laser microdissection was coupled with Illumina RNA sequencing to search for differential regulation of predicted target genes that may be controlled by miR390, miR166 and/or ta-siARF. This research furthers our understanding of leaf development in maize.

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