mop2 Is Required for Multiple Silencing Processes Table 2. Mop2-1/Mop2-1 prevents establishment of p1 paramutationa. Mop2-1 genotypeb Mop2-1/+ Mop2-1/Mop2-1c Red pericarp Orange pericarp Patterned or colorless pericarp Total number of ears 5 1 2 24 25 7 a Paramutation at p1 is observed in the pericarp (seed coat), where the paramutable P1-rr allele specifies dark red pericarp pigment. When paramutated, by exposure to paramutagenic P1-rr’ and the P1.2b::GUS transgene (transgenic event P2P147-37, [13]), pigmentation of paramutable P1-rr is reduced to colorless or light patterned pericarp. Details of experiment are in Figure S4. b Molecular genotyping was used to determine the Mop2-1 genotype. c The negative pleiotropic developmental phenotypes in Mop2-1 homozygotes result in frequent abortion of ears resulting in only a small number of Mop2-1/Mop2-1 homozygous ears that can be pollinated and produce sufficient seed to score the p1 pigment in mature ears. doi:10.1371/journal.pgen.1000725.t002 transgene siRNAs. One possibility is that Mop2-1 can act downstream of siRNA production to relieve sterility. As Pol-V in Arabidopsis acts downstream of siRNA biogenesis, this hypothesis is consistent with Mop2-1 also disrupting a Pol-V-like complex in maize. Alternative explanations are presented in the Discussion. Mop2-1 plants exhibit deleterious pleiotropic developmental phenotypes Mutations in Arabidopsis Pol-IV/Pol-V complexes have not been reported to display major developmental phenotypes, except for a delay in flowering [32,41]. In contrast, Mop2-1 displays a number of developmental phenotypes, but these are less dramatic and more variable than the developmental phenotypes observed with mutations in the rmr6 gene [21], which encodes the large subunit of a Pol-IV like complex in maize [31]. When propagating the Mop2-1 mutation through numerous generations, a number of phenotypes were routinely observed including reduced transmis- sion, altered flowering time and abnormal developmental phenotypes. This was similar to phenotypes of mop1 mutations [20], but different from rmr6 mutations, as the aberrant phenotypes were not observed in every plant carrying the Mop2-1 or mop1 mutations, but were observed in all plants homozygous for rmr6 mutations [21]. The types of abnormal morphological and development phenotypes that we observed in stocks segregating Mop2-1 were reduced plant height, skinny plant stature, tassel seed, failure to develop an ear, and poor seed set in ears that did develop. These phenotypes are variable and they occur in both heterozygous or homozygous Mop2-1 plants, but they are more frequent and more severe in homozygous Mop2-1/Mop2-1 plants. For example, in one experiment Mop2-1/Mop2-1 plants were on average 12 cm shorter and flowered 4.2 days later then heterozygous siblings, and 22% of Mop2-1/Mop2-1 versus 10% of Mop2-1/+ siblings failed to differentiate ears. These negative pleiotropic phenotypes on plant health and reproduction influenced how the stocks could be maintained such that self pollinations were rarely successful and large numbers of crosses were required to obtain sufficient numbers of homozygous Mop2-1 plants with mature ears and reasonable seed set for our experiments. To determine the transmission of Mop2-1, 231 plants were genotyped for the Mop2-1 mutation from a family that would be expected to segregate equal numbers of Mop2-1 homozygous and heterozygous plants if there was no reduction in Mop2-1 transmission. This experiment revealed that the number of Mop2-1 homozygotes was reduced (39%) (x = 211.2, P = 0.0001). To test whether this was due to reduced transmission or reduced germination frequency, we genotyped 94 seeds directly and observed a similar reduced number of Mop2-1 homozygotes, only 39% instead of the 50% expected for normal transmission. This strongly suggested reduced transmission was contributing to the reduced number of Mop2-1 homozygotes. The observation of developmental phenotypes with Mop2-1 differs from the recessive loss-of-function truncation mutations, which lack developmental phenotypes (Stonaker et al., this issue). This is not simply because Mop2-1 is dominant and the mutations isolated in the Hollick lab are recessive as we also see developmental phenotypes with our recessive mop2-2 allele. Although we haven’t grown mop2-2 plants for as many generations as Mop2-1 plants, homozygous mop2-2 plants are very skinny and rarely set seed. One possibility is that our two missense mutations are having broader effects relative to the mutations isolated in the Hollick lab. Another possibility is that the genetic background of the mutants is suppressing developmental phenotypes, or the genetic background of our mutants is enhancing developmental phenotypes. It has long been known that different genetic backgrounds can enhance or suppress developmental phenotypes in maize [71]. A third possibility is that the more extreme environmental growth conditions in Arizona relative to Northern California are enhancing the developmental phenotypes. Discussion Maize contains multiple nrpd2/e2 genes with overlapping expression patterns Our results demonstrate that mop2, a key gene involved in paramutation at multiple loci, encodes a protein closely related to the second largest subunit of the plant-specific RNA polymerase complexes, Pol-IV/Pol-V, first described in Arabidopsis. Unlike Arabidopsis, which encodes only one functional protein (NRPD2/ E2), which is in both the Pol-IV and Pol-V complexes [32,41,42,58], maize encodes three closely related genes. These three genes are likely to encode functional proteins as they are full length, have all of the polymerase domains conserved and are expressed in multiple tissues. This observation suggests that multiple Pol-IV/Pol-V-like complexes and potentially even novel complexes may exist in maize. Potentially these could have diversified to function at different loci, different develop- mental stages, or under different environmental stresses. Multiple Pol-IV/Pol-V related complexes could confer greater complexity and epigenetic regulatory capacity to maize as compared to Arabidopsis. Our expression analyzes revealed that all three maize genes are widely expressed in multiple organs and tissues, similar to that of NRPD2/E2 in Arabidopsis [42,70]. All three maize genes are most highly expressed in maize reproductive organs such as tassels and immature ears. The most diverged gene, nrpd2/e2c, is the major gene expressed in pollen and it is also more highly expressed in PLoS Genetics | www.plosgenetics.org 11 November 2009 | Volume 5 | Issue 11 | e1000725